Bull. Agric. Coll. Vol. IV. Pl, T. ution in the full day- ol the addition of ammonium salts. The red lines to those treated with 0.59 ammonium chloride solution, um in the full day-light. . to shoots treated with half saturated gypsum s te shows the increase of organic bases (N. in phos. tungstic ppt.), especially the shoots of Pinus Thunbergii by FA@RATHE PEOPLE FOR EDVCATION FOR SCIENGE THE AMERICAN MUSEUM OF NATURAL HISTORY THE BULLETIN # ollege of yqriculture, TOKYO IMPERIAL UNIVERSITY, JAPAN. Vol. IV. 1Q00— 1902. Le | MM Aa, SWANN ut Yhoreni, AUTEN Ta = rie a) cay bh a a 6 rs id i ie = ‘ 7 eT : Wh i. i oT el ae ee Rages! hs ri ra pita tie! os" a ae) a= re), CONTENTS*OF VOLUME, The A contribution to the knowledge ofarginin. By U. Suzuxt. On the formation of arginin in coniferous plants. (Plates I-VI.) Vy U. Suzux:. : Can strontium and balium eepieee caleanem in eu aeneeanns ? (Plates. VII.) By U. Suzuxt. The chemical composition of the spores of Reyes orizae. By K. Aso. Die Unt erscheidunvemerkrante der winhtieeronel in can wachsenden Laubholzer. (Tafeln VIII—XVI.) VonS. Kawat. Die Gatiung Tilia in Japan. (Tafeln XVII—XVIII.) Von H. SHIRASAWA Be: Report of ipeesneations| ¢ en the Tmaliberes ARARE Panis a disease widely spread in Japan. By U. Suzuxr. Zur Physiologie des Bacillus pyocyaneus. Von O. Loew und Y. Kozat. eo Ueber die Bestimmung ven | Ere in aes Drecrerde. Von K. Brerer und K. Aso. Ueber die Aufsahme von Stickstoff el BRaenhereaare durch versehiedene Kulturpflanzen (8 Cerealien und 2 Cruciferen) in drei Vegetaticnspericden. Von K. BirELer und K. Aso... On the role of aie in Ge preparation of Commercial tea. By K. Aso. : < On the occurrence of organic iron comments in plants! By U. Suzuk. Investigations on 7 coulbercy- Aayaee wets” a Aineaee widely spread in Japan. By U. Suzuxt. Contributions to the physiological knoeedee of ine a plant. By U. Suzuxt. ole oe: On the localization of sneha in the ia lenwes. Bre We Suzoux1. Ueber die agente dee Niavise place eaten fea Mereahe kowsky’schen Mausetyphusbacillus. Von Y. Kozar. Ueber die Bildung des Pyocyanolysins unter vershiedenen Bedingungen. Von O. Loew und Y. Kosar. i303" es Ae eee Zz PaGcE Ueber die coagulirende Wirkung des Chloroforms. Von O. Borwaundeke ASO; ee B27. On Kaki-shibu, a fouitjaices in fecnieats epellcationnt in Tepe. By M. Tsukamoto. .. 120: Investigations on the digesta enzymes of some benidien: tera. By S. Sawamura. .. , Ons aye On the occurrence of cane sugar in pe peeds of Gingko biloba and Camellia theifera. By U. Suzuxt. Ng . 349. On the formation of asparagin in the ane ieinsiboa of piicate! By U. Suzuxr.. Ame se SET On the composition of the anne of Gere miler By U. SUZUKIe ee em) PAM ae 3 i7< Observations on the mauieeeey, Gerad Paulos (Schrumpf- Krankheit), a disease widely spread in Japan. By U. SAWS 566 sx 2350: On the influence of different ration of iene aaa magnesia on the dévelopment of plants. By K. Aso.. - eS eS ore To what extent should a soil be limed? By T. EURUTAR ee) ee Sie On the lime-factor for different crops. Remarks on the fore- going communications of Mr. Aso and Mr. Furuta. By ©, Ilomiy 5. 1 ee PA eso On the lime content of pienereamic Daraeeea By K. Aso... 387. On the amount of soluble albumin in different parts of plants. ByebieeUNOs en. | te “nS eOOO: Note on the enzymes aoa tae Srapanese spine one By T. TAKAHASHI... 3 0Se On the juice of the paeuaeernl of Musa Banine ‘Sieb. in winter time. ByS. Sawa. .. Bs 309> On ae volatile oil in the wood of Cryptomerat japonica. By A ISGOI Go c SOPs Tee RA OSs On ae poisonous nenion bq quinone. By T. Funes Be ie 5 CO. Are coffeine and antipyrin in high degree pomnaane for plants? By S Sawa. mn a ope Chit Tin Has urea any poisonous aeniente on nheenugaria? 2 By Si sine 413. On the poisonous action of potassium persulphate on plants. By S. Sawa..: .. mee mAb. roe 805% On Hamana-natto, a rind of Sonera cHeese! a, S. SAWA. .. 419. hi Ai wex<« ff & IGE IIE SOEME | OTR KH go ek do oe 1 Ho MIM IESG SME | HOTS A Contribution to the Knowledge of Arginin. BY U. Suzuki, Nogakushi. Lecturer in Agricultural Chemistry. The occurrence of organic bases in the seeds as well as in the germinating shoots of many plants has already been shown many years ago by E. Schulze and_ others. Thus in the year 1886 E. Schulze and Steiger found arginin in the cotyledons of germinating Lupinus, and at the same time, cholin was found by E. Schulze in the-seeds and in the etiolated shoots of Lupinus luteus, Soja hispida and Cucurbita pepo. Still afterwards he® found guanidin, cholin and betain in the germinating shoots of Vicia faba. But these bases were present in such small quantities that they appeared hardly worth a closer investigation. Inthe year 1894 S. G. Hedin™ discovered arginin in the decomposition products of horn and it was soon asserted by him that arginin is a normal constituent of the decomposition products of proteids. A still more interesting result was soon afterwards obtained by A. Kossel™ on his protamine, in the decomposition products of which he found, almost exclusively, the basic compounds, especially arginin. About the same time, E. Schulze also found a considerable quantity of organic bases (chiefly arginin) in the shoots of coniferous plants as well as in the decomposition products of the proteids prepared from the seeds of several plants, (Picea ex- celsa, Abies pectinata, and Pinus sylvestris), Further, it was (1) E. Schulze und FE. Steiger, Zeits. f, Physiol. Chem. Bd. XJ, S. 43. (2) E. Schulze. ibid. Bd. XI, S. 365. (3) E. Schulze. ibid. Bd. XVII, S. 193. (4) S. G, Hedin. ibid. Bd. XX, S. 186. (5) A. Kossel. ibid, Bd. XXII, S.176. A. Kossel and A. Mathews. ibid. Bd. XXII, S. 190. (6) E. Schulze, Zeits. f. Physiol. Chem. Bd. XXII, S. 435. and Bd, XXIV, S, 276. 2 U. SUZUKI. shown by Kutscher™ that arginin results from the artificial trypsin digestion of proteids; and recently Ellinger™ consider- ed it to be the source of putrescins. Thus, the physiological importance of organic bases and especially of arginin has been more and more clearly brought to light by these authors, and consequently its study is now considered as one of the most important subjects in the whole domain of physiological chemistry. Although arginin has so often been made the object of investigation, yet many questions are left unsettled; and, among others, its behaviour, formation and transformation in the plant cells, its relation to the regeneration of proteids and the influence of light and other agents upon these processes have been studied very little. It was with the object of contributing something on these points that I made the present investigation. I. On the decomposition product of the proteids prepared from the seeds of Japanese coniferous plants. A) CRYPTOMERIA JAPONICA. Proteids from the seeds of Cryptomeria japonica were pre- pared according to Ritthausen’s method :—Powdered seeds“ were first extracted with absolute alcohol and ether to free them (1) Fr. Kutscher. ibid. Bd. XXV, S. 195. (2) Ber. d. deutsch. chem, Ges. Bd. XXXIJ, S. 3183. (3) Recently it was found that arginin, prepared from plants, differs somewhat, from that of animal origin and it was considered as isomerides, compare—WI. Gulewitsch :—iiber das Arginin :—Zeits. f. Physiol. Chem. Bd. XXVII. S. 178. (4) Seeds of Cryptomeria contain almost no other nitrogen compounds, Thus a fat-free sample yielded 1.809% nitrogen, of which 1.789% was albuminoid nitrogen. It is also very important to see the chemical nature of the proteids contained in the seeds :—For this purpose 10 grams of the fat-free powder was mixed with 30c.c. of a 10% sodium chloride solution, well stirred and after a few hours standing, filtered off, the clear filtrate gave by boiling, a white curdy precipitate, owing to the presence of some coagulable globulin. This precipitate was collected, while hot, on a filter washed with hot sodium chloride, dried and subjected to Kjeldahl’s method for nitrogen deter- mination, The filtrate was acidified with sulphuric acid and some phospho-tungstic acid was added, to precipitate the globulin not coagulable by boiling and this precipitate was also subjected to the method for nitrogen determination. The following results were obtained : A CONTRIBUTION TO THE KNOWLEDGE OF ARGININ. 3 from fat and other impurities; then a dilute solution of caustic potash (0.2-0 4%) was added, and the whole was well stirred, and after 24 hours the upper portion of the solution, was slowly decanted. The remaining portion was once more extractec with potash solution and the upper portion decanted. This opera- tion was repeated 3 times and the residue filterd with fine cloth, and the filtrate was mixed with the decanted solution. On neutralizing the potash extract thus obtained with dilute acetic acid, a brown flocculent precipitate was produced in a large quantity. This precipitate was first washed by decantation and afterwards collected on a filter, where it was again washed with water. The crude proteids thus obtained, were once more dissolved in the potash solution and reprecipitated with acetic acid, collected on a filter, washed with water, dilute alcohol, absolute alcohol and ether and dried over sulphuric acid. The crude proteids prepared in this way still contained some impuri- ties of a reddish brown colour, which could be removed with difficulty. The dry proteids yielded 8.03% nitrogen, therefore if must have been still mixed with a considerable quantity of non-nitrogenous substances. The impure proteids were finely powdered and boiled with 20% hydrochloric acid (sp. gr 1.10) and some stannous chloride in a reflux cooler, at first very gently and afterwards directly over the flame. After 30 hours boiling, they were cooled and filtered. The filtrate was diluted with about 5 times its own bulk of water, the dissolved tin was precipitated with sulphuretted hydrogen, and the solution filter- ed once more. The filtrate was evaporated to a small volume to expell the greater part of hydrochloric acid, and then diluted I. 10g. air dry=8.28 g. dry sample yielded :— a) Soluble in 109% NaCl, and coagulable by boiling ......... 0.00093 g N. b) is Ps 5 SEDO Niss 9 aoe Lamectesane 0.01414 g N. 2. log. air dry=8.28 g. dry sample yielded :— a) Soluble in 109% NaCl, and coagulable by boiling ......... 0.00112 g N. b) 3 A PA eenlOk 5, cp fo hereon 0.0141 ¢gN. We have on the average 0.189% nitrogen in the form of proteids soluble in 10% sodium chloride. As the fat free sample contains 1.89% nitrogen so we see that nearly 109% of the total proteids is soluble in 10 9 sodium chloride. Therefore we must conclude that the greater part of the bases come from the proteids, which are not soluble in sodium chloride. A U. SUZUKI, with water to a certain volume, and the determination of nitrogen was made. The results were as follows. a). 19.75 grams dry crude proteids (=1.5825 g. N.) boiled with 209% HCl for 30 hours. Total dissolved nitrogen. ISA Onesies 100.0 Nitrogen, in copper hydrate precipitate. OLTOAGs. sense nO Nitrogen, directly precipitated by phospho-tungstic acid. OLG HOO sce ilectts 35-6 Nitrogen in phospho-tungstic precipitate in the filtrate of copper hydrate precipitate. O.431Oresde8 28.6 Nitrogen in ammonia. OT QB OUes-ee Bea Nitrogen in organic bases. Ol2 36 Oseenceee 15.5 b). 3.32 g. dry crude proteids (=0.2632 g. N.) boiled with 20% HCI for 96 hours. Total dissolved nitrogen. O26O4 er ostee 100.0 Nitrogen in copper hydrate precipitate. O:ON 5 Onis ce 6.1 Nitrogen directly precipitated by phospho-tungstic acid. O108 84s .ncs-0-e SAO Nitrogen in phospho-tungstic precipitate in the filtrate of copper hydrate. OLOF2Onreiaccas 27.9 Nitrogen in ammonia. OlOZ Bb cose. elere Nitrogen in organic bases. OMOLTON fgnaeanbos 1 We see from the above results that nearly 15% of the nitrogen in the proteids are found in the form of organic bases (by the action of hydrochloric acid). No difference was observed between 30 and 96 hours boiling, both yielding the same result. Note:—The total amount of nitrogen was determind by Kjeldahl’s method. Nitrogen in copper hydrate precipitate was determined to see whether some proteids or peptone still remain undecomposed, for this purpose the hydrochloric acid extract was exactly neutralized with caustic soda and then a few drops of freshly precipitated copper hydrate were added. The precipitate was collected on a filter, washed well, dried, and the nitrogen determined. In this way, I always found that a small quantity of nitrogen A CONTRIBUTION TO THE KNOWLEDGE OF ARGININ. 5 I have tried also to see whether the splitting of organic bases is performed by a very dilute acid instead of a strong one. For this purpose 0.5% hydrochloric acid (1.25¢.c. conc. HCl to 100 c.c. H,O) was mixed with powdered proteids and boiled for 96 hours. But as I found still some peptone remaining unde- composed, the boiling was continued for 50 hours more until no trace of peptone was present. c). 10.27 g. crude proteids (0.822 g N) boiled with 0.599 hydrochloric acid for 146 hours (with addition of 1% SnCl.). Total dissolved nitrogen. OW88S:..20120- 100.0 Nitrogen in copper hydrate precipitate. GLOW ASK es eens 5.0 Nitrogen directly precipitated by phospho-tungstic acid. O2COSc se oes 34.2 Nitrogen in phospho-tungstic precipitate in the filtrate of copper hydrate. OVD eee 28.8 Nitrogen in ammonia. ONO G aaesee ste eye Nitrogen in organic bases. OND Oe. 2: 167 Thus by boiling with 0.5% hydrochloric acid we obtain nearly the same result as with 209% hydrochloric acid. was present in the copper hydrate precipitate. But I afterwards satished myself that there are neither proteids nor peptone present ; so it must be something else. Phospho-tungstic acid precipitates both ammonia and organic bases ; so we must subtract the nitrogen in ammonia from the nitrogen in phospho-tungstic precipitate and the difference is to be considered to belong to the organic bases. Ammonia in the hydrochloric acid extract was distilled off by magnesia in the usual way. For the determination of nitrogen in phospho-tungstic acid precipitate, the pre- cipitate was collected on a filter, washed with 59% sulphuric acid, dried and subjected to Kjeldahl’s method. (1) It is absolutely necessary to prove the absence of peptone in this case, because it is precipitated by phospho-tungstic acid and would thus give a higher percentage of nitrogen, Tor this purpose the original hydrochloric acid extract was mixed with an excess of strong caustic soda and a few drops of very dilute solution of copper sulphate was added; but no violet colouration was observed. Further the phospho-tungstic precipitate was decomposed with caustic baryta, the excess of baryta removed by carbonic acid, filtered and evaporated and filtered once more. The concentrated filtrate gave no biuret reaction. Therefore peptone must have been entirely absent. 6 U. SUZUKI. B) GINGKO BILOBA. Fresh seeds of Gingko biloba were freed from the hard shell and the juicy kernel was crushed in a mortar and extracted with a 0.2-0.4% solution of caustic soda. The soda extract was neutralized with dilute acetic acid and the crude prepared in the same way as was described before. proteids were The prepa- ration was comparatively pure and contained 15.196 nitrogen. a). 18.775 g. air dry=15.045 g. dry crude proteids (=2.2718 g. N.) boiled with 209% hydrochloric acid for 30 hours :— Total dissolved nitrogen. 2. 2200mE Nitrogen in copper hydrate precipitate. 0.037 2-0. Nitrogen in phospho-tungstic precipitate. 0.8184... Nitrogen in phospho-tungstic precipitate in the filtrate of copper hydrate. 0.7588... Nitrogen in ammonia. ©2230... Nitrogen in organic bases. 0.5 350.6. b). 10.143 g. air dry=8.135 g. dry crude proteids (=1.2285 ¢ N) boiled with 0.5% hydrochloric acid for 146 hours. Total dissolved nitrogen. 1.2270... Nitrogen in copper hydrate precipitate. 0.0372... Nitrogen in phospho-tungstic precipitate. O.AB8 ener Nitrogen in phospho-tungstic precipitate in the filtrate of copper hydrate. 0.4000... Nitrogen in ammonia, OUZOSaer Nitrogen in organic bases. O27, 2 bere (1) Recent investigation has shown that Gingko biloba does not belong to the Coniferae and must be considered to form an independent family, Gingoaceae, This plant is peculiar to Japan. A CONTRIBUTION TO:THE KNOWLEDGE OF ARGININ. 7 C) PINUS THUNBERGII. Preparation of crude proteids in the same way as before, was tolerably pure, and contained 15.6% nitrogen. a). 12.36 g. crude proteids (=1.928 g N) boiled for 30 hours with 20% hydrochloric acid. Total dissolved nitrogen. IOOZO nsec 100.0 Nitrogen in copper hydrate precipitate. OLO4OOr vases 2.1 Nitrogen in phospho-tungstic precipitate. ONZO5 Os a8 27d Nitrogen in phospho-tungstic precipitate in the filtrate of copper hydrate. OLOR Sas aceene 34.6 Nitrogen in ammonia. OunG4iee ns Ree ao Nitrogen in organic bases. ONS O4 One aaa. 26.5 b). 7.52 g. crude proteids (=1.173 g N) boiled with 0.5% hydrochloric acid for 146 hours. Total dissolved nitrogen. BaD GOL stecn conn 100.0 Nitrogen in copper hydrate precipitate. COLO ZN fons Aaescid 1.8 Nitrogen in phospho-tungstic precipitate. OVAAG Neer case 38.5 Nitrogen in phospho-tungstic precipitate in the filtrate of copper hydrate. OVA Gye asters 36.2 Nitrogen in ammonia. OOO Si vecen ee: 8.5 Nitrogen in organic bases. OBZO2 ice. <: 27.7 From the above results, we see that the organic bases are splitted off very easily by the action of dilute acid, the quantity of the bases produced by the action of dilute acid is always‘the same with that of strong acid. 8 U. SUZUKI. II. Organic bases especially arginin in the shoots of coniferous plants. I. CRYPTOMERIA JAPONICA. Since E. Schulze found much organic bases especially arginin in the shoots of Picea excelsa, Abies pectinata and Pinus sylvestris, it is naturally to be expected that the shoots of other Coniferae also contain the same substance. Seeds of Cryptomeria japonica were sown in the purified sea sand and kept in the dark in a warm house, where the tem- perature ranged between 15°C and 35°C. After one month they began to germinate and after three weeks more, the shoots were 6.-7cm. high. Hereupon they were removed from the sand, washed well, dried and analyzed :— a). In 100 parts of dry matter. Total nitrogen. GG scat ge 100.0 Albuminoid nitrogen. BOL ie. aaa 49.2 Asparagine nitrogen. OU Ose eee 2 Nitrogen in phospho-tungstic precipitate. 1653. c 05 21.2 Other nitrogen. TA coe nee 22.4 b). Another sample gave :— Albuminoid nitrogen. 3-77. Nitrogen in phospho-tungstic precipitate. 1.44 We see here that nearly } of the total nitrogen in the shoots belongs to the phospho-tungstic precipitate. 2. GINGKO BILOBA. Seeds of Gingko biloba were sown in the purified sea sand and kept in the dark in a warm house, where the temperature ranged between 15°C and 35°C. When the etiolated shoots were’ 30cm. high on the average, and the roots 10-15cm. long, there being no open leaves yet, one portion of the shoots was removed from the sand, washed, dried and analyzed. The other portion was then exposed to day-light and a solution A CONTRIBUTION TO THE KNOWLEDGE OF ARGININ. 9 containing 0.19% K,HPO,, KH:PO,, MgSO, and trace of FeCl, and CaSO, to half saturation, was added. After twenty one days, the shoots were 36cm. high, with many green open leaves, the diameter of which was on the average 5cm. This second portion was then removed from the sand, washed, dried and analyzed. a). Etiolated shoots of (so many days) growth. In 100 parts of dry matter. Total nitrogen. E MOON caesar 100.0 Albuminoid nitrogen. Whgavera ae 48.0 Asparagine nitrogen. O14 Geenbe sae 16.0 Nitrogen in phospho-tungstic precipitate. Osa 4.7 Other nitrogen. ONOB IE ease 31.3 Every 100 shoots contain :— Total nitrogen. 0.5775 Albuminoid nitrogen. 0.2772 Asparagine nitrogen. 0.0924. Nitrogen in phospho-tungstic precipitate. 0.0270 Other nitrogen. 0.1809 b). Etiolated shoots of (so many days) growth exposed to day-light for the last 3 weeks. In 100 parts of dry matter. Total nitrogen. 2. Oifeasecorn 100.0 Albuminoid nitrogen. TiOOe eet 67.0 Asparagine nitrogen. O:3OF cocaine 10.1 Nitrogen in phospho-tungstic precipitate. ONG men se... 5.4 Other nitrogen. Ong 2racccans 175 Every 100 shoots contain ; — Total nitrogen. 1.1234 Albuminoid nitrogen. 0.7393 (1) The increase of the absolute quantity of nitrogen in the shoots of b) was due to the migration of the reserve materials from the seeds to the shoots, 10 U. SUZUKI. Asp2ragine nitrogen. O.1115 Nitrogen in phospho-tungstic precipitate. 0.0594 Other nitrogen. 0.2132 In this case, contrary to my expectation, I found very little organic bases both in the etiolated shoots and in the green plants. 3. PINUS THUNBERGII. Seeds were sown in the purified sea sand, and kept in the dark in a warm house where the temperature varied between 15°C and 30°C. On the 24th February, they began to germi- nate, and after 28 days they were 10-15cm. high.. They were then removed from the sand, washed, dried and analyzed. In 100 parts of dry matter. Total nitrogen. O15 feeeee ee 100.0 Albuminoid nitrogen. 2OAnscaawhe 30.9 Asparagine nitrogen. 2i8 Ost Ren 24.7 Nitrogen in phospho-tungstic precipitate. 1 Wo: ane 19.7 Other nitrogen. 226) eee 25.7 Ill. Isolation of arginin. I. CRYPTOMERIA JAPONICA. a). Isolation of arginin from the decomposition products, of the proteids prepared from the seeds by the action of hydrochloric acid :—The hydrochloric extract was at first freed from dissolved tin by hydrogen sulphide. The filtrate was then evaporated off to a small volume, to expell the greater part of hydrochloric acid, and diluted with water. Phospho-tungstic acid solution was then added in excess, when a white precipitate was formed in considerable quantities. This was collected on a filter, washed with 5% sulphuric acid until no trace of hydrochloric A CONTRIBUTION TO THE KNOWLEDGE OF ARGININ. II acid was present,“ strongly pressed between the filter, to remove the excess of sulphuric acid, then put ina large porcelain basin, and mixed with water to the consistency of paste. Some caustic lime and baryta were then added, and the whole well rubbed in the basin (during the operation, the basin must be cooled with water), and filtered after a few hours standing. The dissolved lime and baryta in the filtrate were precipitated by carbonic acid. The filtrate from the insoluble carbonates was evaporated to drive off the dissolved carbonic acid, which dissolves some calcium and barium carbonate, and after 24 hours standing, filtered. The filtrate was neutralized with a known quantity of sulphuric acid and further evaporated to a small volume. After cooling, the sulphuric acid, formerly added, was precipitated with a calculated quantity of baryta water, and the insoluble barium sulphate filtered off (during the filtration, the vessels were well covered to prevent the absorption of carbonic acid by the bases). The clear filtrate which contains free bases, was treated according to S. G. Hedin’s method, with a concentrated silver nitrate solution, by which some white flocculent precipitate was formed. This was removed by filtration, and the clear filtrate was evaporated on the water bath to a small volume, filtered once more, and left standing in® a dessicator. After a short time, fine crystals of arginin silver nitrate was formed, which increased very much after two or three days. The crystals were then separated from the mother liquor, well washed with cold water, and recrystallized from a hot water solution. The crystals thus obtained were dried over sulphuric acid and the percentage of silver was determined by direct ignition :— ‘0.083 g. crystals yielded by ignition 0.026 g. Ag= 31.3% Calculated as C;5H,,N,O,, AgNO;+43H,0O...... Ag = 30.59% A portion of the crystals was dissolved in hot water and, after the precipitation of silver by hydrogen sulphide, evaporated (1) As it is very difficult to remove hydrochloric acid by simple washing, the precipi- tate was once more mixed with a large quantity of 59% sulphuric acid, well rubbed in a mortar, and then collected on a filter and well washed with 59% sulphuric acid. In this way all traces of hydrochloric acid was removed. (2) Asa small quantity of silver is reduced during the ecrystalization, it was very difficult to prepare absolutely pure crystals, 12 U. SUZUKI. to a small volume. The solution gave the following reac- Hons) With phospho-tungstic acid...... Voluminous white precipi- tate. ,, phospho-molybdic acid...... Yellow precipitate, solu- ble in excess. potassio-mercury iodide eres eien=nessens-5: No precipitate, on addition of much caustic soda, white precipitate was formed. 5, potassio-bismuth jodid es Ate res: =. 7.4400 Red precipitate. The solution was further concentrated and after several days standing over sulphuric acid, fine white prismatic crystals, were obtained, which agree perfectly well with E. Schulze’s description of arginin nitrate. A solution of these arginin- nitrate crystals was mixed with some copper hydrate, and on being warmed a portion of the copper hydrate was dissolved with a dark blue colour. The solution was filtered while hot, and the filtrate was evaporated to a small volume and let stand over sulphuric acid. After three or four days, dark blue prismatic crystals were obtained, which after careful washing, were dried over sulphuric acid and analyzed. 0.1075 g. crystals became on drying at 100°C. 0.0975 g. =9.3%% water of crystallization. 0.1075 g. crystals yielded 0.c16 g. CuO=0.0126 g. Cu =11.7% CuO Calculated as (C,H yN,O,), Cu(NO.), 3H.O ewceee SY ie eer water of crystallization. Sef ee 2 ae boas ns a ERE Ss ee von ceceseeent 9B Ca atear A somewhat higher percentage of copper in my preparation (1) For the determination of copper the crystals were first ignited and weighed. The CuO thus obtained was dissolved in sulphuric acid and precipitated with a dilute soda solution. The copper hydrate precipitate was collected on a filter, washed very well, dried, ignited and weighed. No difference was observed between the two weighings, A CONTRIBUTION TO THE KNOWLEDGE OF ARGININ. 13 may be due to experimental error; so there is no doubt that the crystals obtained were those of argznin. The quantity of the arginin crystals obtained amounted to about 5% of the proteids used, (calculated as pure proteids), that is to say, nearly 10% of the nitrogen in the proteids was contained in the arginin. As I found that about 15% of the nitrogen in the proteids are contained in the phospho-tungstic precipitate (organic bases) formed by the action of hydrochloric acid, we may conclude that nearly 2 of the organic bases formed, consist of argznin. The mother liquor of basic arginin silver nitrate crystals was diluted with water, and silver was precipitated with hydrogen sulphide. The filtrate from the silver sulphide was evaporated and concentrated, and precipitated with phospho-tungstic acid. The precipitate was washed with 5% sulphuric acid, strongly pressed between the filters and then decomposed with caustic baryta. The dissolved baryta was removed by carbonic acid, evaporated, once more filtered, and a mercuric chloride solution was added, whereby a white precipitate was obtained, perhaps due to histidin (?) but too little to confirm. The filtrate from the mercuric chloride precipitate was diluted with water and the dissolved mercury was removed by hydrogen sulphide, filtered, and the filtrate was evaporated and concentrated. No crystals were obtained after long standing. Therefore lysin was probably not present. b). Etiolated shoots of Cryptomeria japonica. Etiolated shoots 6-8cm. high, grown in the purified sea sand, were dried and powdered. 40 grams of this dry powder were extracted with about one litre of water at 50-60°C. After two hours, the extract was cooled and filtered. To the filtrate, an excess of basic lead acetate was added until no more precipitate was formed, and filtered. The excess of lead in the filtrate was removed by sulphuric acid. The filtrate from the lead sulphate, was precipitated by phospho-tungstic acid, whereby a white precipitate was formed in considerable quantities. The precipi- tate was treated in the same way as described before, and crystals of basic arginin silver nitrate were obtained in a to- lerably large quantity. But in this case it was too difficult to get the crystals pure, owing to the excessive reduction of silver. Therefore it was impossible to decide how much arginin was 14 U. SUZUKI. present in the shoots. But any how I have no doubt that the chief product is arginin. 2. GINGKO BILOBA. The isolation of arginin according to the usual method was in this case encountered with much difficulties by the strong reduction of silver and the separation of pure crystals was almost impossible. SoI tried to convert it to the neutral silver salts according to WI. Gulewitsch’s method.” The latter com- pound was rather stable and it proved to be successful. For this purpose, the basic solution containing free bases, was at first neutralized exactly with dilute nitric acid, and then some concentrated silver nitrate solution was added. The evaporated and concentrated syrupy liquid became soon after cooling a white crystalline mass which after recrystalization became uniform long needles, most of them being united to bundles. It was easily soluble in water, but little soluble in absolute alcohol. The microscopical examination proved it to be exactly identical with Gulewitsch’s description. After drying over sulphuric acid, the crystals were subjected to the following determinations. I. 0.0808 g. crystals yielded on ignition . 0.0220 g=27.2% Ag. 2. 0.090 g. crystals of another preparation yielded on ignition 0.0245 g=27.5% Ag. 3. On drying at 100°C, no loss in weight....... no water of crystallization. Calculated as C,HyN,O2, HNO;+ AgNO, =26.5% Ag. 4. Melting point was found in one case to be 181-183°C. and in the other 182-184°C. Above 130-140°C, the crystals be- came gradually brownish black, but did not melt until 181°C exactly. This result exactly coincides with Gulewitsch’s observation. 5. Apart of the neutral salt was dissolved in water and so much baryta water was added as to remove exactly half of the nitric acid to convert it into the basic salts. Evaporated to a small volume and let stand over sulphuric acid, whereby ~ (1) Wl. Gulewitsch. “Ueber das Arginin ’’ :—Zeits. f. Physiol. Chem. Bd. XX VI. Heft. 3. 1899, s. 178. A CONTRIBUTION TO THE KNOWLEDGE OF ARGININ. 15 the basic salt was formed which after recrystallization became fine needles, uniting to bundles. 0.113 g dry crystals yielded on ignition 0.0345 g= 30.53% Ag. Calculated as C;H,,N,O,, AgNO;+4H,O == 3015 Soe. We may now safely conclude that the base was arginin. I did not calculate how much arginin was present in the decomposition products of the proteids, yet I am sure that the greater part of the bases was arginin. It is not yet decided whether some other bases can be isolated or not. I hope to take up this question later on. 3. PINUS THUNBERGII. In the decomposition products of the proteids and in the etiolated shoots, arginin was isolated just in the same way as in Cryptomeria japonica and was found to amount also to more than half of the entire bases. IV. Organic bases in the decomposition products of the proteids prepared from the shoots of Pinus Thunbergii. > It will be very interesting to see whether the chemical nature of the proteids in the shoots of coniferous plants is differ- ent from that of the proteids in seeds or not. For this purpose, the shoots of Pinus Thunbergii 10-12cm. high grown in the full day-light in the purified sea sand were dried and powdered and its composition was determined first :— Average dry weight of 1000 shoots. 6.54 gram. In 100 parts of dry matter. Total nitrogen. Wages Albuminoid nitrogen. 3.40 Asparagine nitrogen. 1.42 Nitrogen in phospho-tungstic precipitate. 0.34 Other nitrogen. 2.58 . About 50 grams of the finely powdered sample were mixed with about 1000¢.c. of 0.2-0.39% caustic soda solution, well stirred and let stand for 24 hours, then filtered with clean cloth, and (1) By long exposure to day-light, organic bases had been reduced to the minimum quantity. 16 U. SUZUKI. the residue once more extracted with soda solution. The soda extract was neutralized with dilute acetic acid, whereby a flocculent precipitate was formed in abundance. This was washed at first by decantation and afterwards collected on a filter, washed with water, alcohol and ether, and dried over sulphuric acid. The crude proteids thus obtained were still mixed with chlorophyll and other impurities. The dried and powdered proteids were then boiled with 20% hydrochloric acid (sp. gr. 1.10) for 20 hours (with addition of some Sn(€l,) in the reflux cooler, and the solution filtered. The filtrate was treated in the usual way, and the following result was obtained. Total dissolved nitrogen. O:2700 i436 LOO Nitrogen directly precipitated by phospho-tungstic acid. (¢) Om8ON...s-serr 33.0 Nitrogen in ammonia. (3) ©0203 uae Weis (a)—()...Nitrogen in organic bases. 0.0688....... ». 25.5 We see that this result nearly coincides with the one obtained for the seeds; therefore it is very probabie that the chemical nature of the proteids in the shoots is the same as in the seeds. * From want of time, I could not isolate the bases, but I am sure that the chief base is arginin. I suppose also that the proteids in the shoots of other coniferous plants can produce the bases in the same way. I hope to make some further communi- cation on this subject. SUMMARY OF RESULTS. 1. The proteids prepared from the seeds of Cryptomeria japonica, Pinus Thunbergii and Gingko biloba, produce much organic bases by the action of acids, the chief of which is arginin. 2. Organic bases are produced by the action of even very dilute acids. 3. The etiolated shoots of Cryptomeria and Pinus, contain much organic bases, especially arginin; but the shoots of Gingko contain only a very small quantity of the bases. 4. The chemical nature of the proteids prepared from the shoots of coniferous plants are most probably the same as that of the seed proteids, inasmuch as they give the same decom- position products as those of the seeds. A CONTRIBUTION TO THE KNOWLEDGE OF ARGININ. 17 MN ICAL DATA, I. Cryptomeria japonica. (a). Boiled with 209% hydrochloric acid for 30 hours. 50 grams air dry=30.50 g. dry crude proteids (=1.582 g N) were boiled with hydrochloric acid. The extract was diluted to 400 c.c. from which the following determinations were made :— Absolute Solutions | Baryta | Nitrogen | quantity used, water found. in total Ratio. replaced. solution, cc fe) 20.6 ) Mlotalimitrogeny \.s-....c.esossess § 0.0387 1.548 100.0 lone: 208 | Nitrogen in copper hydrate + TS, } DLECIPIEALS ecm naming sovesne 0.0026 0.104 6.7 ” 1.5 j Nitrogen directly precipitated 5 7.4 by phospho-tungstic acid... 0,0138 0.551 35.6 ” 7-4 Nitrogen in phospho-tungstic 5 5.6 precipitate in the filtrate o =0.0108 0.432 28.0 copper hydrate, .......<..-.s6. ¥ 6.0 » 2.5 Nitrogen in ammonia ......... § l 0.0048 0.194 12.5 l ” 2 | Nitrogen in organic bases...... | fi = 0.238 15:5 ” Fs I c.c. baryta water =0.00186 ¢. Nv 18 U. SUZUKI. (b). Boiled with 209% hydrochloric acid for 96 hours. 4.2 g. air dry=3.32 g. dry crude proteids (=0.263 g. N) were boiled with hydrochloric acid of 20%. The extract was diluted to 400 c.c. from which the following determinations were made :— Baryta Absolute Solutions See Nitrogen | quantity : used. é we ie 1 found. in total Ratio. eae solution. OCee : ; 40 13.8 Total VERO GED) Meee iste cleetelebiee ) is 14.2 t .100.0 Nitrogen in copper hydrate | { ” 0.8 | precipitate eetcecae eee Cs 0.9 i 6.1 Nitrogen directly precipitated }{ —» 4.6 | by phospho-tungstic acid... | | fs 4.9 j 34.0 Nitrogen in phospho-tungstic [ ~@ 3.7 } precipitate in the filtrate of = 4.1 i 27.9 Copper hydrate teccas-seccsee E: ‘ee eh Nitrogen in ammonia ......... ye oe 1.9 j 12.8 Nitrogen in organic bases ... j ¥ " | 1 se > 1 Hs 28 j 15.1 I c.c. baryta water =0.00186 g..N. (c). Boiled with 0.5% hydrochloric acid for 146 hours. 0.27 g. dry crude proteids (=0.822 g N) were boiled with 0.5% hydrochloric acid. The extract was diluted to 400 c.c < Absolute Solutions ey la Nitrogen | quantity ‘ water 5 ; Ratio. used, replaced found. in total P : solution, c.c E a ; Io 10.6 ) : Motalitrocen| sees sseee seen } M 10.6 { 9.0197 0.7888 100.0 Nitrogen in copper hydrate}{ » 25 g 6 PICCIPILALE A aren ascn aseeceaee | 0.7 a 104 5 Nitrogen directly precipitated | § 2? 3.6 l 608 2 by phospho-tungstic acid...| | 3: i Sece7, OZ? 34.2 Nitrogen in phospho-tungstic | ( ¥ 3.0 precipitate in the filtrate of 1 3.1 t 0.0057 0.2272 28.8 copper hydrate ............... % ae Nitrogen in ammonia ......... ae 1.3 t 0.0026 0.1033 13.1 Salis meee »” = l Nitrogen in organic bases ... ae we fo 0.1239 ris I c.c. baryta water=0.00186 g. N. A CONTRIBUTION TO THE KNOWLEDGE OF ARGININ. II. Gingko biloba. Boiled for 30 hours with 209% hydrochloric acid. (a). 19 18.775 g. air dry=15.045 g. dry crude proteids (=2.2718 g. N) were boiled with 20% hydrochloric acid. The extract diluted to 400 c.c. A bsolute : Baryta oe g : Solutions ae Nitrogen | quantity Ratio. used, Ticed found. in total coo solution. Ge é (eee 30.4 ehotalemitvoven! eeecsneaea-csee-s 1 29.8 2.2396 100.0 Nitrogen in copper hydrate | § ” f [OAXOWONANCE co canconeconoeeeseo0n 7 0.0372 1.7 Nitrogen directly precipitated | ” by phospho-tungtic acid ... i 0.8184 36.5 Nitrogen in phospho-tungstic - precipitate in the filtrate of | 0.7588 33.9 copper hydrate............... « Nitrogen in ammonia ......... | 0.2236 10.0 Nitrogen in organic bases ... } " 0.5356 23.9 I c.c. baryta water=0.00186 g. N. (b). Boiled with 0.5% hydrochloric acid for 146 hours. 10.143 g. air dry=8.128 g. dry crude proteids (=1.229 g. N) were boiled with 0.5% hydrochloric acid. Nitrogen in organic bases ... The extract diluted to 400 c.c. x Baryta Absolute olutions i Nitrogen | quantity - used. wale, | found. | in total ue S\eLehS S09 solution. CC. : 10 16.5 Mioralimitro genes nye. unease 16.5 1.2276 100.0 Nitrogen in copper hydrate 0.5 ] PECCIpItatenemea-se-peieaenaee 0.5 j Chey 3° Witrogen directly precipitated | | 5.8 | by phospho-tungstic acid... 6.0 j 35-7 Nitrogen in phospho-tungstic 53 ) precipitate in the filtrate of f 32-9 copper hydrates......4.s6s-0. a , ; ah | Nitrogen in ammonia ......... 1.7 fi 10.3 l j 20 U. SUZUKI. Ill. Pinus Thunbergii. (a). Boiled for 30 hours with 20% hydrochloric acid. 12.36 g. dry crude proteids (=1.928 g. N) were boiled for 30 hours with 209% hydrochloric acid. The extract diluted to AGO ICC. Baryta Absolute Solutions aa Nitrogen | quantity yor used. r ty Ste found. in total Ratio oe eae solution. c.c Total nitrogen ee ae l 6 a ie{eW8) Sonseongeee oopcna. ey 25.4 ( 0.047 1.902 100.0 Nitrogen in copper hydrate | § ” o5 ) PLECIpltateneese-eeeeeeeeeeeeees - 0.6 i 0.0010 0.040 2.1 Nitrogen directly precipitated |{ » 93 by phospho-tungstic acid... | | » 9-7 t cere pues 37-1 Nitrogen in phospho-tungstic|( _,, 8.8 precipitate in the filtrate of | ) 8.9 (ee o165 0.6581 34.6 copper hydrate............... i ey ” 0 Nitrogen in ammonia ......... | 2.1 t 0.0039 0.1541 8.1 Nitrogen in organic bases ... ‘ + t = ©.5040 26.5 I c.c. baryta water=0,.00186 g. N. (b). Boiled with 0.5% hydrochloric acid for 146 hours. 7.52 g. dry crude proteids (=1.173 g. N) were boiled with 0.5% hydrochloric acid. The extract diluted to 400 c.c. —— i CR | crs: (hota ian il RASolutCn ain Seo Baryta +: F Solutions ges Nitrogen | quantity : used. water found, in total ae replaced. salkeion. He Total nitr 1 ane ee otalinitro gent eeersssess-seeeeen | 15.6 t 0.0289 1.156 100.0 Nitrogen in copper hydrate | § ” Ce [BISONS 5 Sodnonsdenencesoce ae @.30" ii o:eoes p02 te 1.8 Nitrogen directly precipitated |{ » 595 |) by phospho-tungsticacid...|{ —_,, Gio) if oases O4455% 38.5 Nitrogen in phospho-tungstic ( a ly precipitate in the filtrate of ) 5.7 t 0.0105 0.4185 36.2 copper hydrate ............... a : a ; ” Wo) Nitrogen in ammonia ......... % ret t 0.0025 0.0983 8.5 Nitrogen i ichases ...){ ” = itrogen in organic bases a e t a oni Ba I c.c. baryta water =0.00186 ¢. N. A CONTRIBUTION TO THE KNOWLEDGE OF ARGININ. 21 II. Etiolated Shoots. I, (a). Cryptomeria japonica. Dry matter | Baryta water Nitrogen Percentage of used. replaced. found, nitrogen, Cc: : I. 0.462 19.2 ) } Total nitrogen ......... 0.0359 aaah 2. ” 19.4 I. 0.462 9.4 Albuminoid nitrogen. | 0.0177 3.82 25 is 9.6 \ is 0.7 ] Asparagine nitrogen... ( 0.0012 0.28 By TS 0.6 =4 Asp. N Nitrogen in phospho- | ( 1 % 2 } tungstic precipitate. f 0.0076 1.65 (b) Dry matter | Baryta water Nitrogen Percentage of used. replaced. found. nitrogen, CC. es ie { Nia OHS) 10.4 ) Albuminoid nitrogen. - 0.0195 3.77 A 10.6 Nitrogen in phospho-| ( 1. 5, 3.8 tungstic precipitate. 0.00744 1.44 2 . 2 22 U. SUZUKI. II. Gingko biloba. (a). Etiolated shoots (dried on the 15th May). Dry matter Baryta water Nitrogen Percentage of used, replaced. found. nitrogen. c.c I. 0.387 10.7 ] Total nitrogen ......... 0.0117 3.00 2 rp 10.9 { I.. 0.774 10.2 Albuminoid nitrogen. O.O1II 1.44 2 ” 10.4 ae (2.008: 1.7 ) Asparagine nitrogen... 0.0018 0.24 H 2. 5 Te = Asp. N. Nitrogen in phospho- ( iN + et tungstic precipitate. 0.0011 0.14 | fy, 55 0.8 (b). Etiolated shoots were exposed to the day-light (from 15th May to 5th June). | Dry matter | Baryta water Nitrogen Percentage of used. replaced. found. nitrogen, Cie: I. 0.473 132% Total nitrogen ......... 0.0140 2.97 De > 12.9 pa : i I. 0.946 17-3 Albuminoid nitrogen. 0.0188 1.99 (2 7 175 | 4 , I. ” 13 Asparagine nitrogen... 0.0014 0.15 2. 3 1.3 =} Asp. N. Nitrogen in phospho- | ( 1. D 1.2 tungstic precipitate. 0.0015 0.16 2. xy 1.5 I c.c. baryta water=0.00108 g. N. A CONTRIBUTION TO THE KNOWLEDGE OF ARGININ. 23 1) Pinus Thunbergii. Dry matter used. Baryta water replaced. Total nitrogen ......... Albuminoid nitrogen. Asparagine nitrogen... Nitrogen in phospho- | (1. tungstic precipitate. 24.2 24.4 25.5 26.0 10.1 10.6 16.8 16.1 Nitrogen Percentage of found. nitrogen. 0.0452 9.53 0.0279 2.64 0.0112 1.17 = Asp. N o'o178 1.88 I c.c. baryta water=0.00108 g. N. IV. Decomposition products of the proteids prepared from the shoots of Pinus Thunbergii. Boiled with 209% hydrochloric acid for 20 hours (with addi- tion of 1% SnCl,). The extract diluted to 250 c.c. Total nitrogen Nitrogen directly precipitated by phospho-tungstic acid, Nitrogen in ammonia seat eeeee Nitrogen in organic bases ... Solutions used, Baryta water replaced. Absolute | Nitrogen | quantity : found. in total Raa solution: 0.0270 0.2700 100.0 0,0089 0.0891 33.0 0.0020 0.0205 75 I c.c. baryta water=o0.00108 g. N. —— ve th a f abity: On the Formation of Arginin in Coniferous Plants. BY U. Suzuki, Nogakushi. Lecturer in Agricultural Chemistry. (PLATES. I.—VI.) In my last article I have proved that the seeds of some coniferous plants contain certain proteids which produce by the action of acids large quantities of organic bases, especially, arginin; and also that the shoots of these plants contain arginin in considerable quantities. The distribution of this substance seems to be very wide, and as far as our experience goes, it is found in almost all coniferous plants. So it is highly probable that this substance plays an important rdle in the metabolism of nitrogen compounds in coniferous plants, especial- ly during germination. E. Schulze supposes that this substance arises not only from the hydrolytic decomposition of reserve proteids in the seeds during germination, but also from the transformation of other decomposition products.* So we may assume that this substance has an intimate relation to the decomposition and regeneration of proteids in the plant cells. As it seems to be very interesting to make a closer observation on this substance and to explain its fate more fully, I have directed my endeavours to the solution of the following ques- tions :— * Compare E. Schulze:—“Ueber die beim Umsatz der Proteinstoffe in den Keimpflanzen einiger Coniferae-Arten entstehenden Stickstoffyverbindungen”’ (Zeitsch. f. Physiol. Chem. XXII. s. 445.) He says ;—“ Man muss vielmehr annehmen, dass die starke Anhiufung des Arginins erst eine Folge der Umwandlungen ist, denen die beim Proteinzerfall zuerst entstandenen Produkte im Stoffwechsel der Keimpflanzen unter- lagen. Dass fir das Stattfinden solcher Umwandlungen, welche selbstverstandlich auch mit der Regeneration von Eiweissstoffen im Zusammenhang stehen kénnen, auch die an anderen Keimpflanzen gemachten Beobachtungen sprechen, ist aus der vorher- gehenden Abhandlung zu ersehen.”’ 26 U. SUZUKI. 1. Is arginin synthetically formed from ammonium salts or nitrates offered to the coniferous plants, in a similar way as asparagine is formed in the other plants ? 2. Isit also synthetically formed from ammonium salts or nitrates in plants not belonging to the Coniferae ? 3. What is the effect of light and mineral nutriments upon the formation and transformation of this substance ? 4. Isit the primary decomposition product of proteids only or is it also formed from other decomposition products ? 5. Can it be used directly for the regeneration of proteids in the plant cells ? I. Arginin as Synthetical Product from Ammonium Salts. The following experiments were made to settle the question whether arginin can be formed synthetically from the am- monium salts offered to the plants. The first three experiments were made with coniferous plants and the next four with plants not belonging to the Coniferae, to see whether there is any difference between the two sets. A) CONERBEROUS: PIPANGS: 1. PINUS THUNBERGII. Seeds of Pinus Thunbergii were sown in three wooden boxes containing purified sea sand, moistened with distilled water. On the 25th February they began to germinate and from that time on a half saturated gypsum solution was occasionally added to each box, to keep it constantly moist. On the 15th March, the shoots of one box were carefully removed from the sand, washed well, dried and analyzed. The other two were treated with the following solutions :— 1) Half saturated gypsum solution. The shoots were kept in a warm house in full day-light, the temperature varying between 30° and 15°c. ON THE FORMATION OF ARGININ IN CONIFEROUS PLANTS. 27 2) 2% ammonium chloride solution, half saturated with gypsum.* After twelve days (on the 27th), they were removed from the sand, washed very well, until entirely free of ammonia, dried and analyzed. As the temperature was too high, and the ammonium salt too concentrated, some of the shoots treated with the ammonium salt began to show signs of suffering. These affected shoots were rejected and only the healthy ones were analyzed. a) b) oP Shoots treated with Shoot treated 29 ammonium Shoots cried with 4 sat. chloride, half satu- on the gypsum for rated with gypsum 15th March. 12 days for 12 days (15th-27th). (15th-27th). Number of shoots. 710 1656 1340 Length 6-8cm. 10-15 10-15 Total dry weight. 4.250¢. 15.051 E2353 Dry weight of 100 shoots. 0.599¢. 0.909 0.922 In 100 parts of dry matter. a) b) c) Total nitrogen. 8.73 6.90 8.45 Albuminoid nitrogen, 3.42 3.18 235 Asparagine nitrogen. 1.24 Tes 1.20 Nitrogen in phospho- tungstic precipitate. 1.81 0.38 1.55 Other nitrogen 2.26 2.19 Dis In 100 parts of total nitrogen. a) b) c) Total nitrogen. 100.0 100.0 100.0 Albuminoid nitrogen. 30.2 46.1 39.6 * A 29 ammonium chloride solution is evidently too strong for the young shoots, but I prefered it, because a more dilute solution is too favorable to the growth of the shoots, and the absorbed ammonium salts may be converted into proteids too rapidly, the consequence being that no intermediate product can be detected. I had failed twice in this way ; so it is necessary to give a concentrated solution, and to make the shoots suffer a little, so that the regeneration of proteids may be retarded. Otherwise it is too difficult to find out the intermediate product, stored up as such in the plant cells. 28 U. SUZUKI. Asparagine nitrogen. 14.2 16.7 14.2 Nitrogen in phospho- tungstic precipitate. 20.7 5.5 18.3 Other nitrogen. 25.9 2137 27.9 Every 100 shoots contain :— a) b) c) Total nitrogen. 0.0523 0.0627 0.0779 Albuminoid nitrogen. 0.0210 0.0289 0.0309 Asparagine nitrogen. 0.0074 0.0105 O.OIII Nitrogen in phospho- tungstic precipitate. 0.0109 0.0035 0.0143 Other nitrogen. 0.0135 0.0199 0.0217 The shoots dried on the 15th March a) were not fully developed, and the reserve materials in the seeds were not yet completely transported to the growing shoots. Therefore the absolute quantity of every 100 shoots is considerably lower in a), but after 12 days, that is in b) and c), almost all reserve ma- terial was transported to the shoots. The considerable increase of the absolute quantity of nitrogen in c) compared with b) may be due to the absorption of the ammonium salt offered to the shoots. Anyhow, we see from the above tables that even when exposed to full day-light much organic bases* (that is, nitrogen precipitated by phospho-tungstic acid) are accumulated in the first stage of germination a), while they disappear very rapidly on further exposure to day-light, the disappearance being most probably due to their being directly used for the regeneration of proteids, as we see in the shoots b). But we observe that the shoots treated with ammonium chloride solution still contain a considerable quantity of organic bases c). The greater part of these organic bases must be synthetically formed from the ammonium salt absorbed by the shoots. We shall see the rela- tion more clearly if we calculate the total nitrogen of a) as 100. Thus we have :— * About 209% of the nitrogen must have been transported from the seeds to the shoots during the experiments (15th to 27th), and we see that in c) about 299% of the nitrogen had been absorbed from the ammonium salts offered. ON THE FORMATION OF ARGININ IN CONIFEROUS PLANTS. 29 a) b) e) Total nitrogen. 100.0 120.0 149.0 Albuminoid nitrogen. 39.2 55.3 59.6 Asparagine nitrogen. 14.2 20.0 Zien Nitrogen in phospho- tungstic precipitate. 20.7 6.6 27.3 Other nitrogen. AERO) 38.1 41.4 We can now safely conclude that the greater part of the nitrogen absorbed as ammonium salts, is accumulated as organic bases in the shoots, and as we found no noticeable increase of asparagine even when treated with ammonium chloride solution, it is highly probable that in coniferous plants, organic bases (arginin !) are synthetically formed, instead of asparagine. Plate I. shows the increase of organic bases (N. in phos. tungstic ppt.), especially arginin, in the shoots of Pinus Thun- bergii by the addition of ammonium salts. The black lines refer to shoots treated with half saturated gypsum solution in the full day-light. The red lines refer to shoots treated with 0.5% ammonium chloride solution, half saturated with gypsum in the full day-light. Vote :—The total nitrogen was determined by Kjeldahl’s method, and albuminoid nitrogen by that of Stutzer’s. The filtrate of copper hydrate precipitate was acidified with sulphuric acid and some excess of phospho-tungstic acid solution was added, when a strong white precipitate was formed. The precipitate was collected on a filter, washed with 59% sulphuric acid, dried and subjected to Kjeldahl’s method for nitrogen determina- tion. The nitrogen in phospho-tungstic precipitate chiefly consists of that of organic bases. The filtrate of phospho-tungstic precipitate served for the determination of asparagine nitrogen, For this purpose caustic baryta was added to the filtrate until the solution became alkaline and caused the phospho-tungstic acid in solution to precipitate. The solution was then filtered, and the filtrate was acidified with sulphuric acid to precipitate the excess of baryta as barium sulphate. To the filtrate from barium sulphate, was added some pure concentrated hydrochloric acid (10 c.c. cone. hydrochloric acid to each Too c.c. of the solution), and the mixture boiled in a reflux cooler for 2 hours and distilled in the usual way, care being taken to neutralize the solution with caustic soda until it became faintly acid and then to make it slightly alkaline by the addition of a little magnesia. It is also quite important to prove the absence of ammonia in the shoots, either mechanically adhering to them or preserved unchanged in the plant cells, because phospho-tungstic acid precipitates it together with the organic bases, and thus becomes a source of error. For the detection of ammonia, the powdered sample was extracted with cold water, and to the extract, a few drops of Nessler’s reagent was added. But the 30 U. SUZUKI It is also quite important to investigate what organic base is formed by the assimilation of ammonium salts. As the germi- nating shoots of Coniferae produce large quantities of arginin, it is highly probable that it is the synthetical product in this case. For isolation of the base, a powdered sample of the shoots (weighing 8.8 grams) treated with ammonium chloride solu- tion, was extracted with about 300 c.c. of warm water, to the extract was added an excess of basic lead acetate, until no more precipitate was formed, filtered, and to the clear filtrate was added some sulphuric acid to remove the excess of lead; this filtrate of lead sulphate may contain bases. It was precipitated with phospho-tungstic acid, the precipitate was collected ona filter, washed with 5% sulphuric acid, pressed between the filter and the precipitate then decomposed with baryta, and the base was crystallized as basic arginin silver nitrate, according to S.G. Hedin’s method. It was thus found that there is formed a toler- ably large quantity of arginin silver nitrate. Its quantity was found to be more than 0.4 grams, that is, more than half of the total organic bases consists of arginin.* presence of a little sugar and other reducing compounds in the plants interferes with the reaction ; and as no other certain test could be made, I adopted the following proce- dure :—The phospho-tungstic precipitate was decomposed at the ordinary temperature with caustic baryta, filtered, and the baryta in the filtrate was removed by carbonic acid, and filtered, the filtrate was tested for ammonia by Nessler’s reagents, but no colouration !; so we may be sure that no ammonia was present in the phospho-tungstic precipitate. Also, the filtrate was tested for peptone by adding an excess of strong caustic soda and one drop of copper sulphate ; but no violet colouration! Therefore we may conclude safely that the phospho-tungstic precipitate consisted of organic bases only. Besides the above tests, I made also quantitative determination of ammonia (though the absence of it was formerly proved!) in the following way ;—a sample of 1-2 grams was warmed in so c.c. distilled water at 50-60° C. for half an hour, cooled, and after cooling, basic lead acetate was added in excess, filtered, to the filtrate was added some sulphuric acid to remove the dissolved lead, filtered, the filtrate was nearly neutralized with dilute caustic soda and some magnesia was added until it was slightly-alkaline, and distilled off. I found in this way that a little ammonia always comes off, but its quantity being less than 0.19 of the dry matter. I found afterwards that this ammonia comes from the slight decomposition of bases and amides and was not present previously. * As the basic arginin silver nitrate easily undergoes reduction and changes to the neutral silver nitrate salt, which is twelve times more soluble in water than the basic salt, it is certain that a tolerable quantity of arginin still remains in the mother liquor, We may therefore assume that almost 3 or more consists of arginin, ON THE FORMATION OF ARGININ IN CONIFEROUS PLANTS. 31 I have also tried to isolate arginin from the shoots treated with gypsum solution only (b), but I found only a very small quantity of arginin, while in the shoots dried on the 15th March (a), there was a tolerably large quantity. In both cases, however, it was found to be less than in the shoots treated with ammonium salts (c). 2. SECOND EXPERIMENT WITH THE SHOOTS OF PINUS THUNBERGII. Young shoots, grown in the purified sea sand, were carefully removed from the sand ; one portion was directly dried, and the other portion was divided into 2 parts and put in the following solutions :— , 1) Half saturated gypsum solution. 2) 0.5% ammonium choride solution, half saturated with gypsum. This second portion was kept in the laboratory in diffuse day-light, at the temperature ranging between 10° C and 16° C. After 12 days (from 27th March to 8th April), the shoots were removed from the solutions, washed very well, until no trace ofammonia was left, dried and analyzed. During the experi- ment, the solutions were renewed four times to prevent bacterial turbidity to appear. But, as toward the end of the experiment, the shoots put in the ammonium chloride solution began to suffer, while those kept in the gypsum solution were still healthy, I could not keep them any longer. The analysis gave the following results :— a) b) s) Shoots Shoots kept in Shoots kept in dried on — gypsum solution ammonium chloride the for 12 days solution for 12 days 27th (27th March- (27th March- March, 8th April). 8th April). Number of shoots. 1656 400 446 Length. 12-15cim. 12-15 12-15 Total dry weight. 15.051¢. 3.955 4.296 Dry weight of every 100 shoots. 0.909¢. 0.989 0.963 Oo bo U. SUZUKI. In 100 parts of dry matter. a) b) Total nitrogen. 6.g0 6.64 Albuminoid nitrogen. 3629 ars Asparagine nitrogen. T.15 1.16 Nitrogen in phospho- tungstic precipitate, 0.88 0.45 Other nitrogen. 2.08 1.90 In 100 parts of total nitrogen. a) b) Total nitrogen. 100.0 100.0 Albuminoid niirogen. LOG 47.1 Asparagine nitrogen. 16.7 17.5 Nitrogen in phospho- tungstic precipitate. 5.5 6.8 Other nitrogen. 30.1 28.6 Every 100 shoots contain :— a) b) Total nitrogen. 0.0647 0.0656 Albuminoid nitrogen. 0.0309 0.0310 Asparagine nitrogen. 0.0108 O.O115 Nitrogen in phospho- tungstic precipitate. 0.0036 0.0045 Other nitrogen. 0.0195 0.0187 c) 0.0759 0.0306 0.0137 0.0132 0.0185 Calculating the total nitrogen of a) as 100, we have :— a) b) Total nitrogen. 100.0 101.4 Albuminoid nitrogen. 47.7 47.9 Asparagine nitrogen. 16.7 ray, Nitrogen in phospho- tungstic precipitate 5.5 6.9 Other nitrogen. 30.1 28.9 c) 117.3 47.2 Zed 20.4 28.6 ON THE FORMATION OF ARGININe IN CONIFEROUS PLANTS. 33 We shall see the difference more clearly in the foillowing table :— a) b) Cc) Total nitrogen. 100.0 101.4 [l7.8 Albuminoid nitrogen. 100.0 100. I 99.0 Asparagine nitrogen. 100.0 106.3 127.0 Nitrogen in phospho- tungstic precipitate.* 100.0 125.0 370.2 Other nitrogen. 100.0 96.0 94.8 This result is more decisive than that of the former experi- ment. Almost all the ammonia absorbed by the shoots c), amounting to 17% of the total nitrogen, was converted into the organic base, and there was only a little increase of asparagine. Plate II. shows the increase of organic bases, especially arginin, in the shoots of Pinus Thunbergii by the addition of ammonium salts. The black lines refer to shoots cultured in the half saturated gypsum solution, in the diffused day-light. The red lines refer to shoots cultured in 0.5% ammonium chloride solution, half saturated with gypsum, in the diffused day-light. 3. CRYPTOMERIA JAPONICA. Etiolated shoots of Cryptomeria japonica, grown in the purified sea sand in a warm house, and 6-8cm. high (10th April), were exposed to direct sun-light in two lots. To one lot, a half saturated gypsum solution was added and to the other a 0.5% ammonium chloride solution half saturated with gypsum. After * In this case also, the absence of ammonia and peptone in the phospho-tungstic precipitate was proved in the same way as in the former experiment. As the sample was too little, the isolation of arginin was impossible; but the following examination was made;—From 1.5 grams of each of the three samples phospho-tungstic precipitate was obtained in the usual way, and the basic arginin silver salt was microscopically examined. I found only a very little of arginin crystals in a) and b), but three or four times more in c). So I conclude that the arginin was synthetically formed from ammonium salts in c). 34 We SUZUKL. 10 days (on the 2oth April) they were carefully removed from the sand, washed well to free them from all trace of ammonia, dried and analyzed.* a) b) Shoots, treated with Shoots, treated with gypsum ammonium chloride solu- for Io days tion for Io days (10-20th April). (10-20th April). Number of shoots. 727 667 Length. 8-10 cm. 8-10 cm. Total dry weight. 1.590 ¢. 1.538 Dry weight of 100 shoots. 0.219 g. 0.231 In 100 parts of dry matter. a) b) Total nitrogen. 6.69 7.62 Albuminoid nitrogen. 3517. 3.05 Asparagine nitrogen. 0.42 0.62 Nitrogen in phospho- tungstic precipitate. 1.12 Lee Other nitrogen. 1.98 2.18 In 100 parts of total nitrogen. a) b) Total nitrogen. 100.0 100.0 Albuminoid nitrogen. 474 40.0 Asparagine nitrogen. 6.3 8.1 Nitrogen in phospho- tungstic precipitate. 16.7 23.3 Other nitrogen. 29.6 28.6 Every 100 shoots contained :— Ratio. a) b) a) b) Total nitrogen. 0.0147 0.0176 100: 120.0 * During the experiment, the temperature varied between 15° C. and 38°C. The shoots treated with the ammonium chloride solution began to suffer, (perhaps owing to the concentration of the solution due to evaporation) and so they could not be kept any longer. ON THE FORMATION OF ARGININ IN CONIFEROUS PLANTS. 35 Albuminoid nitrogen. 0.0069 0.0070 100: 101.5 Asparagine nitrogen. 0.0099 0.0014 100: 155.7 Nitrogen in phospho- tungstic precipitate.* 0.0025 0.0041 100: 166.7 Other nitrogen. 0.0043 0,0050 100: 116.1 In this case, some increase of asparagine nitrogen was also observed, but the absolute increase was only 4 as compared with the increase of organic bases. From the above three experiments we may safely conclude that the ammonium salts offered to the coniferous plants are chiefly converted into arginin and only a small quantity of asparagine is formed synthetically, which latter is the chief product of the assimilation of ammonium salts in other plants.f Now let us try to see whether arginin can be synthetically formed in other plants, not belonging to the Coniferae. BP LLANES NOL BELONGING GO THe CONIPERAE. 1. BRASSICA RAPA. On the 7th November, young plants, 10-12 cm. high, grown in the field was carefully removed, washed well, a portion of it was directly dried and the other portion was divided into 2 parts and grown in the following solutions :— 1) Half saturated gypsum solution. 2) 0.5% ammonium sulphate solution, half saturated with gypsum. * Neither ammonia nor peptone was likewise found in this case. + Compare my article “On the Formation of Asparagine etc.’’? Bull. II. No. 7, of the Coll. of Agri., Tokyo. I also made the same experiment twice with Pinus Thunbergii, adding 0.29% sodium nitrate instead of the 0.59% ammonium chloride solution, but failed to prove the increase of arginin, because by exposure to direct sun-light the nitrate offered to the shoots was too quickly transformed into proteids, and no intermediate product remained in. the plant cells. 36 U. SUZUKI. They were kept in the laboratory in diffuse day-light, the temperature varying between 10°C. and 15°C. After 7 days, they were removed from the solutions, washed well, dried and analyzed. The solutions were renewed twice and no bacterial growth was observed. Toward the end of the experiment, some of the leaves began to suffer, and the top became yellow. These weaklings were removed. The analysis gave the following results :-— In 100 parts of dry matter :— a) by c)* Plants, dried Plants kept in Plants kept in am- on ‘gypsum solution monium sulphate 7th Nov. (7-14th). solution (7-14th), Total nitrogen. 4.90 6.12 8.35 Albuminoid nitrogen. 4e21 2.88 3.68 Asparagine nitrogen. 0.18 1.48 2.52 Nitrogen in phospho- ; tungstic precipitate. 0.28 0.30 0.36 Other nitrogen. G23 1.46 179 Here we found no increase of organic bases ; on the contrary, much increase of asparagine was observed. 2. HORDEUM DISTICHON. On the 7th November, young barley plants 10cm. high, were carefully removed from the field and washed well. A portion was directly dried, while the other portion was divided into two lots and grown in the following solutions :— 1) Half saturated gypsum solution. 2) 0.5% ammonium sulphate solution, half saturated with gypsum. * During the experiment, much decomposition of proteids took place, in consequence of which much increase of asparagine was observed in the control plants b). The increase was, however, much greater in c), which must be due partly to synthetical formation from the ammonium salt. The higher percentage of total nitrogen in b) was due to the decrease of dry matter during the experiment and in c) and partly to the absorption of ammonium salt by the roots, Here we need not calculate the absolute quantity of 100 shoots etc., the result*being too decisive. ON THE FORMATION OF ARGININ IN CONIFEROUS PLANTS. 37 They were kept in the laboratory in diffuse day-light, the temperature ranging between 10° C. and 15° C. After 7 days they- were taken out from the solutions, washed, dried and analyzed. In 100 parts of dry matter :-— a) b)* er Plants, keptin Plants, kept in Plants, dried gypsum solution ammonium sul- on the 7th. for 7 days phate solution for (7th-14th). 7 days (7th-14th). Total nitrogen. 5.40 5.50 6.60 Albuminoid nitrogen. 4.52 3.04 4.27 Asparagine nitrogen. 0.30 1.52 1.42 Nitrogen in phospho- tungstic precipitate. 0.42 0.52 0.64 Other nitrogen. 0.16 0.48 0.27 We found again no increase of organic bases in this case. 3. SECOND EXPERIMENT WITH THE SHOOTS OF BARLEY. (Hordeum distichon.) On the 2nd November, grains of barley were sown in four wooden boxes containing purified saw dust. Two were kept in the dark and the other two in full day-light in a warm house. Germination had fairly set in and on the 14th the shoots were 6-8 cm. high. One of the boxes of either set was then treated with a half saturated gypsum solution and the other with a 0.5% ammonium sulphate solution half saturated with gypsum. After 7 days (on the 21st) the shoots were carefully removed from the saw dust, washed well, dried and analyzed. No ammonia was stored up in the shoots. The analysis gave the following results :— In 100 parts of dry matter. Plants kept in :— * During the experiment much decomposition of proteids took place, in consequence of which much asparagine was formed. But we found no difference in the amount of asparagine in b) and c); the asparagine once formed being perhaps transformed into proteids. 38 Ue) SUZUKA. Dark. Light. ——SSeN — a) b) a) b) Treated Treated with Treated Treated with with ammonium with ammonium gypsum. sulphate. gypsum. sulphate. Total nitrogen. 3.82 4.58 272 4.40 Albuminoid nitrogen. 2.48 2.56 2.21 2.40 Asparagine nitrogen. 0.7 0.90 0.94 1.52 Nitrogen in phospho- tungstic precipitate. 0.26 0.36 0.24 0.27 Other Nitrogen. 0.38 0.76 0.33 0.21 We see that the shoots treated with the ammonium sulphate solution, produced both in the dark and in day light, much asparagine, but no organic bases. 4. CUCURBITA MELOPEPO (pumpkin). On the 2nd November, pumpkin seeds were sown in purified sea sand; one portion was kept in the dark and the other portion in day light in a warm house, where the temperature ranged between 15° C. and 35° C. On the 16th, the shoots kept in the dark were 5-13 cm. high and those kept in day light 4-8.5 cm. high; then one-half of the shoots in both plots was removed from the sand, washed and analysed, and the remaining half was treated with a 0.59% ammonium sulphate solution, half saturated with gypsum. After 7 days (22nd) the shoots in the dark were 18 cm. high, while those in day light were 12 cm. high. Both were then removed from the sand, washed well, dried and analyzed. In 100 parts of dry matter. Shoots kept in :— Dark. Light. Control Treated with Treated with (dried on ammonium Control. ammonium the 16th). sulphate sulphate (16-22). (16-22). Total nitrogen. 6.74 8.65 6.96 8.62 Albuminoid nitrogen. 5.85 5.59 52 4.87 Asparagine nitrogen. 0.28 1.36 0.38 2.09 ON THE FORMATION OF ARGININ IN CONIFEROUS PLANTS. 39 Nitrogen in phospho- tungstic precipitate. 0.17 0.49 0.30 0.60 Other nitrogen. 0.44 [.21 0.56 1.06 During the experiment, the shoots treated with the am- monium sulphate solution grew rapidly, in consequence of which much decomposition of proteids and accumulation of asparagine must have taken place. Therefore the considerable increase of asparagine both in the dark and in day-light may be due partly to the decomposition of proteids and partly to the direct assimi- lation of the ammonium salt. Only a- little increase of organic bases in the shoots treated with the ammonium salt was observed. The above four experiments suffice to prove that the plants, not belonging to the Coniferae, can not produce arginin from ammonium salts, and asparagine alone seems to play an import- ant role in the metabolism of nitrogen compounds. This result agrees exactly with that of my former investigation (Compare my article, ‘‘On the Formation of Asparagine in Plants, etc.” Bull. Aor: Coll, Tokyo. Vol. II. No.-7.) IJ. On the Influence of Light and Mineral Nutriments upon the Formation and Transformation of Arginin. Since the investigation of E. Schulze and of myself have shown that in the shoots of Coniferae, a considerable amount of organic bases, especially arginin, is formed, and since further my last investigation has demonstrated that the bases are not only formed by the decomposition of reserve proteids, but also synthetically from ammonium salts, it seems highly probable that arginin in Coniferae plays an important role in the formation of proteids. SoItried to observe more closely the behaviour of it in the shoots during the germination process, and further the influence of light and mineral nutriments on it. As the object of the experiment, young shoots of Pinus Thunbergii were used. The seeds were soaked with water for 3 days and then sown in purified sea sand moistened with distilled water, and kept in a warm house, where the temperature ranged between 15° C. and 30°C. The sand was 40 U. SUZUKI. occasionally irrigated with a half saturated gypsum solution to keep it constantly moist. On the 24th February the germination had fairly set in, and the shoots were subjected to the following treatments : 1. The shoots were kept in perfect darkness until the 23rd March (4 weeks after germination) and the gradual change in nitrogen compounds was observed. a) b) 2 weeks shoots (dried 4 weeks shoots (dried on the 9th March). on the 23rd March). Number of Shoots. 1220 1224 Length. 6-8 cm. 10-15 cm. Total dry weight. 7.437 &. 7.730 Dry weight of 100 shoots. 0.610 g. 0.631 In 100 parts of dry matter. a) b) Total nitrogen. 0.13 9.53 Albuminoid nitrogen. oma 2.94 Asparagine nitrogen. 2230, 2.35 Nitrogen in phospho- tungstic precipitate. 1.60 1.88 Other nitrogen. 212 2.30 In 100 parts of total nitrogen. a) b) Total nitrogen. 100.0 100.0 Albuminoid nitrogen. 24a 30.9 Asparagine nitrogen. 25.2 24.7 Nitrogen in phospho- tungstic precipitate. 17.5 18.7 Other nitrogen. 23.2 PASH) Every 100 shoots contained :— a) b) Total nitrogen. 0.0557 0.0601 Albuminoid nitrogen. 0.01GO 0.0186 Asparagine nitrogen. 0.0140 0.0148 Nitrogen in phospho- tungstic precipitate. 0.0098 0.0119 Other nitrogen. 0.0129 0.0149 ON THE FORMATION OF ARGININ IN CONIFEROUS PLANTS. 4I Calculating the total nitrogen of a) as 100 we have :— a) b) Total nitrogen. 100.0 108.0 Albuminoid nitrogen. 34.1 3303 Asparagine nitrogen. 2D 26.6 Nitrogen in phospho- tungstic precipitate. 17.5 21.3 Other nitrogen. 222 26,3 In the 2 weeks shoots, the reserve material in the seed has not yet been completely transported to the shoots and a portion still remained undissolved. Therefore we find a little increase of total nitrogen in the 4 weeks shoots. We see from the above results that a considerable amount of organic bases is already formed in the first 2 weeks and after-wards increases only very gradually. Plate III. shows the change of various nitrogenous compounds (especially nitrogen in phospho-tungstic precipitate) during the developement of the shoots of Pinus Thunbergii in perfect darkness. 2. The shoots were kept in /w// day-light and harvested in 4 different periods to see the change in nitrogen compounds. a) b) c) d) Shoots dried after 14 19 28 32 days (oth March) (14th) (23rd) (27th) Number of shoots. 1348 710 780 1656 Length. 6-8 8—Io IO-I5 12-16 Total dry weight. 8.672 A750 7085 15.533 Dry weight of 100 shoots. 0.643 0.669 0.921 0.938 In 100 parts of dry matter. a) b) Cc) d) Total nitrogen. 8.81 8.73 6.95 6.90 Albuminoid nitrogen. 3.51 3.81 a7 3.29 Asparagine nitrogen. 1.32 1.24 1.26 TBS Nitrogen in phospho- tungstic precipitate. 1.54 1.62 0.44 0.38 Other nitrogen. 2.44 2.06 1.98 2.08 42 U. SUZUKI. In 100 parts of total nitrogen a) b) Cc) d) Total nitrogen. 100.0 100.0 100.0 100.0 Albuminoid nitrogen. 39.8 43.9 47.1 47.7 Asparagine nitrogen. 15.0 14.2 18.1 16.7 Nitrogen in phospho- tungstic precipitate. 17.5 18.6 6.3 5.5 Other nitrogen. 27.7, 23.6 28.5 30.1 Every 100 shoots contained :— a) b) c) d) Total nitrogen. 0.0567 0.0584 0.0640 0.0647 Albuminoid nitrogen. 0.0226 0.0255 0.0301 0.0309 Asparagine nitrogen. 0.0085 0.0083 0.0116 0.0108 Nitrogen in phospho- tungstic precipitate. 0.0099 0.0107 0.0040 0.0036 Other nitrogen. 0.0157 0.0137 0.0182 0.0195 Calculating the total nitrogen of a) as 100 we have :— a) b) c) d) Total nitrogen. 100.0 103.0 TI20 114.2 Albuminoid nitrogen. 39.8 45.0 53.2 54.3 Asparagine nitrogen. 15.0 14.6 20.5 19.0 Nitrogen in phospho- tungstic precipitate. 17.5 19.2 ik 6.3 Other nitrogen. PEGI 24.2 322 34.2 We see frem the above results that the shoots produce, even in full day-light, in the first stage of germination much organic bases, the quantity of which is almost equal to that which is produced in the dark. They increased for 19 days after germi- nation, but afterwards decreased rapidly with the increase of proteids. The transformation of the bases is far greater and guicker than that of asparagin.* * Prianischnikow found in his experiment (“Zur Kenntniss der Keimungsvorgange bei Vicia sativa,’ Landw. Vers. Stationen Bd, XLV, 1896, S. 247), that the organic bases in the germinating shoots of Vicia sativa undergo no noticeable change both in the dark and in day-light. Thus, he says, “Die in Phosphorwolframsaure-Niederschlag eingegangene Stickstofimenge zeigt keine besondere Schwankungen, indem sie etwa 5 ON THE FORMATION OF ARGININ IN CONIFEROUS PLANTS. 43 Plate IV. shows the change ,of various nitrogenous com- pounds during the development of the shoots of Pinus Thun- bergii, under the full day-light. 3. The shoots were at first kept in the dark and afterwards exposed to full day-light, and the change in nitrogen com- pounds observed. a) Etiolated shoots dried on the oth March......... 14 days after germination. b) Etiolated shoots dried on the 23rd March......... 28 days after germination. c) Etiolated shoots were exposed to full day-light from gth to 23rd March, that is, kept in dark the first 2 weeks and for the next 2 weeks exposed to day-light and treated with half saturated gypsum solution. d) Treated in the same way as c) and with a solution con- taining 0.1% K,HPO, 0.1% KH.PO, 0.199 MgSQO,, half saturated with gypsum. Analysis gave the following results :— a) b) c) d) Number of shoots. 1220 122 1500 1342 Length. 8-10 15-17 10-12 10-12 Total dry weight. easy 7.730 11668 10.284 Dry weight of 100 shoots. 0.610 0.631 0.778 0.766 des Gesamtstickstoffs betragt.”’ The chief organic bases in Vicia sativa are cholin, betain and guanidin etc. We may also suppose that the organic bases, except arginin, play no important réle in coniferous plants, and the principal cause of the increase or decrease of nitrogen in phospho-tungstic precipitate is chiefly due to the change of arginin. Prianischnikow also found that during the first stage of germination (first 10 days) the greater part of proteids decomposes, and amido-compounds, especially asparagine, accumulate in large quantities, but that afterwards, both the decomposition of proteids and the accumulation of asparagine etc. are only very gradual. The change in nitrogen compounds proceeds in the first period almost equally both in the dark and in day-light, If we compare my result with that of Prianischnikow, we shall find out a closer resem- blance between asparagine and arginin in the mode of formation and transformation into proteids and in its behavior toward light. 44 In 100 parts of dry matter. U. SUZUKA: a) b) c) d) Total nitrogen. 9.13 9.53 8.11 8.19 Albuminoid nitrogen. 3.11 2.94 3.18 Bo Asparagine nitrogen. 2.30 2air 1.64 1.60 Nitrogen in phospho- tungstic precipitate. 1.60 1.88 1.12 0.96 Other nitrogen. . BET? 2.36 2.18 2.38 In 100 parts of total nitrogen. a) b) c) d) Total nitrogen. 100.0 100.0 100.0 100.0 Albuminoid nitrogen. 34.1 30.9 30.2 30-7 Asparagine nitrogen, 25.2 DAL 20.2 19.5 Nitrogen in phospho- tungstic precipitate. 17.5 18.7 13.7 11.7 Other nitrogen. 23.2 ASG) 26.9 29.1 Every 100 shoots contained :— a) b) c) d) Total nitrogen. 0.0557 0.0601 0.0621 0.0637 Albuminoid nitrogen. 0.0190 0.0186 0.0244 0.0249 Asparagine nitrogen. 0.0140 0.0148 0.0127 0.0123 Nitrogen in phospho- tungstic precipitate. 0.0098 0.0119 0.0085 0.0075 Other nitrogen. @.0129 0.0149 ©0167 | O.OIOI Calculating the total nitrogen of a) as 100 we have :— a) b) c) d) Total nitrogen. 100.0 108.0 I11.6 114.5 Albuminoid nitrogen. 34.1 3353 43.8 44.7 Asparagine nitrogen. 25.2 26.6 22.5 22.0 Nitrogen in phospho- tungstic precipitate. 17.5 21.8 15.3 13.4 Other nitrogen. 23.2 26.8 30.0 34.4 Here we observe that when the etiolated shoots were exposed to full day-light organic bases decreased with the increase of proteids; but in this case, the decrease of the bases ON THE FORMATION OF ARGININ-IN CONIFEROUS PLANTS. 45 was not so remarkable as in the 2 former experiments. This may be explained by considering that the shoots, having at first been kept in the dark, lost the greater part of carbohydrates by respiration and also that the chlorophyll bodies being decom- posed by keeping in the dark, the assimilation of the carbonic acid gas did not proceed so energetically as to prepare enough carbohydrates necessary for the regeneration of arginin into proteids. Also the energy of living protoplasm might have been greatly diminished by keeping it in the dark. Therefore if I had kept them still longer then, the shoots would have recover- ed from the abnormal condition and much decrease of the bases would have been observed! We found also that the shoots treated with mineral nutriments transformed much organic bases into proteids. Plate V. shows the influence of light upon the change of nitrogenous compounds in the shoots of Pinus Thunbergii. The shoots were kept for 14 days in perfect darkness and a portion was afterwards, exposed to the day-light for 14 days, while the other portion was still kept in the dark. The black lines refer to shoots kept in the dark until the end of the experiment. The red lines refer to shoots, previously kept in the dark, was exposed to the full day-light for 14 days. 4. The shoots which was first exposed to full day-light, were afterwards kept in the dark, and the change in nitrogen compounds observed :— a) The shoots kept in full day-light until 15th March (18 days after germination). b) The shoots were afterwards kept in the dark, from 15th to 27th, with a half saturated gypsum solution. a) b) Number of shoots. 710 560 Length. 8-10 cm. 10-12 Total dry weight. 4.750 3.819 Dry weight of 100 shoots. 0.669 0.681 In 100 parts of dry matter: | a) b) Total nitrogen. 8.73 9.16 46 U. SUZUKI. * Albuminoid nitrogen. 3.81 3-13 Asparagine nitrogen. 1.24 1.58 Nitrogen in phospho- tungstic precipitate. 1.62 1.59 Other nitrogen. 2.06 2.81 In 100 parts of total nitrogen. _ a) b) Total nitrogen. 100.0 100.0 Albuminoid nitrogen. 43.6 34.2 Asparagine nitrogen. 14.2 172 Nitrogen in phospho- tungstic precipitate. 18.6 17.4 Other nitrogen. 23.6 21.2 Every 100 shoots contained :— 2) b) Total nitrogen. 0.0584 0.0624 Albuminoid nitrogen. 0.0255 0.0213 Asparagine nitrogen. 0.0083 0.0108 Nitrogen in phospho- tungstic precipitate. 0.0109 0.0108 Other nitrogen. 0.0137 0.0191 Calculating the total nitrogen of a) as 100, we have :— a) b) Total nitrogen. 100.0 106.8 Albuminoid nitrogen. 43-6 36.5 Asparagine nitrogen. 14.2 18.4 Nitrogen in phospho- tungstic precipitate. 18.6 18.6 Other nitrogen, 23.6 32:7 Here, by keeping in the dark, some decrease of proteids and increase of asparagine and other amido-compounds was observed but no increase of organic bases. ON THE FORMATION OF ARGININ IN CONIFEROUS PLANTS. 47 Plate VI. shows the influence of light upon the change of nitrogenous compounds. Shoots were at first exposed to the full day-light for 19 days, then a portion was kept in the dark for 9 day, while the other portion was still exposed to the day- light. The black lines refer to shoots exposed to the full day- light. The red lines refer to shoots afterwards kept in the dark for 9 days. The chief results obtained from the above four experiments are :— 1). Arginin accumulates in a large quantity, in the dark as well as in full day-light, in the first stage of germination but disappears quickly when the shoots are further exposed to day- light, most probably being directly used for the regeneration of proteids. It, however, increases gradually when the shoots are kept still further in the dark. 2). The formation and transformation of arginin in conifer- ous plants are far greater and quicker than those of asparagine. 3). The addition of mineral nutriments accelerates the transformation of arginin. 4). Asparagine is also present in a tolerably large quantity in the shoots of coniferous plants. It must have also an import- ant function. Although we have not been able to prove experimentally the transformation of other amido-compounds into arginin, yet we can deduce its probability from the following facts :— a) Inthe shoots, we find sometimes so much arginin that we can not regard it as merely the primary decomposition product. Thus I found in the shoots of Cryptomeria japonica about 219% of the total nitrogen in the form of organic bases ; while, when the proteids of the seed are treated with dilute or strong hydrochloric acid, the nitrogen of the organic bases amounts to only 15.5% of the whole. Therefore if arginin came only from the hydrolytic decomposition of proteids, then we should never find more than 15.59 of the total nitrogen in the shoots. Further, I found that the proteids prepared from the shoots of Pinus Thunbergii, have also the same chemical nature, and produce, by the action of hydrochloric acid, almost the same quantity of organic bases as those of the seeds. This fact may hold good also in the shoots of other Coniferae. Now 48 U. SUZUKI. in the shoots of Cryptomeria I found still 49% of the total nitrogen in the form of albuminoid nitrogen, from which we can obtain at least 49x 15.59% =7.6% organic bases. If we suppose that the entire proteids in the shoots were split, then we must have at least 21.+7.6=28.6% of organic bases. Such a large quantity of the bases can never come from mere hydrolytic decomposition !* b) As I have already shown that the coniferous plants can converts ammonium salts into arginin, so it is highly probable that the ammonia formed by the decomposition of other amido- compounds in the plant cells may be easily converted into arginin, just in the same way as asparagine is formed in the other plants. The question whether arginin is formed at the cost of asparagine or whether the former can be converted into asparagine before it is used for the regeneration of proteids, is still left open. SUMMARY AND CONCLUSIONS. 1. Arginin in coniferous plants not only comes from the decomposition of proteids but also can be synthetically formed from the ammonium salts (and also from nitrates !) offered to the plants. 2. Plants not belonging to the Coniferae do not produce arginin by the assimilation of ammonium salts, asparagine being the only product. 3. The synthetical formation of arginin proceeds in full as well as in diffuse day-light. But whether it is formed in the dark is not yet proved. 4. Arginin accumulates in a large quantity in the shoots of Coniferae in the dark as well as in full day-light in the first * E. Schulze found in the etiolated shoots of Picea excelsa 54.6% albuminoid nitrogen and 29.3% nitrogen in phospho-tungsti¢ precipitate. But by the action of hydrochloric acid, he found that ahout 30% of the total nitrogen comes as the nitrogen in phospho-tungstic precipitate ; so we can get about 309% nitrogen in phospho-tungstic precipitate only when all proteids are decomposed, if we assume that the bases are formed only by the hydrolytic decomposition. But we have here still 54.69% of nitrogen in the form of proteids which can split off at least 54.6X30%=16.49% nitrogen in phospho-tungstic precipitate. (Compare E. Schulze :—Zeits. f. Physiol. Chem. XXIL 1896. S. 441.) ON THE FORMATION OF ARGININ IN CONIFEROUS PLANTS. 49 stage of germination, but soon diminishes on further exposure to light and gradually increases on further sojourn in the dark. Its transformation into proteids under the influence of light can be accelerated by the addition of mineral nutriments. 5. Although the greater part of arginin in the shoots of Coniferae comes from the hydrolytic decomposition of reserve proteids, yet a portion of it must come also from the trans- formation of other amido-compounds, that is, it is not onlya primary product, but also a secondary or transitory product. 6. Arginin is probably directly used for the regeneration of proteids; but its relation to other amido-compounds still needs further elucidation. ANMEVTICAL DATA J. Arginin as the Assimilation product from Ammonium salts. Ay GONIREROUS PEANTS: 1. PINUS THUNBERGII. TOTAL NITROGEN. Dry matter Baryta water Nitrogen Percentage of used. replaced. found. nitrogen. 50 U. SUZUKI. ALBUMINOID NITROGEN. Dry matter Baryta water Nitrogen Percentage of used, replaced. found. nitrogen. OG { I. 0.886 16.2 l a) 0.0303 3.42 le. 03 16.4 j I. 0.960 16.5 b) 0.0305 3.18 a 3 16.3 } I. 0.938 16.9 | c) 0.0314 3:35 2 53 16.9 ASPARAGINE NITROGEN. Dry matter Baryta water Nitrogen pire a used, replaced. found. —1 Asp. N. I. 0.886 } a) 0.0056 0.62 2. ” ( I. 0.960 b)* | 0.0055 0.57 2. ” I. 0.938 Cc) 0.0056 0.60 2. > NITROGEN IN PHOSPHO-TUNGSTIC PRECIPITATE. Dry matter Baryta water Nitrogen Percentage of used, replaced. found. nitrogen, c.c. 1. 0.886 8.5 ) a) > 0.0160 1.81 Za 87 I. 0.960 3:5 b)* 0.0037 0.38 2 ” 33 I. 0.938 5 c) 0.0145 1.55 2. ” 7-4 I c.c. baryta water =0.00189 gram nitrogen. I c.c, baryta water =0.00108 gram nitrogen. ON THE FORMATION OF ARGININ IN CONIFEROUS PLANTS. 51 2. PINUS THUNBERGII. TOTAL NITROGEN. Dry matter Baryta water Nitrogen Percentage of used. replaced. found. Nitrogen. ALBUMINOID NITROGEN. Dry matter Baryta water Nitrogen Percentage of used, replaced. found, nitrogen. Glee . I. 0.960 29.0 | ee = 3 3:29 zis) 20.4 z I. 0.932 26.8 ) nied bt ) 0.0292 3:13 2. 5 27.2 j I. 0.918 26.8 c) | l 0.0292 3.17 a 27.2 ASPARAGINE NITROGEN. Percentage of Dry matter Baryta water Nitrogen nitrogen used. replaced. found. = Asp. N. c.c, (I. 0.960 [5.2 Za 5.0 2, 35 5.0 c) | | I, 0.918 6.0 Ge 3 6.1 52 U. SUZUKI. NITROGEN IN PHOSPHO-TUNGSTIC PRECIPITATE. Dry matter Baryta water Nitrogen Percentage of used. replaced. found. nitrogen. CIC: f I. 0.960 BES a) / 0.0037 0.38 oF, » 3.3 I. 0.932 3.8 ) b) 0.0042 0.45 L 2. ” 4.0 ( I. 0.918 11.4 ) Cc) OLOl2 1.37 2: 9 11.0 } I c.c, baryta water =o0.00108 gram nitrogen. 3. CRYPTOMERIA JAPONICA. TOTAL NITROGEN, Dry matter Baryta water Nitrogen Percentage of used. replaced. found. nitrogen. c.c ip CODY 10.8 a) 0.0118 6.69 2 53 11.0 I. 0.182 12.9 ) b) > 0.0138 7.62 2. ” 12.7 ) ALBUMINOID NITROGEN. Dry matter 3aryta water Nitrogen Percentage of used. replaced. found. nitrogen. Ce I. 0.433 WAL j a) f 0.0137 3-17 2 » 12:7 TO:42u 11.5 ) b) 0.0129 3.05 2 ” 12.3 j ON THE FORMATION OF ARGININ IN CONIFEROUS PLANTS. 53 ASPARAGINE NITROGEN. Dry matter Baryta water Nitrogen Seca ee of used, replaced. found. ie fee = Asp. N. CO ( I) 0433 0.8 a) 4 0.0009 0.21 l De 0.8 ( I. 0.421 1.1 } b) 5 0.0013 0.31 aie 1.3 NITROGEN IN PHOSPHO-TUNGSTIC PRECIPITATE. Dry matter Baryta water Nitrogen Percentage of used. replaced. found. nitrogen. c.c ( I. 0.433 4.3 a) 4 7 0.0049 1.12 len Is 4-7 ( I. 0.421 6.8 } b) | j 0.0075 1.77 2. as 7.0 I c.c, baryta water =o.00108 gram nitrogen. By PLANTS NOT BELONGING TO THE CONIFERAE. 1. BRASSICA RAPA. TOTAL NITROGEN. Dry matter Baryta water Nitrogen Percentage of used. replaced. found. nitrogen. Cc ( I. 0.434 8.6 | a) 4 a 0.0212 4.90 | 2. 99 8.6 | Te 1O:432 10.5 b) § : | 0.0264 6,12 ckewass 10.9 I. 0.438 14.9 c) i) 0.0366 8.35 | Pp, 4 14.7 j 54 U. SUZUKI. ALBUMINOID NITROGEN. Dry matter Baryta water Nitrogen Percentage of used, replaced. found. nitrogen. Cc. | I. 0.868 14.6 ) a) 0.0366 4.21 ie 14.9 ) f I. 0.866 10.0 ) b) 0.0250 2.88 \ 2: : 10.2 ( I. 0.876 13.0 c) 4 0.0322 3.68 | 2 P 13.1 ASPARAGINE NITROGEN. - E : = Percentage of Dry matter Baryta water Nitrogen nitrogen used. replaced. found. =} Asp. N NITROGEN IN PHOSPHO-TUNGSTIC PRECIPITATE. Nitrogen Percentage of Baryta water found. nitrogen. replaced. Dry matter used. I c.c. baryta water =0.00247 gram nitrogen. ON THE FORMATION OF ARGININ IN CONIFEROUS PLANTS. 55 é 2. HORDEUM DISTICHON. TOTAL NITROGEN. Dry matter Baryta water Nitrogen Percentage of used. replaced. found. nitrogen. ALBUMINOID NITROGEN. Dry matter Baryta water Nitrogen Percentage of used, replaced. found, nitrogen, CH I. 0.880 16.0 a) 2. “5 16.2 Mie, oystys 10.8 b) ? 10.8 ASPARAGINE NITROGEN. Percentage of Dry matter Baryta water Nitrogen nitrogen used. replaced. found, =} Asp. N. Gc: 1.760 1.0 | a) ( 0.0027 0.15 2 ” 1.755 56 U. SUZUKI. NITROGEN IN PHOSPHO-TUNGSTIC PRECIPITATE. Dry matter Baryta water Nitrogen Percentage of used. replaced. found. nitrogen. ( I. 1.760 3-1 a) | 0.42 2 + 2 f ie, Sigpisre’ 3.6 b 4 0.52 ) | 2. 55 3.8 I. 76. 5 c) § roe a 0.64 (2. 5 4.6 1 c.c. baryta water =0.00247 gram nitrogen, 3. HORDEUM DISTICHON. TOTAL NITROGEN. Dry matter Baryta water Nitrogen Percentage of used, replaced. found. nitrogen. ' c.c. a) ee a 0.0175 382 20s 72 j Dark I. 0.458 8.8 b) | A | 0.0210 4.58 i I. 0.458 6.9 a) 0.0170 3-72 | 2: 3 6.9 Light I. 0.454 8.0 b) 3 § 0.0200 4.40 2 Ps 8.2 ON THE FORMATION OF ARGININ IN CONIFEROUS PLANTS. 57 ALBUMINOID NITROGEN. Dry matter Baryta water Nitrogen Percentage of used, replaced. found, nitrogen. c.C, | Paone) 7 9.0 ) a) 0.0227 2.48 2. ” 9.4 J Dark I. 0.916 95 b) | l 0.0235 2.56 2. ” 95 I. 0.915 8.5 ) a) - 0.0202 2.21 2. ” 8.7 Light 7 I. 0.907 8.7 ( 5 0.0217 2.40 2. is 8.9 ASPARAGINE NITROGEN, D RB 0.0411 9.13 22.3 { 24.2 ) b)* j 0.0452 9-53 24.4. * 1 c.c. baryta water =0,00186 gram nitrogen. ON THE FORMATION OF ARGININ IN CONIFEROUS PLANTS. 61 ALBUMINOID NITROGEN. Dry matter Baryta water Nitrogen Percentage of used. replaced, found, nitrogen, Cc I. 0,900 25.4 } y a) 0.0280 Bo 2 26.4 j I. 0.948 25.4 b) 0.0279 2.94 2 Ay 26.1 \ ASPARAGINE NITROGEN. Dry matter Baryta water Nitrogen Percentage of used replaced found mLOEeE < placed. : =} Asp. N Gis j I. 0.9CcO 95 a) 3. 0.0104 1.15 | ae a 9.8 I. 0.948 10.1 b) 0.0112 1.17 2 es 10.6 NITROGEN IN PHOSPHO-TUNGSTIC PRECIPITATE. Dry matter Baryta water Nitrogen Percentage o used. replaced. found. nitrogen. C.c I. 0.900 13.7 } a) 00144 1.60 | 2. » 12.9 I. 0.948 16.8 } b) - 0.0178 1.88 Gy Fs 16.1 \ I c.c, baryta water =0,00108 gram nitrogen. 62 U. SUZUKI. TOTAL NITROGEN. Dry matter Baryta water Nitrogen Percentage of used, replaced. found, nitrogen. Gic: 21.5 a) 0.0404 8.81 21.9 20.5 b) 4 ( 0.0387 8.73 20. 18.0 c) 0.0339 6.95 18.4 18.0 d) 0.0331 6.90 17-5 } ALBUMINOID NITROGEN. Dry matter Baryta water Nitrogen Percentage of used. replaced. found. nitrogen. | GG, ( I; 0.916 17.0 0.0322 a) 351 | 2. 5 qe7 ; ( I. 0.886 18.0 0.0338 P b) 3.51 f 2: 5 18.3 s ‘ \e ( I. 0.974 29.7 0.0319 ~ c)* 4 3.2 | 2: 55 29.2 : ay ( I. 0.960 29.0 0.0316 * 4 3.29 re 29.4 5 ON THE FORMATION OF ARGININ IN CONIFEROUS PLANTS. 63 ASPARAGINE NITROGEN, Dry matter Baryta water Nitrogen Percentage of used. replaced. found. ae 'N I. 0.916 a) | 0.0061 0.66 (I. 0.886 b) J 0.0056 0.62 lear, } I. 0.974 ©) 5 @ | 0.0062 0,63 [zee 2 (I. 0.960 d)* 0.0055 0.57 NITROGEN IN PHOSPHO-TUNGSTIC PRECIPITATE. Dry matter Baryta water Nitrogen Percentage of used. replaced. found. nitrogen. Cac. I. 0.916 7.8 ) a) - 0.0141 eh mens 74 j I. 0.886 7.8 b) § 0.0144 | ae Py) 7.6 i I. 0.974 4.1 ) c)= 0.0043 i Oe 3-9 I. 0.960 35 d)* 0.0037 25 ” 33 I c.c, baryta water =o0.00186 gram nitrogen. * rc.c, baryta water =o.oo108 gram nitrogen, 64 U. SUZUKI. ° 7 9) TOTAL NITROGEN. Dry matter Baryta water Nitrogen Percentage of used. replaced. found. nitrogen. CHE 2 I. 0.450 22.0 a) 0.0411 9.13 25 aS 22.3 | p I. 0.474 24.2 } b) 0.0452 9-53 ae za J I. 0.486 21.3 ) c) - 0.0394. 8.11 2. * 21.0 \ : { I. 0.479 21.2 i d) 0.0393 8.19 l De 7 21.0 J ALBUMINOID NITROGEN. Dry matter Baryta water Nitrogen Percentage of used, replaced. found, nitrogen. ON TIIE FORMATION OF ARGININ IN CONIFEROUS PLANTS. 65 ASPARAGINE NITROGEN. Dry matter Baryta water Nitrogen Rereneaes used, replaced, found. ae ies I. 0.900 95 ) a) 0.0104. 1.15 2: a 9.8 \ I. 0.948 10.1 ) b)* 0.0112 ety) 2, op 10,6 \ I. 0.972 2 ) c)* 0.0080 ~ 0.82 2. » 4-4 I. 0.958 7.0 d) ( 0.0077 0.80 2. 5 7:2 NITROGEN IN PHOSPHO-TUNGSTIC PRECIPITATE. Dry matter Baryta water Nitrogen Percentage of used. replaced. found. _ nitrogen, ( 0.0144 1.60 0.0108 I.1L 0.0092 0.96 _b) | : es ot 0.0178 1.88 \ | | | Ds Pr) 8.2 I c.c. baryta water =o.00108 gram nitrogen, * 1 c.c. baryta water =0.00186 gram nitrogen. 66 U. SUZUKI. 4. TOTAL NITROGEN, Dry matter Baryta water Nitrogen Percentage of used. replaced, found. nitrogen. G:C: (1- 0.443 20.5 a) - 0.0387 8.73 {2 20.8 (3 0.481 23.6 } b) 4 - 0.0441 9.16 | 2. # 23.8 | ALBUMINOID NITROGEN. Dry matter Baryta water Nitrogen Percentage of used, replaced. found, nitrogen. Ee { I. 0.886 18.0 a) 4 - 0.0338 3.81 | 2. £ 18.3 ( I. 0.962 16.0 he b) : > 0.0301 353 dace oe 16.5 } ASPARAGINE NITROGEN, Dry matter Baryta water Nitrogen eee = Bt used, replaced. found. ax Ae N. ( I. 0.886 3.1 ) a) 0.0056 0.62 | 2 $3 2.8 { I. 0.962 z b) 4 0.0076 0.79 l 2. 2? 4.0 ON THE FORMATION OF ARGININ IN CONIFEROUS PLANTS. 67 NITROGEN IN PHOSPHO-TUNGSTIC PRECIPITATE. Percentage of Dry matter Baryta water Nitrogen nitrogen used. replaced. found. =) ey N. CHe (1. 0.886 78 a) j 0.0144 1.62 | casnanaes 76 I. 0.962 8.0 ) b) - 0.0153 1.59 Br x 8.3 I c.c. baryta water =0.00186 gram nitrogen. ol. IV. Pl. VII. Bull. Agric. Coll. V ys Uhl = Ra ESAS ee Gh a a Sisal iO DS a BE = + : > +} rs} Pa (eR ost aE 4 5! jt et ie = ++ 1 > eecieoes ate ate Hel : sogae (nee Hert te J FEREY ar eeecauee, _ ‘ “| + } ; 5 HH = tlt - ia & Pie ISSN a a seenae . 4 tte } oo = eet it Poteet oo = jt < ‘ i + ees =| NO ye a SOSA Soe Tee eSeee ee Sl pat) =e Ppt 1ST lee a + +—+ =! | j—t—t-- -H4 7 ++ —+ + +4 Ub | } } et ill ME eed a 4 i Ae ee ptt Ht tt 4 SPE ie Es a that j—+—t + + +} 4 f } 4 PE CE GR VE be Wl | (toad RAE Jodo by EES ede | Ly tet RY LS) ec ead teal TER Et Ea] | ee eh oe a ED EPP im | }—+ + + +} 14 CUE (ES IES a | im Ci] [eal \- | CEE tt tooo ao + 4-4 | t——4- 4-- CI i+ 1--t +444 ‘eee [mee [SSE T —- ‘ab +14 4 AO tte a aes { na aeceeatuas SD ae ot a | H i 4 eel ba +4 t+ 4 : + Toletet onl + | isle] aa | tH tw ++ 4 oo Po Ee eal i : ol a | i CP il etd Bal ee ee ee rr es gigel is Pere T | + I} Tt 4 +—+ a is TT rer EERE EEE EEE EEE Ss SERRE EEE 44 Fee EE eee Ee eee eet coy EE ad HE —_—+}— abe oe Sal $f. ft —4j—j}—} ft ppisistclatals (tal Ie] Oe a S| ait im ip “Biba ts ct | | Se as Tet Sree + - - ae ae 7 {++ j++ SHEE EEE ERP A iE OS a SG aE | San + : —+ ' | [a | | | | | | Resse NT [cade Pe | eh wae ei The black lines refer to shoots s, especially arginin, in the shoots This plate shows the increase of organic base of Pinus Thunbergii by the addition of ammonium salts. lines refer to those cultured in 0.59% ammonium chloride solution, half saturated with cultured in the half saturated gypsum solution, in the diffused day-light, and the red gypsum, in the diffused day-light. wae i S p - Be 3 5 = 1 an = ~~ ie : . a t= : _ i he el Bull. Agric. Coll. Vol. TV. Pl. IIT. Beet EEC rae JESS aes Bee CODES aS we Sees matey e Beep Se ON Eee (25005 20050 ee eR iaam Piseispstal Ne | a marae teil ee Bey Ht AT tt mites htt | CONE Beegese eh eae Po et eee rt meine elo et pty ie Fe Revd Ps Pleiadat “ TGo0 Sine meee cere eet JUS SE Do SoS 064 sees mime eto esta tp T | aici ile) 3 Beers Pe ae = al ) mistebeiat tay CLA SENBAD GEER fever al Pera sei ctestostes = ivin ako 59 27,7 OO imap lle) ET eet Serr Titi peer elated mere iiepotteh il. Crt Le at _SaeC0REm BP COE Ce eae eT Iaiat Pa patel lal ied Tmt | meas ECC en Meenimen Seer CECE Peet Segucoeae aes eS mit iat ( Pts SE eageae | ro ion aauE | late iatet Vale Be e518) Hiatt a iS} j PEL OLL ra mere 558 o i: od eee reitaetel PEC | | ge a Sees eee eee Po peter) oa Beeee ee a soisssscesseniitesreers Sees memiciet aj) isl LC es fae | eae e Beeeeeee eee BERS Sessa i pesee ta i.) 7350000000 BE Co Sel Bees See ets SMI aro ttt pt an SRoueeee “ies rt Li [heer fee fa ere a pe a Jeet] aan beable) . . [ea Poca oa |_| Porcine! Li CCG ateiatetiale alcatel tail ater This plate shows the eae Sei various Pnitiogenots compounds (especially nitrogen phospho-tungstic precipitate) during the development of the shoots of Pinus Thunbergii in perfect darkness, - Weare ee did’ de by 3) re Ry ' A Us a i e Ohm der ey aioe F2NE a ae : e a a a a ad i at a a Bull. Agric, Coll. Vol. 1V. Pl. IV. j = i inical seasce FEEEPEEEHE PEC EE EEE EERE EEE eon 4 BESPESa [Ty ane ror am BSS Salt Seceaugeoe PEEP Eee HEEESSEEEEE EEE oeeae de 4 Y fo 2? sic HP amis ee [| (cb Siea bahay ne SROEMECEAZERA RRS This plate shows the change of various nitrogenous compounds during the develop- ment of the shoots of Pinus Thunbergii, under the full day-light. — a a ee oa i hale aed Seve a ee ig a int be 9 ogee > “ah, ate * Bull, Agric, Coll. Vol. IV. Pl. V. ag Total, N WZ Totel WV 1007, 0 IO GO 70 CoO ao Alb. N => , ainn 4O ae ES Ft , maze _A ma. iV = >= Ab. N dO Prat AE “ Amd. N foot alt Sy ts av es ay LY ae JBI: 557 DE tangstic LPC co 7 {== Var phos, Catupstic FPE L0 m7) S 70 WS. 20 26 a BO This plate shows the influence of light upon the change of nitrogenous compounds in the shoots of Pinus lhunbergii. The shoots were kept for 14 days in perfect darkness and a portion was afterwards, exposed to the day-light for 14 days, while the other portion was still kept in the dark, The black lines refer to shoots kept in the dark until the end of the experiment, and the red lines to those, previously kept in the dark, and afterwards exposed to the full day-light for 14 days. * ae | Sip7es 1. er pe 1 7 <= a ‘ one ; a 4 - : oe ; a 7 : Oe a " oa : Sear eo 3 ae mast ie” ; ae Ry Piet — a : » . ies 2 Ss ay <=, 5 * ps ail angie, 5 , yerit & Bull, Agric, Coll. Vol. IV. Pl. VI. Fotat N Fotat-N \ 90 Nt X \ \ 80 \ ; x | \ | \ J : 7O a \ ‘ } | Co - \ TF? \ \ 2d 46 \ 1% \ \ St 4 \ ae a 40 Ven Nae A i 44) 5 30! ee lala Zz — m4) ——_ : prt —— sil 7 ped to > phe ae ———=— So — sat = V REa so ——— hsp, Asp. A 7 A ow ae a _ DS “Sm : “—\in phos tangsttc £Pt 10 75 20 25 30 IS : 4 on Lys This plate shows the influence of light upon the change of nitrogenous compound: Shoots were at first exposed to the full day-light for 19 days, then a portion was kept in the dark for 9 days, while the other portion was still exposed to the day-light. The black lines refer to shoots exposed to the full day-light, and the red lines to those after- wards kept in the dark for g days. ? Can Strontium and Barium Replace Calcium in Phaenogams ? BY U. Suzuki, Mogakushi. It is generally believed that the principal function of lime salts in plants consists in neutralizing oxalic acid or precipitat- ing oxalic acid from soluble oxalates produced by the metabolism of the plant cells. The poisonous oxalic acid is thus removed from the solutions as insoluble oxalate of lime. But it may be objected that many plants do not produce oxalic acid, although lime salts are absolutely necessaay for them, Further the quan- tity of lime needed by the plants does not always correspond with the quantity of oxalic acid produced by them. These facts naturally lead us to look for the physiological function of lime salts in another direction. Recently O. Loew* has propounded the view that the nucleus and chlorophyll bodies contain lime in organized protein com- pounds. Hence if deprived of lime or if lime is substituted by other elements, then those structures will suffer since the capaci- ty for imbibition would change, which would lead to serious disturbances of structure. If the principal function of lime salts merely consists in * O. Loew : Ueber die Physiologischen Functionen der Calciumsalze :—Botanisches Centrbltt Bd. LX XIV. 1898.—His conclusion is as follows :— ; “ Meine Ansicht, dass Zellkerne bei einer gewissen Hohe der Entwickelung, und Chlorophyllkorper, sofern sie nicht auf primitiver Entwickelungsstufe stehen, des Kalks bediirfen und Kalk-Proteinverbindungen sich an deren Organisation betheiligen ist nicht wiederlegt. Sie ist im Gegentheil wahrscheinlicher, als die von Anderen vertheidigte Ansicht, nach welche den Kalksalzen bloss die Besorgung von Stoffwechselvorgangen zukame.” “Strontiumsalze sind so lange den Pflanzen unschadlich, als diesen hinreichend Calciumsalze zur Verfiigung stehen. Jenseits aber eines gewissen Verhaltnises sind schadliche Wirkungen unverkennbar, Eine physiologische Vertretung von Verbindungen des Calcipm durch solche des Strontiums findet nicht statt,” 70 U. SUZUKI. precipitating the oxalic acid produced in the cells, then such elements as strontium and barium, which form insoluble salts with oxalic acid, must prove equally available as calcium. On the contrary, if lime is an integral part of the nucleus and chlorophyll-bodies, then, as Loew says, it can never be replaced by other elements. This is a point which needs a thorough elucidation. In 1893, some experiments on this lime was made by Haselhoff,* who concluded that strontium can replace calcium in phaenogams. But he cultivated his plants in a fertile soil, which contained naturally much lime, and also in his water culture, he added strontium-salts together with lime. Hence we Can not be surprised that he observed no injurious effects. Loew holds the view that when strontium is used together with lime, then the plants will by elective absorption take up much more lime than strontium, and can exchange small doses of strontium taken into organic structures always against the lime present in solution. His observations on the poisonous action of oxalates upon plant-cells under the microscope led him to the view that the calcium is present in the nucleus and chlorophyll bodies in organized protein compounds, as above mentioned. But if strontium-salts alone are offered to the plant, then the effect must be quite different. In accordance with this view, he made two experiments with young branches of Tradescantia repens, and found that the poisonous action of strontium is, to a large extent, masked by the presence of lime, but that as soon as the lime content is diminished to a certain degree, then the poisonous effect of strontium-salts becomes at once apparent As this question appeared very interesting I have made also experiments with five species of phaenogams. The results of these experiments will be described in the following pages :— * Haselhoff ;—(Landw. Jahrbiicher. Bd. XXII. 1893. S. 851.) His conclusion is as follows :— 1) Das Strontium wirkt nicht schadlich auf die Pflanzenentwickelung. 2) Das Strontium wird von der Pflanze anfgenommen und scheint bet der Ernahr- ung die Stelle des Kalks zu vertreten. Diese Substitution des Kalks durch Strontium bei der Pflanzenernahrung scheint aber erst dann einzutreten, wann der Vorrath an Kalk und anderen Niahrstoffen nicht mehr zum Aufbau der pflanzlichen Organismen ausreichen. w — CAN STRONTIUM AND BARIUM REPLACE CALCIUM. 71 san DreULTURE: I. BUCKWHEAT. (Polygonum fagopyrum.) Common sea sand was boiled with strong hydrochloric acid (sp. gr 1.15) for about two hours and then well washed at first with hot water and afterwards with distilled water until no trace of hydrochloric acid was present. The purified sand thus pre- pared was then transfered into four large funnels, of the diameter of about 25 cm., each of which was placed upon a tall glass cylinder, so that the excess of the solution added to the sand may pass through the funnel and drop down into the cylinder, without leaving any excess in it and yet keeping all equally moist. Seeds of buckwheat were previously soaked with water and on March 26, they were equally distributed into the four funnels and kept ina warm house. By April 5, the germinating plants reached 3-8 cm. high. They were then treated with the follow- ing solutions prepared exactly after Haselhoff. \ 2.02 g. KNO, with 0.94 g. K,O0+1.08 g.N,O5]) | ul 1) in 1 litre water. | 3.28 g. Ca(NO,), » 115g. CaO+2.16 g.N,O; 5 j 2.02 g. KNO, » 094g. K,0+1.08 g. N,O; | 4.23 g. Sr(NO,), » 207g. SrO +2.16 g. N,O, a a { 2.02 g. KNO, » 094g. K,O+1.08 g. NO, i 3) | 5.22 g. Ba(NO,), » 306g. BaO+2.16 g. N,O, ‘ ty 2.02 g. KNO, » 094g. K,0+1.08 g.N,O, 4) 1.64 g. Ca(NO,), » 0.575 g. Ca0+1.08 g. NO, | = ze 2.12 g. Sr(NO,), » 1.04g. SrO +1.08 g. N,0, 5.26 g. MgSO,+7H,0 ,, 086g. MgO+1.70 g. SO, 5) 3.36 g. K,HPO, » 1.82g. K,0+1.08 g. PO, | : Traces of FeCl, From April 5, each funnel was treated with the solutions 1), 2), 3) and 4), respectively, and once in every 4-5 days, the solution 5) was added to each funnel, so that the plants in all the funnels may enjoy an equal amount of mineral salts except 72 U. SUZUKI. Ca, Sr, and Ba (I shall hereafter call, for convenience, the plants treated with solution 1), Ca plants, with solution 2), Sr plants etc.). The solutions were given almost every day and the details noticed. At first, there was no difference among the plants, but afterwards the Sr and Ba plants gradually showed signs of suffering, while the Ca and Ca+Sr plants grew on equally well. Toward the end of the experiment, the suffering of the Sr and Ba plants became very serious, many plants beginning to die off, many leaves falling down, and the stems unable to stand upright, so that I could not keep them any longer. On the 24th, the plants were carefully removed from the sand, washed well, and the following measurements made: PLANTS TREATED WITE SOLUTION. 1) 2) 3) 4) Ca plants Sr plants Ba plants |Ca+Sr plants Numiberjof plantsizs.caecsssnasedeee 20 15 28 17 Wenothror stemaec seater 12.9cm I + 10.8 11.4 Wenethyofpetiole es nsessteeesseree 2.4-3.3cm 0.6-0.9 1.5-1.8 2.1-3.0 Menpthiolrootsimseesaneeeeee ee eee 6.0-10.5em 6.0 4.5-7-5 9.0 Total weight of fresh stems ...... 7.0452 3.805 5.245 5-625 Total weight of fresh leaves ...... 3.9508 1.195 1.395 3.285 Weight of 1 fresh stem ............ 0.352¢ 0.260 0.187 0.331 Weight of 1 fresh leaf ............ 0198¢ 0.080 0.050 0193 Diameter of cotyledon ............ 3.6cm 2.4 2.4 3.0-3.6 In the Ca and Ca+Sr plants two or three new leaves develop- ed ineach plant, the diameter of which was in the former 2.1—3.6 em. and in the latter 1.5-3.0cm., while in the Sr and Ba plants no development of new leaves was observed. We see from the above results that the Ca plants were best, the Ca+Sr plants being nearly on the same footing with them, but somewhat inferior; Sr and Ba plants were nearly equal in length among themselves, but the stems were very small and slender, so that they could not stand upright. The growth of leaves was CAN STRONTIUM AND BARIUM REPLACE CALCIUM. 73 especially bad in the Sr and Ba plants, no new leaves developing and the cotyledons remaining very small and afterwards beginning to fall down. The roots were not developed in both plants. 2) BARLEY. (Hordeum distichon.) (See Pl. VII) The treatment was exactly the same with buckwheat. The seeds were sown in purified sea sand contained in four large funneis and kept in a warm house. On the 5th April, the plants were 3-8 cm. high, and the solutions were then given. During the experiment, no obstacles were encountered. Until the 16th, no difference was observed among the plants, but afterwards the Sr and Ba plants began to suffer slowly. This was especial- ly the case with the Ba plants. The tips of the leaves became yellow and afterwards chlorophyll was entirely destroyed, the leaves becoming white, as if the plants had been kept in the dark for several weeks, and no growth was observed. The Sr plants preserved their green colour a little longer, but afterwards they gradually became dirty green and the tips of the leaves turned yellow. The development of the roots was remarkably bad in both plants. The Ca plants grew most energetically, the roots reached their maximum length, and the leaves were a dark green in colour. The Ca+Sr plants also grew very well and at first showed no difference from the Ca plants, but afterwards the growth became rather slow and toward the end of the experi- ment,*he difference between the two was quite distinct, especially as regards the development of the roots. The Ba and Sr plants suffered more and more as days passed on, and as I could not keep them any longer, the experiment was stopped on the 11th, and the plants were carefully removed from the sand, washed well, and the following measurements were made :— 74 U. SUZUKI. PLANTS TREATED WITH SOLUTION. 1) 2) 3) 4) (Ca plants) | (Sr plants) | (Ba plants) |(Ca+Sr plants) Number of plants ............08. 62 60 57 46 mength! of leaves awcneeeasees pees, ne an 5 fms eng thlof rootSya.sssssse eee ees 15—30cm pee 13 pee | 24-15 Breadth of leaves .....,........- 0-9cm 0.6 0.45 0.75 Total fresh weight............... 93. 40. 20. 62. Fresh weight of r plant ...... 15g 0-67 0.51 1.35 Total dry weight ............... 6.673 3.026 1.955 4.522 Dry weight of 1 plant ......... 0.108 0.050 0.034 0.098 Ratio of dry weight ............ 100-0 46.3 31-5 90.7 We see from the above results that strontium and barium salts are strongly poisonous, stopping all growth and gradually causing death, Barium salt destroys chiorophyll bodies and no assimilation can therefore take place.* The poisonous action of * One of the reasons why barium salts are poisonous to plants may be the precipita- tion of sulphates in the plant cells which makes it impossible for them to enter into the organization of the protoplasm and chlorophyll bodies, etc. The presence of barium in the plants treated with barium-salts was easily shown by dissolving the plant ash in hydrochloric acid, and adding some sulphuric acid to the hydrochloric extract, when a strong white precipitate of barium sulphate was formed. In Ca plants I found no white precipitate formed on a similar treatment. Barium-salt is sometimes found asa normal constituent in some plants. Compare R. Hornberger :—Uber das Vorkommen des Bariums in der Pflanze und im Boden (Landw. Vers. Stationen. Miinchen. Bd. LI. Heft VI. S. 473.) Compare also R. Kobert :—Kann ein in einem Pflanzenpulyer gefundenes abnorm héher Barytgehalt erklirt werden durch direkte Aufnahme von Baryumsalz durch die lebende Pflanze aus dem Boden? :—(Chem. Ztg. 10. Juni 1899. Nr. 46). He says as follows :— “ Fast alle Pflanzen sind im Stande, gelegentlich aus dem Boden Baryumverbind- ungen aufzunehmen. In meinem Lehrbuche der Intoxicationen, heisst es. “Strontian, Baryt und Mangan werden ohne Nachtheil fiir den Pflanzenorganisms von Pflanzen auf- genommen, und die so barythaltiggewordenen Pflanzen kénnen auf Menschen und Thiere giftig wirken.’ ‘Bei mikroscopischen Untersuchung des von Dr. Jonscher er- wahnten Paprikapulvers muss sich ja zeigen, ob die Barytmenge nur zugemischt sind, oder, in der Pflanzen enthalten sind.” CAN STRONTIUM AND BARIUM REPLACE CALCIUM. 75 strontium was very much neutralized (?) by the presence of calci- um-salt. This is the reason why Haselhoff was led to erroneous conclusion. B) WATER CULTURE: 1) PHLOX PANICULATA. (Polemoni.) Young plants of Phlox paniculata were cut off and put in the following solutions : 1) 3.28 g. Ca(NO,), in 1 litre of water .........1.12 g. CaO+2.16 g. N,O, 2) 4.23 g. Sr(NO3),_,, A pe wenetaed eee 2.07 g. SrO+2.16 g. N,O, 3) 5.22 g. Ba(NO,). 5, o REMY cassia 3.06 g. BaO+2.16 g. N,O, 1.64 g. Ca(NO,),_,, 35 sie | SBeatooees 0.56 g. CaO+1.08 g. NO, Ha Poh (INO Fs. ss 2 BAN e! bacco 1.04 g. SrO +1.08 g. N,O, 1.64 g. Ca(NO,)._,, = oj. eee 0.56 g. CaO+1.08 g. N,O; os g. Ba(NO,), 5 p = apreceee 1.53 g- BaO+1.08 g. N,O, The experiment was commenced on the 19th April, and by the 22nd Ba plants had already begun to show signs of suffering, the upper soft part of the stems turning at first brownish black and drying up, and the leaves could not flourish any longer, becoming yellow or brownish black, and on the 24th nearly all the leaves withered away. Sr plants fared somewhat better than Ba plants, but on the 24th they began to show the same signs as Ba plants, and no leaves remained alive until the 27th. Ca plants were quite healthy and showed no signs of suffering until the end of the experiment (1st May), on the contrary, many new leaves appeared during the experiment. Ca+Sr and Ca+Ba plants were far better than Sr or Ba plants. By the 24th they had shown only slight signs of suffering, many leaves remaining alive until the 27th ; no new leaves, however, appeared, and on the 29th nearly all leaves except a few on the top, were dead. Nevertheless it is quite evident that the poisonous action of strontium and barium salts was much neutralized by the pre- sence of calcium salt. 76 U. SUZUKI. 2) RUBUS IDAEUS L, VAR. STRIGASUS MAXIM. The experiment was carried on exactly in the same way as with Phlox paniculata. But in this case, control experiment was also made with distilled water. The result was almost the same as in the former experiment, the only difference was that the Sr plants suffered most. They died already on the 24th and no leaf remained alive until the 27th, all having turned brownish black. The Ba plants also died, and nothing remained until the 24th, The Ca and control plants were quite healthy until the end of the experiment and no difference was observed between them. The Ca+Sr and Ca+Ba plants also remained alive until the end of the experiment (Ist May), but by this time the edges of the leaves turned brownish black. 3) COREOPSIS TINCTORIA NUTL. The same experiment was repeated with the young plants of this species having roots, and the same result as above was obtained. The Ca and control plants were the best; the Ca+Sr and Ca+Ba plants were also very good; but toward the end of the experiment (1st May) they began to show signs of suffering. The Sr and Ba plants, on the contrary, began to suffer already on the 24th, being unable to stand upright, and the leaves, with the exception of those on the upper part of the stems, nearly all dying off by the end of the experiment. The above five experiments are enough to prove that strontium and barium salts are strongly poisonous to the higher plants and can never replace calcium salts. Their poisonous action can, however, be neutralized to a large extent in the presence of lime salts. In the experiment with Rubus and Coreopsis we observe that control plants, kept in distilled water, were equally healthy; so it is quite evident that the bad condition of the Sr and Ba plants in these species was due not to deficiency of the necessary lime salts, but to the poisonous action of strontium and barium salts. Ifthe principal function of lime salts consists merely in neutralizing the oxalic acid formed during metabolism in the plant cells, and if it never enters into closer combination with the protoplasm, then we must have naturally quite different results from those above obtained ! CAN STRONTIUM AND BARIUM REPLACE CALCIUM. WY. My results exactly agree with those of Loew,* and as he has already discussed fully the physiological function of lime salts, I will not repeat it here. SUMMARY. 1. Strontium and barium can never replace calcium in phaenogams; they are strongly poisonous and the poisonous action may, to a certain degree, be lessened by the addition of lime salts. 2. Haselhoff’s view that strontium can replace calcium is incorrect. 3. Loew’s view that calcium is contained in the nucleus and chlorophyll-bodies in organized compounds would agree best with my observations. OBSERVATIONS DURING THE EXPERIMENTS. 1) BUCKWHEAT. ee bem: aed Ca+Sr Ba SA a ed Date. | Weather. (max.)| plants. | plants, plants. Remarks. April 5th Rain | 16°C 40 c.c. of the solution added, 3-8 cm high, - No difference yet observed 1o | Cloudy| 21 =n - > 1 6-14 cm. =e No difference yet observed 1 i ine 31 ”» ” ” ” 6-15 cm, Ca plants became a little 13 »” 25 ” ” ”» » better. 15 » 32 »” ” ” » Ca plants were very well, 16 5 20 40 c.c, of the solution 5) added. small new leaves appeared, but none on other plants. 18 | Cloudy | 20 4o c.c, of the solution added. 20 Fine 30 Distilled water added. New leaves also appeared in Ca+Sr plants, Sr and 5 Ba plants suffered much, 3 HY zo ‘3 ? a many leaves falling down from Sr and Ba plants. 24 5 25 - *s és Experiment stopped. * Cf: Die chemische Energie der lebenden Zellen, Chapt. 3 und 4. 78 | Date | 5th April 27 29 39 Ist May Oo 10 U, SUZUKI. 4oc.c. of the solution added. ” 4oc.c. of the sol. 5) added. 8oc.c. of each solution added. 4oc.c. of each solution and some distilled water added. 8oc.c. of solution 5) and some distilled water added. 4oc.c. of each solution and some distilled water added. 8oc.c. of each solution and some distilled water added. 100€.¢. of each solution and some distilled water added. Dark green colour, little 100c.c. of each solution and some distilled water added. Iooc.c, of solution 5) and some distilled water added. 1ooc.c. of each solution and some distilled water added. Pure water, growth slower. Weather. nee Ca plants. Ca+5r plants. ' | Rain 10°C. | 40 c.c. of the solution added, Cloudy 21 | »”» » >’ > ” ” J | line 31 »” » ” ” ” ” » 25 » » »” ” ”» ” ”» 32 ” » »” ” » »” » 20 | 4oc.c. of the sol. 7) added. Cloudy 20 \80c.c. ot each solution added. Fine ie) ” » ” ” ” »” ” 20 ” »” ” ” ” »” ” 25 »” ” ” ” »” ” +: 24 | Distilled water added. Distilled water added. » a 4coc.c. of each solution and 9 some distilled water added. a 25 ” ” »” ” ” ” 8oc.c. of solution and 20 5a 5 ” = | some distilled water added. 1 «|: 400.€. of each solution and a S) _ some distilled water added, | _ | 8oc.c. of each solution and 2 33 some distilled water added. ; , 1ooc.c, of each solution and Cloudy 25 some distilled water added. Rain be Dark green colour, growth : aa best. inferior. - 100¢.¢. of each solution and 2 20 some distilled water added. a 1ooc.c, of solution 5) and 22 aie some distilled water added. > tooc.c. of each solution and Fine a5 some distilled water added. 5 25 Growth best, dark green, Growth little slower. ” 23 fe 34 Pure water added. Aone Healthiest, Healthy. CAN STRONTIUM AND BARIUM REPLACE CALCIUM. LEY. Sr plants. 4oc.c. of the solution added, 4oc.c, of the sol. 5) added. Soc.c. of each solution added. Distilled water added. 4oc.c. of each solution and some distilled water added. 8oc.c. of solution 5) and some distilled water added. 40€.c. of each solution and some distilled water added. 8oc.c. of each solution and some distilled water added. 1ooc.c. of each solution and some distilled water added. No growth, top of the leaves turned yellow. | tooc.c. of each solution and some distilled water added. 1ooc.c. of solution 5) and some distilled water added, tooc.c, of each solution and some distilled water added, Top yellow, no growth. Pure water added. Suffered much, half of the leaves died off. Ba plants. 79 Remarks. 4oc.c. of the solution added, Distilled water added. 4oc.c. of each solution and some distilled water added. 8oc.c. of solution 5) and some distilled water added. 4oc.c. of each solution and some distilled water added. 8oc.c. of each solution and some distilled water added. tooc.c, of each solution and some distilled water added, No growth, chlorophyll destroyed. 1ooc.c. of each solution and some distilled water added, 1ooc.c. of solution 5) and some distilled water added. 1oo0c.c. of each solution and some distilled water added. Leaves white and almost died off, 3-8cm. growth equal. 7-15cm. growth equal, 7.5-16.5 no difference yet. Ca plants a little better. Ca and Ca +Sr plants equally well, Sr and Ba plants be- came very bad, Ba plants suffered very much, difference very remarkable. Ba plants nearly died off, Sr plants also began to die off. Experiment stopped. + [cee Ame 1 Cer pian ig Va: fee ee h J ee ~ . ae li 7 bj *Agpreq UO uINIIeq pur wWNIZUOIs Jo sjoaya snoranfur ay} sMoys ayeyd siy 7, ‘ONMOL “VM VDO “MN AT AARLOTIOO i i . ee. =. és IIA ‘Id ‘Al ‘JOA *]/00 ‘ols *]/Ng The Chemical Composition of the Spores of Aspergillus Oryzae. IBY K. Aso, Nogakushi. Our knowledge of the chemical ccmposition of the spores of fungi is still very scanty, and as regards their ash, no analysis whatever has thus far been made. There are however questions of physiological interest, suggesting a comparison of the spores of fungi with the seeds of the higher plants, that have induced me to determine, as accurately as possible, the composition of the spores of Aspergillus oryzae. These spores can easily be obtained in any quantity in Japan, as the rice grains covered with the mycelium and the spores of this fungus form an article of commerce under the name of Tane-kdji or Koji-dane I have preferred however to collect the spores myself, in order to obtain them in a pure state. Aspergillus oryzae plays a great part in several industries in Japan, being used not only in the manufacture of Sake or rice wine, but also in that of Shoyu or Soja-sauce,™ and of Miso. This fungus contains powerful enzymes, a diastase, a maltase, invertase and a peptase. Oxydases appear to be present only in traces. I extracted Tane-kdji with 309% alcohol, (1) The composition of KGji, z.e. rice grains covered with the mycelium of A. oryzae was investigated by Kellner, Mori and Nagaoka, (Bull. College of Agric., Toky6 ; vol. I, No. 5), and Tane-kdji was analysed by Saté (see the article by Okumura, This Bull. Vol. III. No. 3). (2) Okumura, Bull. College of Agric,, Toéky6 ; Vol. III. No. 3. (3) Y. Nishimura, this Bull. Vol. III. No. 3. (4) Kellner, Mori and Nagaoka, this Bull, Vol. I. No. 6. Besides Koji is used for manufacturing Mirin (a sweet alcoholic drink) and many other Japanese beverages and articles of food. (5) Kellner, Mori and Nagaoka, Bull. College of Agric., Tokyé, Vol. I. No. 5. Okumura, this Bull. Vol. IIT. No. 3. Takamuku, Journal of the Tokyo Chemical Society, Vol. XIX. No. 8. 82 K. ASO. from which the enzymes were precipitated with strong alcohol and ether. After filtering and washing with strong alcohol, they were dissolvod in a small quantity of water, precipitated and washed with alcohol twice more. The enzymes thus obtain- ed were dissolved in a small quantity of water, and to this solution, a few drops of freshly prepared guaiacum tincture and diluted hydrogen peroxide were added; only a slightly bluish colouration was obtained at first, but it became more and more distinct after a few minutes. Prof. O. Loew, lately of this college, found some years ago, that oxydase is not present, and peroxydase present only in traces, in Koji. T. Takamuku of this college found the presence of cytase in a small quantity in Soja-koji. I. Preparation of Samples. I collected the spores of the fungus in the following way: Tane-koji was prepared with roughly milled rice without mixing any ash; and when the rice grains became covered with innumerable spores, they were brought out from the cellar and the spores were separated from the rice grains and the mycelium by tapping the bottom of the culture boxes inverted on a sheet of paper. By this means, most of the spores were obtained on the paper, and they were then sifted with a very fine silk sieve to remove all impurities. The sample™ thus obtained is of course not absolutely, but only tolerably pure. This sample was exposed to the air in a balance room for two weeks and kept in a bottle. It was used for organic analysis. In the same manner, I collected a second sample, which was used for ash analysis. A third sample, which was used for special purposes, was (1) Tane-kdji used in the Sake-factories is commonly prepared by mixing the ash of oak-leaves and the spores with steamed rice. On the details of the preparation, see J. Okumura’s article in this Bulletin, Vol. III, No. 3. (2) The wooden boxes are commonly called K6ji-buta. (3) One K6ji-buta gave about three grams of the sample. (4) I examined several portions of the sample under the microscope, but I did not find any piece of the mycelium at all. THE CHEMICAL COMPOSITION OF THE SPORES. 83 collected from a commercial Tane-koji prepared in a Sake- factory at Osaka. II. Organic Constituents of the Spores of Aspergillus Oryzae. The result of my analysis regarding the organic constituents of the spores of the fungus was as follows: In 100 parts of air-dry spores, NIN GNESy sean Pere? BAcRer re SHE See eee ocr 42.515 RAVE Ua ELC Teta ete ots cies caine « Ear eeee 57.485 In 100 parts of dry matter, Mota mithoMe Mirren. sdgarcer ce ccesse occaedeahact 6.380 WrNde PLObeiny ant een. aces cn ss teiiecae tenes 39.875 Ee ReaMescef ae Eee enee cats. seus ade cigtucne 0.377 Alcoholic extract after extracting with SUMEIs sar coe Meee Sect on side davinee eens 27, OOO Wrideriies aacnscsetieec nts ban lecuctsabecnairce 8.994 Total carbohydrates (as dextrose) ...... 20.017 GIT pte Ce oer Ae SAE oe aE 2d Sanaa 5.151 1. DRY MATTER AND WATER. I determined the dry matter by drying the spores to con- stant weight at 100°C. 2. NITROGENOUS SUBSTANCES, The total nitogen was determined by Kjeldahl’s method, and the crude protein was calculated by multiplying 6.25 by the quantity of nitrogen obtained. I determined the indigestible part of the proteids in the (1) The commercial Tane-kGji is generally prepared by mixing some ash with the rice, I determined the proportion of ash in the third sample and obtained 6.11% of dry matter, while that in the first sample was 5.159%. (2) The first and the second samples were prepared in the Miso-factory of Kagaya at Yotsuya in Tékys. (3) The first sample. 84 K. ASO. following way: 16.69 grams of the dry spores” were extracted with dilute caustic soda after extracting with ether and alcohol, and some dilute hydrochloric acid was added until the solution gave a faintly acid reaction. After filtering and well washing with dilute hydrochloric acid (about 0.2%), the dark brown precipitate was mixed with 250 c.c. of gastric juice and digested in the usual way in a water bath for eighteen hours at 40°C.; then filtered and thoroughly washed with dilute hydrochloric acid. The residue was dis- solved in dilute ammonia, and again precipitated by making the ammoniacal solution faintly acid with dilute hydrochloric acid; then filtered. The precipitate was collected on a weighed filter and thoroughly washing first with dilute hydrochloric acid and then with water, washed with boiling alcohol and ether. Here- upon I obtained a brown mass which was weighed after drying at 100°C. 16.69 grams_of the dry spores yielded 0.59 grams of the brown mass, that is, 3.535% in the dry spore. This brown mass contained 10.8% nitrogen. It seemed to me of interest to determine the presence of some bases, if any, in the spores. About Io grams of the dry spores® were extracted with boiling water, and from this aque- ous solution a precipitate was obtained with basic lead acetate. After filtering, the excess of lead in the filtrate was precipitated by sulphuretted hydrogen, and filtered. After evaporating to a small volume, adding a little sulphuric acid, phospho-tungstic acid was added to the filtrate, whereby the characteristic pre- cipitate was obtained. The phospho-tungstic precipitate was first washed with cold water containing some sulphuric acid, and then decomposed with caustic baryta, and the filtrate evaporated after removal of the excess of baryta by means of carbonic acid. The evaporated solution was treated with an ammoniacal solution of silver nitrate, whereby a white precipitate was obtained. The precipitate thus obtained was collected on a (1) The third sample. (2) The spores could not be digested completely unless I treated them first with alkalies, because some of them floated on the surface of the gastric juice and could not be brought into intimate contact with the juice. (3) The third sample. (4) I applied the biuret test for peptone, but no reaction was obtained. THE CHEMICAL COMPOSITION OF THE SPORES. 85 filter and washed with a diluted ammoniacal solution of silver nitrate and afterwards with cold water, then dissolved in a warm nitric acid of the specific gravity of 1.1, after the addition ofa little urea, and upon cooling, microscopical needles were obtain- ed which were analogous to the silver compounds of the xanthin- bases. Though the crystals obtained were too small in quantity, yet I made some further researches. After separating the crystals by filtration, they were washed with cold water, and suspended in some water slightly acidified with hydrochloric acid, and the silver was removed with sulphuretted hydrogon. The filtrate from the silver sulphide was then neutralized with ammonia and evaporated to dryness. The residue was treated with ammonia, upon which one part was dissolved and left an insoluble residue. The latter gave the reaction of Capranica for guanin, and also produced the characteristic change of colour when treated with nitric acid and caustic soda. The ammoniacal solution was evaporated and from it some powder of a faintly yellow colour was obtained. This substance did not give the reaction of Weidel, and when evaporated with nitric acid, left a yellowish residue which turned dark reddish when treated with caustic soda and on heating. It is probable that this colour- ation was caused by other bases than hypoxanthin, because I did not purify the latter by repeated recrystallization.© The nitric acid solution from which the needle-shaped crystals were separated, was made slightly alkaline with am- monia, whereby a very small quantity of a brownish yellow flocculent precipitate was obtained, from which the silver was removed by sulphuretted hydrogen, and filtered. The filtrate was then evaporated to dryness, and a yellowish powder was obtained, which was slightly soluble in water but insoluble in alcohol and ether. On applying Weidel’s test, it gave a dark reddish colour, but not Hoppe-Seyler’s reaction. The change of colour with nitric acid and caustic soda was also observed. Although the amount of the sample used was too small to afford any positive proof for the presence of xanthin bases in the spores, yet it is likely that they are contained in the spores. It appears to me that many other bases besides those of the xanthin group (1) Compare ‘On the Nitrogenous Non-albuminous Constituents of Bamboo Shoots,’ by Kozai. (Bull. College of Argic., Téky6, Vol. I. No. 7). 86 kK. ASO. are contained in the spores; but this question must be deferred to future investigation. 3. (ETHEREAL EXTRACT. The ethereal extract was obtained by the extraction of dried spores® with absolute ether in Soxhlet’s extraction apparatus. After evaporating the ether, the residue was weighed in the usual way. The ethereal solution was colourless, but the residue was slightly yellow. In this extract, there were contain- ed cholesterin and lecithin besides the common fats. About 17 grams of the dry spores were extracted with ether, and after evaporation, the residue was saponified with alcoholic potash, and the alcohol evaporated. The residue thus obtained was treated with water and then put into a cylinder. Some ether was poured into this cylinder, and after shaking repeatedly, the etherial solution was drawn off with a pipette. The evapo- ration-residue of this ethereal solution was dissolved with some warm alcohol and left for some time. Some tabular crytals were observed in the alcoholic solution under the microscope. These crystals were dissolved in chloroform, and to it some concentrated sulphuric acid was added, whereby a red colour- ation was produced, and upon adding some water a green precipitate appeared. The presence of lecithin was confirmed in the following manner: several grams of spores“ were separately extracted with ether and alcohol, and the two were mixed. After heating the evaporation-residue with a mixture of sodium carbonate and some potassium nitrate, the ash was dissolved in nitric acid and the presence of phosphoric acid in the solution was tested with ammonium molybdate. 4. ABCOHOEIC EXTRAET, After extracting with ether, the spores were extracted (1) The first sample. (2) The third sample. (3) E. Gérard, Jahresber. f. Agric. Chem. XIX. 1896. S. 274. (4) The second sample. (5) The first sample. THE CHEMICAL COMPOSITION OF THE SPORES. 87 with alcohol (989) by means of a reverted cooler, and after evaporating, the residue was dried to constant weight at 100° C, and weighed. The alcoholic solution was neutral™ and yellow or brownish yellow according to the quantity of the spores used. The evaporation-residue was brownish and had a pungent smell and a sweet taste. On cooling, innumerable needle-shaped crystals formed in bushes or bundles from the hot alcoholic solution. This substance left no ash upon ignition and was sweet, without smell, soluble in water and hot alcohol, very slightly so in cold alcohol and insoluble in ether. I determined the melting point of the crystals in the capillary tube and obtained 163.5° C on the average. Besides, the lustre of the somewhat purified crystals was brilliant. Hence, there could be no doubt that this substance was mannitol. I determined roughly its proportion in the alcoholic extract by weighing the crystals somewhat purified by recrystallization, and obtained 44.752%. Besides mannit, there were contained trehalose,© and some resinous matters. 5. CRUDE FIBRES. The quantity of the crude fibres was determined according to Gabriel’s method by deducting the ash from the raw fibres found. 6. CARBOHYDRATES. The spores” were mixed with water and steamed in a digester for three hours in just the same manner as for the determination of starch, and then filtered. The filtrate did not reduce Fehling’s solution, formed no osazone, and upon boiling with concentrated sulphuric acid no dark colouration appeared. Hence, I concluded that this extract did not contain glucose, maltose or cane sugar. The extract was next boiled for about three hours with dilute hydrochloric acid (about 2.5%), and after neutralizing it with (1) I tested with litmus paper, (2) Of course, these crystals were not absolutely pure. (3) About this I shall speak soon afterwards. (4) The first sample. 88 K. ASO, sodium hydrate and filtering, the quantity of sugar was deter- mined as dextrose by Allihn’s method, and I obtained 20.017% of it in the dry matter. To see how much sugar is contained in the alcoholic ex- tract, I extracted the spores with alcohol (93%) and the evaporation-residue was dissolved in water, and after boiling this for about three hours with dilute hydrochloric acid (about 2.59%), the quantity of sugar was determined by the above- mentioned method. Here I obtained 6.2259 sugar as dextrose in the dry matter. The difference between these two quantities perhaps cor- responds to that of the carbohydrates, which are soluble in hot water under some pressure and insoluble in boiling alcehol. Cramer™ determined carbohydrates in the spores of Penicillium glaucum under the name of starch by inverting the carbohyd- rates with dilute sulphuric acid, but he did not investigate their properties. Marschall determined starch in the mycelium of some fungi by converting it into glucose with an acid, but he could not obtain any positive proof of its presence. It appeared to me of some interest to investigate the carbohydrates in the spores of Aspergillus oryzae. I tested the spores as well as the mycelium of the fungus with iodine solution under the microscope ; no blue, but the characteristic dark brown colour for glycogen was observed. Itis then clear that the spores as well as the mycellium contained no starch, but glycogen. Indeed, starch has never been found in fungi, while glycogen is spread widely among them. When the alcoholic extract was left for some days, some tabular crystals were obtained besides the needle-shaped crystals of mannit. They were rhombic prisms, colourless and transparent, carbonized on heating and left no ash on ignition. It was sweet, without smell, insoluble in ether and chloroform, nearly so in cold alcohol, soluble in hot alcohol, easily so in water, melted at 100° C, even in a strongly boiling water bath, and solidified to a glassy mass on cooling. Its aqueous solution was neutral and could reduce Fehling’s solution only after (1) Centralbl. f. Bak, I. Abtheilung, Bd. I. (2) Archiv f. Hygiene Bd, XXVIII. 1896. (3) The temperature in this was about gg.° 8 C. THE CHEMICAL COMPOSITION OF THE SPORES. 89 boiling with dilute hydrochloric acid. It produced oxalic acid upon boiling with strong nitric acid. There can hardly be any doubt that this substance was trehalose (mycose). The quanti- ty of dextrose obtained after boiling the alcoholic extract might be equivalent to that of glucose, which was split from trehalose. III. Mineral Constituents of the Spores of Aspergillus Oryzae. OA CANS The ash of the spores was estimated after incineration and determination of the carbon and carbonic acid contained therein, which were deducted from the raw ash. The result is shown in the following table: First sample. Second sample. Third sample. Total ash in dry BeLbL% 4.844% 6.112% matter. 2. MINERAL CONSTITUENTS. The ash-analysis“ was carried out by the usual method for vegetable ash with the following result : In 100 parts of the ash, LO can BUA ARet 050 eee 45.964 Naz Om oasis ERC eat vie.s saicisieigloes 4.131 CA OM ye tome ens oon s8-s seeks 1.038 VO We ators eet tii via aes aplezsen 4.304 Bes OF sick ae eh cis sek awe 4.916 Fg ©) Pence MAaMC Gs cha\tentols asics + dates 39.640 S) Oh otesrebhoovece. >. Cte a aEe ean Eee 2.000 SI Ota sedtotl: bodeo i: ae ae eee 0.409 The test for chlorine was made and its presence in the ash distinctly proved. (1) The ash of the second sample was here used. (ele) K. ASO. DISCUSSION OF RESULTS: A. THE ORGANIC CONSTIZUENTS: tT. WATER. The high percentage of water is a striking feature; it indicates the presence of a very hygroscopic substance, which in other kinds of spores is absent. Thus, Reinke found that the spores of Aethalium septicum contain only 7.13% water in the air-dry state. According to Cramer, Penicillium spores contain a very high percentage of dry matter, and give up all the water on drying, which they again take up in moist air. Planta found that a fresh sample of the pollen-grains of hazelnut gave up 4.219 of water on drying over sulphuric acid, and then 4.98% more on drying at 100° C, making a total of 9.19%. 2. CARBOHYDRATES. Glycogen is very widely spread as reserve material™ in the higher as well as the lower fungi, forming as much as 30% of the dry matter of beer-yeast. Starch is never formed in spores. Further, trehalose and mannit have been found in a number of fungi, either together or separately. It is of interest to observe the differences in the chemical activities of the higher plants and fungi: thus, the starch of green plants is replaced by glycogen in the fungi and the cane sugar of phanerogams is here replaced by trehalose. ‘The chief difference between trehalose (C,.H»Oy +2H,0O) and cane sugar (C,,H.O,,) is that the former is split by hydrolysis into two molecules of glucose, and the latter into glucose and fructose ; trehalose is also less easily invertible than cane sugar. Remarkable in this respect is the high percentage of cane sugar in the pollen-grains of pine (11.24%) and of hazelunt (14.70%), as found by Planta. Of special interest is (1) Pfeffer: Pflanzenphysiologie, I Bd. 1897. S. 474. (2) According to Miintz, Penicillium glaucum, Agaricus campestris &c. contain always mannitol, but no trehalose, while Agaricus muscaris produce trehalose, but no mannit. (The same book.) (3) Zeit. Physiol. Chem., 1894., Winterstein: Zur Kenntniss der Trehalose. (4) Landw. Versuchs-Stat. 1885. XXXI. and 1886 XXXII. THE CHEMICAL COMPOSITION OF THE SPORES. ony the frequent occurence of mannitol in the fungi. It serves doubt- less in the germination of the spores, and is probably, in the first stage of oxidation, transformed into a hexose (mannose, glucose or fructose). It seems to me that the crude fibres of the spores contain some chitin-like substance, the study of which will be deferred to future. 3. NITROGENOUS SUBSTANCE, The percentage of crude protein was high, that of the mycelium of Aspergillus niger being 30.49, as found by Mar- schall,® and of the pollen-grains of hazelnut 31.63% of the dry matter, according to Planta. In general, fructification-organs contain more nitrogen than others. Stiitzer found much nuclein in the mycelium of fungi, there being 40.75 parts of nuclein-nitrogen in 100 parts of the total nitrogen. Perhaps, the brown mass which remained as an indigestible residue of the spores of Aspergillus oryzae, may be a mixture of some colour- ing matters and nucleo-proteids. I tested for the presence of phosphorus, sulphur and iron in the ash of the brown mass and proved their presence, phosphorus and iron being especially predominant. Hence this substance has a close relation to the haematogen studied recently by Stoklasa. The bases of the xanthin group as described before, may perhaps be partly derived from nuclein by a partial decomposi- tion of it on heating with water. 4. FATTY MATTERS. The amount of fatty matters including some lecithin and cholesterin in fungi fluctuates between wide limits, viz. 0.29 in Agaricus and 35% in Claviceps. The following table gives some figures as regards the contents of fat. (1) The average quantity of crude protein in the mycelium of the three fungi investigated by him was 38.000%. (2) Zeit. Physiol. Chem., VI. (3) Compt. rend., 1898, 128. 92 K. ASO Fats (ethereal extract) in dry matter, : Spores of Pollen-grains | Pollen-grains Seeds abores of | Penicillium of of Barley. of i Pe oe glaucum. hazelnut, pine. rape. ee (a) (b) (b) (c) (c) 0.377% 7.34.% 4.20% 10.60% 2.90% 48.20% (a) According to Cramer, (b) according to Planta, (c) ac- cording to Wolffs Chemico-Agric., Tables. 5. ALCOHOLIC EXTRACT. As far as my knowledge goes, the percentage of the sub- stances soluble in aicohol is higher in the spores than in the mycelium of fungi, as the following table shows: In 100 parts dry matter, Spores Spores Mycelium Mycelium | Mycelium of of of of of Aspergillus | Penicillium | Aspergillus | Penicillium Mucor oryzae. glaucum. niger. glaucum. stolonifer. Alcoholic 27.67 30.46 18.50 11.80 11.80 extract. Se ee———ee It is probable that such substances as mannitol are ac- cumulated in the spores as reserve materials, which are soluble in aicohol. Finally, I give in the following table some figures showing relations regarding to the composition of the spores, the pollen- grains and the seeds of some plants: Penicillium-spores were analysed by Cramer, its mycelium by Marschall and the pollen-grains by Planta, as mentioned before, THE CHEMICAL COMPOSITION OF THE SPORES. 93 Spores of In 100 parts Pollen-grains of of Soy-bean.* dry matter, Aspergillus Penicillium hazelnut. oryzae. glaucum. Crude protein...... 39.88 28.44 31.63 372DL N-free substances. 54.97 69.65 64.36 57-33 ENS ieen sees cseseotiees 5-15 1.91 4.01 5-56 Bo CHE MINERAL CONSTITUENTS: fe eOmAT, ASH: Though the quantity of ash in the spores of fungi depends partly upon the medium in which they were cultivated, yet it seems to me singular that such a great difference as is shown in the foilowing table should be found between the ash of Asper- gillus oryzae and of Penicillium glaucum. Spores Mycelium of of fungi. — Aspergillus. Penicillium. In the average. In average. Ash in dry matter, 5-37.% 1.91% 6.37% It will prove interesting to analyse many other kinds of spores and deduce some general conclusion on this point. 2. ASH CONSTITUENTS. Marschall made a qualitative analysis of the ash of the mycelium of fungi and found iron, phosphoric acid, chlorine, sodium and potassium etc ; besides some other ingredients which were not remarkable. I determined the ash ingredients of the spores of Aspergillus oryzae quantitatively and found all com- mon ingredients except manganese.” Very interesting was the high percentage of oxide of iron found in this ash ; it is probably present in the spores as a nuclein compound; thus haematogen * This is the average composition of many sorts of soy-beans. (1) I made a qualitative test for this, 04. K, ASO. in the bulb of Allium cepa obtained by Stoklasa contained 1.68% of iron. According to Molisch iron is an essential constituent of fungi; but others only admit that it is useful. Traces of iron, sufficient for a large amount of mycelium, are often contained in the nutriments furnished to the fungi, which naturally take it up. K.Yabe™ of this college states that he observed, during his studies on the developement of this Asper- gillus under different conditions, that only in those solutions which contained some iron, spores were developed; which shows, in accordance with the view of Molisch, the importance of this element for the fungus. The percentage of oxide of iron in the common rice grains (not whitened) in Japan is 1.63 in the ash ; and according to the recent investigation by Bunge, rice grains contain 1-2 milligrams of iron in 100 parts of dry matter. Aspergillus-spores contained relatively more iron than rice grains. Doubtless, this iron must have been derived from the rice grains. The sulphuric acid in the ash is derived perhaps exclusively from the sulphur of the proteids, by oxidation during the in- cineration process. The presence of silica and lime in the ash is perhaps just as accidental as that of chlorine and soda. These four substances are not necessary for the developement of the lower fungi. Nor do the more complex members of the higher fungi probably require silica or soda ; but as to lime, we can not yet say anything definite as to whether it is required by such complicated fungi as Phallus, Agaricus, Morchella. Of great interest is the high percentage of potash and phosphoric acid; and on this point, there is a close analogy with the seeds of phanerogams. The following table shows the comparison of K,O and P.O, in the ash of the seeds of higher plants and that of the spores of the fungus: (1) Bull. of Agric. College. Vol. III. No. 3. (2) This Bull. Vol. I. No. 12., Kellner and Nagaoka, (3) Zeit. Physiol. Chem., 1898, 25. THE CHEMICAL COMPOSITION OF THE SPORES, 95 In too parts || Winter | Not whiten- Not Spores of wheat. ed paddy : whitened of dry matter. (1) rice. (1) rice, (2) | Aspergillus. Ash. 1.68 0.87 A f ; 4.844 K,O 0.52 : . . ; 2.227 120) 1,920 In too parts of ash, K,0 30.95 22.99 17.93 44.52 22.47 45-964 12 (0) 47.02 52.87 25.47 36.75 48.31 39.640 The particularly striking relation between the spores and beans is here evident, but as to its significance we can not make any assertion without further investigations. Of special interest is, further, the presence of 4.264% of magnesia. This base plays evidently an important role in the assimilation of phosphoric acid. Wherever proteids are formed with accompanying development, and phosphates are present, there is magnesia always found. .From magnesium-phosphate, ‘phosphoric acid can more easily be assimilated than from any other phosphates, as the dissociation (hydrolysis) of magne- sium salts is easily accomplished. It may be safely assumed that, about as much magnesia as was found here, is contained in the spores of the related Penicillium glaucum. Indeed, we may say, no seed or spore without magnesium ! Nevertheless, it is a fact that the germination of the Penicil- lium-spores takes place in solutions containing, as organic nutriment, ammonium acetate alone, only when traces of magne- sium salts are present. It seems, that only a good nutrition brings on those changes in the spores, which make the magne- sium phosphate soluble and available for the protoplasm. (1) According to Wolf's Chemico-Agric. Tables. (2) Kellner and Nagaoka: Bull. College of Agric. Vol. I. No. 12. 96 K. ASO. Though no ash anayslis of the spores of Aethalium have yet been made, its plasmodium has been analysed by Reinke. According to him the ash contained 27.7% of calcium carbonate, 6.49% of phosphoric acid, 1.429 of potash, 0.139% of iron oxide and 0.719% of magnesia. j In conclusion, I must express much thanks to Dr. O. Loew, formerly professor in this college, for giving me the present subject for investigation and for many valuable suggestions during its progress. ED hil) hai) i ui. < 43 Xs 58. SAE 124. XI, XIV. 165, nn: XIV. Wang SVE oe VIII. 16. VIII. 4. X. 44, 45, 46. VI Michelia compressa. Morus alba var. stylosa. indica. Myrica rubra. Myrsine capitellata. Nephelium Longana. Orixa japonica. Osmanthus Aquifolium. fragrans. Ostrya japonica. Pasania cuspidata. glabra. Paulownia tomentosa. Phellodendron amurense. Photinia glabra. Picrasma quassioides. Pirus aria var. kamaonensis. Aucuparia var. japonica. Calleryana. Miyabei. sinensis. Toringo. Tschonoskii. Pittosporum Tobira. REGISTER. 142. 103. XIII. XII. XBL VE XVI. VII. 93- REGISTER. VII S. Taf. Fig. Platycarya strobilacea. BIOs IS 30. Populus balsamifera var. suaveolens. PAT. XI, MVE 77 120! tremula var. villosa. HAO: XV. 128. Pourthiaea villosa. 136. Prunus Buergeriana. a7, | Xai D1 3. Cerasoidos. L277: communis. 105. Grayana. Lee) XIVe 112. incisa. 127, Mume. 104. VIII. Tk: persica var. vulgaris. 105. pseudocerasus var. spontanea. 126. XI, XIV. 56, III. pseudocerasus var. Sieboldi. [26 Xi, IVES Pterocarya rhoifolia. 129; XI. 59. Quercus acuta. 146. UE 87. dentata. ES. Exe 28. gilva. 140. glandulifera. DES. Ax: 20. glauca. 148. grossesserata. In6. XII 97, 98. myrsinaefolia. 147. phyllireoides. 147. serrata. D4. EX: Dif sessilifolia. 148. thalassica. 148. variabilis. DES. Vibrayeana. TA7. Xie 88. Rhododendron dilatatum. 136. Metternichii. 136;. Xe 125% Rhus semialata. 103. VIII. 9. silvestris. 103. VIII REGISTER. S. succedanea. 103. tricocarpa. 102. vernicifera. 102. sp. 103 Robinia pseudacacia. 100. Salix Caprea. I4I. Urodaniana. IAI. Sambucus racemosa. 128. Sapindus Mukurosi. 114. Sophora japonica. Die platycarpa. Li sp. 123: Spondias ? 101. Staphylea Bumalda. 126. Sterculia platanifolia. 108. Stewartia monadelpha. 132 pseudo-Camellia. 132. Styrax japonica. 130. Obassia. Die Symplocos crataegoides. var. pallida. 140. Tamarix juniperina. 100. Ternstroemia japonica. 135 Thea japonica. 135 Sasanqua. Igce Tilia cordata var. japonica. 125 Taf. VIIl. VIil. XII. IX. IX. X. VIII, XIII. I, STI XV. XI, SVs XIV. N 6 26. 2. OG. 66, 119. 120. 62, 63, 645 1146 115. 69. Miqueliana. 2p Trochodendron aralioides. 152 Ulmus campestris. 110. montana. var. laciniata. LIO. parvifolia. PLT. Viburnum dilatatum. 17 furcatum. 137. odoratissimum. sy Opulus. 141. Zanthoxylum ailanthoides. 1232 piperitum. 132: _ schinifolium. 132 Zelkowa acuminata. 109. Zizyphus vulgaris var. inermis. 138. REGISTER. Mats XIII. IX. Fig. 19. Ubersicht. I. Ringporige Hoizer. A. Die Gefasse ausserhalb des Porenkreises, d.i. des inneren Oe ur ~~ 16. 18. 19. Gefiissreicheren Holztheiles in einem Jahrringe, sind gleich- massig vertheilt, cder doch zuweilen nur bei den breiten Jahr- ringen in der Nahe der ausseren Jahrringsgrenze zu kurzen periphkerischen oder mehr schragen Linien vereinigt. a) Die Markstrahlen fein. 1. Die Gefasse im Porenkreise sehr weit. a. Die Gefasse an der Aussengrenze des Jahrringes sind fast nie zu Strichen vereinigt. Cedrela chinensis Juss. (Chanchin.) Spondias sp.? (Kaname.) Albizzia Julibrissin Boiy. (Nemu-no-ki.) 6. Die Gefiisse an der Aussengrenze des Jahrringes sind fast immer zu Strichen, wenn auch nur zu kurzen, vereinet. Melia japonica Don. (Sendan.) Hovenia dulcis Thunb. (Kemponashi.) Ehretia acuminata R. Br. (Chisha-no-ki.) 2. Die Gefisse im Porenkreise weit. a. Die Gefasse an der Aussengrenze des Jahrringes sind fast nie zu Strichen vereinigt. Khus vernicifera DC, (Urushi.); Rhus tricocarpa Miq. (Yama-urushi.) Rhus silvestris S. et Z. (Yamahaze.) Rhus succedanea L. (Haze.) Rhus sp. (Yamahaze ?) 6, Die Gefasse an der Aussengrenze des Jahrringes sind fast immer zu Strichen, wenn auch nur kurzen, vereinist. Rhus semialata Marr, (Nurude.) Picrasma quassioides Benn, (Nigaki.) Broussonetia papyritera Vent. (Kaji-no-ki.) Broussonetia Kasinoki Sieb. (KGzo.) 3. Die Gcfasse in Porenkreise fein. Die Gefiisse ausserhalb des Porenkreises cleichmiissig zerstreut, Eleagnus macrophylla Thunb. (Natsugumi.) Prunus Mume S. et Z. (Ume.) Prunus communis IHluds. (Sumomo.); Prunus Persica S. et Z, var. vulgaris Max. (Momo.) Acanthopanax sciadophylloides Fr. et Sav. (KKoshiabura.) Dendropanax japonicum Seem, (Kakuremino.) Edgeworthia chrysantha Lind]. (Mitsumata.) b) Die Markstrahlen sehr fein. 1. Die Gefasse im Porenkreise weit. | Die Gefisse an der Aussengrenze des Jahrringes hiiufig zu Strichen vereinigt. 20. tN _ to ty N oe) Fraxinus Sieboldiana Bl. (Shioji.) Fraxinus mandshurica Rupr. (Yachidamo.} Catalpa Kaempferi S, et Z. (Ki-sasage.) Robinia pseudacacia I.. (Inu-akashia.) 2. Die Gefisse im Porenkreise fein. Fraxinus Bungeana DC. var. pubinervis Wg. (Toneriko.) Ligustrum Ibota Sieb. (Ibota-no-ki.) ec.) Die Markstrahlen dusserst fein. Die Gefiisse im Porenkreise weit. Mallotus japonicus Muell. Arg. (Akamegashiwa.) Aleurites cordata Muell. Arg. (Aburagiri.) B. Die Gefisse ausserhalb des Porenkreises sind zu peripherischen, Os GF O Owe OI GW a 2 ol AAR $b Ono Cc. nN ww zuweilen etwas verzweigten Wellenlinien vereinigt. a.) Die Markstrahlen breit. 1. Die Gefasse im Porenkreise weit. Sterculia platanifolia L. (Ao-giri.) Celtis sinensis Pers. (Enoki.) 2. Die Gefasse im Porenkreise fein. Berberis Thunbergii DC. (Megi.) Tamarix juniperina Bge. (Gyoriu.) b.) Die Markstrahlen fein. Die Gefasse im Porenkreise weit. Zelkowa acuminata Pl. (Keyaki.) Acanthopanax ricinifolium S. et Z. (Hari-giri.) Ulmus campestris Sm. var. laevis Planch. (Haru-nire.) Ulmus montana Sm. var. laciniata Trautv. (Ohyo.) Ulmus parvifolia Jacq. (Akinire.) Cladrastis amurensis B. et H. var. floribunda Maxim. (Inu-enju.) Sophora japonica L. (Enju.) Sophora platycarpa Maxim. (Fujiki.) Phellodendron amurense Rupr. (Kiwada.) Gleditschia japonica Miq. (Saikachi.) Morus alba L. var. stylosa Bur. (Yama-guwa.) Ailanthus glandulosa Desf. (Shinju.) Paulownia tomentosa H. Bn. (Kiri.) Clerodendron tricotomum Thunb. (Kusagi.) c.) Die Markstrahlen sehr fein. Aphananthe aspera Planch. (Mukunoki.) Sapindus Mukurosi Gaertn. (Mukuroji.) Die Gefasse ausserhalb des Porenkreises sind in einer radial verlaufenden, oft auch verzweigten Linie vereinigt. a.) Einzelne Markstrahlen sehr breit, andere aber ausserst fein. Quercus serrata Thunb. (Kunugi.) Quercus variabilis Bl. (Abemaki.) Quercus dentata Thunb. (Kashiwa.) b.) Einzelne Markstrahlen breit, andere aber ausserst fein. Quercus glandulifera Bl. (Konara.) Il. al N 54. 13 Quercus grosseserrata Bl. (O-nara.) ce.) Die Markstrahlen sehr fein. Platycarya strobilacea S. et Z. (Nobu-no-ki.) d.) Die Markstrthlen dusserst fein. Castanea vulgaris Lam. var. japonica DC, (Kuri.) Zerstreutporige Holzer. A. Ein Theil der Markstrahlen sehr breit, anderer aber ausserst fein Alnus japonica S. ct Z. (Mannoki.) Alnus incana Willd. var. glauca Ait. (Yamahannoki.) Alnus viridis DC. var sibirica Rgl. (Miyama-hannoki.) Alnus glutinosa Willd. (Kawara-hannoki.) B. Ein Theil der Markstrahlen breit. 59- 60. 61. 62. 63. a.) Die breiten Markstrahlen sind sehr zahlreich, nicht glanzend und verlaufen etwas krummlinig. Zwischen denselben sind die feineren vertreten, aber die feinsten fehlen. Ardisia Sieboldi Miq. (Mokutachibana.) Myrsine capitellata Wall. (Hichi-no-ki.) Aucuba japonica Thunb. (Aoki.) b.) Die breiten atlasglanzenden Markstlahlen sind haufig und veilaufen gerade. Zweischen denselben sind die feineren und feisten vertreten. Fagus sylvatica L. var. Sieboldi Maxim. (Buna.) Fagus japonica Maxim. (Inu-buna.) C. Die meisten der Markstrahlen sind fein, aber deutlich und scharf, so dass man deren Verlauf auf dem Querschnitt leicht mit un- bewaffnetem Auge verfolgen kann. a.) Die Markstrahlen sind auf dem Tangentialschnitt als Spindel deutlich sichtbayr. 1. Die Markstrahlen sind theils hoch, theils niedrig. Euptelaea polyandra S, et Z. (Fusazakura.) Meliosma pungens Wall. (Yama-biwa.) 2. Die Markstrahlen sind niedrig und fast gleich hoch. Cornus Kousa Buerg, (Yamabdshi.) Hex crenata Thunb. (Inu-tsuge.) Ilex integra Thunb. (Mochi-no-ki) ; Ilex rotunda Thunb, (Kuroganemochi.) Tex latifolia Thunb. (Taray3.) liex macropoda Mig. (Aohada.) Tiex pedunculosa Mig. (Soyogo.) Euscaphis japonica Pax. (Gonzui.) 14 “a 4 lo sIosy “I obs b.) Die Markstrahlen sind aufdem Tangentialschnitt sehr schwer erkonnbar. 1. Die Gefisse sind arm und fcin, aber anf dem Querschnitt gut sichtbar. a. Die Markstrah!en sind fast gleich fein. Melicsma myniantha S. et Z. (Awabuki.} Die meisten Markstrahlea fein, dazwischen die sehr feinen bemerkbar. Morus indica L. (Shima-guwa.) | Sophora sp. ? Zanthoxylum ailanthoides S. et Z. (Karasu-zansho.) 2. Die Gelfasse sind so fein, dass sie auf dem Querschnitt schwer, oder fast nicht mehr, auf dem Langsschnitt dagegen als verticite Linien deutlich sichtbar sind. a. Ohne Kern. ~ - Acer pictam Thunb. var. Mono Maxim. (ltaya-kayede.) Acer palmatum Thunb. (Yamamomiji.) Acer purpurascens Fr. et Sav. (Kajikayede.) Acer carpinifolium S. et Z. (Chidori-no-ki.) Acer nikoense Maxim. (Choja-no-ki.) Acer argutum Maxim. (Asanoha-kayede.) Acer rafnerve S.et Z. (Uri-kayede.) Acer crataegifoliam S. et Z. (Meuri-kayede.) Acer japonicum Thunb. (Hauchiwa-kayede.): Acer Sieboldianum Mig. var. microphyllum Maxim. (Kohauchiwa-kayede.) Acer distylum S. et Z. (Maruba-kayede.) ‘Tilia cordata Mill. var. japonica Mig. (Shina-no-ki_), Tilia Miqueliana Maxim. (Bodaiju.) Comus macrophylla Wall, (Mizuki) Cornus ignorata C. Koch. (Sawa-mizuki} b. Mit Kern. Staphylea Bumalda S.et Z. (Mitsuba-uisugi_) Pronus Pseudo-Cerasus Lindl. var. spontanea Maxim. (Yama-zakura.); Pranus Pseudo-Cerasus var. Sieboldi Maxim. ( Yoshino-zakura.} Pronus Grayana Maxim. {Uwamizu-z2kuiz.) Pronus Buergeriana Miq. (Iau-zakura.) Pranus Ceraseidos Maxim. (Mejiro-zakura.) Pronus incisa Thunb. (Mame-zakura.) Michelia compresa Maxim. (Ogatama-no-ki.) Sambucus racemosa L. (Niwatoko.) Deutzia scabra Thunb. (Utsugi.) D. Die meisten Markstrahlien sind sehr fein. 98- 99. a-) Die Gefasse weit, zuweilen sehr weit, sind auf dem Querschnitt deutlich als Locher sichthes, und stehen nicht dicht zusammen. _ Diospyros Kaki L. f. (Kaki_) Diospyros Lotus L. (Mamegaki.) 100. 101. 102. 103. 104. 105. 126. Yi -_ -_ Lael Ww Ww N ) KS) es ~ I “ ~ OW 2 Wd Lo BSS Geo CO vt iS) Oe “ 15 Juglans Sieboldiana Maxim. (Onigurumi.) Pterocarya rhoifolia S. et Z. (Sawa-gurumi.} Hernandia peltata Meisn. (Ilasunohagiri.) Cinnamomum Camphora Nees. (Kusu.) Machilus Thunbergii S. et Z. (Tabu-no-ki.) Ficus retusa L. var. nitida Miq. (Gazumaru.) b.) Die Gefasse sind auf dem Querschnitt mit unbewaffnetem Auge nur schwer als Locher, auf dem Langsschnitt aber gut als vertiefte Linien sichtbar. Styrax japonica S. et Z. (Yego-no-ki.) Sryrax Obassia S. et Z. (Hakuumboku.) Idesia polycarpa Maxim. (Ii-giri.) Magnolia hypoleuca S. et Z. (H6-no-ki.) Magnolia Kobus DC. (Kobushi.) Magnolia salicifolia Maxim. (Tamushiba.) Zanthoxylum piperitam DC. (Sanshd): Zanthoxylum schinifolium §. et Z. (Inuzansho.) Stewartia monadelpha S. et Z. (Saruta.) Stewartia pseadocamellia Maxim, (Natsutsubaki.} Alnus firma S. et Z. (Yashabushi.) . Cercidiphyllum japonicum S. et Z. (Katsura.) Machilus Thunbergii S. et Z. var japonica Yatabe. (Baribari.) Ficus erecta Thunb. (Inu-biwa.) Litsea glauca Sieb. (Shirodamo.) Actinopaphne lancifolia Meisn. (IKago-no-ki.) Cinnamomum pedunculatum Nees. (Yabu-nikkei.) Lindera praecox Bl. (Aburachan.) Lindera sericea Bl. (Kuromoji.) Lindera triloba Bl. (Shiromoji); Lindera obtusiloba Bl. (Dankobai.) Lindera umbellata Thunb. (Kanakugi.) c.) Die Gefasse sind mit undewaffuetem Auge weder auf den Querschnitt, noch auf dem Liangsschnitt sichtbar. Myrica rubra S. et Z..(Yamamomo.) Ternstroemia japonica Thunb. (Mokkoku.) Thea japonica Nois. (Tsubaki.) : Thea Sazanqua Nois, (Sazan-kwa.) Clethra barbinervis S. et Z. (Rydbu.) Cydonia sinensis Thourin. (Kwarin.) Lyonia ovalifolia Don. (Kashioshimi.) Rhododendron Metternichii S. et Z. (Shakunagi.) Rhododendron dilatatum Miq. (Mitsubatsutsuji.) Pourthiaea villosa Dene. (Ushikoroshi.) Ligustrum japopicum Thunb. (Nezumimochi.) Viburnum odoratissimum Ker, (Sangoju.) Viburnum dilatatum Thunb. (Gamazami.) ;Viburnum farcatum B], (Ogame- no-ki.) 16 E. Alle Markstrahlen sind ausserst fein, ohne Lupe nicht mehr sichtbar. a.) Die Gefasse weit, sind aufdem Querschnitt deutlich als. Locher sichtbar und stehen nicht zusammen, 139. Nephelium Longana Camb. (Rytgan.) b.) Die Gefasse sind mit unbewatinetem Auge auf dem Querschnitt nur sehr schwer als Locher, auf dem Langsschnitt aber gut als vertiefte Linie sichtbar. 1. Die Gefiisse, mit Lupe betrachtet, sind arm und klar, stehen nicht dicht zusammen. a. Harte Holzer. 140, Ostrya japonica Sargent. (Asada.) 141. Zizyphus vulgaris Lam. var. inermis Bge. (Natsume.) 142. Citrus trifoliata L. (Karatachi.) 143. Lagerstroemia indica L. (Hyakujikko.) 144. Betula Bhojpattra Wall. var. typica Rgl. (Ono-ore.); Betula globispica Shirai. .. (Jizo-kamba.) 145- Betula ulmifolia S. et Z. (Yoguso-minebari.) 146. Betula corylifolia Rgl. et Max. (Urajiro-kamba.); Betula grossa S. et Z- (Mizume.) B. Weiche Hdlzer. 147. Betula Maximowicziana Rgl. (Saihada.) 148. Betula alba L. var. vulgaris DC, (Shira-kamba.) 149- Betula alba L. var. communis Rgl- (Makamba.) 150. Betula alba L. var. cordifolia Rgl. (Aka-kamba.) 2. Die Gefasse sind, mit Lupe betrachtet, zahlreich und stehen dicht zusammen. a. Harte: Holz. 151. Symplocos crataegoides Ham. var. pallida Fr. et Sav. (Shiro-tsuge.) B. Weiche Hdlzer. 152. Populus tremula L. var. villosa Wesm. (Yamanarashi.) 153. Populus baisamifera 1.. var. suaveolens Loud. (Dero.) 154. , Salix Caprea L. (Saruyanagi.) 155. Salix Urbaniana v. Seemann. (Akay.nagi.) 156. Malesia corymbosa B, et H. (Asagara); Halesia hispida B. et U1. (Oba-asagara.) 157- Viburnum Opulus L. (Kamboku.) ce.) Die Gefasse sind mit freiem Auge weder -auf dem Querschnitt noch auf dem Liangsschniit sichtbar. a Harte Iélzer. 158. Distylium racemosum S. et Z. (Isu.) 159. Diervilla grandiflora S, et Z. (Hakone-utsugi.) 160. Tlydrangea paniculala Sieb. (Nori-no-ki.) 161. Eriobotrya japonica Lindl. (Biwa.) 162. Photinia glabra Thunb, (Kanamemochi.) 17 163. Eurya ochnacea Szysz. (Sakaki.); Eurya japonica Thunb, (Hisakaki.) 164. Illicium Anisatum L. (Shikimi.) 165. Andromeda japonica ‘Vhunb. (Asebi.) 166. Tirus Toringo Sieb. (Zumi.) 167. Pirus sinensis Lindl. (Nashi.) 168. Pirus Calleryana Dene. (Konashi.) 169. Kuonymus europaea L, var. Hamiltoniana Maxim. (Mayumi.) 170. Euonymus oxyphylla Miq. (Tsuribana.) 171. Euonymus japonica Thunb. (Masaki.) 172. Buxus sempervirens L. (Tsuge.) 173. Daphniphyllum macropodum Miq. (Yuzuriha.) 174. Hamamelis japonica S. et Z. (Mansaku.) 175. Pirus aucuparia Gaertn. var. japonica Maxim. (Nanakamado.) 176. Pirus Miyabei Sargent. (Azukinashi.) 177. Pirus Tschonoski Maxim (Miyama-nashizumi.) 178. Pirus aria Ebrh. var. kamaonensis Wall. (Urajiro-no-ki.) B. Weiches Holz. 179. Aesculus turbinata Bl. (Vochi.) lll. Die Holzer, bei welchen die alle Gefasse in radialer und nicht selten verzweigter Linie gruppirt sind. Diese Linie wollen wir die radiale Gefasslinie nennen. A. Die einzelnen Markstrahlen sind sehr breit, die meisten ausserst fein, kaum sichtbar. 180. (Quercus acuta Thunb, (Akagashi.) 181. Quercus gilva Bl. (Ichii-gashi.) 182. Quercus Vibrayeana Fr, et Say. (Shira-gashi.) 183. Quercus myrsinaefolia Bl. (Urajiro-gashi.) 184. Quercus phyllireoides A. Gr. (Ubamegashi.) 185. Quercus glauca Thunb. (Aragashi.) 186. Quercus thalassica Hee. (Shirifuka-gashi.) 187. Quercus sessilifolia Bl. (Tsukubane-gashi.) 188. Pasania cuspidata Oerst. (Shii.) 189. Pasania glabra Oerst. (Mateba-shii.) B. Die Markstrahlen ausserst fein. 1go. Hibiscus syriacus L. (Mukuge.) C. Die breiten Markstrahlen entsthehen durch das Zusammentreten zahlreicher, schmaler Markstrahlen zu einem componirten, sogenannten falschen Markstrahle, Es fehlt ihnen an Glanz und scharfer Begrenzung. tot. Carpinus laxiflora Bl. (Akashide.) 192. Carpinus yedoensis Maxim. (Inu-shide.) 193. Carpinus japonica Bl. (Kuma-shide.) 194. Carpinus cordata Bl. (Sawashiba.) 18 lV. 1Q5. Corylus heirophylla Fisch. (Hashibami.) Die Hélzer, bei welchen die Gefasse sich zu Figuren verbinden und auf dem Querschnitt nur als helle Fiquren sichtbar sind. Die Markstrahien aussersi fein. 196. 197. 198. 199. Osmanthus Aquifolium B, et H. (Hiiragi.) Osmanthus fragrans Lour. (Mokusei.) Pittosporum Tobira Ait. (Tobera.) Orixa japonica Thunb, (Ko-kusagi.) Das Hoiz welches in seinem secundaren Holze kein Gefass besitzt. 200. Trochodendron aralioides S. et Z. (Yamaguruma.) TABE ES VIL. Fig. 1. Cedrela chinensis Juss. (Chanchin.) Fig. 2. Spondias sp? (Kaname.) Fig. 3. Albizzia Julibrissin Boiv. (Nemu-no-ki.) Fig. 4. Melia japonica Don. (Sendan.) Fig. 5. MHovenia dulcis Thunb. (Kemponashi.) Fig. 6. Ehretia acuminata R. Br. (Chisha-no-ki.) Rica. 7. dehus verniciteras D6.) (Urushi.) Fig. 8. Rhus sp. (Yamahaze ?) Bis.) 9: ,» semialata Murr. (Nurude.) Fig. 10. Picrasma quassioides Benn. (Nigaki.) Fig, 11. Prunus Mume S. et Z. (Ume.) Fig. 12. Acanthopanax sciadophylloides Fr. et Sav. (Koshi- abura.) Fig. 13. Fraxinus Sieboldiana Bl. (Shioji.) Fig. 14. Catalpa Kaempferi S. et Z. (Ki-sasage.) Fig. 15. Fraxinus Bungeana D.C. var. pubinervis Wg. (To- neriko.) Fig. 16. Mallotus japonicus Muel. Arg. (Akamegashiwa.) its atau a flung i: a > iho rat ; cere i Raye? Mica Bull. Agric. Coll. Vol. IV. Pl. VIII, als ee a Gene A TS paar Ae, Celtis sinensis Pers. (Enoki.) Berberis Thunbergii DC. (Megi.) Zelkowa acuminata Pl. (Keyaki.) Acanthopanax ricinifolium S. et Z. (Hari-giri.) Ulmus campestris Sm. var. laevis Planch. (Haru- nire.) Ulmus palvifolia Jacq. (Aki-nire.) Sophora platycarpa Maxim. (Fujiki.) Phellodendron amurense Rupr. (Kiwada.) Paulownia tomentosa H. Bn. (Kiri.) Sapindus Mukurosi Gaertn. (Mukuroji.) Quercus serrata Thunb. (Kunugi.) - dentata Thunb. (Kashiwa.) af glandulifera Bl. (Konara.) Platycarya strobilacea S.et Z. (Nobu-no-ki.) Castanea vulgaris Lam. var. japonica D.C. (Kuri.) Alnus japonica S, et Z. (Hannoki.) ? ory OES ae © ike)... load os. war. es oa ‘ a] =e | ae ee eS . 7 = ee BA aay se es vane a joreaa eiss - Tn Be oy Et hata IV. Pl. 1X van dbedad Lettie r 8 AUR ENE, Sees LOANS APLAR y ry sare rp a abd oEnS Fa idg eerie a Lacan ‘ Be OT cAI ahi wae a ain sth Dewan WW ar 5 Bion ie SRE Ua eaai 33 + 34. Se 30: BEY 5 BS 5 Zion + 40; aA: Az. AG. AA: AS: = AG; Ar wo: a AG; E50: « 51. we 2: TARE. Xx. Alnus incana Willd. var. glauca Ait. (Yama- hannoki.) Ardisia Sieboldi Mig. (Mokutachibana.) Fagus sylvatica L. vaz. Sieboldi Maxim. (Buna.) y japonica Maxim. (Inu-buna.) Euptelaea polyaudra S. et Z. (Fusazakura.) 9 » » ” Cornus Kousa Buerg. (Yamabdshi.) Ilex integra Thunb. (Mochi-no-ki.) Meliosma myriantha S, et Z. (Awabuki.) ” yy ” ” » ” » » Morus indica L. (Shima-guwa.) Sophora spe ? Zanthoxglum ailanthoides S. et Z. (Karasu+zansho.) Acer pictum Thunb. var. Mono Maxim. (Itaya-kayede.) yy ” ” ” ” ” » ” ” ” ” ” ” ” yee. ' fi = ‘4 mf I Nth TH NOAH yO VA eetenalar v ial | ey a i if l) i} i == —— = a3 Fig. sae esse Ware , XI, Acer rufinerve S, et Z. (Uri-kayede.) », japonicum Thunb. (Hauchiwa-kayede.) Cornus macrophylla Wall. (Mizuki.) Prunus pseudo-cerasus Lindl. var. spontanea Maxim. (Yama-zakura), Prunus pseudo-cerasus var. Sie- boldi Maxim. (Yoshino-zakura.) Diospyros Kaki L. f. (Kaki.) Juglans Sieboldiana Maxim. (Onigurumi.) Pterocarya rhoifolia S. et Z. (Sawa-gurumi.) Cinnamomum Camphora Nees. (Kusu.) Ficus retusa var, nitida Mig. (Gazumaru.) Styrax japonica S. et Z. (Yego-no-ki.) Magnolia hypoleuca S. et Z. (H6-no-ki.) Stewartia monadelpha S. et Z. (Saruta.) Cercidiphyllum japonicum S. et Z. (Kutsura.) Myrica rubra S, et Z. (Yamamomo.) Ternstoroemia japonica Thunb. (Mokkoku.) Bull. Agric. Coll. Vol. IV. PI. XI. Fig. 80. Bis. Si. Bigs (52, Fis. 83. Fig. 84. Fig. 85. Fig. 86. Fig. 87 ibe XT Clethra barbinervis S. et Z. (Riydbu.) Ostrya japonica Surgent. (Asada.) Betula Bhojpattra Wall. var. typica Rgl. (Onoore), Betula glopispica Shirai. (Jizokamba.) Betula alba L. var. vulgaris D.C. (Shira-kamba.) Symplocos crataegoides Ham. var. pallida Fr. et Sav. (Shiro-tsuge.) Populus balsamifera L. var. suaveolens Loud. (Dero.) Salix Caprea L. (Saruyanagi.) Halesia corymbosa B. et HI. (Asagara), MHalesia hispida B. et H. (Oba-asagara.) Distylium racemosum S. et Z. (Isu.) Eurya japonica Thunb. (Hisakaki.) Euonymus europza var, Hamiltoniana Maxim, (Ma- yumi.) Buxus sempervirens L. (Tsuge.) Aesculus turbinata Bl. (Tochi.) Quercus acuta Thunb. (Akagashi.) an Coit a Aer nay? Lt J , . . Ti ae our >» 7 fe + " 3! l ‘ee ld ni ‘6] fees ye ; eultl Me a i : ‘\ 24) Me ve J . 2 P| 87 Fig. 86. Fig. 85. Fig. 84. Fig. Fig. Fig. 7 5 Fig. Fig. Fig. Fig D>° Fig. Fig. c Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. 101, 102, 103. AL EE XIIl, Quercus Vibrayeana Tr. et Tav. (Shira-gashi.) Pasania cuspidata Oerst. (Shii.) ” ” ” ” Carpinus laxiflora Bl. (Akashide.) - cordata Bl. (Sawashiba.) Osmanthus Aguifolium Bet. H. (Hiiragi.) Trochodendron aralioides S. et Z, (Yamaguruma.) Cedrela chinensis Juss. (Chanchin.) Spondias sp.? (Kaname.) Quercus grosserrata Bl. (O-nara.) Alnus japonicus S. et Z. (Hannoki.) » incana Willd. var. glauca Ait. (Yamahanno- ki.) Fagus sylvatica L. var. Sieboldi Maxim. (Buna.) “6 japonica Maxim. (Inu-buna,) Acer pictum Thunb. var Mono Maxim. (Itaya- kaede.) P : F HaaTAT | - (itenieetide) ¥sT ie 4 sts: ue eile ties ot art ce .£ +n Pale SUS s Posies , , ee ; a ax ; “ $2 i eis J) .> J) 5s) 3 . ety oak) : Pal EPs Li 4 a ; nani Fl . ¥ Bnidtip ame eh Geae jonni) 8 Jo aa iit: ee ae . Caithustilem Wane? iens® eres | dak Be) Se tt amol) Aste age earns) eee aL Tone es 27 SE of a, Pa HL Soon =) ee a etn nett ae = tr G1 JIS OSe inyiffe F 7% » = - ¥ @ rir assite Cy ne aoe mas ic //) Sis 1 UE + Hik odoin oo a au j obimetga “ells sty ‘nig # ea fstind-a ee Ts i iM Bas ils HEBE mn ~swnt?) .nizab’ onc? Gage eOiaiT see ee (aboru Bull. Agric. Coll. Vol. IV. Pl. XIII. 95. Fig. 92. Fig. 99. Fig. 98. Fig. WY. Fig. 9%. Fig. 2 I 103. Fig. » 10) Fig. 101. Fig. 100, Fig. Fig. Fig. Fig. Fig. Fig, Fig. Fig. Fig. Fig. Fig. Fig. Big. Fig. Fig. Fig. Fig. TAPEL XIV, Acer palmatum Thunb, (Yamamomiji.) », purpurascens Fr, et Sav. (Kajikayede.) ,, carpinifolium S. et Z. (Chidori-no-ki.) 5, nikoense Maxim. (Choja-no-ki.) », Yrufinerve S, et Z. (Uri-kayede.) », Japonicum Thunb, (Hauchiwa-kayede.) » distylum S et Z, (Maruba-kayede.) Prunus pseudo-cerasus Lindl. var, spontanea Maxim, (Yama-zakura), Prunus pseudo-cerasus Lindl. var. Sieboldi Maxim, (Yoshino-zakura.) Prunus Grayana Maxim. (Uwamizu-zakura.) », Buergeriana Miq. (Inu-zakura.) Styrax japonica S. et Z. (Yego-no-ki.) Obassia S. et Z. (Hakuunboku.) Magnolia hypoleuca S, et Z. (H6-no-ki.) - Kobus D.C. (Kobushi.) Pe salicifolia Maxim. (Tamushiba.) Stewartia monadelpha S, et Z, (Saruta.) ¥ Eek: 8255 & . 76 (iit ogre j ant musoeleg ot C Peas A ) hae ok 2037 idoeho bo) ee. 9 ieee Lil. i oh: See Cohe"at eee a bos 2 -2 Seas ay i weiiast! t “cH = i (obo tediiay o> Soa ne crTA Seti eget Bee wie bre bil «ent.ros-c Uc ree 7 Eup iRmph eige / ( wherein it ae ( hadrS eee sterie) Countoonn ty tt ry a8 3 g¥j Bull. Agric. Coll. Vol. IV. Pl. XIV. 115. Fig. 114, Fig. 113. Fig. 2) Il Fig. 1/7. Fig. 116. ig. F Ar XOVe Stewartia pseudocamellia Maxim. (Natsutsubaki.) Thea japonica Nois. (Tsubaki.) », sazanqua Nois. (Sazan-kwa.) Cydonia sinensis Thourin. (Kwarin.) Lyonia ovalifolia Don. (Kashioshimi.) Rhododendron Metteynichii S.et Z. (Shakunagi.) Betula Bhojpattra Wahl. var. typica Rgl. (Onoore), Betula globispica Shirai. (Zizokamba.) Betuta alba L. var. vulgaris D.C. (Shira-kamba.) Populus tremula L. var, villosa Wesm. (Yama- narashi.) Populus balsamifera L. var. suaveolens Loud. (Dero.) Halesia corymbosa B. et H. (Asagara), H. hispida B. et H. (Oba-asagara.) Pirus Toringo Sieb. (Zuini.) 5, Calleryana Decne. (Konashi.) Euonymus europaea L. var. Hamiltoniana Maxim. (Mayumi.) Euonymus oxyphylla Mig. (Tsuribana.) Pirus aucuparia Gaertn. var. japonica Maxim, (Nana- kamado.) es tae — eo or eee a a ie 130: =» 137: gon 3130; . 140. = Mie mAs TAFEL XVI. Pirus Miyabei Surgent. (Azukinashi.) Pasania cuspidata Oerst. (Shii.) glabra Oerst. Carpinus laxiflora BI. ” ” ) (Mateba-shii.) (Akashide.) yedoensis Maxim. (Inu-shide.) japonica Bl. cordata Bl. (Kuma-shide.) (Sawa-shiba.) Ivn JER © (idene a a= ge ee 4 { es hilar A heey at, m- (.2diiie-saiey ae ' ; = sol}, onixele: Sa + : htgaai"® me epine'd ELS in, ee OT ACI a ART ha Bho y ¥ i P. Bull. A gric. Coll. Vol. IV. Pl. XVI. 136. Fig. 39. k Fig. 38. I Fig. 140. iq. F 142. Fig. Die Gattung Tilia in Japan. VON Homi Shirasawa, Rimgakusht. Die Gattung Tilia, deren Bliiten mit sehr merkwiirdigen und eigenthtimlich ausgebildeten Organen (Fliigelblatter, Stami- nodien) versehen sind, besteht nur an baumartigen Gewachsen, welche als Zierbiume, sowie auch als Waldbaume geschatzt werden. Man beschreibt zunachst, als typische Formen, ueber 1o Arten auf der nérdlichen Hemisphire. Doch ist diese Gattung bis jetzt in Japan und China oft vernachlassigt worden. Maximowicz beschrieb erst zwei chinesische Arten 7. Chinensis* und T. paucicostata,* und, obwohl bei uns mehr Arten als in anderen Landern vorkommen, hat doch bisher niemand eine eingehende Bearbeitung derselben unternommen, ausser Miquel und Maximowicz. Die erste Beschreibung tiber die Tiliaarten Japans lieferte Miquel, (Prol. Fl. Jap. 1865-1867), nach ihm Franchet et Savatier (Enum. Plantarum Japonicarum), Maximowicz, der lange Jahre‘ iiber die Flora Ostasiens sehr werthvolle Studien gemacht hatte, schrieb im Jahre 1880, (Mélanges Biologiques), tiber die Tilia- arten Japans eine Verbesserung, hauptsdchlich tber die T. Mandschurica Mig. Obwohl unsere Tilia-arten durch Maxi- mowicz’s Arbeit zu Klarheit gelangt sind, liegen doch, veranlasst durch das mangelhafte Material in den Handen der Fremden, mehr oder weniger Liicken vor, die fiir die Wissenschaft von Bedeutung sind. Diesen Mangel zu ergainzen habe ich mich seit einigen Jahren bestrebt, geniigendes Material zu sammeln, und da jetzt alles notige vorhanden ist, um auch itiber die Gebiete, in welchen die obigen Arten vorkommen, klar zu werden, habe ich die vorliegende Arbeit ausgefiihrt. Endlich fithle ich mich ver- pflichtet meinen wirmsten und herzlichsten Dank Herrn Prof. * Plantae Chinensis, 154 SHIRASAWA. Rigakuhakushi, J. Matsumura und auch Herrn T. Makino aus- zusprechen, welche mir ihre freundliche und giitige Hiilfe bei der voliegenden Arbeit hatten zu Theil werden lassen. LITERATUR, Endlicher, Genera Plantarum. Gray, Synoptical Flora of North-America. Sargent, Forest Flora of Japan. De Candolle, Prodromus. I. Maximowicz, Primitae Florae Amurensis. iy, Mélanges Biologiques, 2. sp) 99 10. Miguel, Prolusio florae Japonicae. Franchet et Savatier, Enumeratio plantarum Japonicarum. Maximowicz, Plantae Chinensis. C. Koch, Dendrologie. Dippel, Handbuch der Laubholzkunde, Vol. 3. Schwarz, Forstbotanik. Hempel Die Baume und Straucher des Waldes. Engleru. Plantl, Die Natiir. Pflanzenfamilien. Franchet, Plantae Davidianae. Ledebour u. Plant, Flora Rossica. etc, Hin 4 Tt lal KF AS HE HAR bid Be Th BK Bib (AE SE Boi Fs ae a oF DIE GATTUNG TILIA IN JAPAN, rs fe I, Die Beschreibung der einzelnen Arten. GRinere A, Tit1a KiusIANA MAKINO ET SHIRASAWA, NOv. SP. Nom. JAP. HERANOKL KAMA BZA. Bt (Taf. XVII. Fig. 1). Bliitenknospen eipyramidenformig, abgestumpft, an der Basis abgestutzt. Bliten hangend, schwach duftend, 43-5 mm lang, 5-6 mm im Durchmesser, Kelch finfblatterig, Kelchblat- ter eilanzettformig, allmahlich zugespitzt, ganzrandig, circa 33 mm lang, nach innen gewolbt, am Grunde der [nnenseite dicht, doch oben am Rande und aussen diinn behaart; Blumenblatter 5, an den Kelchblattern abwechselnd stehend, langer als die- selben, circa 44 mm _ lang, eilanzettformig, zugespitzt, kahl, weissgelblich gefarbt ; Staubgefasse 15-20, an den Staminodien verwachsen, fiinf in Biindeln, kiirzer als die Staminodien, ungleich lang ; Staminodien fiinf, kiirzer als die Blumenblatter, langlich, gekielt, gestumpft, an des Basis verschmialert, weiss- gelblich ; Fruchtknoten fast kugelig, 14 mm Durchmesser, be- haart, fiinffacherig, in jedem Fache mit zwei centralstandigen Samenknospen; Griffel 1, cylinderisch, gerade, kahl; dop- pelt so lang als der Fruchtknoten, Narbe mit 5 Vertiefungen ; Staubfaden bandférming, kahl—; Staubbeutel 2, getrennt, der eine verlangert, umgekehrt-eiformig, der andere schmal-eiférmig, oft beide zusammengesetzt; Frucht niisschenartig, kugelig, fiinf mm Durchmesser, graubraun behaart, am Grunde undeutlich fiinfrippig, kurz geschnabelt, meist einsamig ; Blitenstand mit einem Fliigelblatt versehen, tragt 20-36 Bliiten; Bliitenstiel meist widerholt dreigabelig, Fliigelblatter langlich, 5-7 cm lang, 0,8—1.2 cm breit, haufig riickwarts gewendet, hellbraun gelblich, mit unregelmassig verbreiteten Nerven anastomosiert. Gemeinschaftlicher Blitenstiel 1-14 cm lang, und Bliitenstielchen 4-2 cm lang. Knospen wechselstindig, sitzend, eif6rmig, etwas flach zugespitzt, kahl, die aiisseren zwei Schuppen sichtbar, im Winter udnkelréthlich. 156 SHIRASAWA. Blatter diinn, papierartig, langlich, eiformig, zugespitzt, am Grunde unsymetrisch schiefherzformig oder abgestutzt, einfach unregelmassig, kurz kerbgesiagt, oberseits dunkelgriin, unterseits hellgriinlich, auf der Unterseite meist sechs Nerven auf einer Seite der Ader sichtbar erhaben, Blattstiel braun, diinn behaart. Die Rinde ist in der Jugend graubraun, glatt, weisslich ge- fleckt, bildet spater eine Borke, langrissig und lést sich in klein- schuppigen Blattern ab. Das Holz ist gelblichweiss, Kern und Splint ziemlich vers- chieden gefarbt, weich, leicht spaltbar, und die Gefiisse sind im ganzen Jahrringe gleichmassig vertheilt. Das specifische Gewi- cht im Lufttrockenzustande ist 0,44, das Absoluttrockengewicht 0,36. Es eignet sich nicht gut zur Feuerung und hat bis jezt keine andere Nutzverwendung gefunden, als zu hélzernen Schuhen (Geta) und Zitudholzern verarbeitet zu werden. Der Stamm ist geradschaftig mit kurzkegelformiger Krone, erreicht 60cm Durch- messer und 18 m Hohe; man findet aber grosse Baume nur selten in einzelnen Orten, da sie meist schon in der Jugend abgetrieben worden. Zur Bastfaserbereitung wird das 15-20 zahrige Holz im Walde alljahrlich abgefallt, es liefert bei langer Dauer der Mutterst6cke immer mehr reichliche und kraftigere Ausschlage, so kommen oft 10-15 am einem Stocke vor. Mitte Mai schabt man die Rinde von den 15-20 cm im Durchmesser und 7 m_ hohen Stammen ab, legt sie zwei Wochen in Teichwasser, wascht sie dann mit frischem Wasser bis die Bastfaser fast gereinigt ist. Die Faser ist sehr zahe und wird zur Mattenweberei, sowie zu Flechtwerkmaterial und Binden verwendet. Dieser Baum findet sich nur auf der Kiusiu-insel (woher der Name T. Kiusiana stammt), dem siidlichen Theil Japans und besonders in dem Grenzgebirge der Provinz Hiuga, Bungo, Buzen, Chikugo und Higo von 100 m bis auf 600 m wtber der Meeresflache, wo er vermischt mit anderen winterkahlen Laub- hédlzern, am haufigsten mit Carpinus laxifera Bl., Quercus glandulifera BI., Quercus serrata Thunb, und Castanea. vulgaris Lam. var. japonica DC. vorkommt, hie und da wohl auch fast reine Bestande von geringerer Ausdehnung bildet. Er eignet DIE GATTUNG TILIA IN JAPAN, 157 sich mehr fiir siidliche als ftir nérdliche Exposition, zieht aber im Allgemeinen tiefgriindige, fruchtbare Berghange vor. Seit einigen Jahren habe ich mich bemiht die wissenschaft- lichen Untersuchungsmerkmale zwischen dieser Holzart und Tilia cordata Mill. var japonica Miq. festzustellen. Im Jahre 1897 kam, nach den Anweisungen der Herrn Yoshio Tanaka, gliicklicherweise das notige Material fiir diese Arbeit in meine Hinde. In Ubereinstimmung mit meinem Collegen Herrn T. Makino nannten wir die Art T, Kiusiana, jedoch erst auf meiner diesjahrigen Reise nach Kiusiu habe ich vollstandige Klarheit tiber Standort, Verbreitung, etc. erlangt. TILIA CORDATA MILL. DICT, N. I, VAR. JAPONICA MIQUEL, PROL. FL. JAP. 207; Fr. ET SAV., ENUM. wPAP. 1. P. 66; Nom. JAP. SHINANOK1. Bliiten 20-40 in einem Stande, hangend schwach duftend. Blitenknospen kurzeiformig ; Kelchblatter und Blumenblatter 5, die ersteren ovallanzettformig, zugespitzt, nach innen gew6lbt, 3-4 mm_ breit, 5-6 mm lang, am _ Grunde filzig, am Rande diinn und an der Riickseite kleinwarzig behaart, die letzteren circa 4 mm breit, 7 mm lang, an der Basis verschmialert, gelblich; Staubgefasse kiirzer als die Staminodien, 25-30, je 5-6 in Biindeln vereinigt, ungleich lang, Staubbeutel 2, meist getrennt, flach elliptisch, auf fast gleicher Hohe stehend, Staminodien verlangert, linealisch, haben ungefahr dieselbe Lange wie die Blumenblatter, Fruchtknoten kugelig, langlich 2 mm Durchmesser, behaart, fiinf-facherig. Griffel 6 mm lang, behaart. Frucht oval oder umgekehrt eif6rmig, 5 mm Durchmesser, grau- braun behaart, kurz geschnabelt, ohne Rippen; Schale zerbrech- lich, Fliigelblatter gestielt, circa 5-8 cm lang, 1-1} cm breit, Bliitenstiel 3$ cm lang, Bliitenstielchen 4-1 cm lang, kahl. Blatter mit 2-4 cm, diinnbehaarten Stielen, papierartig, ei- oder schiefherzf6rmig, plotzlichin eine langere schmale Spitze ausgehend, circa 3-6 cm breit, 4-7 cm lang, spitz gesagt, kahl, doch in den Winkeln der Adern und der von Grunde austrah- lenden Hauptnerven dicht braun behaart, oberseits dunkel- gelblichgriin, unterseits hellblaugriin. Knospen eiformig, zugespitzt, im Winter dunkelréthlich und 158 SHIRASAWA. die einjahrigen Zweige braunréthlich. Rinde dunkelgraubraun, Borke lang gerippt, Bastfaser sehr zahe. Holz leicht, weich, Splint mit 7-9 Jahrringen, hellgelblich, mit den im ganzen Jahrringe fast gleichmassig zerstreuten Gefassen ; in frischem Zustande hat es ein specifisches Gewicht von 50, im lufttrockenen ein Gewicht von 40 und absolut-trocken- en Zustande von 33. Diese in Japan meist gemeine Lindenart ist als Waldbaum fast in allen Provinzen verbreitet, so siidlich von den Kiusiu- inseln bis nérdlich nach Hokkaidd, und zwar in dem siidlichen Theil der Hauptinsel (Honshu) in der Héhe von 350-2500 m, im Norden von Honshi in 250-1800 m und in Hokkaido uber 10O m. Sie verlangt mehr frischen, maissig feuchten Boden in Niede- rungen, Thalern oder geschiitzten Lagen wie seichtgriindig trockenen Boden an Berghangen, und erreicht haufig eine Hohe von 20 m. Bei der kraftigen Stockausschlagfahigkeit treibt sie zahlrei- che Stamme auf einem Stocke. Das Holz ist zur Verfertigung von Mébeln, besonders jedoch zur Bastfaserbereitung zu Seilen und Weberei geeignet. Es wird aber unter Umstainden auch nur als Brennmaterial benutzt. Bemerkung: C. S. Sargent’s Forest Plora of Japan p. 20,—This (T. cordata var Japonica) is the only Linden cultivated by the Japanese, who occa- sionally plant it in temple gardens, especially in the interior and mountainous part of the Empire.—Hs scheint mir, daher er diese Holzart mit T. Miqueliana Max. verwechselt hat, GRUPPE 3B: TILIA MAXIMOWICZIANA MIIII. Syn. T. MIQUELIANA FOLIIS ROTUNDIORIBUS MAx., 1M BRIEFE AN PROF. Dr. K. MIYABE (1887)., MATSU- MURA’S SHOKUBUTZUMEII P. 294. N. 3121. Nom. JAP. OBABODAIJU. (Taf. XVIII. fig. 2) Hinsichtlich seines Habitus hat diese Art die gréssten Organe (Blatter, Bliiten, etc.) unter den japanischen Tilia-Arten, Bliiten hangend, 10-18 an einem gemeinschaftlichen Stiele, stark duftend, circa 10-11 mm Durchmesser, 10 mm lang; Kelchblatter DIE GATTUNG TILIA IN JAPAN. 159 5, innen gewolbt, rautenformig, zugespitzt, an der Basis etwas breit, 9 mm lang, 5 mm breit, dick, gelblichgriin, aussen und innen behaart, am Grunde der Innenseite mit weisslich glanzend- er dichter Behaarung, Kronenblatter 5, rinnenformig, gekielt, diinn, 10 mm:lang, 4 mm breit, kahl, hellyelblich, Staubgefasse 65-75, ungleich lang und undeutlich in finf Phalangen vereinigt, kiirzer als die Kronenblatter; Staubfaden zweiastig, Staub- beutel verschmalert ovalf6rmig, und sehr verschieden in der Grésse, Staminodien fiinf, an der Spitze deutlich gezaihnt, stark gekielt, gleich lang mit den Kronenblattern, doch schmiler als dieselben, Fruchtknoten rundlich eiformig, 4 mm Durchmesser, fiinffacherig, in jedem Fache zweisamig, Griffel cylinderisch, kahl, zur Bliitezeit 5 mm lang und spater weit verlangert bis 9 mm. Narbe mit fiinf Furchen, Fligelblatter sitzend, beiderseits grau, diinn behaart, zur Bliitezeit hellgriinlich, circa 1$-2 cm breit 7-10 cm lang, Pedunculus .3-5 cm lang, Pedicelle 1-13 cm lang, behaart. Frucht rundlich ovalférmig, circa 8 mm Durchmesser, mit holziger, dicker, und grau behaarter, mehr oder weniger deutlich fiinfrippiger Schale. Blatter dick, hautartig, rundlichherzférmig, einfach, ziem- lich gleichmassig, 9-12 cm breit und lang, mit 3-5 cm langen Stielen, oberseits dunkelgriin, unterseits grau behaart. Die Blatter an den fruchtbaren Zweigen und bei jungen Ausschlagen sind viel grésser, fast 25 cm breit und lang. Die jungen Zweige, Blattstiele und Blattnerven graubraun filzig behaart. Rinde dick, dunkelvioletgrau, lange Jahre glatt, Borke weisslichgrau, seicht, langrippig, Bastfaser nicht so stark wie die der vorigen Arten. Holz weich, leicht spaltbar, Kern braunlich gelblich weiss, Splint mehr hell gefarbt; specifisches Gewicht in frischem Zustande ist 0,52, in lufttrockenem 5,35 und in absolut trockenem 5,31. Das Gebiet, in dem diese Art vorkommt, ist Mitteljapan nordlich bis Hokkaido. Ich habe vor einigen Jahren diesen Baum in Thal des Tone-Flusses in der Provinz Kotsuke, und am Fusse des Berges Hakkoda in der Provinz Mutsu, und auch in der Provinz Rikuchu gefunden, wo er in einer Héhe von 250-500 m iiber der Meeresfliche mit Alnus incana, Willd. var. glauca Ait. Populas tremula le. var. villosa Wesm. Quercus grosseserrata Bl. ets, einen gemischten Bestand bildet. Die weit ausgebreiteten 160 SHIRASAWA, starken Aeste, die dicken unterseits weisslichgrau behaarten Blatter und die gelbgriinlichen grossen Fliigelblatter lassen uns leicht sein Vorhandensein erkennen. Er liebt mehr tiefgriindige Thaler als trockene Berghange, wo er haufig 70 cm Durchmesser und 18 m Ho6he erreicht. In Hokkaido, nach? Prof. Dr. K. Miyabe und Prof. J. Matsumura, kommt er auf oben erwahnten Boden mit der voher erwahnten Tilia-Art (T. cordata var. jap- onica), mit Quercus grosseserrata, Bl. Cercidiphyllum japonicum S. et Z. Ulmus montana Sm. var. laciniata Trautv., Magnolia hypoleuca S. et Z. und Magnolia Kobus DC. vermischt vor, und erreicht 1 m Durchmesser. Zur Verwendung des Holzes findet man keine charakteris- tischen schatzbaren Eigenschaften, und wird es nur als Brenn- material benutzt. Aus der Rinde wird Bastfaser fiir Seilenmaterial bereitet, sie ist aber minder zahe als dieselbe der vorigen Arten. C. S, Sargent beschrieb diese Holzart unter dem Namen T. Miqueliana, Forest Flora of Japan p. 19. (1894). TILIA MIQUELIANA MAxIM, MEL BIOL. X. p. 584. Syn. T. MANDSCHURICA MIQ. PROL. 206; FR. ET SAV2 ENUM. PL. JAP: Ip: 675 NON RupR. ET MAXIM. Nom. JAp. BODAIJU. Bliiten stark duftend, 10-22 auf einem regelmassig wiederholt dreigabeligen Stande, 8 mm Durchmesser und 8 mm in Hohe, Bliitenknospen halboval, am Grunde fiinf, tief gefurcht, Kelch- blatter 5, oben gelblichgriin, unten in der Basis griin, 7 mm lang 3 mm breit,am Rande und Grunde fein behaart, Kronenblatter 5-zahlig, langer als die Kelchblatter, hellgelblich, Staubgefasse 60-75,zu fiinf Biindeln vereinigt, an der Spitze zweidstig und fast gleichlang, Staubbeutel ovalférmig in der Mitte ziemlich versch- mialert, Staminodien schmialer und kiirzer als Kronenblatter, Furchtknoten halbelliptisch, fiinf-facherig, fiinffach gefurcht, und rippig, Griffel cylindrisch, zur Bliitezeit kiirzer als Staubgefasse und weiter nach vorne hervortretend, Narbe mehr oder weniger tief fiinfrissig, Fliigelblatter gestielt, 2 cm breit, 5-9 cm lang, gelblichgriin, Bliitenstiele circa 44 cm lang, Stielchen circa 1 cm lang, grauweisslich, weich-haarig. DI£ GATIUNG TILIA IN JAPAN. 161 Frucht fast kugelig, ziemlich lang, 8 mm Durchmesser, diinn behaart, mit 5 Rippen, die Schale sehr hart. Blatter dicklich, breit deltaformig, zugespitzt, am Grunde unsymmetrisch, herzformig oder abgestutzt, fast. gleichmassig gezihnt, 6 cm breit, und gleich lang, auf der oberen Seite glinzend dunkelgriin, auf der unteren Seite grau, Blattstiel und einjahrige Zweige kleinwarzig behaart. Blatter an den jungen Ausschlagen auffallend gross, fast 15 cm breit und 22 cm lang. Knospen kugelig, zugespitzt, etwas flach, im Winter gelblich- grin gefirbt. Junge Zweige griin, altere grau, Borke lang gerippt. Die Bastfaser hat weniger Zahigkeit als die der vorigen Gruppe. Holz weich, Kern hellbraungelblich weiss, Splint gelbweiss, die Gefaiss englumig, spec, lLufttrocken-Gewicht 0,63 und Absoluttrocken-Gewicht 0,54. Rinde dunkelvioletgrau, lange Zeit glatt, spatcr treten Langsrisse auf. Diese erst nach Miquel im Jahre 1867 als in Japan einhei- misch beschriebene Holzart, welche iiberall in Mitteljapan ver- breitet ist, wachst sehr ippig, und erreicht 12 m Hohe mit locker austretenden Aesten, Besonders in den Tempelhainen des Buddha wird sie angepflanzt, wo siealsein heiliger Baum betrachtet wird. Sie ist in Japan unter dem Namen Bodaiju (+ #2 fi} Ficus religiosa) bekannt, ist jedoch eine andere Art als die gleich- namige, welche in Indien vorkommt. Hinsichtlich ihres Habitus, Wachstums und ihbres haufigen Vorkommens, schien es Miquel eine japanische Art zu sein, die jedoch nicht von Anfang anin Japan einheimisch war, sondern erst vor 710 Jahren durcheinen buddhistischen Priester aus China eingefiihrt wurde. II. Gruppierung der japanischen Tilia-arten. Fiir eine Gattung, welche zahlreiche Arten umfasst, ist es notwendig die Untergattungen oder Sectionen festzustellen, welche alle untereinander ahnliche und in allen wesentlichen Merkmalen wbereinstimmende Arten einschliessen. Die Gat- tung Tilia, die schon beschrieben worden ist, umfasst melir als 162 SHIRASAWA., 15 Arten, welche nur in dernérdlichen Hemisphare vorkom- men. In Bezug auf ihre Bliitenbildung ist das Vorhandensein oder Nichtvorhandensein der Staminodien ein entscheidendes Merk- mal, wodurch sie in zwei Untergattungen oder Sectionen eingetheilt werden kann. Alle japanischen Tilia-arten weisen jedoch Staminodien auf, also bilden diese fiir uns kein Unterscheidungsmerkmal. Obwohl die japanischen Tilia-arten in der Entwickelung der Blatterbildungen 4 selbstandige Arten darstellen, glaube ich doch, das sie in directer Verwandschaft mit denselben Arten des Continentes Ostasiens stehen. Desshalb habe ich in meiner Arbeit unsere Tilia-Arten in zwei Gruppen classificiert, deren typische Formen 7. cordata und T. Mandschurica auch im Continente vorkommen. In nachfolgendem sind die wesenttich characteristischen Ejigenschaften der beiden Gruppen auf- eefthrt. GRUPPE Ae (T. cordata) Bliten Klein, schwach duftend, zahlreich, 20-40 in einem Stande. Blumenblétter ungekielt, die Spitze derselben und Stamino- dien fast ganzrandig. Staubgefisse 15-30; Staubbeutel elliptisch. Friichte Fruchtschale diinn, zerbrechlich, fast rippenlos, geschnabelt. Knospen Dunkelr6éthlich, kahl. Blatter Diinn, papierartig, schmal, kahl, hellgefarbt. Zweige Schlank, dunkelréthlich, kahl. GRUPPE B. (T. Mandschurica) Bliten Gross, stark duftend, wenig, 10-20 in einem Stande. DIE GATTUNG TILIA IN JAPAN, 163 Blumenblatter Gekielt, die Spitze derselben und Staminodien gezahnt. Staubgefisse 60-75, Staubbeutel langlich oval. Frucht Fruchtschale stark, mit schwachen Rippen, nicht ge- schnabelt. Kuospen Griin, behaart. Blatter Dick, hautartig, breit, dicht behaart, dunkelgefarbt. Zwetge Dick, grunlich, behaart. Zur ersten Gruppe geh6éren Tilia cordata var. japonica und Tilia Kiusiana, und die zweite Gruppe umfasst Tilia Maximowic- ziana und Tilia Miqueliana. lll. Das Verbreitungsgebiet der beiden Gruppen. I. DIE GUEPARGORDATA GRUPPE: Die typische Art diese Gruppe T. cordata ist in ganz Europa, namentlich in Mittel-und Nordeuropa, und im Ural, Kaukasischen Gebirge vorhanden; in Sibirien tritt sie, nach Maximowicz, zuerst im stidlichen Theil des nérdlichen Amur auf und kommt anfangs nur auf Flachland in lichten Gehdlzern in der Gesellschaft von Populus tremula vor, ist an dem mit- tleren Amur sehr haufig an den Randern der Laubwalder anzutref- fen, und scheint hier eine grosse Entwicklung zu erreichen, ist jedoch auch am unteren Amur und langs der Ussuri ein gew6ohn- licher Waldbaum. Von hier aus zieht sich die Art an der Kiiste von Korea entlang nach Siidosten bis sie endlich Japan erreicht, wo sie in einer Form der Varietat (var. japonica) auftritt, und fast in ganzem Lande verbreitet ist. Hier nimmt sie eine Veranderung des Habitus an, und zwar ist die Entwickelungs- geschichte der Staminodien in ihren Bliiten sehr interessant. In Europa, sowie in Sibirien entwickeln ihre Bliiten keine Stamino- dien, wahrend sie in Korea nur selten, doch in Japan ganz voll- standig ausgebildet sind. Es muss jedoch erwahnt werden, dass die Staminodienbildungen der T. cordata var, japonica 164 SHIRASAWA. und T, Kiusiana in gewisser Beziehung zu einander stehen. T. Kiusiana entwickelt zwar breitere und vollstandigere Stami- nodien als T. cordata var. japonica, und die Zahl der Staubge- fasse ist bedeutend kleiner, jedoch scheint es mir, dass T. cordata var. japonica vielleicht ein Verbindungsglied zwischen der in Sibirien und Europa vorkommenden Art (T. cordata) und der T. Kiusiana sein diirfte; denn es ist die Beobachtung gemacht worden, dass die Staminodienbildung dieser Tilia-Gruppe sich je mehr wir nach Osten gehen, immer mehr entwickelt, bis sie in T. Kiusiana, welche hier in Japan, in einem beschrankten Gebiete vorkommt, ihre héchste Entwickelung erreicht, 2; DIE TILIASEAN DSCHURICA GRUEEE. Tilia Mandschurica Maxim. stellt eine gute Representantin dieser Gruppe vor. Nach Miximowicz ist ihre Heimat Mandschuria, sowie das Amur-und Ussuri-gebiet und zwar wachst sie an Berghangen oder Waldrandern in dieser Gegend haufig mit mehreren Stammen auf einem Stocke and mit weit ausgestreckten Aesten bis 40’ Hohe und 6” Dicke. Tilia Maximowicziana kommt in no6rdlichen Theil des Japanischen Reiches vor, besonders in Hokkaido wachst er hoéchst tippig. Die in der Bliittenbildung der T, Maximowiczi- ana sehr ahnliche T. Miqueliana, welche ich noch nicht in Freien vorkommenden Zustande beobachtet habe, wachst nach alten chinesischen Beschreibungen im ostlichen mittlerem Theile Chinas und erreicht eine grosse Héhe. In Bezug hierauf scheint es mir, dass das eigentliche Gebiet dieser Gruppe auf die noérdlich kithlere Region beschrankt sei, und dass die in Hokkaido vorkommende Art (T. Maxim.) eine Fortsetzung der des Continents (T. Mands,) darstellt. Die Blatter der T. Maximowiczana haben eine grosse Aehnlichkeit mit denen der T, Mandschurica, und ihre Bliten und Friichte sind fast ebenso wie die der T. Miqueliana, also ist die T. Maximowicziana eine Zwischenform der beiden Arten, und so méchte ich sie im Gegensatz zu Maximowicz’s Benennung, T. Miqueliana Max. fol. volundioribus, T. Maximowicziana benennen, Tokio, Sept. 1899. DIE GATTUNG TILIA IN JAPAN. 165 Figuren-erklarung. eA V IT, (TILIA KIUSIANA.) Fig. 1. Zweige mit Bliitenstanden in der natur. Gr. », 2 Bliitenknospe. » 3,4 Aufbliihende Bliiten. » 5- Kelchblatt voninnen gesehen. 6. Kronenblatt. 7. Staminodus von innen gesehen. 8. Derselbe von aussen gesehen. » 9,10. Staubgefasse. » i1, Langsschnitt des Fruchtknotens, », 12. Querschnitt desselben. F, 13. Querschnitt der Bliite. » 14. Zweige mit reifen Friichten, im natiir. Gr. 3» 15. Frucht, zweifach vererossert, 16. Langsschnitt der Frucht. » 17.18. Zweige im Winterkahlzustande. eee XVIII. (T. Maximowicziana.) Fig. 1. Zweige mit drei Blitenstanden. », 2.3. Aufblithende Bliten. » 4 Kelchblatt von innen gesehen, » 5+ Kronenblatt von innen gesehen. ,, 6, Staminodie voninnen gesehen. ,, 7. Dieselbe von aussen, 8,9. Staubgefasse. », 10. Querschnitt des Fruchtknotens. 5 LL. Langschnitt “desselben. » 12. Querschnitt der Bliite. y 13. Fruchte in einem Stande, natir. Gr. » 14. Frucht, doppelt vergréssert. », 15. Zweige im Winterzustande. Cae: AY 1 Wie ie f i Pa * ai oF a VW 2 i 07) a fir > igay (END aia bai are ce $ aie 4° 7 i aa fe ol wens =iy aed ae | 7 eT a ha aide . Ona y ae add Ve Pe ‘ 4 3 fa ony i al * ; j = sal a, uf? Ge my Bull. Agric. Coll. Vol, IV. Pl. XVIl. Tafel |. Tafel Il Bull. Agric. Coll. Vol. IV. Pl. XVIII. [7a . Jf P ; 3 CPA. : J ilea : Uaximouicyima DS pttitbill (36.6 Average fresh weight of I leaf 0.954 0.168 100: 17.6 Average dry weight of 1 leaf 0.275 0.052 100: 18.8 Moisture in fresh leaves 74.87 70.88 “AOO: Dry matter in fresh leaves 25.13 29.12 100: 116.0 MULBERRY-DWARF TROUBLES IN JAPAN. 185 Average composition of leaves. Healthy. Diseased. Ratio, Moisture 74.87 70.88 100 : 94.7 Dry matter Ase 29.12 100 : 116.0 In 100 parts of dry matter, Crude proteids 31.47 25.76 100) “8158 Crude fats 4.42 3.80 100 : 86.0 Crude fibres 10.00 8.14 100: 81.4 Nitrogen-free extracts 47.97 57.60 100 : 120.0 Ash (free from carbon and sand) 8.52 oie 100: 91.0 Total nitrogen 5.02 4,12 100: 81.8 Albuminoid nitrogen 3.85 3,34 100: 86.6 Non-albuminoid nitrogen 1,17 0.78 100 : 66.6 In 100 parts of ash (free from carbon and sand), SiO, 2.68 3.03 TOO = F131 SO; 3.18 3.09 LOO" 2) (O72 P.O; 12.48 12.69 100) > 1016 K,O Dy mis 25.05 TOG 3 O2-3 CaO 26.63 28.09 160) 7 105.5 MgO LOSES 12.25 100 : 120.6 Average composition of stems. Healthy. Diseased. Ratio, Moisture 80.14 78.68 100 : 98.2 Dry matter 19.86 2532 100 : 107.3 In 100 parts of dry matter, Crude proteids 13.51 15.43 TOO.7 Hi4:2 Crude fats 1.50 226 TOO : 151.0 Crude fibres 49,16 43.64 100: 88.8 186 U. SUZUKI. Healthy. Diseased. Ratio, Nitrogen-free extracts 32.08 33:55 100 : 104.6 Ash (free from carbon and sand) 6.92 7.19 100 : 103.9 Total nitrogen 2.16 2.47 LOO: 3 He4e2 Albuminoid nitrogen 1.02 1.19 LOO, 7 HEZ.0 Non-albuminoid nitrogen 1.14 1.28 LOO); -bE2 In 100 parts of ash (free from carbon and sand), SiO, 1.14 252 100 : 186.0 SO; 3.40 4.48 100 : 132.0 P.O; 9.86 12.07 100'*! 12310 K,O 34.85 32.59 FOO?s> 9335 CaO 2Y.23 23.30 100 : 110.0 MgO r-6O 13.90 100 : 126.0 We see from the above tables that the average length of the diseased stems is nearly half that of the healthy ones, and the fresh weight nearly 1. Both the diseased stems and leaves con- tain a little less moisture, but the difference is not so remarkable, being only 29 in the stems and 5% in the leaves. The total dry weight of the leaves of one diseased stem is about 4 of those of a healthy one, while the average dry weight of one diseased leaf is far less than that of a healthy one, being only # of the latter. Comparing the chemical constituents of the leaves, we find that nitrogen compounds, fibres, and fats are remarkably deficient in the diseased ones, being nearly 4 of the healthy, while, on the contrary, nitrogen-free extracts are more abundant. The reason why the leaves remain so small and shrivel may be due to the deficiency of nitrogen compounds in the living cells, in conse- quence of which the chemical activity of the living protoplasm must be much retarded, and also to the bad development of fibres. The assimilation products in the leaves remain unchang- ed, and the conversion by the living protoplasm, of soluble sugars into cellulose seems to be especially retarded. It is a most remarkable fact that the diseased leaves con- tain only 2 of the non-albuminoid nitrogen compounds, compared with the healthy ones. The decrease of non-albuminoid nitrogen MULBERRY-DWARF TROUBLES IN JAPAN. 187 in the leaves is a clear evidence of the retardation of vital activity. The deficiency of nitrogen compounds in the diseased leaves is not caused by an insufficient supply of nitrogen manures, since the disease is always rampant on fertile soils and on soils treat- ed with soluble manures. The principal cause of the retarda- tion of vital activity must be either the cutting of the stemsin the growing season, or the exhaustion of reserve materials in the roots and stems by excessive drain of the leaves. This question will be discussed fully in the next chapter. As regards the ash contents, we find on the average less in the diseased leaves, but sometimes there are exceptions, so that we can not draw any definite conclusion on this point. In the ash of the dis- eased leaves we generally find less silica, sulphuric acid and phos- phoric acid and more lime and magnesia, but the difference is not very regular. At any rate, we may conclude that the disease is not caused by the deficiency of some special mineral nutriments in the soil, and the difference in the ash ingredients may be simply an effect of the disease. The diseased stems contain also less fibres, but the percent- age of nitrogen is apparently higher ; this may be explained by the fact that in the healthy stems the development of fibres is enormous, in consequence of which the percentage of nitrogen seems apparently lowered. There must of course be remarkable difference in the absolute quantity of nitrogen contained in one stem. No regular difference is found in the ash ingredients of the diseased stems. Chapter II. Qn the Reserve Materials of the Mulberry Tree and their Relation to the Disease. In this chapter, the reserve materials of the mulberry tree, their migration, and their relation to cutting is fully discussed. The samples used for analysis were all taken from the mulberry farm of the Tokyo Sericultural Experiment Station at Nishigahara. Three varieties, Takasuke, Tsuruta, and Jamonji, were selected for this purpose. They were all under the same treatment in the same farm, and eight years old (low cutting). Five plants of 188 U. SUZUKI. each variety which were very similar in all respects, were analysed at different seasons of the year, and the quantity of the reserve materials, the rapidity of their migration, etc. were com- pared. As the cutting in the growing season has a special relation to the disease, it was my chief aim to find out the relation of the reserve materials to the period of cutting, and finally the conclusion was arrived at that the cutting in the growing season is the principal cause of the disease. I. TAKASUKE. (1). April 28, 1899. Fresh Dry Dry Moisture weight. weight. matter % % wood 2805 ¢. on ae 864 240.7 27.90 72al wood 812 oes hee 420 132.6 31.6 68.4 Leaves “© 282 48.2 174 82.9 © As the time of gathering was a little too late, the leaves began to develop. In 100 parts of dry matter, Root bark. Stem bark. Leaves. Crude proteids 16.0 071 40.0 Crude fats 8.3 au1 — Crude fibres 27.3 35-9 — Total carbohydrates (as starch) 18.4 — — Crude ash 8.2 6.7 10.9 Nitrogen-free extracts 40.2 3722 — Total nitrogen 2.56 2.74 6.40 Albuminoid nitrogen 1.38 1.60 4.10 Non-albuminoid nitrogen 1.18 1.14 2.30 MULBERRY-DWARF TROUBLES IN JAPAN. (2) May 18, 1899. Fresh Dry weight, weight. wood 3500.0 oor ee 1174.5 341-3 wood 1064.2 Stems rae 456.5 146.2 New stems 305.2 43.0 Leaves 1220.7 266.8 In 100 parts of dry matter, Root bark, Stem bark, Crude proteids 10.8 11.9 Crude fats Q.1 355 Crude fibres 30.2 5327 Total carbohydrates 14.0 _ Crude ash 73 6.2 Nitrogen-free extracts 42.6 45.7 Total nitrogen 5.73 1.QI Albuminoid nitrogen i12 1.30 Non-albuminoid _ nitro- gen 0.61 0.61 (3). July 11, 1899. Number of new shoots 32; average length of I shoot 50cm. Fresh Dry weight. weight. wood 2000.0 HORI oe 717.2 186.5 New shoots 116.3 19.6 Leaves 214.1 55:4 Dry matter %. 20.5 32.0 14.1 21.9 New stem. 19.38 Dry matter %%. 26.0 16.8 25-9 189 Moisture ie Leaves. Bm.25 Moisture %. 74:9 $3.2 74.1 190 U. SUZUKI. In 100 parts of dry matter, Root bark. New shoots. Leaves Crude proteid 8.0 10.77 33.0 Crude fats 10.5 — — Crude fibres 63.1 — = Total carbohydrates 12.5 —_ —_ Crude ash Get 10.7 II.0 Nitrogen-free extracts 40.7 — —_— Total nitrogen 1.28 1.72 5.28 Albuminoid nitrogen 0.95 = = Non-albuminoid nitrogen 0.33 — = (4). November 28, 1899. Number of stems 21. Fresh Dry Dry Moisture, weight, weight. matter %. %. wood 2207.5 Roots ees 660.8 269.2 40.7 59.3 wood 35765 stems ee 172.1 VOs7 |, hrO. BB In 100 parts of dry matter, Root bark. Stem bark. Crude proteids 9.3 16.4 Crude fats 6.6 5.3 Crude fibres 254 33-4 Total carbohydrates 28.2 18.6 Crude ash 7a 6.7 Nitrogen-free extracts 51.9 38.2 Total nitrogen 1.49 2.62 Albuminoid nitrogen 0.92 1.79 Non-albuminoid nitrogen 0.57 0.83 MULBERRY-DWARF TROUBLES IN JAPAN. I9QI (5). December 2, 1899. Number of stems 24. (no-cutting) Fresh Dry Dry moister weight. weight. matter %. %. R wood 4962.4 ots 1 bark 17Ol3 5QI.1 33.0 67.0 St wood 5229.4 oe | bark aso O10.3 53.7 46.3 In 100 parts of dry matter, Root bark, Stem bark, Crude proteids 12.3 14.3 Crude fats 6.8 5.6 Crude fibres 27.9 36.3 Total carbohydrates 21.6 12.9 Crude ash 7.8 5.6 Nitrogen-free extracts 45.2 38.2 Total nitrogen 1.07 2.28 Albuminoid nitrogen 1.16 1-55 Non-albuminoid nitrogen 0.81 O77 TSURUTA. (i): April 28, 1899. Fresh Dry Dry Moisture weight. weight. matter %. %. R | wood 3120.0 pets late 1000.0 338.3 33.8 66.2 St | wood 610.0 ems | bark 285.0 Gy:8 — 34.2 65.8 Leaves Zao 40.8 ity fey) 82.3 In 100 parts of dry matter, Root bark, Stem bark Leaves. Crude proteids 13.4 25 42.5 Crude fats 10.5 4.4 a Crude fibres 25.8 34.2 — 2 U. SUZUKI. Total carbohydrates 20.0 — — Crude ash 77, 7.0 10.0 Nitrogen-free extracts 42.6 41.9 o= Total nitrogen 2.15 2.00 6.80 Albuminoid nitrogen 1.20 1.40 — Non-albuminoid nitrogen 0.95 0.60 — (2). May 28, 1899° Fresh Dry Dry Moisture weight. weight. matter 9%. ie wood 2520.0 SOG oe 866.2 293.4 661 33.9 wood 127-1127 Stems (oer 2709 12516 66.0" ete New stems 415.2 80.5 80.6 19.4 Leaves 1473.7 3771 74.4 25.6 In 100 parts of dry matter, Root bark. Stem bark. New bark. Leaves. Crude proteids 8.6 9.3 13.75 26.9 Crude fats Lie 4.5 — — Crude fibres 30.0 31.8 — — Total carbohydrates 14.4 — — — Crude ash 8.7 6.9 7.0 8.6 Nitrogen-free extracts 41.2 47.5 — — Total nitrogen 137 1.48 2.2 4.3 Albuminoid nitrogen 0.95 1.00 teh — Non-albuminoid nitrogen 0.42 0.48 tat — (3). July 11, 1899. Number of new shoots 27; average length of one shoot 60 cm. Fresh Dry Dry Moisture weight. weight. matter %, %. ( wood 3380.0 Leer i bark 945.8 2688 284 71.6 New shoots 311.5 50.4 LOW 83.8 Leaves 433.6 62:1 212 78.8 MULBERRY-DWARF TROUBLES IN JAPAN. In 100 parts of dry matter, Root bark. Crude proteids 8.9 Crude fats 12.6 Crude fibres 34.0 Total carbohydrates 10.9 Crude ash 10.1 Nitrogen-free extracts 34.6 Total nitrogen 1.42 Albuminoid nitrogen 0.92 Non-albuminoid nitrogen 0.50 (4). December 2, Number of stems 31. Fresh weight. wood 4558.5 Roots eee 1493.1 S wood 742.4 tems} bark 313.4 New shoots. Leaves. 10.4 Bou; We, 10.2 1.67 5 23 1899. Dry Dry Moisture weight. matter %. %. 602.6 41.7 58.3 172.3- 54.9 45-1 In 100 parts of dry matter, Root bark. Stem bark Crude proteids 10.7 12.9 Crude fats Q.1 5.9 Crude fibres 26.5 20,7, Total carbohydrates 24.0 16.4 Crude ash 7.4 6.3 Nitrogen-free extract 40.3 44.2 Total nitrogen 171 2.07 Albuminoid nitrogen 1.02 1.48 Non-albuminoid nitrogen 0.69 0.59 193 194 (5). December 6, 1899. U. SUZUKI. Number of stems 10. (zo cutting.) Fresh Dry Dry Moisture weight. weight. matter %. 9%. R | wood 4135.1 opts | bark 1495.7 534.0 Bey) 64.3 wood 2308.4 Sieus | bark 806.9 381.0 472 528 In 100 parts of dry matter, Root bark. Stem bark, Crude proteids 13.3 13.4 Crude fats 11.2 5-9 Crude fibres 27.8 Ey Ail Total carbohydrates 17.4 14.7 Crude ash 6.6 6.4 Nitrogen-free extracts 41.1 372 Total nitrogen 213 2.BAi Albuminoid nitrogen 0.99 1,36 Non-albuminoid nitrogen jig) 0.78 Ill. JUMONJL. (1). April 28, 1899. Fresh Dry Dry Moisture weight. weight. matter %. 6 wood 3158.0 OBS ee 778.0 311.1 40.0 60.0 wood 660.0 Stems Re, 295.0 90.2 30.6 694 Leaves 130.0 255 19.6 80.4 In 100 parts of dry matter, Root bark, Stem bark, Leaves Crude proteids 11.3 T5et 36.0 Crude fats 10.4 5.5 _ Crude fibres 25.4 36.3 — Total carbohydrates 23.6 — — MULBERRY-DWARF TROUBLES IN JAPAN. 195 Crude ash Win. Fk 10.7 Nitrogen-free extracts 45.2 35.6 — Total nitrogen 1.80 2.41 5.70 Albuminoid nitrogen 1.10 1.82 4.00 Non-albuminoid nitrogen 0.70 0.59 1.76 (2). May 29, 1899. Fresh Dry Dry Moisture weight. weight. matter 9% % wood 1180.0 GOS Nees ‘ 1018.5 382.3 elena 62.5 |} wood 1170.1 =temis ion Agel 149.6 34.4 65.6 New stems Bibel 50.3 16.0 84.6 Leaves 1200.5 316.4 26.4 73.6 In 100 parts of dry matter, Root bark, Stem bark. New stems. Leaves. Crude proteids 8.1 10.2 14.4 25.0 Crude fats 10.6 a3 — = Crude fibres 30.8 28.6 — — Total carbohydrates 16.9 = = — Crude ash Fed 6.3 8.0 10.7 Nitrogen-free extracts 43.1 49.0 — — Total nitrogen 1.29 162 2.30 4.00 Albuminoid nitrogen 0.95 iG, 1.30 3.30 Non-albuminoid nitrogen 0.34 0.53 1,00 0.70 (3). July 11, 1899. Number of new shoots 27; average length of one shoot 55 cm. Fresh Dry Dry Moisture weight. weight, matter %. %. wood 5650.0 Roots F bark 1307.8 412.2 255 68.5 New shoots 198.5 28.4 14.3 85.7 Leaves 255.3 49:9 19.5 80.5 U. SUZUKI. In 100 parts of dry matter, Root bark, New shoots. Leaves. Crude proteids 8.0 12.88 32.75 Crude fats 1G ths — — Crude fibres 34.9 —_— == Total carbohydrates 14.6 — -— Crude ash 9.2 g.10 11.9 Nitrogen-free extracts 30.4 — _ Total nitrogen 1.28 2.06 5.24 Albuminoid nitrogen 0.79 1.03 — Non-albuminoid nitrogen 0.49 1.03 — (4). December 2, 1899. Number of stems 21. Fresh Dry Dry Moisture weight. weight, matter 9%. %. \ wood 6233.0 HOGS (bark yee Com 4a 86,7 c { wood 793.0 pons | bark 383.7 183.0 Nebel Bong In 100 parts of dry matter, Root bark, Stem bark. Crude proteids Q.7 12.9 Crude fats 10.0 6.2 Crude fibres 28,4 29.1 Total carbohydrates Poke Is 17@ Crude ash 7.0 6.0 Nitrogen-free extracts 44.9 45.8 Total nitrogen 1.55 2.07 Albuminoid nitrogen 0.92 1.48 Non-albuminoid nitrogen 0.63 0.59 From the above mentioned data, we have prepared the fol- lowing tables, from which one can clearly see that the reserve materials in the barks of roots and stems rapidly decrease with the development of the leaves, and that at the time of cutting, i.e. May 18-28, they reach the minimum. Compare also Plates XIX-— XXX, MULBERRY-DWARF TROUBLES IN JAPAN. 197 Il. TAKASUKE. A) Root bark. 9 May 18. | July rr. NOTES Es Tape 28 =! (no cutting) Moisture merce mets. cscnaniroeances 72.1 795 74.0 59,3 67.0 ID Jay PALE? ooeoonneacoendesbOsg OOD 27.9 20.5 26.0 40.7 33-0 In 100 parts of dry matter, Crudeiproteldsi.cces a: son .cs-s 0: 21.1 10.8 8.0 93 12.3 Grad etfats eesscaccuisenusaceesees 8.3 9.1 10.5 6.6 6.8 Grud epGbres! Sites. .eenccesaesooee 27.3 30.2 33.1 25.1 27.9 Total carbohydrates ............ 18.4 14.0 12.5 28.2 21.6 Crd eraShMerree ce sticsnceasccvenees 8.2 73 7.7 7.1 7.8 Nitrogen-free extracts ......... 40.2 42.6 40.7 51.9 45.2 SR Gfalnitrogenl rcss.sccsersse-n 3.38 1.73 1.28 1.49 1.97 Albuminoid nitrogen............ 1.82 1.12 0.95 0.92 1.16 Non-albuminoid nitrogen...... 1.56 0.61 0.33 0.57 0.81 Motalinitrogensensenesreseeee 100.0 51.2 37.9 44.1 58.3 Albuminoid nitrogen,........... 53.9 33.1 28.1 27.2 34.3 Non-albuminoid nitrogen...... 46.1 18.1 9,8 16.9 24.0 Motaly nitrogen .erees.sssss4e. 100.0 51.2 37.9 44,1 58.3 Albuminoid nitrogen............ 100.0 61.5 52.2 50.6 63.7 Non-albuminoid nitrogen...... 100.0 39.1 21.2 36.5 51.9 198 U. SUZUKI. B) Stem bark. April 28. | (May 18. | November oe “= ‘ (no cutting) Moistare 2), ..c. ccccteosecccstacteoeee 68.4 68.0 57:8 46.3 Dry matter [st ..-ccacdcaccseccomeer 31.6 32.0 42.2 Bae In 100 parts of dry matter, Crudejproterdsisces-seeeas se ereeee 22.3 I1.9 16.4 14.3 Grade dats’ 4...0. va ae 3.1 3-5 5-3 5.6 (rude fibress tteea-.-o-seseeereeeeee 35-9 32.7 33-4 36.3 Total carbohydrates ............... — — 18.6 12.9 Grudeé‘ash: S..ccscs.cteese-meeeeeeceee 6.7 6.2 6.7 5-6 Nitrogen-free extracts ............ 37-2 45-7 38.2 38.2 Totalmitrogen: 2 es--sssseeses sees 3.56 1.91 2.62 2.28 Albuminoid nitrogen............... 2.08 1.30 1.79 1.51 Non-albuminoid nitrogen......... 1.48 0.60 0.83 0.77 dotalimitrogens) --s---e- a eeeeeeeeee 100.0 53.7 73.6 64.0 A-Suminoid nitrogen............... 58.4 36.5 50.3 42.4 Non-albuminoid nitrogen......... 41.6 17.2 23.3 21.6 Rotalinitroyens sarees ee eee 100.0 53.7 73.6 64.0 Albuminoid nitrogen............... 100.0 62°5 86.6 72.6 Non-albuminoid nitrogen......... 100.0 41.3 56.1 52.0 eS MULBERRY-DWARF TROUBLES IN JAPAN. II. TSURUTA. A) Root bark. December 109 ecember April 28. | May 29. | July 11. ee om (no cutting) RGIS NOS iad gaa tere eee 66.2 66.1 71.6 58.3 64.3 Dry In atten re 5 28 aeeldcee emcee sain 33.8 33-9 28.4 41.7 35-7 In 100 parts of dry matter, @rude proleidsh +s,.-e.e..+-2-e0e- 17.4 8.6 8.9 10.7 Tees Grudeytats! Viasecssctessecencsceaes 10.5 11.5 12.6 g.1 Nyt p (CHGS HOES, connseneeseoeunedsen: 25.8 30.0 34.0 26.5, 27.8 Total carbohydrates ............ 200 14.4 10.9 24.0 17.4. Craderashe sexcaectessieteaccecensen Heal 8.7 10.1 7.4 6.6 Nitrogen-free extracts ......... 42.6 41.2 34.6 46.3 41.1 otalmitrocenmensscuessessecceere 2.79 1.37 1.42 ier 2.13 Albuminoid nitrogen............ 1.56 0.95 0.92 1.02 0.99 Non-albuminoid nitrogen....., 1.28 0.42 0.50 0.69 1.14 Srotalimitrogenty..sweceee-ee eee 100.0 49.1 50.9 61.3 763 Albuminoid nitrogen............ 55.8 34.1 33.0 . 36.6 35.5 Non-albuminoid nitrogen....., 44.2 15.0 17.9 24.7 40.8 Wotalimitrogen J. /iasess.cceoees- 100.0 49.1 50.9 61.3 76.3 Albuminoid nitrogen............ 100.0 60.9 59.0 65.4 63.5 Non-albuminoid nitrogen...... 100.0 34.1 40.7 56.1 92.7 U. SUZUKI. Stem bark. December 6. April 28. May 29. | December 2. (no cutting) 65.8 66.0 52.8 34.2 « 34.0 47.2 In 100 parts of dry matter, Grudesprotetds) ye seee- eee eee 16.5 93 12.9 13.4 Crudésfats: cin5c.t ee ae 4.4 4.5 5:9 5.9 Crude} ‘fibres: 225.0520 34.2 31.8 30.7 37-1 Total carbohydrates ............... _ = 16.4 14.7 Crudé: ash ® 5.00 secures eee 7.0 6.9 6.3 6.4 Nitrogen-free extracts ............ 41.9 47:5 44.2 37-2 Motall nitrogeny y5.2sse--e-e eee 2.64 1.48 2.07 2.14 Albuminoid nitrogen............... 1.85 1.00 1.48 1.36 Non-albuminoid nitrogen......... 0.79 0.48 0.59 0.78 Motalimitrosenh yee seeps eeeeeeee 1000 | 56.6 78.4 81.1 Albuminoid nitrogen,.............. 700 37.9 56.6 51.5 Non-albuminoid nitrogen......... 30.0 | 18.7 21.8 29.6 Totalmitrogenys....c5-.06-¢ee emer 1000 56.6 78.4 81.1 Albuminoid nitrogen............... 100.0 540 80.0 73.5 Non-albuminoid nitrogen......... 100.0 60.8 74.7 98.7 MULBERRY-DWARF TROUBLES IN JAPAN. Erde PKOteIAS Mee). scssceescecseen GrudeHtatsieeessccssschiacsaevecorwes Total carbohydrates ............... ROI CFAS I ea. c on ee cnpene oxietnavere Nitrogen-free extracts ............ Motalimitrocentenenasssesarenecer eee Albuminoid nitrogen............... Non-albuminoid nitrogen,........ Motalimitrogen) tees: cadences encase Albuminoid nitrogen............... Non-albuminoid nitrogen......... GGtAlMitKO GEN, sercrssece hese seers: Albuminoid nitrogen............... Non-albuminoid nitrogen......... A) Ill April 28. 100.0 100.0 100.0 JUMONJI. Root bark. 201 May 20. July 11. December 3. 62.5 68.5 56.1 SES 43-9 8.1 8.0 9.7 10.6 11.5 10.0 30.8 34-9 28.4 16.9 14.6 25.5 7.4 9.2 7.0 43-1 36-4 44.9 1.29 1.28 1.55 0.95 0.79 0.92 0.34 0 49 0.63 59.7 59.1 ales 44.0 36.6 42.6 15.7 22.5 29.2 59.7 59.1 71.8 72.0 60.0 70.0 40.5 53.8 75.0 202 U. SUZUKI. B) Stem bark, April 28. May 20. December 3. Moisture 225.2039. s2 se -aee cae 69.4 65.6 52.3 Dry matter. 2... sce<2-0--2-o sae 30.6 34°4 47-7 Grude proteids2)-:-.--- see -eeeee 18.3 102 12.9 @rude fats. .iic.css.cstescese comers 5.5 53 6.2 Crude fibreési.ccescese oo. - eee 36.3 28.6 29.1 Total carbohydrates ............ _ _— 17.9 Crude@-ashi2®,.sc0.. eckson eee 7.5 6.3 6.0 Nitrogen-free extracts.............. 35-6 49 6 45.8 Tiotalnitrogenten-cs.s---eesee 2.77 1.63 2.07 Albuminoid nitrogen ......... .... 2.09 1105 1.48 Non-albuminoid nitrogen ........ 0.68 0.53 0.59 Motalimitrogenseeseeee cesses eee 100.0 58.8 74.7 Albuminoid nitrogen ..............- 75.5 40.0 53.4 Non-albuminoid nitrogen ........ 24.5 18.8 21.3 Total nitrogen 74.7 70.8 Albuminoid nitrogen Non-albuminoid nitrogen MULBERRY-DWARF TROUBLES IN JAPAN. 203 The above tables clearly show that the reserve materials in the roots and stems undergo a very remarkable change during the development of leaves in spring. The greater part is trans- ported to the growing parts, especially to the leaves, and there used partly for the formation of new cells and partly for the respiration process. Thus we see that in summer, when the leaves are in full growth, the roots and stems contain only very little reserve materials, but in autumn, the assimilation products formed in the leaves, again come down to the stems and roots and are there stored up to provide for future growth. Such a process is quite natural and every plant follows the same course; still there obtains a great difference in different plants, as to the quantity of the reserve materials and the rapidity of their migration. Some plants store up large quantities, while others contain very little. These differences must naturally exist not only among plants of different species, but also among different plants of the same species. The reserve materials in plants are generally fats, carbohy- drates and nitrogen compounds, but in the case of the mulberry, fats seem to play no important rdOle. It is a remarkable fact that non-albuminous nitrogen com- pounds (most probably amido-compounds) are contained as reserve materials in large quantities, corresponding, as they do, to nearly one-half of the total nitrogen. As amido-compounds are soluble in water and easily trans- portable they may be conveniently used during the energetic development of the plant. Here we must not forget that, not the whole of the nitrogen compounds contained in the stems and roots, serves as reserve materials, but only some portions of them are capable of transformation; since there ts a not inconsider- able quantity of nitrogen in the form of insoluble compounds The greater part of the reserve carbohydrates in the mul- berry consists of starch, the presence of which may be easily demonstrated under the microscope. Further, we can easily prove the migration of starch during development without the aid of quantitative analysis. Thus, if we examine the bark of stems or roots under the microscope, we shall soon discover that in winter and in early spring, the bark is remarkably rich in starch grains, which, on the application of iodine, cover the entire field with violet spots. But in summer, when the leaves are fully 204 U. SUZUKI. developed, we can hardly find a single starch grain under the same treatment. The importance of reserve materials in the first stages of development is self-evident, and hardly needs an explanation, since the youngest buds can only develop by the aid of reserve materials, and even the green leaves, in their youngest stages, have very little assimilative faculty and must chiefly rely upon reserve materials until they become tolerably large. No plants can develop their leaves without the aid of reserve materials But the precise extent to which the mulberry stands in need of reserve materials for proper development, is still open to ques- tion and must be discussed fully. There is no doubt that differ- ent plants needs different quantities of reserve materials, and ifa plant contains a large quantity of reserve materials, it shows that the plant wants so much until it becomes able to assimilate the necessary nutriments from outside. Otherwise the plant would never keep in store an unnecessarily large amount of reserve materials. As already shown in the preceding tables, a considerable amount of reserve materials is stored up in the stems and roots of the mulberry, the greater part of which is consumed during the development of the leaves; nence there is no doubt that this plant has need of a large amount of reserve materials, and it may naturally be expected that when the reserve materials are in- sufficient, the plant can not attain normal development. It is an evident fact that an enormous quantity of reserve materials moves toward the growing points of the buds and roots to build up new cells, and a still larger quantity of fats and carbohy- drates must be consumed there by the energetic respiration which commences “long before the new leaves are unfolded, The necessity of reserve materials will be the greater the quicker the development of young leaves, and the nutriments absorbed by the roots must be insufficient to meet the demand, as the absorptive power of the roots depends greatly upon the intensity of transpiration; and it may naturally be expected that in the first stages of development, when the leaves are very small the absorption of the nutriments must be very slow and thus makes the necessity of reserve materials still more urgent. — According to some recent investigations, proteids can be formed from amido-compounds or other inorganic nitrogen com- MULBERRY-DWARF TROUBLES IN JAPAN. 205 pounds (ammonium salts or nitrates) in the absence of light, although much more slowly than in full day-light ; the amido- compounds seeming, when the light is insufficient, to be far more conveniently used than the nitrates for the synthesis of proteids. It is therefore very probable that the amido-compounds, which are present in the stems and roots of the mulberry, are far more efficiently and quickly used for the formation of new proteids in the growing roots and in the buds where light has no access, and can never be replaced by the inorganic nitrogen compounds absorbed from the soil, As can be seen from the tables, the root-bark of Takasuke and that of Tsuruta and Jamonji contained on May 18 and 29 respectively (the dates of cutting) only very little reserve materials. The new shoots are therefore compelled to com- mence their development with a very scanty source of reserve materials, so that it may sometimes happen that the reserve materials become exhausted before the new leaves can duly perform their function. The natural result in such a case would be an imperfect development of the leaves, as a consequence of which the assimilation process would be retarded and_ the nourishment of the new roots become insufficient and their de- velopment be more and more retarded until at Jast they would be compelled to die off, and the consequent deficient absorption of nutriments from the soil would react injuriously on the leaves. This may be compared to babies deprived of milk and nourished only with solid food, before the digestive organs have developed their powers. Only those plants which have effected a certain degree of development before the exhaution of reserve materials can attain normal growth. We can now clearly understand why the variety Jamonji, which requires the least amount of reserve materials as compar- ed with the other varieties, is so rarely subject to the disease. The following calculations will bring out this point more clearly ;— A) Takasuke, on April 28, had already developed new leaves, the fresh weight of which was 282 grams and the dry weight 48.2 grams for one stock, the nitrogen amounting to about 3.072 grams. This nitrogen must have come almost entirely from reserve materials in the stems and roots and not from the soil. Notwithstanding so much con- 206 U. SUZUKI. sumption of reserve nitrogen for the development of new leaves, not an inconsiderable amount of nitrogen was, still afterwards, transported from the stems and roots to the leaves. That is*tevsay, on April “28, the” bark ofthe roots still contained 2.569% and that of the stems 2.74% of nitrogen; this, however, gradually decreased, and on May 18, we found only 1.73% in the bark of the roots and 1.919% in that of the stems. As the dry matter of the bark of the roots was nearly 250 grams and that of the stems 140 grams, we can calculate the total amount of the nitrogen transported after April 28 ; thus :— 250 » (2.56 —de73)= 250 x 0.83% =2.075 SkatnS mci - transported from the roots 140 X (2.74 — 91) = 140 X 0,83 % = 1.162 oramSmen nen transported from the stems. Sum total =3,237 grams. If now we add the nitrogen consumed before April 28, then we have :— 3.237 + 3.072=6.381 grams nitrogen, transported during the development of the leaves, of which nearly 4,15 grams was in the roots. B) Tsuruta. On April 28, new leaves contained already sap. eric ac eee tees) «rd tiers 2.79 gram. After the 28th. transported from the roots 2.34 fromthestems 0.52 ” a” ” Sulinibbotallley ros sss oper . 5,65 grams nitrogen Nearly 4.26 grams therefore was in the roots. C) Jumonji. On April 28, the new leaves contained.. 1.45 grams. After the 28th. transported from the roots 1.50 y 58 53 from the stems 1.00 Sume@otalifpedss.05..8% 3.95 grams nitrogen. Of this nearly 2.60 grams was in the roots. MULBERRY-DWARF TROUBLES IN JAPAN. 207 The following table shows the decreased percentage of nitrogen in the dry matter. | Takasuke. | ‘Tsuruta. | Jumonji. | | | | { in the bark of roots | 1.65% | 1.42 | 0.87 Total nitrogen | | E stems | 1.65 1.16 | 1.14 roots | 0.70 | 0.61 0.37 Albuminoid nitrogen | | | > stems | 0.78 0.85 0.99 = roots 0.95 | 0.81 0.50 Non-albuminoid nitrogen - | a stems 0.84 | 0.31 0.15 Let us now try tosee how much nitrogen was contained in the leaves and the new stems on May 18 and 28 respectively, viz. at the time of the full development of leaves. Takasuke Turuta Jumonji (May 18.) (May 28.) (May 28.) Nitrocemini the new Stems” —. see sseanaeaeeeeee 1.330 ¢ 1.782 ¢ 1.150 ¢ leaves Maatacmee peeatcn cee 12-340) 5; ROOM | L2tO4 Ome. J Aa EMRE 5 5. 14.670,,| 17.992,,| 13.790,, Su totaly... ane The quantity of nitrogen transported from the roots and stems to the leaves is :— | Takasuke. | suruta. Jumonji. | ie 6.31) ¢ | 5.65 g.| 3.95 g. J | rm ee 208 U. SUZUKI. That is to say, Takasuke consumed 6.31 grams of reserve nitrogen to develop the new stems and leaves containing 14,670 grams nitrogen. 6.310 an : Therefore 4.676 of the nitrogen in the new stems and ieaves came from the reserve materials. .65 Fog3 = 81% 17.992 In Jamonji. _3:95°_ 99 0 13.790 It is surprising to find that the mulberry consumes a far greater quantity of reserve nitrogen than we supposed. We observe at the same time that Takasuke consumes a far greater quantity of reserve nitrogen than Tsuruta or Jumonji. This is especially interesting, since Takasuke is more liable to suffer from the disease than the other two varieties. In Tsuruta Of course, the above mentioned figures represent only ap- proximate values, because there are individual differences among the same varieties, and absolutely exact comparisons are impos- sible. Further, the reserve nitrogen does not only migrate into the leaves, but a portion of it must, of course, be consumed in the roots. Nevertheless, the above is sufficient to show how im- portant a part the reserve materials play during the develop- ment of new leaves in the mulberry. We shall now turn our attention ‘to the question, whether the new shoots that come out from the stump after cutting can be sufficiently nourished with the reserve materials remaining in the bark of the roots. At the period of cutting, the bark of the roots contained the following quantities of nitrogen :— Takasuke Tsaruta Jamonji. (May 18.) (May 28.) (May 28.) Total nitrogen.......... - Ses 1-73.% 1.37 1.29 Albuminoid nitrogen ...... eek. 1.12 0.95 0.95 Non-albuminoid nitrogen ......... 0.61 0.42 0.34 MULBERRY-DWARF TROUBLES IN JAPAN. 209 Absolute quantities in the root-bark of one stock. Takasuke. Tsuruta. Jamonji. (May 18.) (May 28.) (May 28.) Dry matter in the bark of roots... 341 grams 293 38.2 meal ANOS EM. ese pian -/en eer oeenee 5.900 4.020 4.930 Albuminoid nitrogen ............... 3.820 2.780 3-630 Non-albuminoid nitrogen ........ 2.080 1.240 1.300 Although the above are only approximate values yet there will be no great error in assuming that nearly 4-6 grams nitrogen is still contained in the roots after cutting ; but this quantity of nitrogen is not entirely available for the young shoots, because a portion of it is in the form of insoluble compounds, and only a portion is capable of being transported to the growing parts. If we assume the quantity of non-albuminoid nitrogen as represent- ing the quantity of available nitrogen, there will be no great error; and in that case 1.3-2,0 grams or at most 3 grams can be utilized by the new developing shoots. Let us now see wheth- er this quantity is sufficient for the energetic development of the new shoots. On July 11, when the new shoots had reached the height of 4c-60 cm., they were analyzed, and the following results were obtained : 1) Takasuke. Total dry Nitrogen Sum total of matter. in same. nitrogen. New stems... 3.2% Pa9;6 0.337 3.969 o Leaves. . os DD 2.925 : ea 2) Tsuruta. New: stems... ..). aaa 50.4 0.842 5.66 ¢. WMeaves: 5... 5.2 eee 92.1 4.817 3) Jamoniji. New stems. ee Os: 0.585 3.20 g. CAVES) 2 <<: tenn 50.0 2.620 : We see now that when the young shoots had reached the height of about 50 cm and the dry matter of the leaves amount- ed to about 50 grams, there were more than 3 grams of nitrogen 210 U. SUZUKI. in the latter. The reserve nitrogen is therefore hardly sufficient for the normal development of the shoots, and some plants may suffer in consequence and become diseased. One may ask now, how can we explain the fact that some varieties, like Takasuke, become diseased more easily than others, like Jamonji. This [| shall explain fully in the following pages. As we see from the results of the analyses, the decrease of nitrogen in the bark of the roots of Jamonji during the growing period, is smaller than in Takasuke and _ Tsuruta, and the total quantity of reserve nitrogen is also far smaller in Jamonji than in the other two. This means that Jamonji needs a far smaller quantity of reserve nitrogen to build up the same quantity of leaves and new shoots. The roots of Jumonji seem to be especially fitted for the absorption of soil nutriments, and to make good the deficiency of the reserve materials already pre- sent. If now we compare the nitrogen contents of the leaves in the three varieties we find: Takasuke. Tsuruta. Jamon ji. April 28, 6.40 6.80 5.76 May 18, 5.00 4.30 4.00 From this it is clear that Jamonjit can build up the same quantity of leaves with a far smaller quantity of nitrogen, that is, the necessity for nitrogen must be far smaller than in the other two varieties. Further, we have calculated that Takasuke consumes 6.31 grams reserve nitrogen to produce 1220 grams fresh leaves (=266.8 grams dry matter, containing 13.34 grams nitrogen), while in Jamonji only 3.95 grams reserve nitrogen is spent to produce the same amount of fresh leaves (=316 grams dry matter, containing 12.64 grams nitrogen). Thus we see that Jumonji needs only 62% or nearly 3 of the reserve nitrogen re- quired by the other two varieties to produce the same quantity of leaves. This evidently shows that Jumonji has a stronger absorptive power for the nitrogen in the soil and manures, and does not rely upon reserve nitrogen so muchas the other two. It is therefore not to be wondered at that, Juimonji is less liable to attack by the disease, since the comparatively small quantity of reserve materials in the roots is soon made good by the newly absorbed nutriments. MULBERRY-DWARF TROUBLES IN JAPAN, ZU But we must here remember that the power of resistance to the disease is not absolutely confined to certain fixed varieties, but may be gradually changed by climatic conditions, soil, and treatment. Thus we always observe that, when certain varieties are induced to rapid growth by the addition of soluble manures in excess, and then cut, then even the favored varieties may be- come diseased, while on the contrary, such varieties, as Takasuke, will never become diseased if they are cultivated in infertile soil with poor manuring. Thus, one and the same variety may undergo a very remarkable change in its character, especially as regards its conduct towards the disease. Over- growth accelerates the migration of reserve materials. If the de- velopment of new shoots after cutting is very energetic, then the want for reserve materials must be correspondingly great, and the absorbed nutriments must be insufficient for the demand, the re- sult being an emaciated condition of the shoots. Over manuring will never directly increase the quantity of reserve materials in the roots and stems in the growing periods, the nutriments supplied as manures being all transported to the growing parts. So if we cut such over-grown plants, the new shoots will develop more energetically and the reserve materials will soon be exhausted, just the natural condition for the disease. Thus we can understand why the power of resistance to the disease is not absolutely confined to certain fixed varieties. Young plants are rarely attacked ; this may be explained by the fact that the cells of young plants are more active, and after cutting can develop small rootlets more easily, and thus gain a stronger absorptive power, than old plants, so that they can easily recover from the deficiency of reserve materials. Old plants are less active, and after cutting, the develop- ment of new rootlets takes place with more difficulty, and accord- ingly, their absorptive power must remain comparatively station- ary and the recovery will consequently be slower. For the same reason, the disease appears oftener after cutting in late summer or autumn, while it is less frequent when the plants are cut earlier. The development of new rootlets and the consequent recovery after cutting, must be much more difficult in autumn. The young rootlets must once lose their activity after cutting, and the entire root-system will become gradually ineffici- 212 U. SUZUKI. ent, unless the development of the new rootlets is exceptionally good; and after successive cuttings, the small roots must entirely die off. So it is quite natural that we always find de- cayed roots when the disease is much advanced. But in the earlier stages of the disease, we find only small rootlets dead. The roots and leaves are closely correlated to each other, a bad condition in the one accelerating the degeneration of the other; and the primary cause of the disease must be sought in the practice of cutting in the growing period. We see [Plates XIX-XXVI] that the quantity of reserve nitrogen in the roots of Takasuke, Tsuruta etc. is not restored to its norm evenin December. This evidently shows a gradual wearing of the plants, and after two or three years, they must become diseased unless they are kept from cutting. In the foregoing pages, we have considered only the nitro- gen compounds; but we must remember that the carbohydrates, especially starch, have also the same physiological importance during the first stages of development That the cutting in the growing period, when the starch contents of the roots have reached the minimum, has a specially injurious effect upon the new shoots hardly requires any further explanation. The view above developed finds an additional support from: the following facts :— = (1). The disease does not affect those plants which are not cut. This is true throughout the whole country and there is no exception to it. We have observed this in such provinces as Fukushima, Yamagata, Akita, Hyogo,etc., where the cutting meth- od is not practiced. The plants in these provinces attain their maximum height and are 30-40 years, or sometimes even more than 100 years old. Further, we have observed a very instructive case in these provinces, viz. some of the farmers adopted the method of cutting, and all suffered from the disease, while in the neighbouring farms where the method was not adopted the plants were perfectly healthy. A more striking fact was observed in Tanba. A farmer cut the same plants 3 times in one ummer, in consequence of which, all the plants in a farm with- out exception became diseased, while in the neighbouring farm in which the plants were cut only once, the disease did not appear. That the cutting is the principal cause of the disease may also be shown by the fact that, by keeping the diseased plants for. MULBERRY-DWARF TROUBLES IN JAPAN. 243 several years from cutting, most of them can be made to re- cuperate. Many experiments have been made to prove this fact. Thus we see :— Result. —" —— c_r- Number |Number of} Became Remained Date of Variety. cutting. auc br exper flee Diseased. Healthy: Death. Jumonji I 5 I I 3 ra Obata ms I 5 I 2 2 — Hikojiro 55 I 5 I 1 3 — Tsuruta = I 5 I 3 I — Takowase 3 I 5 — 4 I — Hosoye August 19. I 50 6 9 23 12 Late autumn enecre|| 2mm] s | we loo | — ” ” 2 15 5 4 5 I A August 26. I 30 3 13 6 8 Yotsume = 28% 1 30 4 6 16 4 Hosoye Oe I 15 4 6 3 2 yee 1 15 — 12 3 — Total 215 35 72 81 27 Ratio 100 16 34 38 12 The disease did not appear in the control (not cut) plants. A similar result has been reported by Mr. Ichikawa. Though he does not mention the age of the experimented plants, yet I donot doubt that they were moderately old; young plants do not show, in my opinion, such a remarkable effect. We have also made a similar experiment in 1898 with the variety Nezumigayeshi, eight years old :— 214 U. SUZUKI. Date of cutting. ee Se Healthy. ees Diseased. May 16. I 28 26 _ BD » I Il II = as » I II | II — = June 1 I 20 | 20 -- = + I II II — a I II II — = A I 20 | 14 = 6 I II 9 — 2 | The same experiment was repeated in 1899 with the same variety in the same farm :— Date of cutting. pee eee c | Healthy. | Diseased. June 1 I 20 | 16 | 4 | REV EMETOR Skcaraeectaoob ace I 20 13 | df i sedpslea eect I | I 3 5 fol kW te ode anecene ac | | 51 32 19 Percentag ene. ee. | 100 63 | 37 The control plants all remained healthy. Hence it is quite evident that the cutting is the principal cause of the disease. (11). If my assumption be true, then the disease must be in- duced in healthy plants by simply picking off the leaves after their full expansion, as by this means the reserve materials in the roots and stems must as well become exhausted. Following is the result of such an experiment. MULBERRY-DWARF.TROUBLES IN JAPAN. een I I ES Plot. All the leaves taken. A portion of leaves taken. New stems, together | | with leaves taken. | Stem cut off. | Variety. Komaki. Komaki Ros6, Tsuru- ta, Takasuke. Takasuke. Takasuke, Hosoye, Akagi, Number plants. mn 980 122 | R Date of ¢ picking off Result. the leaves, 3 times. Healthy 3 (May 7-Sept 9)| | Diseased 2 3 times. ( Healthy 5 (Apr. 28-Sept. 9)! ) Diseased o Healthy 995 | 2 times. Partly diseas- (May 18-July 15)) ) ed 96 Diseased 198 1 times. Healthy 5 eMiry 28) Diseased 1 Healthy 75 1 times. 5 (May 21) Diseased 34 | | | | | | Dead 13 Healthy 783 Diseased 8322 This table shows that by frequent picking off of the leaves, nearly 4 of the plants may become diseased. Second Experiment. Variety. All the leaves Takasuke, Gen A portion of leaves taken. All the leaves taken. A portion of leaves taken, Roso. Date of picking. August 14 17 14 io} ” 3 August 15 ” Sept. Number plants, 92 82 561 Partly Dead Healthy.|Diseased,| 9: -o3céd. 81 2 9 — 68 2 12 -— | 69 1 14 — | 530 6 20 2 216: za Seo SUZUKI. Third experiment. | | } ; Date Number | — _ Plot. Variety. of of Healthy. Diseased. | picking. plants. SSS a = A portion of the ) |, al leaves on new eon August 16 35 32 3 shoots taken. gay ; All the leaves on new shoots al 3 June 1. a, 28 12 { } |, -June 1. A portion of the | leaves taken 3} | |< Juneto. | 40 37 3 times. | | | \ Sept. 20. June 1. All the leaves} | | ( » . | agulyizo. 40 31 fe) taken 3 times } | } | \ Sept. 20. Fourth experiment. Variety of | | picking “tank a | \ A portion of ed : leaves on new } | legos | 35 28 5 2 shoots taken, ly pesca i A portion of) | ee new shoots pick- | ‘ 40 25 15 =_ ed. New shoots wholly cut. f ; 40 16 24 or | | May 28. 4 A portion of | | leaves picked 3 |< July to. 40 35 5 | = times. | | | | \ Sept. 20. May 28. All the leaves, picked 3 times, July to. 40 20 20 _ | Sept. 20. { MULBERRY-DWARF TROUBLES IN JAPAN. 217 No case of the disease was found in the control plants, which were not deprived of their leaves. See Plates XXXV- XXXVI. The age of the plants, varietal peculiarities and other con- ditions have a great influence upon the disease. It is a well known fact that in those provinces where silk- worm culture was recently gueduced, the disease is especially oceee Seseible crop of te leaves Fron rather small farms, and consequently, over-manuring and frequent picking of the leaves, sometimes followed by cutting, iscommonly practiced. Further, those varieties such as Takasuke, Hosoye, etc., which grow rather rapidly, are mostly preferred. Moreover, silkworm culture in late summer or autumn is especially injurious for the mulberry, since the leaves are taken offin autumn, and the assimilation products in them to be utilized by the stems and roots for the next year’s growth, are neccessarily lost ; and thus the principal cause of the wearing of the plants is brogeue about. One might suppose that by giving an excess of soluble manures, the injurious effect of the frequent picking of the leaves or of cutting, can be counterbalanced ; such a view is erroneous. In the growing period, manuring does not directly nourish the stems or roots; the nutriments being all transported to the growing parts, especially to the leaves ; and in late autumn, the assimilation products in the leaves come down again to the roots and stems and are stored there until the next spring. In the growing period, the roots and stems merely serve as a passage for the nutriments. Therefore if we cut off these over-grown plants in the growing season, the new shoots will grow energetically and the reserve materials must be rapidly exhausted, the disease being the result. Here I shall cite one more example showing that the disease is nothing but the result of the deficiency of the reserve materials. Healthy and diseased plants in the same farm were cut on August 30, and the new shoots coming from the diseased stems showed distinct signs of the disease even in the earliest stage. On October 15, the plants were cut down and analyzed, with the following result :— 218 ire eae U. SUZUKI. Leaves, Stems, Healthy |Diseased. Ratio. Healthy.|Diseased Ratio. Moisture. | 78.90] 73.64|100; 93.0} 87.35 85.05 | 100: 97.38 Dry matter. | 21.10 | 26.36 | 100:125.0/ 12.65 14.95 | 100: 118.0 | In 100 parts of dry matter, Ash, 11.76 7.82 | 100: 66.5] 12.5 8.90 |100; 71.2 Total nitrogen, 5.28 3.70 |100: 70.0 3.16 3.29 | 100: 104.1 Albuminoid =| 390] 290/100: 71.0| 140] 130/100; 92.9 nitrogen. | Non-albuminoid é ‘ nitrogen, 1.48 1.00 |100: 67.9 1.76 1.99 | 100: 1138.0 We see from this that the new shoots coming from the diseased stocks are considerably poorer in nitrogen and ash, which evidently shows that their roots contained very little reserve materials, which were insufficient to meet the demand of the new shoots even in the first stages of development. (III). By grafting diseased shoots on healthy roots or stems, many of them can be made to recover and grow normally. We may therefore infer that the disease is exclusively due to defici- ency of nutriments, and may be prevented by replacing worn- out roots with vigorous ones, The following experiments prove this point. MULBERRY-DWARF TROUBLES IN JAPAN. 219 = First experiment. Plot, Diseased roots of to Healthy stems of Diseased stems of Hosoye on Healthy roots of Roso Variety. Takowase Nezumigayesbi Number of experi-| ments, 20 | | Positive Negative LEUNG results, result, Geen ced oO 18 A Second experiment. Plot. Variety. Diseased stems Of ...........0.0-«- ie akowase | to healthy stems of ............... Nezumigayeshi | ” Diseased stems tohealthy root... Diseased stems ‘to health y Misho.,.,... -Diseased stems on diseased roots.....,.. Healthy stems Diseased roots or stems _ Number of ‘ iti It, experiment, Positive resu 10 15 In the control experiments (grafting of healthy shoots on healthy roots) nearly 60% were successfu. 220 i] 2h SeSUZUKL (IV). By layering i,e. burying a portion of the diseased shoots bent into the earth, as shown in Pl. XXXVII-XXXIII. The new rootlets are produced from the covered part, and absorb nutriments from the soil, and the shoots recover from the disease. Numerous experiments have shown this fact, and even the severet cases of the disease may sometimes be suecessfully treated in this way. See Plates XXXVIII-XXXVIII. (V). By keeping diseased plants from eutting for two or three years and well manuring, many can be made to recover, as has been proved by many experiments, But if these plants are again cut in the growing season they will become again diseased. Further, if we cut down the diseased shoots in early spring before the leaves are developed, the next new shoots will grow normatly. This may be explained by considering that the roots of diseased plants may still contain a moderately large quantity of reserve materials during the winter, if they were kept from cutting; so that the new shoots may develop normally without suffering from the deficiency of reserve materials. Inthe experiment made in 18¢8, in which 22 diseased plants were kept from cutting, 11 plants recovered and 3 more showed signs of imperfect recovery. In another experiment made in the same year 15 recovered out of 22, while those that were subjected to cutting in the growing season, did not only not recover, but became even more severely diseased, The result obtained in 1899 is equally interesting, 13 plants having recovered out of 22 diseased plants ; while in the case of plants subjected to cutting none recovered. eto se (VI). After cutting in the growing season, a considerable amount of the sap flows out from the cut stems for several days, amounting sometimes to several hundred cubic centimenters for one plant. And as this sap has been believed by many to have an intimate relation to the exhaustion of the reserve materials, I have directed my attention to its chemical analysis. For collecting the flowing sap India rubber tubes were connected at one end with .the cut stems and at the other with tightly corked test tubes, a). Sap of Obata. Collected on May 20. Colour.—Slightly turbid; after standing for several hours, a little precipitate was formed at the bottom, MULBERRY-DWARF TROUBLES IN JAPAN. 221 Reaction,—Faintly acid. Specific gravity.—I.col. Qualitative tests :— J. Nitrates.—Diphenylamine reaction, dark blue colouration :— Nitrates present in tolerable quantity. 2. Ammonia.—Nessler’s reagent produced brown precipitate. The solution boiled with a little. caustic potash, ammonia gas evolved, which turned red litmus paper distinctly blue and yellow turmeric paper brown. Doubtful whether the ammonia was present as such in the original fresh solution or was derived from the decomposi- tion of proteids or amido-compounds. Normal sap commonly contains no ammonia. Further, a brown precipitate may also be produced by Nessler’s reagent in the presence of sugars. 3. Lime salts.—A little present, white precipitate by ammonium oxalate. 4. Sulphate.—Doubtful trace. 5. Chlorides.—Tolerably much, white precipitate by silver nitrate. 6. Iron salts.—Absent. 7. Proteids.—Biuret reaction, only faintly violet. No precipitate by nitric acid. Almost no precipitate by potassium ferrocyanide and acetic acid. From these reactions, the presence of proteids is doubtful, or it must have been decomposed during the collecting of the sap. 8. Sugars.—Slightly reduces Fehling’s solution. 100 c.c. of the sap yielded 0.0172 grams nitrogen. — b). Sap of Jimonji. June 2. Colour.—Same as in Obata. Specific graviiy.—1.0007. 100 ¢.c. contained.—0.007 grams nitrogen. : 0.185 grams dry matter, 0.080 grams ash (769§ of which dis- solves in hydrochloric acid.) 0.095 grams organic matter. 222 w. SUZUKA, The sap of Jamonji contained far less nitrogen than that of Obata. The composition of the sap may therefore be very differ- ent according to varieties, time of gathering and other conditions. The nitrogen contained in the sap may come partly from the reserve materials in the roots and partly from the freshly absorbed nutriments. I have not determined the exact quantity of the sap that flows out from one plant, as it is rather difficult. But it is plain enough that Takasuke and Tsuruta furnish a far greater quantity of it than Jamonji, which fact may have some significance for the resisting capacity of these varieties towards the disease, though I do not believe it has so much influence upon the disease as many suppose. The numerous facts above mentioned are in full accord with my view. I must here add a few words on a case that I have observed very often, viz. that the cut ends of the stems decay and the putrefaction preceeds between the woodand the bark towards the inner part, and interrupts the connection between the vascular bundles of the old stems and those of the new shoots, and thus prevents a free circulation of the nutriments into the growing shoots. Foratime I thought that this putrefaction was the Sometimes the disease appears on plants which have never been cut, or on young plants which has not yet attained the age of cutting ; but these cases are very rare and may have some special cause, either attack of fungi or insects, etc., or some physiological abnormality. Ihave analyzed a young plant which has never been cut, but some leaves of which showed signs of the disease, and obtained the following results :— Leaves. Stems. Healthy. _— Diseased, Healthy. Diseased. Total nitropen. sci. s..c.ec-dets 3.42 3.04 2.15 200 Albuminoid nitrogen ......... 2.94 2.37 1.50 1.53 Non-albuminoid nitrogen ... 0.48 0.67 0.65 0.47 ASI fereccnentenncerec eens eres 10.7 8.6 6.0 6.1 Leaves. Stems. Healthy. Diseased. Healthy. Diseased. Total nitrogens.s...cssssseeenes 100.0 88.8 100.0 93.2 Albuminoid nitrogen ........ 5 80.6 3 102.2 Non-albuminoid nitrogen... ,, 140.4 72.3 ASh: iccitisicin eee 5 80.4 s 101.0 Thus we see that the diseased leaves and stems are considerably poorer in nitrogen. So we may conclude that the disease appears when the nitrogen supply is reduced beyond a certain limit, whatever the efficient cause may be. MU!.BERRY-DWARF TROUBLES IN JAPAN. 223 cause of the disease. But as my observation became more ex- tensive, I found that in many diseased plants there was no putre- faction, and also that it was not generally observed in the first stage of the disease. Soitis very probable that this putrefac- tion is only a secondary phenomenon accopanying the disease and not its primary cause. Nevertheless the putrefaction unavoida- bly accelerates the disease and hastens the death of the whole plant. Further, the sap flowing out from the cut stems may be a good nourishment for small orgamsms. As the sap is very abundant in Takasuke, Tsuruta etc, it is natural that these varieties are especially prone to putrefaction. Again many believe that the principal cause of the disease lies in the decay of the roots. It is true that the roots of the diseased plants are generally in a very bad condition, the small roots having almost entirely decayed; but at the beginning of the disease, the roots are generally quite normal. Moreover, if the decay of the roots be the cause of the disease we could not understand why those plants which are not subjected to cutting are entirely free from the disease. It is very interesting to see whether other plants are also liable to the same disease when subjected to repeated cutting or when frequently robbed of their leaves in the growing season, Japanese Salix is cultivated in Tanba and Tango just in the same manner as the mulberry, but the stems are cut in late autumn when the leaves have fallen down. This is avery raticnal mode of precedure and entirely in harmony with the teachings of plant physiology, and we have never observed the disease in this plants. Japanese tea plant is generally subjected to frequent picking of the leaves, sometimes even 3 or 4 times in a year, and we observe very often that in sucha case the leaves be- come smaller and can not develop well, and that sometimes even the whole plant dies ; but the disease is soon healed by good manuring. 224 U. SUZUKI. Summary and Conclusions. 1). The diseased leaves are remarkably poor in nitrogen and the development of fibres is considerably retarded ; no cther peculiarity is regularly found. The shrinking of the leaves and the retardation of growth may be due to the deficiency of nitrogen and the bad development of fibres. The deficiency of nitrogen in the diseased plants is not caused by an insufficient supply of nitrogen manures in the soil, but must be due to a diminution of the absorptive power of the roots, and also of chemical activity in the plant cells; since the disease is always observed in those places where soluble manure is given in excess and over-growth is induced. Further, the diseased plants can never recover on simple application of nitrogen manures. The diminution of chemical activty in the living cells, owing to the deficiency of nitrogen, may also be the cause of the bad development of fibres, since the formation of cellulose from solu- ble carbohydrates is performed by the living protoplasm, 2). During winter a considerable amount of reserve mate- rials (especially nitrogen compounds and starch) is stored up in the bark of stems and roots, and in spring, when the develop- ment of new leaves commences, the greater part is transported to the growing parts, and the assimilation products in the leaves again come down in late autumn, when the leaves begin to fall. Therefore the stems and roots are considerably poorer in reserve materials during the growing season. Now it will be at once evident that the cutting of the plants in the growing season must have a very bad effect on the new developing shoots, since they must be nourished with scanty reserve materials, and moreover, it isnot impossible that the reserve materials may be entirely ex- hausted before the new shoots have attained a certain height and become able to assimilate independently enough nutriments from outside. In such a case the shoots will not grow normally and the disease must be the result. Many facts support this view :— a). The first appearance of the disease is always in the new shoots, after cutting in the growing season. MULBERRY-DWARF TROUBLES IN JAPAN. 225 b). The disease is not observed where the plants are not cut, and even plants already diseased may recover when kept from cutting for some years. c) Many experiments show that cutting in the growing season is followed by the disease, while it is not observed when the plants are left without cutting. d). The disease is not induced when the stems are cut during winter or in early spring before the leaves are un- folded. 3. Not only by cutting in the growing season, but also by frequent picking of the leaves, the reserve materials in the roots and stems, may be exhausted, and the new leaves developed subsequently become diseased, as proved by many experi- ments. 4. Some varieties, such as Takasuke, store up a large quantity of reserve materials, while others, like Jamonji, contain comparatively little. The former need a far greater amount of reserve materials during the devlopment of the leaves, while the latter require only a comparatively small amount. This indicates that Takasuke has a weaker absorptive power for soil nutriments in the first stages of development. So it must be very difficult for it to counterbalance the deficiency of reserve materials with the nutriments absorbed from the soil. The con- sequence is that Takasuke is more liable to the disease than Jumonji. But such a propensity is not absolutely confined to certain varieties ; on the contrary, it must have a wide range of variation even among the same variety and is subject to modicification by various conditions. Generally speaking, a plant becomes more liable to the disease by accelerating its growth with abundant soluble manures, and by some other treatment. Therefore no variety would be absolutely free from the disease ifthey were cut in the growing season, or if the leaves were picked frequently. 5. Young plants become very rarely diseased. This may be due to energetic development of the roots and a large capaci- ty for the absorption of nutriments in the young plants; while old plants have less power of developing new roots, and consequ- 226 MULBERRY-DWARF TROUBLES IN JAPAN. ently the deficiency of reserve materials can not be made good by the nutriments absorbed from outside. For the same reason, cutting in late autumn produces more cases of the disease than cutting in early summer. Since the activity of plant cells is already diminished in late autumn the development of new roots must be more difficult. 6. Micro-organisms are not the cause, because they are not always present in the diseased plants. Further, the decay of the roots of the diseased plants may be a secondary phenomenon, since we can not understand why the disease is not observed when the plants are left without cutting, or why the diseased plants may even recover by being kept from cutting, for some years. Shoots of the diseased plants may develop normally when they are grafted on healthy roots or stems, or when they are covered with earth and propagated by cutting. But there is ne doubt that the mirco-organisms have some accelerating in- fluence upon the disease and at last cause the death of the plants. 7. As regards the methods for the prevention and cure of the disease, we must leave them to future investigations, since we have not succeeded in finding any that promises success. 1. Comparison of healthy and diseased plants. “suruta (1) Ysurata (II May 6. ) ilosoye (I) Oct. 15. May 6. Hosoye (II) Yotsume Takasuke (I) Oct. 15. May 22. Takasuke (II) May 22. Takasuke (III) Jumoyji (1) Jumonji (II) July 9. Sept. 9. July 9. Sept. 9. Average. Ratio. Healthy.|Diseased.| Healthy.|/Diseased,} Healthy .|Diseased.| Healthy.|Diseased.| Healthy.|Diseased.| Healthy.|Diseased.| Healthy.|Diseased.] Healthy.|Diseased.] Healthy |Diseased,| Healthy.|Disesaed.| Healthy.|Diseased.| Healthy.|Diseased | y y y. y y y y y: y y Average length of shoots 96.1 45.0 — - 195.0 82.5 39.0 33.0 66.0 | 45.0 90.00 | 45.00] 30.0 18.0 180.0 90.0 16.50 12.00 | 150.0 75.0 95:80] 49:5 | 100.0) 51.7 Fresh weight of 1 shoot 59.7 15.1 — — 205.0 18.85 2.22 0.904) 29.3 6.00 50.0 78.70 5-45 1.04 | IoI.o 36.3 2.50 1.02 58.5 12.00 | 57.07 1110 | 100.0} 19.4 Dry wei ht of 1 shoot - _ — _ = _ 0,28 0.135) — — — _— 0.87 0.24 29.56 11.06 0.30 O.1I5| 17-19 3:33 9.64 2.98 | 100.0} 31.0 Misture in shoots — —_ —_ — — — | 87.35 85.05 | — — — — 84.04 | 78.00] 70.66] 69.49] 88.04] 8860] 7060] 72.27) 8014] 7868) 100.0} 98.2 Dry matter in shoots - — — — _ — 12.65 14.95 |. — — — _— 15.96 | 22.00| 29.34 | 30.51 11.96] 11.40] 29.40] 72.73 19.86 | 21.32 | 100.0| 107.3 Number of leaves on 1 shoot | 57.0 31.0 — _ 224.0 5.0 10.0 1.30 140.0 | 40.0 136.0 45.0 18.00 19.co | 36.0 26.6 7.00 11.00] 45.00] 37.00] 75.00] 57.00] 100.0) 76,0 Dry matter in leaves of 1 shoot 2.36 0.42 _ _ 5:37 0.52 0.695 0.422| 7.15 0.77: 15.09 2.02 1,622 0.647} 31.02 20.34 0.82 0.28 18.42 4.75 9.17 335 | 100.0} 36.6 Fresh weight of 1 leaf 0.229 0.065 3.22 0.36 O.1IL 0.049] 0.304 0,126) 0.181 0.072 0.376) 0.141 0.39 O11 2.57 0.16 | 0.982 0.249 1.18 0.35 | 0.954 0.168} 100.0) 17.6 Dry weight of 1 leaf 0.041 0.014) 0,964) 0.135) 0,024) 0.010] 0.066 0.033) 0.0513] 0.C195 O.1II 0.045 0.10 0.034 0.87 0.076) | 0.113 0.024) 0.41 0.127 2.275, 0,052) 100.0} 18.8 Moisture in leaves 81.20] 78.14 | 7o.00| 62.60] 79.84} 78.81 | 78.90 73:64 | 71.63 | 72.60 jo.co| 68.00] 76.70 | 69.15 66.23 | 52.70 | | 88.50) 90.0 65.24] 63.12 | 74.87] 70.88 | 100.0] 94.7 100.0 | 116.0 Dry maiter in leaves 18.80 | 21.86 30.00.| 37.40 20,16 21.19 | 21.10 26.36 | 28.37 27.40 29.50] 32.00] 23.30 | 60.85 33-77 47-30 11.50 10.0 4) a } » % ’ . oa were? hy ane * te ai in by ab A . Lae? IL. Comparison of healthy and diseased plants (Leaves). | Tsuruta (I) Tsuruta (IT) Hosoye (1) Hosoye (IT) Yotsume May 6. Oct. 15. Takasuke (I) Takasuke (11) Takasuke (III) | Jamonji (1) Jamonji (II) May 6. Oct. 15. May 22. May 22. July 9. Sept. 9. July 9. Sept. 9. Average. Ratio. | Healthy .|Diseased.| Healthy.|Diseased. Healthy.|Diseased.] Healthy.|Diseased.) Healthy .|Diseased.| Mealthy.|/Diseased.| Healthy.|Diseased.| Healthy.|Diseased.| Healthy .|Diseased.| Healthy.|Diseased | Healthy |Diseased, Healthy [Diseased| 78.81 78.90 73.64 71.63 70.50 68.00 76.70 69.15 66.23 52.70 88.50 90.00 65.24 63.12 74.87 | 70.88 | 100.0] 94.7 Moisture | 81.20 78.14 70.00 62.60 Dry matter 18.80 | 21.86] 70.00] 27.40] 20.16 | 21.19] 21.10] 25.36] 28.37 | 27.40 | 29.50] 32.00] 23.30] 30.85] 33.77] 47.30] 11.50] 10.00] 34.76) 36.88| 25.13) 29.12 | 100.0] 116.0 Crude proteids | 47-94 34.56 26.88 23-13 41.00 38.00 33.00 | 23,13) 28:75 | 27.94 23.75 18.44 | 30.50 23.13 22.88 20.25 36.63 32.5¢ 22.38 16.50 | 31.47 25.76 | 100.0 81.8 Crude fats | 1.92 3.10 5.80 4.40 2.26 | 1.51 | 5-19 | 4.96 3.83 | 3.45 3.46 | 3:34 | 284 3.05 7-34 4.34 4.26 3.08, 7.29 6.72 4,42 3.80 | 100.0) 86.0 Crude fibres | ( 8.74 6.70 ) { 1053 7.26 | | { 9.61 8.60 11.14 8.03 9:59 WAT 11.04 9.56 9.32 9.73 10,00 8.14 | 100.0} 81.4 + 41.27 54.20 49.18 52.33 | 4 59.66 60.07 | - Nitrogen-free extracts | 51.23 57-71 | 30:52 56.83 \ l 56.84 63 40 47:26 | 58.90} 52.42 60.88 | 37.67 45.04, 50.88 | 60.50} 47.97 57-60 | 100.0 | 120.0 | Ash 7.87 8.14 7.35 8.06 7.56 8.16 11.76 7:82 7-76 8.54 6.34 6.13, 8.26 6.89 777 7.42 10.40 9.82 10.13 6.55 8.52 7-75 | 100.0) 91.0 Total nitrogen 7.67 5.53 4.30 3.70 6.56 6.08 5.28 3.70 4.60 4.47 3.80 2.95 4.88 370 3.66 3.24 5.86 5.20 3.58 2.64 5.02 4.12 | 100.0| 81,3 Albuminoid nitrogen 5.15 4.07 3.60 3.c0 4.08 3-94 3.80 2.70 4:34 AAS 3.05 2.79 | 3.85 3:35 3.37 2.98 4.18 4.10 3.10 2.33, 3.85 3-34 | 100.0) 86.6 Non-albuminoid nitrogen | Tsuruta (I) ‘Tsuruta (IT) May 6. Oct. 15. Hosoye (I) Hosoye (11 May 6. ) Oct. 15. Yotsume May 22. Takasuke (1) May 22. Takasuke (11) July 9. Comparison of healthy and diseased plants (Leaves). Takasuke (111) Sept. 9. umonji J (I) July 9. Jamonji (II) Sept. 9. Average. Ratio. Healthy .|Diseased.] Healthy.| Diseased Healthy.) Diseased.} Healthy |D/seased,] Healthy.|Diseased | Healthy.|Diseased. Healthy .| Diseased. Healthy.|Diseased. Healthy. Diseased.| | | | Healthy.|Diseased.| Healthy |Diseased. Healthy. Diseased, Moisture Dry matter | | | 81.20 78.14 70.00 62.60 18.80 21.86 70.00 27.40 69.15 66.23 | 52.70 33-77 47.39 88.50 11.50 90.00 10.00 | 65.24 34-76 74:87 70.88 29.12 100.0| 94.7 100.0 | 116.0 matter, Crude proteids Crude fats Crude fibres Nitrogen-iree extracts Ash Total nitrogen Albuminoid nitrogen Non-albuminoid nitrogen 100.0 100.0; 81.8 100.0, 86.0 100.0) 81.4 100.0 | 120,0 100.0) 91.0 100.0| 81.3 100.0} 86.6 66.6 SiO, SO, Rar. K,0 CaO MgO te ang + F i © fy -- ® > a 5 as « te (3 " oo a ; » oe _ “3 (a ee he eemenate ns fan alas eae ya 4 Hil. Takasuke (I) Oct. 15. Comprison of healthy and diseased plonts (Stems.) Takasuke (11) July 9. Takasuke (III). Sept. 9. Jamonji (1) July 9. Jamonji (II). Sept. °g. Average. Ratio. Healthy. | Diseased. Healthy. Diseased. Healthy. Diseased. Moisture Dry matter 84.04 78.00 15.96 22.00 70.66 69.49 29.34 30.51 parts of Healthy. Diseased. Healthy. | Diseased Healthy. Diseased. Healthy. Diseased. 88.04 88.60 11.96 11.40 70.60 72.27 29.40 27.73 80.14 19.86 78.68 21.32 dry matter, Crude proteids Crude fats Crude fibres Nitrogen-free extracts Crude ash Total nitrogen Albuminoid nitrogen Non-albuminoid nitrogen In 100 parts of pure ash, lV. Comprison of the chemical composition of different varieties (Healthy plants.) Leaves (May 6). Leaves (May 22.) Leaves (July 9.) | f j Stems (July 9.) Tsurata Hosoye Yotsume | Takasuke Average length of shoots Fresh weight of 1 shoot Dry weight of 1 shoot Fresh weight of 1 leaf Dry weight of 1 leaf Moisture Dry matter 0.181 0.051 71.63 28.37 0.376 0.121 70.50 29.50 Takasuke Jumonji Takasuke Jamonji 30.00 16.5 5-45 0.87 2.50 0.30 Leayes (Sept. 9.) ee Stems (Sept. 9). Takasuke | Jamonji Takasuke Jamoniji. 180.0 101.0 29.56 150.0 58.5 17.19 Crude proteids Crude fats Crude fibres Nitrogen-free extracts Ash Total nitrogen Albuminoid nitrogen Non-albuminoid nitrogen In 100 parts 28.75 3.83 59.66 7-76 4.60 4.34 0.26 23-75 3.46 9.61 56.84 6.34 3.80 4.88 3:85 1.03 3:05 0.75 In 100 parts of pure ash, 2.64 4.68 3.64 2.60 17.90 28.51 14,60 21.15 23.64 27.21 10.44 10.13 Showing the variation of Chemical composition according to seasons. Tsurata Hosoye Takasuke Jamonji May 6. Oct. 15. May 6, Oct. 15. | May 22. July 9. Sept. 9. July 9. Sept. 9. July 9. | Sept. 9. July % Sept. 9. Before After —_—_ | Before lAfter cutting Before After After After After After After {After After cutting cutting cutting] at autumn cutting| cutting catttng cutting cutting cutting cutting! cutting cuttir Leaves Leaves Leaves Leaves Leaves Leaves Leaves Stems Stems Leaves Leaves Stems Stems : Average length of shoots 30. 180. 16.5 150. Fresh weight of 1 shoot 5.45 Iol. 2.50 58-5 Dry weight 1 shoot 0.87 29.50 0.30 17.19 Fresh weight of 1 leaf 0.229 3.22 O.1II 0.304 0.39 2.57 0.982 1.18 | Dry weight of 1 leaf 0.041 0,964 0.024 0.060 0.10 0.87 0.113 0.41 Moisture 81.20 70.cO 79.84 78.90 76.70 66.23 84.04 70.66 88.50 65.24 88.04 70-60 } Dry matter 18.80 30.00 20.16 21.10 23.30 33-77 15.96 29.34 11.50 34.76 11.96 29.40 1 In 100 parts of dry matter, Crude proteids 48.94 26,88 41.00 33.00 23.75 30.50 22.88 15.38 5.38 36.63 22.38 21.75 5.31 Crude fats 1.92 5.80 2.26 5:19 3.46 2.84 7:34 0.83 1.10 4.26 7.29 2.20 1.00 Crude fibres 8.74 10.53 9.61 11.14 9.59 46.30 54.60 11.04 9.32 36.72 59.00 } 41.27 } 49.18 Nitrogen-free extracts 51.23 39.52 56.84 47.26 52.42 31.54 34.58 37.67 50.88 31.85 30.36 Ash 7.87 7.35 7:56 11.76 6.34 8.26 1-77 5.95 4.34 10.40 10.13 7.48 4.83 Total nitrogen 7.67 4.30 6.56 5.28 3.80 4.88 3.66 2.46 0.86 5.86 3-58 3-48 0.85 Albuminoid nitrogen 5.15 3.60 4.08 3.80 3:05 3.85 3.37 1.10 0.54 4.18 3-10 1.47 057 Non-albuminoid nitrogen 2.52 0.70 2.48 1.48 O75 1.03 0.29 1.36 0.32 1.68 0.48 2.01 0.28 apna fadinrsd Lie sollgiiay anf paivorne ee ~~ 7 te : ! rit fy { Maeot pote oe a ' at a i of = eee Oe oN or pene al A + 25- ett ( E f 7 28 te! at Le eee ae dv 2 ae ae = nae + i ia tags a = ges. ner Mee eee Me er oS eie wits PP ffetz Ue = . oe srg ine) a 7 { : “oe sprees * Li i i :: ‘ i x ‘ . ! 1 | | : Ree: ow es r ) $ ti 2 ' ¥ Te bie yee “43 H Ley . ‘ef Gu ¥ i t 1 ‘ wie Ne Z » Soom . ce ry 1 2 ; a : s% s s weg. i % he bh, : } o : Pe ae | saa } i ry . ae 1 ae C a ims. 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Jo Jayeut Lap oy} ul adviuao1ag) ‘aveA Buo Ut SayvApAYOGIVI Jo UOBVIARA oy) SuULMOYS WAXX Id “AL 19A 1199 ‘948Y “1/09 "YdDG 100Y *0ynsBYD L ‘BUND JO SUIT], OF 7 T = ‘i _ Sega ioe ate lame . 7 wv 7 e ; my bie ait ~ s32% a 2) a ~~ ¢ — ° baie FS Siro 5 = e 4 > Caen "THAXX ‘Id ‘AL TOA ‘1199 9143 “11 *BUIQ{NS JO DWIT, “yuuo0Ul et ML OF oO OL 0g OF OF -avak auo ut sayeipAyoqsvs Jo uoreiszea dy) SuLMoYyS *YAdg J00N “VAIMANS T of a Na) a j ae See lem £ a j *BuIyyNd Jo awry, *yjuour et UL OF 6 g L Ss “ ¢ N 7 “azah ouo UT sayeapAyoqavs jo UOUALA oy} SuLMOYg *y.ivg 7o0y ‘iluowmns ies “XIXX Id ‘ALTON ‘1/99 ‘91487 “])"g ee Se 8 at vs +" *“ b t i] ? Aare, “BUI}IND JO JUITT, Wuownf saul, MCA A “yarq oon} Ryans pseu, Pay aynseyop saul, yoVL, (x4eq oor Jo Jay; Aup ay} ur advyUedIEg) % savaf ouo ul saytapAyoqAvs jo uoyeItea oy) Jo Aysuajut sania. 94} Burmoys ies ° "YXX ‘Id ‘AL ‘J°A ‘1/99 ‘9148y ‘]/Ng a wa >. ; J _ & ‘sell pote” >. , : a Oe ae PA ~) cu eee Bull. Agric. Coll. Vol. 1V. Pl. XXX1. A healthy plant (low cutting). : ene - a) ’ vo an 7 — { hs : ia : : rv a”), ery 4 i ‘ ~ | | 1 : apie : - La ni foe uty | ; Pt : 7 ) Bull. Agric. Coll. Vol. 1V. Pl. XXX1/. 4 t } | ; } ‘ } f i tee Se 4 A healthy and a diseased plant compared. Bull, Agric. Coll. Vol. IV. Pl. XXXII Diseased plants in winter. ‘ ‘ p a . , i som ny 7 $ , r , . i ‘ y ve f ‘ t ‘7 . P 1 : ‘ ‘A 5 A 4 i i es ‘ a ee ‘ ‘ % ¢ : ; i > i { | i 4 \ i t hie 1 ’ ‘ Wy > H 1 Coll. Vol. IV. Pl, XXXIV. fo Bull, Agric. Normal growth without cutting, Bull. Agric. Coll. Vol. IV. Pl. XXXV. 3 A portion of stems diseased by frequent plucking off of the leaves, ne AV ae PAYS re oh 7 5 Ate ie : Bul. Agric. Coll. Vol. lV. Pl. XXXVI. Plants diseased by plucking off of all the leaves. Bull. Agric. Coll. Vol. IV. Pl. XXXVIT. 7 Two stems of a diseased plant recovered by layering, Bull. Agric. Coll. Vol. 1V. Pl. XXXVIIT- Diseased plants recovered by layering. Bull. Agric. Coll. Vol. 1V. Pl. XXXIX. Nes y, giant enee—te Diseased plants. “Suyyns Aq pasvasip syur[q atm ‘s Ps » Ve 7 a ie ‘ Bull. Agric. Coll. Vol. 1V. Pl. XLI. lant compared, A healthy and a diseased | 4. Hl hi! hi! Wr alk K & Ui ola aa PRE este Ke | bot eS ZR RK ae 1 HP FRE tree NRA | hot ee Zur Physiologie des Bacillus pyocyaneus, VON O. Loew und Y. Kozai. Da der Bacillus pyocyaneus ein betrachtliches Interesse darbietet, haben wir eine Anzahl von Versuchen unternommen, welche itiber Abhangigkeit der Farbstoff- und besonders der Enzymproduction von den jeweiligen Nahrsubstanzen weitere Anhaltspunkte liefern sollten. Ueber seine Entwicklung in verschiedenen Nahrlésungen liegen zwar bereits Mittheilungen vor, besonders von /, Hippe und von Thum,} doch hatten diese nicht speciell die Enzymbildung, sondern hauptsachlich die Farbstoffbildung verfolgt. Ausser dem blauen Pyocyanin (C\, Hy N; O, nach Ledderhose) wurden bekanntlich noch zwei weitere Farbstoffe in Culturen des Pyocyaneus beobachtet, das gelbbraune Pyoxanthin (Fordas) und ein weiterer, dessen am- moniakalische Losung eine griine Fluorescenz zeigt und in durchfallendem Lichte gelb erscheint (Azz). Doch scheinen gelegentlich auch davon verschiedene Farbstoffe aufzutreten, wie durch Babes wahrscheinlich geworden ist. Jedenfalls hat die Varietat des Pyocyaneus einen betrachtlichen Einfluss in dieser Richtung.? Erhohtes Interesse kann aber die Production eines Enzyms beanspruchen, welches in gentigender Concentration nicht nur den Bacillus selbst wieder auflést, sondern in verschiedenem Energiegrade auch Cholera-, Typhus-, Anthrax-, und Diphtherie- 1 Arbeiten aus dem bacteriologischen Institut der technischen Hochschule in Karlsruhe, Bd. I, p. 337 (1897). Zum verwendete Asparagin, Ammonsalze organischer Siuren, sowie Gemische von Harnstoff mit Glucose und mit Mannit. 2 Es ist von wesentlichem Interesse, dass auch die Anwesenheit von Magnesium als Sulfat notwendig ist, die Farbstoffe zu bilden (Voesshe), was mit analogen Beobachtungen am Bac, fluorescens (7/2) und am Bae, prodigiosus (Kwztze) iibereinstimmt. 228 O. LOEW UND Y. KOZATI. bacillen.! Reaction und Concentration des Nahrmediums, sowie die chemische Structur der organischen Nahrsubstanzen tiben einen betrachtlichen Einfluss in dieser Richtung aus. Neutrale oder nur sehr schwach alkalische Reaction sind am giinstigsten. Ferner ist der Luftzutritt in Betracht zu ziehen. Krause? beo- bachtete tippige Entwickelung in einer Wasserstoffatmosphiare, aber keine Farbstoffbildung. Diese erfolgt erst, wenn man Luft zutreten lasst. In einer Kohlensaureatmosphare gedeitht nach ihm dieser Bacillus nicht, ja soll er nach 24 Stunden absterben. Dass auch die Toxinbildung bei verschiedenen Baeterienarten durch manche Umstinde bedeutend beeinflusst wird, ist bekannt. Dasselbe gilt aber auch ftir die Bildung von ,, Schleim,‘‘ welche beim Bac..pyocyaneus auftreten oder ausbleiben kann. Die Schleimbildung ist bekanntlich nach Magelz Folge einer weit getriebenen Aufschwellung der Zellmembranen. Man kann also folgern, dass bei ausbleibender Schleimbildung weit weniger Membransubstanz gebildet wird, oder dass in der betreffenden Nahrlésung Enzyme gebildet werden, welche die Membranen véllig wieder auflésen. MWarfiann? schreibt in Bezug auf die Membranbildung (Centr. Blatt. f. Bakt. I] Abt. 6, 674) :,, Es steht somit fest, dass die Substanz der Hiille, resp. Kapsel, nicht bei allen Spaltpilzen aus denselben Stoffen gebildet ist und dass die Stoffe nach Art der chemischen Zusammensetzung des N&ahrbo- dens sehr variiren und endlich, dass diese Hiillen bei besonderen Verhaltnissen von Temperatur und Nahrboden iiberhaupt nicht gebildet werden.“ Was die Enzymbildung betrifft, sohat Auerbach? mitgetheilt, dass bei Bacterium vulgare die Bildung des Gelatine verfliis- sigenden Enzyms durch Gegenwart von Zucker verhindert wird. Aehnliches haben wir beim B. pyocyaneus beobachtet betreffs 1 Emmerich und Loew, Zeitschzift fir Hygiene Bd. 31 (1899). Von der erhaltenen Pyocyanase-Lésung konnte 1 cc. in 2 Stunden iiber 4 Millionen Milzbrandbacillen, in 6 Stunden 27 Millionen Pestbacillen, in 24 Stunden 23 Millionen Staphylococcen, in 3 Tagen 29 Millionen Typhusbacillen und in 4 Tagen 20 Millionen Diphtheriebacillen auflésen. In neuester Zeit wurde ein noch weit starkeres Enzym erhalten. 2 Centralbl. fiir Bakt. Bd. 27, p. 769 (1900). 3 Archiv f. Hygiene 1897, 4 Auch Anaérobiose scheint manchmal Einfluss auf die Enzymlbidung zu aussern, So verliert nach San Felice der B, pyocyaneus bei anaérober Kultur seine Fahigkeit zu peptonisiren, ZUR PHYSIOLOGIE DES BACILLUS PYOCYANEUS, 229 der Bildung des bacteriolytischen Enzyms. Die Bildung lasst sich leicht schon daran erkennen, dass nach anfanglich reichlicher Entwickelung bald ein Zeitpunkt eintritt, wo nicht nur die weitere Entwickelung sistirt wird, sondern die vorhandenen Bacterienmassen Agglutination zeigen, alle Bacterien absterben und bis auf minimale Granula und Krystéllchen das ganze Sediment gelést wird. In manchen Lésungen tritt dieser Zeitpunkt der Wiederauf- loesung bald, in andern sehr spat, in manchen vielleicht gar nicht ein. Wahrend in bloser Peptonlésung mit mineralischen Nahrsalzen dieses Enzym reichlich gehildet wird, entsteht weit weniger, wenn ausserdem (1-29) Glycerin zugezetzt wird, ja in einer Mischung von 1% Pepton mit 2% Rohrzucker blieb das Bakteriensediment selbst nach neun Wochen noch ungelést. In unserer ersten Versuchsreihe wandten wir auf eine relativ grosse Menge stickstofffreien Materials absichtlich nur sehr geringe Mengen stickstoffhaltigen Materials (mit Ausnahme von No, VI.) an. Nach der whblichen dreimaligen Behandlung im Dampftopf wurden die Lésungen mit einer pathogenen Art des B. pyocyaneus, der in Berlin aus Eiter geziichtet worden war, inficirt. Jede Lésung, 1co cc, befand sich in einem ca. 250 cc. fassenden, einen Watteverschluss tragenden Frlenmeyer Kolben. Die anorganischen Nahrsalze bestanden, wo nicht speciell anders bemerkt wird, tiberall aus: 0,29 neutralem Kaliumphosphat, 0,29¢ Kochsalz, 0,19 Natriumsulfat und 0,01 9% Magnesiumsulfat. Letzteres Salz wurde in einer zweiprocentizen Losung separat sterilisirt und bei der Infection zugesetzt, um die Ausfallung des meisten Magnesiums als Phosphat beim Sterilisiren zu vermeiden. Die organischen Nihrsubstanzen bes- tanden aus : No. I. Glycerin 19 ; asparaginsaures Natron 0.1%. No, II. Glycerin 19 ; Pepton 0.1%. No. III. Glucose 19% ; Pepton 0.1%. No. IV. Weinsaures Kali-Natron 1% ; Pepton 0.19%. No. V. Essigsaures Natron 1% ; Pepton 0.1%. No. VI. Glycerin 0.1% ; Pepton 19%. Die im Briitschrank gehaltenen Proben zeigten folgende Erscheinungen : 1 Das verwendete Pepton war nicht das an Albumosen reiche von Witte, sondern war albumosenfreies, von Gehe & Co, bezogenes. 230 ‘O. LOEW UND Y. KOZAI, No. I. Nach 3 Tagen sehr geringe Entwicklung, die auch nach acht weiteren Tagen nur sehr miassig blieb, Farbung schwach gelbgriinlich. No. II. Nach 3 Tagen missige Triibung und _ blaugriine Farbung, welche bei weitererm Aufenthalt im Brut- schrank verschwand, um beim massigen Schwenken des Gefasses immer wieder zu erscheinen. Reaction allmalig spurenweise sauer ; offenbar erméglichte das Glycerin anaérobes Leben, wobei der Farbstoff zu einer Leukoverbindung reducirt wurde, die unter Sauerstoffaufnahme wieder in den Farbstoff iiberging. Eine Wiederaufldsung der gebildeten Bacterien- massen war nach 2 Wochen nicht zu bemerken.! No. III. Hier trat gar keine blaugriine Farbung, sondern nur weisse Triibung auf. Selbst nach 10 Tagen war die Vegetation nur gering und trat selbst beim Um- schwenken des Glases keine Farbung auf; es war also auch keine Leukoverbindung vorhanden. Offenbar kann der Bacillus Glucose nur gut verwerthen, wenn eine grdssere Menge stickstoffhaltigen Materials vorhanden ist. Die Reaction war kaum merklich sauer. No. IV. Hier waren die Erscheinungen wesentlich dieselben wie in III, nur war die Vegetation noch geringer. Auch Zusatz geringer Mengen kohlensauren Natrons brachte in III und IV keine wesentliche Aenderung zustande. Weinsdaure kann daher kaum von diesem Bacillus verwerthet werden, so lange die gleichzeitig gebotene Menge stickstoffhaltiger Nahrsubstanz sehr gering ist. Ueber ihre Verwendbarkeit zur Farbstoff- production gehen die Meinungen noch auseinander ; Thum folgerte, dass die Weinsdure hierzu nicht geeignet sei, wahrend AHzppe die entgegengesetzte Beobachtung machte. Wahrscheinlich verhalten sich verschiedene Varietaten des B. pyocyaneus in dieser Hinsicht nicht gleich. 1 Auch nach Zusatz von geringen Mengen kohlensauren Kalis nicht ; doch trat dann allmalich Agglutination ein. , ZUR PHYSIOLOGIE DES BACILLUS PYOCYANEUS. 231 No. V. Hier war nach fiinf Tagen eine sch6n dunkelgriine Farbung eingetreten, welche sich auch nach tage- langem Stehen nicht verminderte. Hier war also das Nahrmedium der Bildung eine Leukoverbindung nicht gtinstig ; die Eutfarbung fand nicht statt, weil hier keine anaérobe Existenz in den unteren Schich- ten der Fliissigkeit ermdglicht war, wie in No. II. Jeden Tag war eine diinne Haut an der Oberflache gebildet. welche beim Umschiitteln zu Boden fiel. Die Vegetation horte nach 10 Tagen auf, die Losung wurde schleimig, die Bacterienmassen agglutinirten, doch schritt die Wiederauflésung nur sehr langsam fort. Die Schleimbildung erreichte jedoch nicht den starken Grad, wie er in Bouillonculturen beobachtet wird. No. VI. Hier war die Vegetation am lebhaftesten und Farbe und Geruch des Pyocyaneusculturen am_ besten entwickelt. Nach 10 Tagen trat Agglutination und beginnende Wiederaufldsung ein. Reaction schwach alkalisch, doch waren nur Spuren Ammoniak gebildet. Nach 15 Tagen liess eine in Bouillon abgeimpfte Probe erkennen, dass die Bacterien bereits wieder abgestorben waren, es trat keine Spur von Vegeta- tion in dieser Probe auf. Schleimbildung war relativ gering, geringer als bei No. V. Als wesentliche Resultate ergeben sich also : 1. Eine starke Vermehrung stickstoffreier Nahrstoffe, resp. Verminderung stickstoffhaltiger, ist der Enzymbil- dung oder auch der Vegetation im Allgemeinen nicht gunstig. 2. Glycerin ist unter dieser Bedingung ein besseres Nahrmaterial als Glucose, aber ein schlechteres als essigsaures Natron. 3. Die Schleimbildung wurde durch essigsaures Natron mehr befordert als durch die anderen Nahrsubstan- zen, jedoch noch nicht in dem Grade wie durch Bouillon. 4. Am ginstigsten fiir Farbstoff- und Enzymproduction erwies sich die 19% Peptonlésung, mit nur 0.19% Glycerin, auch der specifische Geruch war am besten entwickelt, die Schleimbildung war gering. 232 O. LOEW UND Y. KOZAT. In der zweiten Versuchsreihe wurden sowohl verschicdene Stickstoffquellen, als auch einige stickstofffreie Substanzen betreffs ihres Nahrwerthes mit ecinander verglichen. Die orga- nischen Nahrstoffe waren : No. VII. Asparagin 0,5% ; Glycerin 1,09 ; Pepton 0,5%. No. VIII. Asparagin 0,5% ; Glycerin 1,0%. No. IX. Asparagin 0,5% ; Glycerin 0,2%. No. X. Asparagin 0,5% ; Essigsaures Natron 0,6%. No. XI. Asparagin 0,5% ; Methylalcohol 1%.! No. XII. Tyrosin 0,29% ; Glycerin 1,0% ; Essigsaures Natron 0,6%. No. XIII. Leucin 0,2%?; Glycerin 1,0% ; Essigsaures Natron 0,6.%. No. XIV. Harnstoff 1,0% ; Glycerin 1,0% ;.Essigsaures Natron 0,6%. Die Mengen der Na&ahrlésungen waren die gleichen wie bei der ersten Reihe, doch war die Temperatur anfanglich niedriger (18—22° C., statt 30—35° C.) Nach drei Tagen ergab sich Folgendes : No. VII. Viele Flocken aber nur geringe griine Farbung. No. VIII. Schéne Haut und etwas mehr Farbung wie bei VII. No. IX. Massige Vegetation, wenig Farbung. No. X. Mehr Vegetation und Farbung als in IX. No. XI. Nur spurenweise weissliche Triibung. No. XII. Wenig entwickelt, nur gelbliche Farbung. No. XIII. Geringe Entwickelung, aber blaugriine Farbung. No. XIV. Spur Triibung, farblos. | No. XII u. XIII wurden nun 2 Tage bei 36° C, gehalten, aber das anderte an dem Resultate nicht wesentlich, in beiden Fallen blieb die Vegetation sehr gering, und zwar war sie bei No. XII (Tyrosin) noch weit geringer als bei No. XIII, (Leucin). Bei No. XII stellte sich ferner nicht einmal eine griinliche 1 Der Methylalcohol wurde nach dem Sterilisiren zugesetzt. 2 Beim Vergleich der Stickstoffquellen sollten diese allerdings in gleichen Stickstoff- mengen entsprechenden Quantitaten verwendet werden, doch scheitert dieses 6fters an der Schwerléslichkeit gewisser Verbindungen, wie Z. B. beim Tyrosin, andererseits wire selbst bei Befolgang jenes Grundsatzes Gfters schon desshalb eine strenge Beurtheilung ausgeschlossen, weil die Mengen des gleichzeitig mit den Stickstoffquellen eingefiihrten Kohlen- und Wasserstoffs zu grosse Differenzen aufweisen wiirden. Man vergleiche Z. B. Leucin mit Asparagin in dieser Hinsicht. ZUR PHYSIOLOGIE DES BACILLUS PYOCYANEUS. 233 Farbung ein, es war lediglich eine schwache schmutzig gelbe Farbung vorhanden, welche auch beim Umschwenken des Gefasses und vermehrter Luftzufuhr sich nicht im Geringsten veriinderte. Es konnte wohl keinem Zweifel bei Vergleich mit No. X unterliegen, dass Leucin und Tyrosin weit weniger giinstige Stickstoffquellen fir den Bac. pyocyaneus sind als Asparagin, denn die Unterschiede waren so auffallend, dass man jene geringe Vegetation nicht etwa nur der geringeren Menge Stickstoff hatte zuschreiben dirfen. Ferner wurde es evident, dass zur Farbstoffbildung Leucin und Asparagin gecignet sind, aber nicht Tyrosin. Auch weiterhin anderte sich an diesem Verhalten nichts und wenn auch beim Leucin etwas mehr Vegetation sich entwickelt hatte als beim Tyrosin, so war doch sclbst nach 10 weiteren Tagen keine gréssere Ent- wickelung zu beobachten. Die Ueberimpfung aus diesen Losungen aber in sterilisirte Bouillon ergab das Vorhandensein von noch lebenden Bacillen. Zehn Tage nach der Impfung zeigte sich bei den wbrigen Proben Folgendes : No. VII. Haut und starker Bodensatz, Farbung gelblich, beim Umschwenken griin. No. VIII. Vegetation schwacher als bei VII, sonst gleiches Verhalten. No. IX. Weniger Entwickeung als in VII. Farblos, beim Umschwenken sich allmalig griin farbend. No. X. Dickere Haut als in IX., griine Farbung. No. XI. Flockige Massen am Boden. Schwach gelblich- griin, sich nicht andernd bein Schiittcln. No. XIV. Diinne weisse Haut, Fliissigkeit farblos, nicht griin werdend beim Schiitteln. Als nun die Proben XI und XIV bei Brutwarme gehalten wurden, zeigten sie bald eine lebhafte griine Farbung. Bei XIV trat allmalich eine lebhafte Vegetation ein und 30 Tage nach der Impfung war diese Probe ziemlich schleimig geworden, das Bacteriensediment zeigte Agglutination, war aber nicht merklich gelést worden, auch enthielt es noch lebende Bacillen wie die Impfung in Bouillon bewies. Miassige Schleimbildung trat auch in den tibrigen Proben cin, Eine véllige Wiederauf- 234 O. LOEW UND Y. KOZAT. losung der Bacterienmassen war nirgends zu bemerken.! Am- moniak war in massiger Menge tiberall vorhanden, wo Asparagin als Nahrstoff diente ; ziemlich gering war die Wesslersche Reaction in XIV, Harnstoff wurde blosin geringem Grade gespalten. Als wesentliche Resultate ergeben sich aus Reihe Il: Leuetn und Tyrosin sind schlechte Néhrstoffe fiir dte pathogene Vartetat des Bacillus pyocyaneus ; Asparagin ist cin bessercr Nahrstoff. Leucin ermiglicht die Farbstoffbildung wahrend Tyrosin hiefiiry ungeeiget scheint. Glycerin erméglcht die Bildung des Leukofarbstoffs und ist etn besseres Nahrinittel als Methylalcohol. In der dritten Versuchsreihe wurden stickstoffhaltige Stoffe ohne Beimengung stickstofffreier im Bezug auf ihren Nahrwerth fir den B. pyocyaneus verglichen. Die Lésungen enthielten je ein Procent der folgenden Stoffe : No. XV. Pepton. No. XVI. Betain (das salzsaure Salz mit kohlensaurem Natron neutralisirt). No. XVII. Asparagin. No. XVIII. Glycocoll. No. XIX. Hydantoin. No. XX. Kreatin. Nach 2 Tagen bei 36° ergab sich (Jan. 14) Folgendes : Pepton : schmutzig gelbe, starke Triibung, weisse Haut ; beim Umschwenken griinliche Farbe. Betain: Starke Triibung und Haut, Farbung schwach griin- lich, starker griinblau werdend beim Schwenken. Asparagin: Gelbgriine Fluorescenz, beim Umschwenken blaugriine Farbung. Starke Triibung und Haut. Glycocoll : Ausserst schwache weisse Triibung. Hydantoin: Miassige. weisse Triibung, nicht verandert beim Schiitteln. Kreatin: Wie bei Hydantoin. Nach zwei weiteren Tagen war nur bei der Asparagin- Nahrlésung eine wesentliche Aenderung zu bemerken. Hier waren die Bacterienmassen zu einer dicken zahen Masse verei- 1 In obigen Lisungen VII u. IX ergab sich jedoch ein wesentlicher Unterschied ; in VIII bedingte der grissere Glyceringehalt eine geringere Enzymbildung als in IX, wo eine partielle Wiederaufésung stattgefunden hatte und schliesslich nur sehr wenig Bacterien- sediment vorhanden war. ZUR PHYSIOLOGIE DES BACILLUS PYOCYANEUS. 235 nigt und keine neue Haut zu bemerken. Dieses auffallend friihe Aufhéren der Vegetation nach der anfangs so tiberaus lebhaften Entwicklung ist allem Anschein nach nur der schad- lichen Wirkung der entstandenen nicht unbetrachtlichen Menge kohlensauren Ammoniaks zuzuschreiben. Das Schleimigwer- den der Fliissigkeit, sowie der flockigen Bacterienmassen erinnert ganz an das Verhalten desselben Bacillus in Bouillon. Zehn Tage nach der Infection : Pepton: Vegetation nahezu abgelaufen, Bodensatz sehr gering, Farbung schmutzig graugrin. Betain : Neue Haut nach dem Umschiitteln allmalig gebildet, viel flockiges Sediment. Farbung gelblich, nach dem Umschiitteln griin. Glycocoll, Hydantoin und Kreatin: Nur geringfiigige weisse Triibung. Dreizehn Tage nach der Infection war bei Pepton die Vegetation abgelaufen ; zwei Tage spater auch bei Betain, wo nun auch die griinc Farbung nicht wieder in die gelbe tiber- ging. Ammoniakbildung hatte in beiden Fallen stattgefunden. Schleimbildung war in beiden ausgeblieben. Ein auffallender Unterschied bestand jedoch im Betreff des Sedimentes ; bei Pepton nur geringer krystallinischer Bodensatz, bei Betain nicht unbetrachtliche Mengen flockiger Massen, welche indessen nur zu kleinem Theile aus unveranderten Bacterien bestanden und ausser amorphen Zerfallsproducten noch lange diinne Krystall- nadeln enthielten. Auffallend blieb der so geringe N&ahrwerth von Glycocoll, verglichen mit dem von Betain und Asparagin. Fassen wir die wesentlichsten Punkte der drei Versuchsreihen zusammen so ergibt sich: 1. Asparaginist ein besserer Nahrstoff ftir den B. pyocya- neus als Leucin, Tyrosin, Glycocoll, Hydantoin und Kreatin. 2. Essigsaure Salze sind giinstigere Nahrstoffe als wein- saure, 3. Essigsaure Salze und Glycerin begiinstigen die Schleimbildung, jedoch nicht in dem Grade wie Bouillon. 4. Pepton ist der giinstigste Nahrstoff fiir die Bildung des bacteriolytischen Enzyms und fiihrt nicht zu einer wesentlichen Schleimbildung. Nachtragliche 236 O. LOEW UND Y. KOZAT. Versuche ergaben, dass ein ganz geringer Glycerin- zusatz zur Peptonlésung ginstig auf die Ent- wickelung des Pyocyaneus wirkt. Eine Frhéhung der Menge der schwefelsauren Magnesia auf 19 brachte keinen Vortheil. SS IS Ueber die Bestimmung von Humus in der Ackererde. VON Dr. K. Bieler und K. Aso. Zur Bestimmung von Humus in der Ackererde sind mehrere Methoden vorgeschlagen und im Gebrauch, keine jedoch liefert ein wirklich richtiges Resultat. Je nach dem Grade der vorge- schrittenen Zersetzung besitzt ferner der Humus eine andere Zusammensetzung, Humus entspricht keinem einheitlichen KGrper. Als zuverlassigste Methode gilt allgemein die Elementar- analyse. Die gefundene Menge Kohlensaure wird mit dem Factor 0.471 multiplicirt, wobei angenommen ist, dass der in Betracht kommende Humus 58% Kohlenstoff enthalt. Es ist klar, dass diese Methode oft zu hohe Resultate liefern wird, weil auch andere Kohlenstoffverbindungen vorhanden sein kénnen. Auf der andern Seite ist es eine wohl bekannte Thatsache, dass die noch oft angewandte Knop’sche Methode zu niedrige Resultate giebt. Eine andere Methode, welche vielfach in Gebrauch ist, ist die Extractionsmethode. Obgleich diese Methode modificiert und verbessert wurde, sind trotzdem die gefundenen Humus- zahlen zu hoch. Die Methode basiert auf der Thatsache, dass die Humussubstanzen in verdtinnten Alkalienl6sungen léslich sind. Es gehen hierbei jedoch auch andere Bestandtheile des Bodens mit in Lésung, Neuerdings wird von Aschmanu und Fader! eine volumet- rische Methode zur Humusbestimmung vorgeschlagen, und zwar wird durch diese Methode der Humus als Humussaure bestimmt. 1 Chem-Zeitg. 1899. Nr. 7. S. 61. 238 DR. K. BIELER UND K. ASO. Keine der angefiihrten Methoden ist also unbedingt zuverlassig. Wir haben nach jeder dieser Methoden den Humusgehalt im Boden unserer Versuchstation bestimmt und geben im Weiteren die von uns gemachten Beobachtungen. Die Probenahme des Bodens geschah im September 1898 in der ublichen Weise aus der oberen Culturschicht. Die gezogene Durchschnittsprobe wurde durch Zerreiben zwischen den Handflachen und mit den Fingern geniigend zerkleinert, darauf an der Sonne getrocknet, im Wagezimmer gelassen bis er lufttrocken war und sodann durch das 1 m.m. Sieb gebracht Der abgesiebte Boden wurde in eine Glasflasche aufbewahrt. J. Elementaranalyse. a. 1.8274 grm. des lufttrockenen Bodens ergaben 0,536 erm.COs=20533 0 6. 1.7829 grm. ergaben 0.522 erm. CO, =29.29%. ¢. 3grm. ergaben 0.883 grm CO,=29.43%. d. 2grm. ergaben 0.5975 grm. CO, =29.87%. Im Mittel: 29.48% CO,. CO, in Form von Karbonaten im Boden wurde im Mittel von 2 Bestimmungen! gefunden: 0.104%. 29.48 — 0.104 = 29.376. 29.376 X 0.471=13.84 Humus. I]. Knop’s Methode. a. 5 grm. des lufttrockenen Bodens ergaben : 0.939 erm. CO, =18.78% Humus. 6. 5 grm. des lufttrockenen Bodens ergaben : 0.9394 grm. CO, = 18.79% Im Mittel: 18.785% CO, 18.785 X 0.471 =8.85% Humus. Ill. Extractionsmethode.? 10 erm. des lufttrockenen Bodens wurden nach vorher- segangener Behandlung mit verdiinnter Salzsaure mit 39% tigem Ammoniakwasser extrahiert, und der Extract zu } Liter auf- 1 Mit verdiinnter Salzsiiure. 2 Harry Snyder ; Chem, Centralbl. Nr. 18. Bd. II. 1897. UEBER DIE BESTIMMUNG VON HUMUS IN DER ACKERERDE, 239 gefiillt. 100 ccm. der alkalischen Loésung wurden sodann in einer gewogenen Platinschale verdampft und gewogen nachdem durch Trockenen bei 100° C. Gewichtsconstanz erreicht worden war, Darauf wurde verascht und das Gewicht der Asche ebenfalls festgestellt. Die Humusmenge wurde bestimmt durch Abzug der Aschenmenge von dem Gewicht der Gesammt- trockensubstanz. a. Trockensubstanz 0.218 Asche 0.026 Humus 0.192 0.192 X 5 X 1o=9,60% Humus. 6. Trockensubstanz 0.2260 Asche 0.0265 Humus 0.1995 0.1995 X 5 X 10=9.98% Humus. Im Mittel: 9.79% Humus. iV. Volumetrische® Methode.' 10 c.c. Oxalsaéure entsprechend, 11.3 c.c. KMnO,-Lésung, und 10 ¢.c. Oxalséure +5 c.c. Humussaurelésung entsprechend 20.6 c.c. KMnO,-Lésung. (die erhaltenen Zahlen waren: 20.5, 2.8, 20.4, 20.5, 20.8 ; im Mittel 20.6 c.c.). 20.6—11.3=9.3 c.c. KMnO,-Lésung entsprechened 5 c.c. Humussaurelésung. 930 c.c. KMnO,-Lésung entsprechend 500 c.c. Humussdurelésung enthaltend 0.125 gr. reine Humus- saure. I. c.c. KMnO,-Lésung=0.0001344 grm. Humussaure. 25 gr. des lufttrockenen Bodens in vorgeschriebener Weise behandelt, und die alkaliche Loésung zu einem halben Liter aufgefillt, Da diese Lésung sich als zu concentriert erwies im Vergleich mit der Humussdureldsung, so wurden 10 ¢.c, zu 150 C.c. verdiinnt. 5 c.c. der so verdiinnten Lésung wurden zur Titration verwandt. Die verbrauchten Volumina der KMnO,-Losung Detrugen ; 20. ¢.c., 19.7 Cegzom c.c: Im-Mittel: 19.92 ¢.c. 1 Vergl, Chem-Zeitg. 1899. Nr. 7, S. 61. 240 DR. K. BIELER UND K. aSo. 19.92—11.3=8.62 c.c. 8.62 X 30 X 5[0= 12930 C.c. 12930 X 0.0001 344= 1.738 er. 1.738 x 2 =6.95% Humus. Die hierzu benutzte Humussaure? wurde von uns analy- siert, die Analyse ergab in 100 Theilen der lufttrockenen Substanz : Hygroscopisches QWasser .....:----.. 10.76 Trockensubstanz age... .. .. seep anes = 80.24. In 100 Theilen der Trockensubstanz : Cy ap EAE.» ee 52.602 ee ne ee 3s + ae 4.19 Ni. 5. See. « se 4.22 Oe. 5. wae 35.03 Asche: “Alec . eS Be 3.904 Die Farbe der Asche war r6thlich und bestand haupt- sachlich aus Eisenoxyd und Thonerde, Schwefelsaure, Phosphor- saure etc. Wenn wir unsere Resultate kurz zusammenfassen, so ist zu bemerken: Die durch die Elementaranalyse erhaltene Zahl war zuhoch, Die von uns benutzte Bodenprobe enthielt trotz der vorsichtigen Vorbehandlung immerhin einige durch das Sieb gegangene Pflanzenreste, mehr oder weniger in Zersetzung begriffen, deren Kohlenstoffgehalt ebenfalls mit zur Berechnung der Humusmenge herangezogen wurde. Die durch die Extrac- tionsmethode erhaltene Zahl betrug nur 719 der mit Hiilfe der Elementaranalyse berechneten Humusmenge. Die Humusmenge, welche durch die volumetrische Methode gefunden wurde, war die kleinste. Es wurden in 100 Theilen lufttrockenen Bodens gefunden bei: Elementaranalyse.2>. . 79 13.84 Theile Humussubstanz. Knop’sche Methode...... S05 Ot ia Extractionsmethode ...... 9:79° “= 4 Volumetrische Methode .. 6.95 __,, 9 Wir sind der Ansicht, dass ahnliche Arbeiten wie die unserige mit den verschiedenartigsten Bodenarten ausgefthrt werden miissen, um weitere Beitrage zu diesem wichtigen Gegenstande zu liefern. - 1 bezogen von Dr. Koenig, chemische Fabrik, Leipzig. Ueber die Aufnahme von Stickstoff und Phosphorsaure durch verschiedene Kulturpflanzen (3 Cerealien und 2 Cruciferen) in drei Vegetationsperioden. VON Dr. K. Bieler und K. Asé. Die hier beschriebenen Versuche hatten den Zweck, die Aufnahme von Stickstoff und Phosphorsaure durch einige Culturgewachse—Winterfriichte in Japan—in 3 verschiedenen Vegetationsstadien zu vergleichen. Obgleich natiirlich nur Durch- schnittszahlen, gewonnen durch 6fters wiederholte Versuche, diesen Vergleich zu illustrieren im Stande waren, so glauben wir doch, dass die Resultate des im Winter 1899 von uns ausgefiihrten Versuches als Beitrag zu diesem Gegenstande gut verwendbar sind, Die Pflanzen wurden in Toépfen im Glashause unter genau denselben Vegetationsbedingungen gezogen und empfingen vollstandige Diingung. Es wurden allen 5 verschiedenen Pflan- zen dieselben Quantitéten verabreicht ; namlich 50 Kgr. P,O, als Doppelsuperphosphat, 50 Ker. K,O als Kaliumcarbonat und 50 Kgr. N. in Gestalt von Ammoniumsulfat per Hektar. Aus- serdem wurde Calcium carbonat angewandt und zwar fiir je 1 Ker Boden, 1 gr. CaCO,;. Die von uns benutzten Toepfe waren aus Porzellan, im Ubrigen jedoch ganz wie die gebrauchlichen Vegetations gefasse beschaffen. Der Durchmesser der Gefasse betrug 25 cm. und die Flache, welche fiir die Pflanzen in Betracht 1 Hectag 20000 angesetzt, so dass fiir jedes der fiinf Culturgewachse im Ganzen 9g Gefiasse bestellt wurden. Die fiir die Oberflache des Bodens kam, betrug circa Jeder Versuch wurde dreifach 242 DR. K. BIELER UND K. ASO. in den Gefaissen berechneten Diingerquantitaten betrugen pro Topf: 0.54 gr. Doppelsuperphosphat enthaltend 0.25 gr. P,O;. 0.39 gr. Kaliumcarbonat 5 3 O25 of Ki: 75 c.c. einer Ammoniumsullatlésung ,, ,, 0.25 gr. N. Das Beschicken der Tépfe, welche mit grobem Kies in der iiblichen Weise auf dasselbe Gewicht gebracht wurden, fand am 9. November 1899 statt. Jedes Gefass wurde mit 6,4 Ker. vesiebten Bodens unserer Versuchsfelder in Komaba gefiillt, welcher vorher mit der bestimmten Menge kohlensauren Kalkes innig gemischt worden war. Am nachsten Tage wurde die Kalidiingung gegeben und zwar in Form einer wasserigen Lésung. Am 13. November wurde die Phosphorsaurediing- ung und zwar in fester Form als Doppelsuperphosphat am Vormittage in die Toepfe gebracht und am Nachmittage desselben Tages die Stickstoffdiingung in fliissiger Form verab- reicht. - Die Samen wurden am Tage darauf, am 14. November, in der fiir Vegetationsversuche iiblichen Weise in die Toepfe gelegt und zwar 20 in jedes einzelne Gefass. Die von uns angewandten Pflanzen waren : 1) Weizen (Sdshu Shirakawa). 2) Gerste (Golden Melon). 3) Hafer (Golden). 4) Raps (Tokyo Wase). 5) Senf (Gew6hnliche Art). Der Aufgang der Pflanzen war nicht gleichmassig, zuerst kamen die Senf- darauf die Rapspflanzen zum Vorschein. In den ersten Tagen des Decembers jedoch waren in allen Toepfen die Pflanzen aufgegangen ; es wurden nun alle iibrigen Pflanzen herausgezogen, so dass in jedem Gefass nur 10 verblieben. Was die Wasserzufuhr anbelangt, so wurden alle Gefasse wahrend des ganzen Versuches in einem fiir die betreffencen Pflanzen giinstigsten Feuchtigkeitszustande erhalten. In der Mitte des Monates Januar 1900 war der Stand der Senfpflanzen am iippigsten, jedoch allmalig gewannen die Rapspflanzen die Oberhand. Die drei Cerealien befanden sich zu derselben Zeit ebenfalls im tippigen Wachsthum ; jedoch gegen Ende des Januars hatten die Gerstenpflanzen einige gelbe Blatter, verursacht durch die Winterkalte, welche sich auch in gewissem Grade im Glashause geltend machte. Bei AUFNAHME VON STICKSTOFF UND PHOSPHORSAURE. 243 den Haferpflanzen war dieses in ganz bedeutend schwicherem Massstabe zu constatieren und beim Weizen nur vereinzelt. In den um Komaba gelegenen Farmen konnten wir allerdings zu derselben Zeit auch ein Gelbwerden von Blittern der Gerste- und auch Weizenpflanzen! und zwar in bedeutend starkerem Masse als bei unsern Versuchen beobachten. Die I Vegetationspcriode endete am 2. Februar, also ca 2} Monate nach der Samenlegung. Die Pflanzen aus je drei Tépfen wurden zusammen mit ihren Wurzelgebilden mit grésster Vorsicht und Sorgfalt, um Substanzverluste so viel es iiberhaupt mézlich, zu verhtiten. Zu diesem Zwecke wurden die Vegetationsgefisse 2 Tage vor dem Abernten aus dem Glashause in einen fiir derartige Zwecke vorgesehenen Raum gebracht, um dort etwas abzutrocken. Die Durchschnittszahl der Blatter betrug bei den einzelnen Pflanzen: Durchschuittszahl. Weizen 5 Gerste 6 Hafer 5 Raps 8 Senf 7 Trockensubstanz der verschiedenen je 30 Pflanzen (3 Tépfe) betrug : Weizen: soe Lo 2.966. grm. Gerste!’ «,.... 2S sae BT Ta as Raters 5, . RE «oo Dtvs by eee. — shel Ore Senf. ......5 eee Fema ASCHE (Rohasche).? Prozente der Trocken- Absolute substanz. Menge. Weizen 9.38 0.278 grm. Gerste 7.58 On362 an Hafer 9.49 O70, 5, Raps 18.04 Tago: i Sent 15.85 0.459 4, 1 Hafer wird in dieser Gegend Japans nicht angebaut. 2 Tie verschiedenen Aschen zeigten verschiedene Farben: beim Weizen : réthlich braun, Gerste: gelblich, Wafer: gréulich braun, Raps: grau und Senf: dunkelgrau, Die Asche der jungen Senfpflanzen liess bei Behandlung mit Salpetersaiure deutlich eine Entwickelung von Schwefelwasserstoft erkennen, 244 DR. K. BIELER UND K. ASO. STICKSTOFF. Prozente der Trocken- Absolute substanz. Menge. Weizen 2.65 0.079 grm. Gerste 3.55 O170's., Hafer S12 0.090 _,, Raps 5.04 0.424 ,, Senf 5.60 0.162 PHOSPHORSAURE. Prozente der Trocken- Absolute substanz. Menge. Weizen 1.18 0.035 grm. Gerste 0.95 0.045 ,, Hafer 0.89 0.026; Raps 0.95 0.080 _,, Senf 0.78 0.0235, Das Verhiltniss zwischen den N- und Phosphorsiuremengen (N=1) war : in den Weizenpflanzen I: 0.443 7 = Gegste = I : 0.265 Hafer I : 0.289 Raps I : 0.189 Senf 7 I O72 ENDE DER II PERIODE. Am ig. Marz 1900, also ungefahr 4 Monate nach der Samenlegung wurden von je drei T6pfen zuerst die tiber der Erdoberflache befindlichen Pflanzentheile abgeschnitten und gesammelt. Die Wurzeln wurden nachher mit grosser Sorgfalt den Tépfen unter Vermeidung von Substanzverlusten entnommen. Um die anhangenden erdigen Theile zu entfernen, wurden die aus je drei Tépfen stammenden Wurzeln 1—2 Tage getrocknet, darauf mit grosser Vorsicht gewaschen und in den Trocken- schrank gebracht. Die Lange der Pflanzen betrug : bis zu beim Weizen 19.5 cm. Gerste 20:5, <5; Hafer 779 Raps 33:05, » Sent 17.3.8 AUFNAHME VON STICKSTOFF UND PHOSPHORSAURE. 245 Die Wurzeln hatten in allen Fallen den Boden der Toepfe erreicht. Trockensubstanz von je 3 Toepfen (30 Pflanzen). Obere Pflanzentheile. Wurzeln. Im Ganzen. Weizen 3.211 grm. 5.098 grm. 8.309 grm. Gerste 5.079) tas AeOlA sy 10.493 ,, Hafer 3-405) ay A370 45 7-724 , Raps 24.605 ,, 8.621 45 339226. 5 Senf 8.240 ,, 4.404 ,, 12.644 ,, ASCHE (Rohasche). Prozente der Absolute Im Trockensubstanz. Menge. Ganzen. Wedzen 4 ee RR ETc gne er. Gert dgeaa ae eaee| ® }u033 Hafer iwetn os seer i | 0874 P ape wean ea He is f 4-745» oat Diwan mee Ea ee STICKSTOFF. Weizen .. ae heile 22, eae an +0.191 grm. Gates He Bar heseo. Haier | Worecln its a Raps = ]wyurela eas oc Seta Were ae Be | 0.466 P PHOSPHORSAURE. Weizen «| Wrrctn eo ooar ob 0.049 gem. Gerste...| Warn att 020 7 $2018 » Halet i | waren ae ore que Cote Eee {Gaede ee: Ou Power. Sei..| woreda 0 ae? Soka. (228 6 246 DR. K. BIELER UND K. ASO. Das Verhiltniss zwischen den N- und Phosphorsiuremengen (N=1) war bei den Weizenpflanzen I: 0.251 » 9», Gersten Be i= OAT 3 ae Sideed a tt O85 7 >) aaps 3 TeOZol 5 sy Seue Hs I 1 10;129 ENDE DER III PERIODE. Die tibriggebliebenen je 3 Toepfe wurden abgeerntet nach dem Abbliihen der Pflanzen. : Beginn der Bliithe. Weizen 4. Mai. Gerste -——--- Hafer 12, Mai. Raps bereits am Ende der II Periode. Senf , 65 April: Ende des Bliithezustandes. Weizen 24. Mai. Gerste i Hafer 20. Mai. Raps tL. Aypril. Senf 16. April. Das Abernten der To6pfe geschahin derselben Weise wie am Ende der II Periode. Die Ernte fand Die ganze Zeit der statt : Vegetation betrug : beim Weizen 25. Mai 6} Monate. on hGrensthes 7. Juni 653 55 a idater®™ 25. Mai 6} * >» kaps ra. A pril 5 * i) Seu 27. April 52 i Die Héhe der Pflanzen betrug im Mittel : Weizen 76 cm. Gerste 40% as Hafer 63 sae, Raps 70 Senf 69 4 1 Das Wachstum der Gerstenpflanzen machte in den letzten beiden Wochen keinerlei Fortschritte. 2 Eine Pflanze war nicht zur vélligen Entwickelung gelangt. AUFNAHME VON STICKSTOFF UND PHOSPHORSAUR. ~ 247 TROCKENSUBSTANZ. Obere Pflanzentheile. Wurzeln. im Ganzen. Weizen 24.877 grm. 7.63 grm. 32.515 grm. Gerste 14.05 oe 10:267 ;, DIS eTU) Lss Hafer 2i2 OOM il COL se 22,500) 5, Raps AOU Saas 6340.8 5O:20n .. Senf 27.899 ,, 1O:790) ==, 38.692 ,, ASCHE (Rohasche). Prozente der Absolute im Trockensubstanz, Menge. Ganzen. Weisen.fQhee Tele B78 2 38y SN 555 gn, Geste...}Svarcein Slee? ros fe ato BC tts bse, Raps {Qn | ee gan 2 46559» Seti] Warzeln Mee 3499 7 45998 » STICKSTOFF. Ween .{ Qere Tele 3969819 Lop gr Gersto...{Qurcin aad ont7 7 fos Hale wocda | mee, «=| G2 > fewag Raps | Word) alee ae Sent | Waren =” ta5 Sree ECS PHOSPHORSAURE. Weizen . as Bone 4 Bee ae | 0.096 grm, fe (ace eee Ce taose Bae {Qe ee SOS dot aed, Wcaiiy 2 ef OES |e ee | Ce fo 248 DR. K. BIELER UND K. ASO. Das Verhialtniss zwischen den N- und Phosphorséuremengen (N=1) war bei den Weizen pflanzen InasOsl 53) i. one ensce c I noo A ga elalieie i sO: 137 ll oes e I: 0.288 pent Ks 1 O;1S0 TABELLE I. VERGLEICHSRESULTATE. Gesammt- | Periode der trocken- Gesammt- | Gesammt- Gesammt- Vegetation. | substanz. | Asche. N. | Oj ———-| Ls IG 2.966 grm. 0.278 grm. 0.079 grm. 0.035 grm. Weizen 101, 8.309 ,, 0.934 5 Gill: 0,049 ,, iDDt, SOE — xp 2.666 ,, O1627e 0.096, ie ALT 77 en rn OS302. i: OMG) 5 0.045 5, Gerste If. 10.493 5 1023) O00 0.043, II. 25.92) BrA07/5 oe 0.609 ,, 0.067 ,, 1 2.884 ,, O27 0.090 ,, 0.026 _ ,, Hafer Il. Wfsikelsh oy 0.874 ,, OSS) es, 0.044 ,, I. 32.566 ,, B.27O, 0.629 ,, 0.086 __,, It 8.416 ,, 0.424 0.080 ,, Raps Il. BQPAD sy 0.644 OST: Ul. 50261 5y- 0.609 Olege ib 2.898 ,, 0.162 leHeya} 5 Senf I, 12.644 ,, 0.466 0.060 ,, 1606, 331002) 4; 0.705 OME e Fy AUFNAHME VON STICKSTOFF UND PHOSPHORSAURE. 249 TABELLE iE Vertheilung des N, der P,O, und der Asche in den oberen Pflanzentheilen und Wurzeln.! Periode der Vegetation VL IIT. II. mi. III. Tl. IIT. Il. II. Obere Theile. re N. PLOE 0.106 grm. 0.028 grm. 0.512 0.083 Obere Theile. N. IR(O)- 0.211 grm. 0.023 grm. 0392 5 0.044 ,, Obere ‘heile. N. BLOF: 0.119 grm, 0.025 grm. GIS OKs 0.073) ie; Obere Theile. N, AOR: 0.519 grm. 0.143 grm. 0.509 ” 0.127 ” Obere Theile. N. LEO) Asche, 0.375 grm. O572 5 0.035 grm. ©:063) 5; Weizen. Asche. N. 0.306 grm. 0.085 yrm. 2.184 OM Gerste. Asche. N, 0.539 grm. 0.095 grm. T:5O2m,, O:2175 ss Hafer. Asche, N. 0.307 grm. 0.066 grm. 219005, O28, Raps. Asche. N. 3.302 grm. 0.125 grm. 4728), 0.100 Senf. N. 1.210 grm. o.CgI grm. 2.497 5 0.133 5 Wurzeln. > P20; 0.021 grm. 0.013 4, Wurzeln. peor. 0,020 grm. 0,023 ,, Wurzeln. P50... 0.019 grm. 0.013 ” Wurzhln. 1 Oke 0.038 grm. 0.048 ,, Wourzeln. P,0;; 0.025 grm. 0.068 ,, Asche. 0.628 grm. 0.482 ,, Asche. 0 484 grm. 1.973» Asche. 0.567 grm. 1.074 5 Asche. 1.443 grm. 1.941 5 Asche. 1.455 grm. 3-499 » 1 Die Pflanzen der I Periode wurden im Ganzen analysiert. 250 DR. K. BIELER UND K. ASO. ~~ TABELLE Ill. VERGLEICH DER PROZENT-ZAHLEN. | | { | Periode. ‘Theile. N. EOF } I. —— 2.65 1.18 Obere. 3-30 0.88 Weizen. Il. Wurzeln. 1.67 0.41 Obere. 2.06 0.34 il. | Wurzeln. 1.50 0.17 L — 3-55 9.95 | Obere. 3-71 Coy. (oa Gerste. II. : [| Wurzeln, 1.98 o.4te al Obere. 2.63 0.39 Ii. Wurzeln, | 2.11 0.22 | E 3-12 0.89 | 2 ' Obere. 3.48 } 0.73 Hafer. Tl. | Wurzeln, 1.52 0.43 ) { Obere. 2.36 0.34 i | | Wurzeln. 1.13 O.11 | + | } | I ae SE 0.95 ( Obere. 2.11 0.58 Raps. I. / Wurzeln. 1.45 0.44 { Obere. 1,22 0,30 ill. | + | | { Waurzeln. 1.20 os7 = | | E& | 5.60 0.78 Obere. 4.55 0.43 | Senf. II. - | | Wurzeln. 2.07 H 0.57 ( Obere. 2.05 | 023 | lil. 2 ee { Wurzeln. 1.23 0.63 | ! AUFNAHME VON STICKSTOFF UND PHOSPHORSAURE. 251 STICKSTOFF UND PHOSPHORSAURE IN DEN SAMEN. Zur Bestimmung von N benutzen wir} je 30 Samen, zur Bestimmung der P,O, jedoch 300 und zwar wurden in allen Fallen nur gute, auserlesene Samen verwandt. 30 Samen (den Pflanzen von 3 T6pfen entsprechend) enthielten : N. P35 Ox. beim Weizen 0.015 grm. 0.006 grm. ., Gerste 91023, QOUZ 9p Eater Goro ,, (GHOSE 5p =. Raps OOO4 ,, O1002ay » Senf 003. ;, O1OO27 Die von je einer Pflinze aufgenommene Phosphor- siure- und N- Menge betrug am Ende des Versuches : N. PO} Aufgenommen. Aufgenommen, beim Weizen 0.021 grm. 0.003 grm. » Gerste @O20 ,,; O1OO2T es, 2 later OO2l jj, OKGOG) op a» aps GO20 ,, CGLOEOM ee Sent G1023 ,; GOo4,, ,; Wenn wir zuerst die Resultute der Cerealienversuche ins Auge fassen, so constatieren wir, dass beim Weizen und Hafer eine Zunihme der Trockensubstanz von Periode zu Periode in ungefahr demselben Verhialtnisse stattgefunden hatte, und von den Gesamntaschenmengen wire dasselbe zu erwahnen. Die Gerstenpflanzen entwickelten sich in der ersten Periode viel tippiger als die andern Cerealien; und auch noch nach einer Vegetationszeit von 4 Monaten (Ende der Periode II) war das Gewicht der Trockensubstanz und ebenso das der Asche bei den 30 Gerstenpflanzen héher als die entsprechenden Zahlen beim Weizen sowohl wie bei dem Hafer. Nach dieser Zeit jedoch entwickelten die Weizen- und Haferpflanzen ein weiteres kraftiges Wachsthum, welches bis zum Schlusse der Versuches anhielt. Bei den Gerstenpflanzen war dieses aber nicht der Fall. Nach einer gewissen Zeit schien die Vegetation der Gerste stillzustchen und nicht eine einzige Pflanze erreichte das Bliitthestadium. Die vollstandig ausgebildeten Gerstenahren 252 DR. K. BIELER UND K. ASO. blieben in der Blattscheide langer als 2 Wochen und in diesem Zustande wurde sodann am 7. Juni nach einer Vegetations- dauer von 64 Monaten abgeerntet. Das Gewicht der Gesammt- asche betrug trotzdem bei den Gerstenpflanzen am Ende des Versuche mehr als das der véllig entwickelten Weizen- und Haferpflanzen. Was die Stickstoffaufnahme anbetrifft, so stellen wir fest, dass am Ende des Versuches die in je 30 Pflanzen der 3 Cerealien enthaltene N- Menge ungefahr dieselbe war. Die Stickstoffmengen, welche am Ende der I und II Periode in den Gerstenpflanzen am h6échsten waren, nahmen bei der Gerste vom Ende der II bis zum Schluss des Versuches im Verhaltnisse von 1:2 zu, wahrend dieses Verhaltniss bei den Weizen- und Haferpflanzen 1 : 3 betrug. In Hinsicht auf die Phosphorsaureaufnahme miissen wir zuerst erwahnen, dass die jungen Gerstenpflanzen mehr Phos- phorsaure aufgenommen hatten als die andern Cerealien. Aber bei den Weizen- und Haferpflanzen nahm die Phosphorsdure von Periode zu Periode zu, was bei den Gerstenpflanzen nicht der Fall war. Von dem Ende der I bis zum Ende der II Periode konnte bei den Gerstenpflanzen tiberhaupt keine Erhéhung der Phos- phorsaureaufnahme constatiert werden. Eine Zunahme der Phosphorsdure vom Ende der II Periode bis zum Ende des Versuches konnte allerdings constatiert werden, war jedoch nur gering. Die aufgenommenen P,O,-Mengen in den Pflanzen der I Periode standen zu diejenigen Mengen der Pflanzen der III bei der Gerste in dem Verhiltnisse von 1: 1.5, wahrend dieses Verhaltniss beim Weizen 1:2.7 und beim Hafer sogar 1: 3.3 betrug. Bei einem weiteren Vergleich der aufgenommenen P,O,- Mengen, erkennt man, dass die Absorptionsfahigkeit fiir P,O, beim Weizen am starksten war. Der Gehalt der Asche an P,O, betrug am Ende der III Periode: beim Weizen 3.6, beim Hafer 2.6 bei der Gerste nur 1.9%. Bei den Pflanzen der ersten Periode betrug der P,O,-Gehalt der Asche bei dem Weizen: 12.6, bei der Gerste 12.4 und beim Hafer 9.6%. Wenn wir alle diese Resultate zusammenfassen, k6nnen wir sagen: 1) 30 Gerstenpflanzen waren im Stande ungefahr dieselbe Menge N aufzunehmen als 30 Weizen resp. Haferpflanzen. 2) Obgleich AUFNAHME VON STICKSTOFF UND PHOSPHORSAURE. 253 die Menge der Gesammtasche bei den Gerstenpflanzen am Ende des Versuches die grésste war, war die aufgenommene P,O, am kleinsten. 3) Die in der Diingung gegebenen und im Boden vorhandenen Nahrstoffe waren im Stande, die 30 Weizen- und Haferpflanzen zur volligen Entwickelung zu bringen, reichten jedoch nicht aus oder vielmehr waren nicht fahig dasselbe bei den Gerstenpflanzen zu vollbringen. In Tabelle II sind die in den oberirdischen Pflanzentheilen und in den Wurzeln enthaltenen N, P,O,; und Aschemengen zusammengefasst. Die Phosphorséurezahlen sind bei den Weizen- und Haferpflanzen interressant und geben ein Bild von der fortschreitenden P,O,- Aufnahme. Was die Gerstenzahlen anbetrifft, so constatieren wir, dass am Ende der III Periode die Gesammtmenge von N in den oberen Pflanzentheilen die kleinste die in den Wurzeln durch die grésste Zahl ausgedriickt wird. Der Prozentgehalt an N (Tabelle III) war bei den Gersten- pflanzen am Ende des Versuches am héchsten und zwar sowohl in den oberen Theilen als in den Wurzeln. Wenn wir hierauf die Resultate der Raps- und Senfversuche betrachten, so fallen uns zuerst die Rapszahlen ins Auge. Die Zahlen fir N und P,O,; (Tabelle 1) zeigen vom Ende der II bis zum Ende der III Periode eine kleine Abnahme, die Zahlen fiir Trockensubstanz und Totalasche jedoch eine Zunahme. Beim Auswahlen der drei Tépfe am Schluss der II Periode nahmen wir die T6pfe mit den best entwickelten Pflanzen, die Raps- pflanzen befanden sich in einem Stadium kurz vor der Biiithe. Da bei dem Abernten und der weiteren Behandlung dusserst vorsichtig verfahren wurde, um so viel wie méglich, Sub- stanzverluste zu verhiiten, mtissen wir annehmen, dass keine weitere Aufnahme von P,O,; und N in den so weit entwickelten Rapspflanzen stattfindet und dass die Zunahme von Trocken- substanz und Asche in den Pflanzen der III Periode durch die Aufnahme von andern, nicht fiir die Ernahrung wesentlichen Stoffen verursacht sei. Beim Vergleich der Raps- und Senfzahlen in Tabelle I sehen wir, dass das Wachsthum der Rapspflanzen bis zum Ende der II Periode kraftiger war, als das der Senfpflanzen, Die obwaltenden Verhialtnisse, unter welchen unsere Versuche aus- gefihrt wurden, schienen von vorneherein giinstiger fir die Rapspflanzen als fiir die Senfpflanzen zu sein, infolgedessen die 254 DR. K. BIELER UND K. ASO. Vegetation der ersteren beschleunrigt, die der letzteren verz6gert wurde. Der Gehalt an P,O, war in den oberen Pflanzentheilen beim Raps am Ende der II und III Periode hGéher als beim Senf und dasselbe war der Fall mit Aschengehalt am Ende des Versuches. Wenn wir jedoch die zu den Wurzeln gehG6rigen Zahlen betrachten, so constatieren wir das Gegentheil: Am Ende der II und ebenso am Ende der III Periode war der Gehalt an Asche sowohl wie an P,O, ger6ésser in den Senf- pflanzen. Was den N-Gehalt anbetrifft, so ist in allen Fallen sowohl am Ende der I Periode (die Zahlen beziehen sich auf die gesammte Pflanzensubstanz) als auch am Ende der II und III Periode der Gehalt an N héher beim Senf als beim Raps und zwar in den oberen Pflanzentheilen und auch in den Wurzeln. Die Zahl, welche die Gesammtmenge von N in den 30 Senfpflanzen am Ende des Versuches angiebt, ist nicht nur hdher als die entsprechende Zahl beim Raps sondern auch hGoher als diejenigen der drei Cerealien. Auf der andern Seite zeigte jedoch der Raps die starkste Absorptionskraft fir P,O,. Zum Schluss fiihren wir an, dass die Cerealien und die beiden Cruciferen wahrend ihres ganzen Wachsthums ungefahr dieselbe Menge von N aufmahmen. Von P,O; nahmen die Cerealien weniger auf als die andern Pflanzen, und dasselbe k6nnen wir von der Gesammtasche sagen, Die siarkste Absorptionsfahigkeit fiir P,O, zeigte der Raps, dann folgte der Senf. Weizen besass von den 3 Cerealien fir P,O, die stirkste Absorptionskraft, die diesbeziigliche Zahl fur Hafer liegt zwischen Weizen und der Gerste. In Beziehung der Aufnahme von Nahrstoffen durch die Rapspflanzen zeigte unser Versuch, dass die starkste Aufnahme stattfund als die Vflanzen die erste Entwickelung hinter sich hatten (Periode I) und andauert bis zu dem Stadium der Blithe. Nach der Biithe scheint keine weitere Aufiahme von Nahr- stoffen stattzufinden. S822 —_—— —_——_ = On the Réle of Oxydase in the Preparation of Commercial Tea. BY K. Aso. It is well known that fresh tea-leaves if steamed after being collected, will preserve their green color, while if exposed toa partial drying in the sun, they will turn gradually brown. ~The former is the first operation in the preparation of the green tea and the latter that of the black tea of commerce. About ten years ago, Prof. Kozai1 found that the tea- leaves if once sufficiently steamed do not undergo further change or the so-called fermentation, but that in the further preparation of black tea, the sacks of rolled and _ pressed tea-leaves showed a gradual rise of temperature to 34.°5 C at an air-temperature of 24° C.2, He further observed that black tea contains considerably less tannin than green tea : In 100 parts of the dry matter, Original Green Black leaves. tea. tea, Tannin. 12.91% 10.64% 4.89% calculated as gallotannic acid The development of the black color seemed to me to be due to the action of an oxydizing enzyme upon the tannin of the tea-leaves, and this has led me to the following tests. The tea leaves were collected in the middle of September, taking only the five uppermost leaves of each branch. They were pulverised and extracted at the ordinary temperature with 1 Kozai, Bull. College of Agric., Toky6. Vol. I, No. 7. 1890. 2 ‘the process of ** fermenting” consists in pressing the rolled leaves into sacks, arranging them, side by side, under a white cloth and placing them in a sunny place. 256 K. ASO. dilute alcohol (30%) for 40 hours. The filtrate was mixed with strong alcohol (93%) and left to stand for several days, where- upon the precipitate was collected on an asbestos filter, dissolved in a small quantity of water and again precipitated with strong alcohol. The product thus obtained from about 300 grams of the fresh leaves was dissolved in 150 c.c. water, and subjected to the following tests: A freshly prepared guaiacum tincture added to the cold solution, gave at once a blue coloration, which soon increased in intensity, while the boiled solution failed to do so. This proves the presence of an oxidase. In order to observe the temperature at which this enzyme is destroyed some tests were made with the following result : Temperature, | Time of heating. Reaction with guaiac. 729:C 5 minutes. Pale blue color after several seconds, 75°C Pa A pale blue color set in after about 10 minutes. 76°C > Slight coloration after about 13 minutes, We may therefore infer, that the oxidase of the tea-leaves is destroyed at about 76°—77?° C. When tea-leaves are extracted directly with water, the solution does not give the blue reaction, since the tannin in the solution prevents it. In order to test also the presence of peroxidase in the tea- leaves, I proceeded as follows : Fresh tea-leaves were finely crushed with addition of some sand and extracted twice with strong alcohol (absolute) at the ordinary temperature in order to remove the tannin,! since it not only prevents the usual guaiac reaction of peroxidase, but in smaller quantities can also bleach out again the blue color after it has made its appearance. The residue was then extracted at the ordinary temperature with water, and this solution? was heated for 5 minutes at 76°C in order to destroy the oxidase. In this liquid the peroxidase reaction was obtained with 1 Not only the guaiac reaction of oxidase and peroxidase, but also the action of myrosin and emulsin is prevented by tannin. (Reynolds Green: Fermentation. p. 154.) 2 This solution produced a brownish coloration_with hydroquinone, ROLE OF OXYDASE IN THE PREPARATION OF TEA. 257 tincture of guaiac and some hydrogen peroxide with great intensity. In the following test, 6¢ c.c. of a cold prepared strong alcoholic extract of the tea-leaves were diluted with four times its own volume of water and separated into two portions. To one, some of the purified enzyme solution above mentioned was added in order to observe the action of the oxidase on the tannin of the tea-leaves while the other portion served for control. Both flasks were only half filled to admit air freely, and some ether was added to prevent bacterial growth and plugged with cotton. The gradual change of color was very evident: Tannin solution Tannin solution containing enzyme. without enzyme. After 24 hours. Somewhat brown. Green. 48, Brown. Yellowish green.! ey ae Dark brown. Brownish green. Hence, an action of the oxidase of tea on its tannin is evident, and there can be no further doubt, that the so-called ‘fermentation’ of the black tea leading to the formation of the black color is due to the action of the oxidizing enzymes in the tea-leaf. Finally also a test for catalase was made, not only with fresh tea-leaves, but also with the residue after extracting leaves at the ordinary temperature with alcohol and water, and its presence recognised by the considerable development of oxygen from dilute hydrogen peroxide solution. Some tests for oxidase and peroxidase were also made with the green as well as the black tea of commerce,? but none of the three enzymes mentioned was found. This is not surprising since in the manufacture of black tea, the leaves are finally heated up to 100° C or even higher. In the green tea of commerce, it is the steaming at the beginning of its preparation, that kills the enzymes, Since Bertrand has observed a small percentage of manga- nese in oxidizing enzymes and attributes to this metal an essential role in their oxydizing actions, and since Lepinois 1 This incomplete change of color is probably due to the acid reaction changing the chlorophyll, 2 Prepared according to Japanese method. 258 K. ASO. found also some iron in one of them, and further, since it has been shown by Hofer, Macallum and others that the enzymes are secreted by the nuclei (at first in a state of zymogen), and moreover as Bunge, Macallum and Stoklasa have observed some nuclecproteids containing iron, I was led to the following tests, which show that there occur in tea-leaves nucleoproteids that contain not only iron but also manganese. Whether one and the same nucleoproteid contains both these metals, or whether there be two nucleoproteids of which one contains iron and the other manganese, was however not decided by my tests. I proceeded as follows: 200 grams of air-dried tea-leaves were powdered and extracted first with ether and then with alcohol, then digested with artificial gastric juice for 24 hours at 38°C. The residue was treated with dilute ammonia and the solution precipitated by slightly acidifying it with acetic acid. The dark brown! gelatinous precipitate thus obtained was washed with dilute acetic acid, then with water, 2ud dried and powdered. This substance was again dissolved in dilute ammonia and precipitated with absolute alcohol (a). The brown precipitate was thoroughly washed with alcohol and ether, and then subjected to d gestion with artificial gastric juice for 3> hours at 37°-38°C. The residue was dissolved in dilute ammonia, and precipitated and well washed with alcohol. The dark brown substance thus obtained, contained phosphorus and yielded for 100 parts of dry matt-r: eres. ae PY secs a 4.91 % Feces fee a -. e slat breton hd deseag e225 Min}, icc 25 ee: - so eee 0.04. % The filtrate of the precipitate (a) was again precipitated with acetic acid, and the dark brown precipitate thus o tained was, after being washed with dilute acetic acid, subjected to artificial digestion for 24 hours at 38°C. The indigestible residue was dissolved in dilute ammonia and again precipitated with acctic acid, washed thoroughly with dilute acetic acid, alcohol, and ether, and dried. This substance contained : 1 The brown color may have been partly due to traces of oxidised tannin. ROLE OF OXYDASE IN THE PREPARATION OF TEA. 259 In 100 parts of dry matter, ING eee occa EES So a cc-oy eae ence renee 8.66 TO ieee 5 55s AOE: & 6 oe og Dae eS 0.18 OMIT eos) 5. east MS cuss antic aes no reaction. SUMMARY. I. The black color of the commercial black tea is produced by the action of oxidase upon tannin. II. The green variety of the commercial tea owes its green color to the destruction of oxidase in the first steps of preparation. III. By the final steps of its preparation, the black tea also loses the oxidizing enzyms. IV. In tea leaves occur proteids containing iron and manganese. On the Occurrence of Organic Iron Compounds in Plants, BY U. Suzuki. Although iron is indispensable for the formation of chlorophyll, yet according to Gautier and to Molisch,? it is not contained in this colouring matter itself. Further J. Stoklasa? found that it is not present in his ‘‘ Chlor lecithin.” Bunge and others had already proved that in plants iron never exists as inorganic compounds. Bunge*® succeeded in isolating an iron- nuclein compound from the yolk of the hen’s egg which he called haematogen to indicate its close relation to haemoglobin. Stoklasa* obtained from the bulb of Allium cepa and from the seed of Pisum sativum, a similar compound. Haematogen contains, besides iron, a small quantity of calcium, magnesium, chlorine and much phosphoric acid. The presence of these is common in nuclein. It was hitherto supposed that the common nuclein does not contain iron; this may be due to the usual mode of its preparation which consists in dissolving it several times in alkaline solution and subsequently precipi- tating it. The ‘‘haematogen” of Bunge and of Stoklasa has. the following composition :— We see that the haematogen of animal and vegetable origin closely resemble in their chemical nature. The only difference being the higher percentage of iron in the vegetable haematogen. Zaleski obtained Bunge’s haematogen from the liver of animals, Macallum also found iron in the nucleus of plant cells. 1 H. Molisch ;—Eisen und ihre Beziehungen zu den Pflanzen,—Jena 1892. 2 J. Stoklasa ;—Ueber die physiologische Funktion des Lecithins in der Pflanze (Akademie der Wissenschaften, Wien 1896). 3 J. Bunge;—Ueber die Assimilation des Eisens (Zeitschrift fiir physiologische Chemie); Strassburg 1885. 4 J. Stoklasa ;—Comptes rendus d.1’Acad. des sciences 127. 282-83. Chem, Labor, des Polytechnikums. Prag. EO UZ UK. 2601 Bunge’s Stoklasa’s Nuclein. IIaematogen Haemoglobin. Haematogen from egg yolk. from plants. G 40.81 42.11 54.26 43.05 H 5.38 6.08 Fi 5.56 N 15.98 14.73 120 15.13 S) 0.38 0.55 0.54 0.28 P 6.19 5.19 O77(esOe)) O21 Fe —- 0.29 0.43 1.68 O 31.26 BOS 20.69 28.08 We see how closely nuclein and haematogen agree in composition.1_ Both chemical and microscopical examination of seeds by Bunge has shown that the iron is localized in the embryo and endosperm only in an organic combination. Most probably the haematogen is used for the formation of the cellular nucleus. Stoklasa tried in vain to isolate haematogen from young maize plants cultivated in a solution containing no iron while those cultivated in a solution containing iron, gave a moderate quantity of it. Lower plants containing no chloro- phyll require nevertheless, according to Molisch, iron just as chlorophyll bearing plants do. Thus, Mucor mucedo or Bacillus megatherium never develop well in a solution free from iron. Certain fungi contain also haematogen; thus from 1000g. of Boletus edulis 3.5g. haematogen was isolated by Stoklasa, and he therefore suggested that iron is an_ essential constituent of nuclein like phosphoric acid, K.Aso® has shown that the spores of Aspergillus oryzae contain relatively much iron (about 5% Fe,O, in the ash). On treating the spores with dilute alkali and neutralizing the alkaline extract with acetic acid, a protein substance was obtained, which, after being subjected to artificial pepsin digestion, yielded an insoluble residue containing iron. This compound was again dissolved in ammonia, the ammoniacal solution again neutralized with hydrochloric acid, and even after this purification process the nuclein obtained was tolerably rich in iron. It is especially interesting that this 1 Recently Hausermann found a small quantity of iron in the serum of blood, (t mg. in 100 c.c.). Dried fibrin from the dogs blood contained 0.01% iron, 2 Bul, Cell. of Agriculture Vol. III No. 1. 262 ORGANIC IRON COMPOUNDS IN PLANTS. fungus grown on a substratum poor in iron like rice (0.005% Fe,O,) store up so much iron in the spores. Since the question whether iron forms a regular constituent of nucleins is of considerable importance, I have made some further investigations with vegetable materials. My attention was first drawn to the richness in iron of the seed of Polygonum tinctorium, and this has led me to analyse more fully the ashes of the seeds and leaves of two indigo plants. Polygonum tinctorium Indigofera tinctoria Seeds. Leaves. Seeds. Leaves. In 100 parts of dry matter + Crude ash 2.84 15.5 4.0 4.3 In 100 parts of ash. SiO 0.35 Te 3.1 6.25 SO 4.10 4.77 2.4 3.80 IPAON, 4I.I 4.13 34.2 5.50 KO 21.5 21.40 18.7 17.80 Na,O 2 6.00 4.2 2.00 HesOr We Zak 12.0 4.8 CaO Ee | 39.10 1I.0 27.0 MgO gee 12.30 9-9 6.4 Total 101.85 83.56 95-50 80.55 We see that about 12% of the ashes, corresponding to 0.34% of the dry matter, of the seeds consists of Fe,O,. Similar results were obtained with other samples. Such a high percentage of iron content in seeds has to my knowledge not yet been observed. On looking through Wolffs tables, I found that of the twenty two species of seeds examined, the richest in iron contains only 2.5% in the ash and 0.08% in the dry matter. Occasionally plants absorb by accident unnecessary substances from the soil and deposit them in the leaves and stems, but this can hardly be the case with the seeds. In the following pages several experiments to isolate the iron compounds from the seeds! are described. (1). Seeds of Polygonum tinctorium were dried and finely pulverized, and treated with ether, absolute alcohol and water, These Extracts contained no iron. Further the water extract 1 All the materials used in this investigation were carefully tested for traces of iron and were found free of them. U. SUZUKI. 263 contained very little proteids. The residue from the water extract, was now treated with 1 sodium chlorid solution. This extract was also almost free from iron. The residue now left, was treated with 0.29% caustic potash solution at the ordinary temperature, frepuently stirred, and after standing for one day, the clear supernatant solution was decanted, since it was difficult to filter, and mixed with dilute acetic acid until a moderately acid reaction was obtained. Hereby a greyish white flocculent precipitate was abundantly produced. This precipitate, con- sisting of several proteids, was washed with water, at first by decantation, then collected on a filter, washed with water, alcohol and ether, dried and pulverized. It contained :— N=10.4% Fe=05% N: Fe=100'3 4.0. Repeated tests have shown that nearly 60—70% of the iron contained in the seeds may thus be obtained in a form soluble in dilute potash and separable from this solution with acetic acid. (2). In this test I applied the method of Stoklasa which consists in extracting the seeds with a large quantity of 0.19¢ hydrochloric acid, evaporating this extract at 30—36°c and subjecting the product thus obtained to artificial pepsin digestion. The residue thus obtained was, however, so little (from 100 grams of the seeds only 0.1—0.2 gram was obtained) that further examination was impossibble. (3). 100g. of the dry seeds were extracted with ether and alcohol and the residue divided into two portions :— One portion (a) was boiled with water for half an hour cooled to 60°C and a small quantity of malt extract added to saccharify the starch. The residue from filtration was washed with water and then subjected to artificial pepsin digestion, whereby a large part of the proteids was dissolved. The remaining residue was now treated with dilute caustic potash 1 Fearing that the extraction with potash might injure the original iron compounds to some extent I used in another case dilute ammonia for extraction ; but I observed that the iron compound is very imperfectly soluble in it. a). Crude ferro-nuclein of Polygonum tinctorium was extracted with dilute ammo- nia and precipitated with alcohol, subjected to artificial digestion the residue once more dissolved in ammonia and precipitated with alcohol, it contained only INES Or ge b). Crude ferro-nuclein of Indigofera tinctoria thus treated, yielded N=6.6% Fe=0.51%. 264 ORGANIC IRON COMPOUNDS IN PLANIS. and the alkaline extract was acidified with acetic acid whereby the crude ferro-nuclein was obtained :— Ash 49% e,O- 1G, Fe 0.91% This precipitate was however probably not yet pure. The other portion (4) was not previously deprived of its starch content, but directly subjected to artificial pepsin diges- tion, the ‘insoluble residue treated with 0.19% caustic potash, acidified with acetic acid and the precipitate thus produced, was washed with water, alcohol and ether. Nearly 7 grams of impure ferro-nuclein resembling closely that of (@) was thus obtained. Thus from 100 grams of dry sample I obtained nearly 10 grams of impure ferro-nuclein with about 0.9% iron. (4). ©.2 grams of the crude ferro-nuclein (containing 0.5% iron and 5% nitrogen) of Polygonum seeds! was treated with 100 CC. of 0.2% hydrochloric acid, and divided into two parts. To one part, was added some pepsin and both were kept for twenty hours at the temperature of 37—40°, whereby a small portion of the iron was evidently splitt off by the action of the acid from the original compound, as the ferro-cyanide test clearly proved. It was further observed that the albumoses and peptones formed by the action of pepsin contained iron in organic combination. These experiments were repeated with several other samples with essentially the same result. Since the haematogens of Bunge and of Stoklasa do not undergo any change by artificial digestion and as they do not so easily splitt off some iron by the action of 0.29 hydrochloric acid, my ferro-nuclein differs evidently from the former, especially since it is not easily soluble in dilute ammonia. The existence of apparently the same ferro-nuclein in the seeds of Indigofera tinctoria, etiolated shoots of Polygonum tinctorium, and in the full grown leaves of Indigofera and Polygonum was shown by my further experiments. Similar 1 and 0.1 grams of that of indigofera seed, U. SUZUKI. 265 ferro-nuclein exists evidently also in other plants, as tests with mulberry leaves! and tea leaves? have shown. Summary of Results. 1). Seeds of Polygonum tinctorium and Indigofera tinctoria are exceedingly rich in iron, as are the leaves of the same plants. The iron does not exist in these plants as inorganic salts. 2). Ethereal, alcoholic and aqueous extracts of the dried and pulverized seeds or leaves contain no iron. Also the sodium chlorid extract contains no iron compound. or only traces of it. However the dilute alkali extract contains a nuclein like substance which can be precipitated with dilute acetic acid. This contains the greater part of the iron of the original material. This precipitate was subjected to artificial, pepsin digestion, whereby a portion of the proteids dissolved, and if this solution is again precipitated with absolute alcohol the products formed still contained iron. The insoluble residue obtained from artificial digestion chiefly consists of a nuclein like substance and contains 0.5—1.09% iron and 5—10% nitrogen according to the methods of preparation. A small portion of iron was liberated during the digestion process. 3). Tests made to isolate the so called haematogen by the methods of Bunge and of Stoklasa from plant seeds, yielded unsatisfactory results. Stoklasa obtained 1.g grams haematogen from 1500 grams of dry Allium cepa, that is to say nearly 0.002% of iron in the dry matter of the original material was found to be in the form of haematogen. With Pisum sativum his result was similar. As these vegetable materials contain on the average more than 0.02% of iron in the dry matter, only about one tenth of the iron in the original materials was obtained in the form of ‘‘ haematogen.’” Therefore we may infer that the greater part of the iron in these cases also exists in other forms than ‘‘ haematogen.” 1 About 300 grams of dry powder were extracted with dilute caustic potash, but the precipitate obtained with acetic acid was exceedingly gelatinous, which made further purification difficult. It could however be easily shown that this precipitate contained iron. 2 Tea leaves are very rich in iron, sometimes 129% of the ash consisting of the oxid. Compare the article of Mr. Aso in this Bulletin. 266 ORGANIC JRON -COMPOUNDS IN PLANTS. 4). The iron compound which I obtained is evidently different from the so called “ haematogen,” since the former is partly soluble by artificial pepsin digestion, and both the residue and the dissolved portion (precipitable with absolute alcohol) contain iron in organic combination and liberate a small quantity of the iron by the action of 0.2% hydrochloric acid while the so called ‘‘ haematogen”’ does not undergo any change by artificial digestion or by the action of 0.2% hydrochloric acid for a short time at the ordinary temperature. The substance obtained by me is also much more difficultly soluble in dilute ammonia. 5). Asimilar iron compound exists in many other plants according to my examination, and its distribution seems to be very wide. Investigations on the Mulberry Dwarf Troubles, a Disease widely spread in Japan. BY U. Suzuki. In a former number of this Bulletin (Vol. LV, No. 3) I have described how widely distributed this disease is and how much ‘damage is caused by it. The general diagnosis was given, numerous analyses were carried on to find out the differences in chemical composition of the healthy and the diseased plants ; the relation of reserve materials to the disease was especially investigated, and extensive field experiments were further made and many facts observed with plants from different provinces. From my studies I came to the conclusion that the disease was not caused by any parasite, but the primary cause was to be sought in the practice now in vogue of subjecting the mulberry tree to repeated cuttings in the growing season. During winter a considerable amount of reserve materials (especially nitrogen compounds and starch) is stored up in the bark of stems and roots, while in spring, when the development of new leaves commences, the greater part of it is transported to the growing shoots. The assimilation products in the leaves again migrate to the stems and roots in late autumn, when the leaves begin to fall. Therefore the stems and roots are rather poor in reserve materials during the growing season, Now it will be at once evident that the cutting of the plants in the growing season must have a very bad effect on the shoots that develop later on, since the supply of reserve materials upon which they depend is but very scanty. Moreover, it is not impossible that the stored materials may be entirely exhausted before the new shoots have attained a certain height and have become able to prepare their own organic food by assimilation. In sucha case 268 U. SUZUKI. the normal growth of the shoots will be seriously impaired and starvation must set in; the leaves remain small and their tissues develop only partly. For the same reason, the disease must be induced by the frequent plucking of the leaves. This view was comfirmed by numerous observations and no doubt could further be entertained about the correctness of my inferences. But there remain still many points to be investigated, in regard to the various pathological phenomena, accompanying the deficiency of reserve materials, and also in regard to other secondary causes which accelerate the disease. Finally methods of check- ing or preventing the disease should be found. The following investigations were carried out with the intention to throw more light upon the pathology of the disease. OXIDIZING ENZYMES. The wide distribution of oxidizing enzymes in the vegetable kingdom has recently attracted the notice of many plant physio- logists and various investigations have been made upon the nature and the physiological actions of these interesting sub- stances. Albert F. Woods? has propounded the view that the change of chlorophyll in the autumnal yellowing of leaves is chiefly due to their action, a view which seems to agree well with the fact that the oxidizing enzymes are abunduntly produced in autumn when the activity of the cells in the leaves decreases. If such leaves are ground in-a mortar with addition of some water, the green colour will soon be changed to reddish brown, while generally the green of the leaves in full vigour is but slowly changed. Woods has also made very interesting obser- vations on the influence of the oxidizing enzymes upon various diseases of plants. Thus he found that the plants diseased either by some physiological causes or by the attacks of insects and fungi or by some mechanical disturbances, produce always an abnormal quantity of oxidizing enzymes. He made especially careful examinations of albinism or variegation of many plants, especially of the so-called ‘‘Mosaic disease’? of tobacco: In the light green parts of the diseased leaves, always much of the oxidizing enzymes has been found, sometimes even five or six times more than in the normal leaves. Further those discolored 1 Centr.-Bltt. fiir Bakt, II. Abt. V. Band 1899 No, 22. MULBERRY-DWARF TROUBLES IN JAPAN. 269 regions contained, in spite of the imperfect developement of chlorophyll, always many starch granules. He founda that the oxidizing enzymes can inhibit the diastatic action, and by making the saccharification of the starch in the cells much more difficult than in the healthy leaves, prevent the migration of it into other parts of the plants.t To prove the correctness of this view, he prepared a concentrated solution of tobacco oxidase, mixed it together with a small quantity of ‘‘ Taka- diastase”’ or malt extract with some diluted starch paste and kept the mixture at 45°C, At the same time a control mixture was prepared, in which the oxidase solution was previously boiled for a few minutes ; After thirty minutes a very remarkable difference was observed. In the former case starch was not at all saccharified, erythrodextrin being the final product, while in the latter (control case) the entire starch was saccharified. Such phenomena may also occur in the diseased leaves. _Beijerinck? supposes that there exists a so-called ‘‘Contagium vivum fluidum” in the extracted and filtered juice of the tobacco leaves suffering from the mosaic disease. This fluid contains no visible organism, still it produces the same disease by injection. Woods rejects this view and thinks that the so called ‘‘contagium vivum fluiduin” is identical with the oxidizing enzymes. He made several experiments to produce the disease by injecting solutions of oxidizing enzymes into the healthy plants and succeeded in some cases, though not always. The question why oxidizing enzymes are produced in an abnormally large quantity in the diseased leaves has not yet been satisfactorily answered, but it is very probable that the decrease of vital activity and the deficiency of nutriment in the cells has some intimate relation with their production. Woods succeeded in producting the mosaic disease artificially by cutting back rapidly growing tobacco plants. Loew also holds the view that partial starvation can cause an increase of these 1 Dr. Smith also observed an increase of the amount of the oxidizing enzymes in the diseased leaves in the “ peach yellow ’’-disease. 1 Science. N.S. Vol. XI., No. 262, page, (January 5, 1900). Beijerinck. Verhandelingen der Koninklijke Akademie van Wetenschappen to Amsterdan. Tweede sect. Deel. VI. 1898, No. 5. 270 U. SUZUKI. enzymes. Brown and Morris! proved that the secretion of diastase is much enhanced by partial starvation. Weak lily plants containing much of oxidizing enzymes, especially peroxidase, are always those which have a very poor root system and are suffering from partial starvation. Church ana- lyzed the albino leaves of [lex aquifolium. Hedera helix and Acer negundo and always found less organic matter, especially nitrogen compounds. Thus in Acer negundo the following differences were observed :— Albino leaves, Green leaves. Water. 82.83 2.40 Organic substances. 15.15 24.22 Ash. 2.02: 3.08 By the puncture of aphides, scale insects, leaf hoppers, etc., the attacked parts show partial starvation, sugar and nitrogenous matter being removed, and thus, as Woods found again, an increase of oxidizing enzymes results. In short, there is no doubt that the oxidizing enzymes stand in close relation with many diseases of plants, This has led me to examine whether in the mulberry dwarf disease, these enzymes are also produced in abnormal quantities and stand in causal relation to it. This will prove of interest not only for further explanations of the phenomena of the mulberry dwarf disease, but also as a further contribution to the knowledge of the oxidizing enzymes. The tests for oxidizing enzymes in’the mulberry plant were made exactly after Loew and Woods, The details are as follows: a) Oxidase. Oct. 2. Several samples were gathered at Komaba, and tested in the fresh as well as in the air dry condition. The diseased leaves contained always more oxidase, sometimes four or five times as much as the healthy ones. The air dry and powdered leaves were at first treated with alcohol and ether, the residue dried, ground in a mortar, and made into a fine paste, with addition of some water. The paste was then diluted and filtered, and to the clear filtrate were added a few drops of guaiac tincture. A difference was at once observed between 1 Journal of the Chemical Society Vol. XIII. London 1893. MULBERRY-DWARF TROUBLES IN JAPAN. 271 the healthy and diseased leaves, thus the filtrate obtained from the latter producing at once a dark blue color, while in the filtrate obtained from the healthy leaves the coloration set in much more slowly and increased but gradually. 2. Oct. 29. Roso variety, gathered at Komaba. The diseased leaves contained nearly two and a half times as much oxidase as the healthy ones. As the leaves were still healthy and the assimilation process was still energetically going on and as there was no noticeable quantity of tannin and albumin, the colorimetric comparison must be considered reliabie. It is also to be noticed here that the diseased leaves turned very quickly reddish brown on being ground in a mortar with addition of some water, chlorophyll and other organic substances being changed in but a few minutes, while in the case of the healthy leaves, the change of color was very gradual, the green color of chlorophyll being retained for a long time. This evidently shows the strong oxidizing power of the diseased leaves. It was generally observed that the growing leaves in full activity and full assimilation energy, contained always less oxidase than the old leaves. Very young plants, however, contained sometimes much more oxidase than the full grown ones. So it is very probable that the activity of oxidizing enzymes is inversely proportional to the energy of assimilation. 3. Oct. 5. Akagi-variety at Nishigahara. The diseased leaves contained much oxidase but the difference from the healthy ones was not so marked here. 4. Oct. 27. Other varieties behaved as follows: Yeijiwase Diseased leaves contained 2-3 times as much oxidase. Ichihei 53 fy 5 6-7 = as Roso “6 3 6 2-25 5 5G Tsuruta os 3 » alittle more but the difference not very marked. Nezumigayeshi ; 3 Ay 2 3 3 Shimanouchi a a a 2 = vs Jamonji - Fr Fh 2, > Akagi 3 x », a little more but the difference not very marked. Yanagita - Fr », difference not very marked, Takasuke 5 5 ,», difference not very marked, Sometimes the difference was not very marked since the extract was too concentrated, but after a proper dilution, the 272 U. SUZUKI. difference became very noticeable, sometimes the difference was heightened when the extract was heated to 70°C for five minutes, thus retarding the activity of oxidase. But except in a few cases, this procedure was hardly necessary, the differences being plainly noticeable. ~ 6) Peroxidase. Peroxidase, as already mentioned, does not produce a blue colour on simple addition of guaiac tincture, but it requires also some hydrogen peroxid, It always accompanies oxidase in the mulberry leaves. Its resisting power against heat is, however greater than that of the latter. After destroying the oxidase at 75—80°C, the peroxidase can still produce a distinct blue coloration with hydrogen peroxid and guaiac tincture. The coloration was always much darker than that of the oxidase and somewhat greenish. We can also prove the presence of peroxidase when mixed with oxidase by properly diluting the extract. Thus, the extract was divided into two parts, to one portion was added only guaiac and to the other guaiac and hydrogen peroxide. After a few minutes, a very marked difference was observed; the blue coloration being several times more intense in the latter. Further, by heating to 70°C, the power of oxidase is largely weakened and the subsequent addition of hydrogen peroxid produces a distinct difference after a few minutes. The opinion of Spitzer, that peroxidase can cause the developement of oxygen from hydrogen peroxide is erroneous, since the activity of catalase, i.e., the power of decomposing hydrogen peroxid, is entirely lost by heating to 7o—75°C for five minutes, while still a distinct blue coloration may be produced by the addition of guaiac and hydrogen peroxid.' In the mulberry leaves I found always a good deal of peroxidase. The comparison of the peroxidase reaction in healthy and diseased leaves showed the following result : Oct. 27. gathered at Nishigahara. 1 Hydrogen peroxide is for our purpose conveniently prepared by dissolving 6 grams of sodium peroxid in 1co c.c. water and exactly neutralizing this solution with dilute sulphuric acid. MULBERRY-DWARF TROUBLES IN JAPAN, 273 Takasuke Diseased leaves contained 2 tim2s as much or more peroxidase, Akagi ” ” ” ” ” Roso i Pa 4-5 times more, healthy ieaves contained only traces. Jumonji i » 2 a Pe Nezumigayeshi i , 2D - *p Tsuruta s s 2 55 * Shimanouchi . FH much, while the healthy contained only traces, Yanagita 3 3 more Yeijiwase af 7 very much Tchihei - * af A Oct. 29. Roso. At Komaba. Diseased leaves contained very much. We see from the above results that the diseased leaves contain always much more peroxidase than the healthy ones ; but here I must mention that all the above samples were taken in October and never earlier in the year. It will be necessary to repeat these experiments once more with the leaves of early summer, though I have no doubt that essentially the same results will be obtained. The root bark of the diseased plants also contains sometimes much more oxidase than that of the healthy ones. Some experiments were also made to see whether zymogens of the oxidizing enzymes exist in the plant cells, but no decisive result has thus far been obtained. As we have already stated, the transportation of starch in the diseased leaves is very slow and sometimes becomes almost insignificant and much of it is therefore retained in the leaves. This ts one of the most characteristic phenomena of the mulberry dwarf disease. This fact was repeatedly observed by Prof. Miyoshi by means of the jodine test. I have also stated in my former report that much of the assimilation products in the diseased leaves remains unchanged there. Miyoshi gathered the leaves in the early morning and at 3 o’clock in the afternoon, and compared the amount of starch. He found in the healthy leaves almost always no starch in the morning but very much in the afternoon ; in the diseased leaves, however, he found no essential difference in the starch content at different hours of the day. This shows that the starch formed in the healthy leaves during the day time is completely dissolved during the night and for the most part transported to other organs, while one portion is consumed by the respiration process. The migration of starch 274 U. SUZUKI. in the healthy leaves is remarkably rapid; thus, I have very often observed in October that in rainy or cloudy weather or even in the evening shortly after the sun had set, starch was no longer visible, and only in fine weather when the leaves were exposed to strong sun light, the accumulation of starch in the leaves was plainly observed. The following table also shows very marked differences between healthy and diseased leaves: Average composition of healthy and diseased leaves (15 samples). In 100 parts of dry matter : Healthy. Diseased. Ratio. Crude protein 31.47 25.76 100 : 81.8 Crude fat 4.42 3.80 100 : 86.0 Crude fibre 10.00 8.14 100 : 81.4 Other compounds, chiefly starch 47.97 57-60 100 : 120.0 Crude ash 8.52 7-75 100 : 91:0 We see from this table that the starch content is markedly increased in the diseased leaves. I have also repeated the experiment of Prof. Miyoshi; 0.1—0.2 grams of dried and pulverized leaves were boiled for a few minutes with 10 c.c. water to pastify the starch. Upon addition of a few drops of iodine tincture, the following results were obtained. Starch content. Healthy. Diseased. Oct. 2 Roso None Much 55 6 Jumonji 35 3 >» 5-6 Akagi Morning 6 o'clock, 25 3 Noon) 25 5,. much , Evening 6 ,, Almost none s » 27- Ichihei none 35 FN » » Nezumigayeshi very little . +» 9» Lakasuke Almost none - 43, 9 33) paAkagi none S » » LYanagita 5 = gathered 10 a.m. cloudy day. » x» Jumonji * & +» 3) Shimanouchi Ba 3 49° oy LSuratal 5 5 ” ” Roso ” > The same experiment was repeated with several other samples, always with the same result. I tried to see how long it would take to use up the starch deposited in the diseased leaves when kept in the dark, the assimilation process MULBERRY-DWARF TROUBLES IN JAPAN. 275 being completely prevented. For this purpose, a healthy and a diseased plant were selected. A portion of the leaves was taken from each plant for control (a) (Oct. 11) while the entire plants were then covered with a large black paper cylinder and thus kept deprived of light for two weeks, when leaves were again gathered (4). The iodine test gave the following results : (@) (2) Healthy very little none Nezumigayeshi (1) 1 Diseased very much Almost none Healthy ’ Almost none none ” 2 | a very much Almost none Ogon (Normal condition) Healthy very little neue (Not kept in the dark) | Diseased very much very much Thus even after two weeks in darkness, traces of starch were still present, while in the healthy leaves, the starch disappeared completely in one night. I have also endeavored to find out whether the migration of nitrogenous compounds in the diseased leaves is also retarded like that of starch. It is a well known fact that the nitrogen salts absorbed from the soil, are converted chiefly in the leaves into proteids. According to my observation! the synthesis of proteids chiefly goes on in the leaves during the day time, whereas during the night they are again split up into soluble amido-compounds which are transported to the other organs, like starch is in the form of sugar. I have also observed that in the young growing leaves the migration of these amido- compounds during one night can clearly be shown by analysis ; the absolute quantity of nitrogen in the leaves gathered in the morning is considerably less than in the evening. As to the mulberry tree my experiment was made a little too late in the season, and the migration intensity was too weak to show a sufficient difference after one night. Hence I adopted the following way: In the late autumn, when the activity of the cells decreased and the absorption of nitrogen compounds became rather slow, a healthy and a diseased plant were selected ; a portion of the leaves was taken from each plant and dried directly in the air bath at 50°C, while another 1 Bulletin of the Coll. of Agriculture. Komaba, Tokyo. Vol, III, No, 3. 276 U. SUZUKI. portion of the leaves was gathered from the same branches 10—14 days later. Both samples were now carefully compared in regard to their nitrogen contents. As in this period nitrogen compounds migrate from the leaves into other organs in larger quantities and more quickly than they are formed by synthesis, the leaves must show a considerable decrease in the amount of nitrogen, and this decrease must be greater if the migration is quicker. This method would, of course not be applicable in the growing season, when the synthesis of proteids in the leaves surpasses the intensity of transportation. Another method of comparison was as follows: In late autumn, portions of leaves from ahealthy and a diseased plant were taken and directly dried at 50°C, while the plants were covered with large black paper cylinders and kept thus in the dark for 10—14 days. Hereby the synthesis of proteid, was suppressed, since glucose, the product of carbonic acid assimilation is hereby absolutely required. The results were as follows :— Oct. 15 —26. Takasuke. Kept normal, not in dark. Healthy. Diseased, Oct. 15 Oct. 26 Oct, 15 Oct. 26 INumiberiof leaves... ..ke-o-e. 26 25 25 25 DEIN sposodbaocodoon 48.8 41.3 18.80 20.6 Diyawel@htreeeeedesressesses 19.711 16.309 6.948 6.816 Moisture eredscrmasstereeccceret 59.6% 60.5 63.1 66.9 Drysmatteys apace 40.4% 39.5 36.9 33.1 Fresh weight of one laef, ... 1.88 1.65 0.75 0.82 DISA OME 25 hy gg oon 0.76 0.65 0.28 0.27 Ratio of dry matter ,,,, ...! 100.0 85.5 100.0 096.4 Rotalenitcocenwese see 0.0293 0.0223 0 00997 0.0103 This table clearly shows that the nitrogen compounds are transported far more quickly from the healthy leaves than from the diseased ones. In the former case nearly 14.5% of the dry matter and 23.9% of the nitrogen disappeared from the leaves during 11 days, while in the latter case only a decrease of 49% of dry matter and no decrease of nitrogen was observed. MULBERRY-DWARF TROUBLES IN JAPAN, Leaves from another variety served fcr a be Octo Tr. similar experiment. Control (a) ; Leaves 14 days in darkness (0). Nezumigayeshi (I). Healthy. Diseased. Pe) 08) 5 a Cat ee iCO) Number of leaves 25 25 25 25 Dry weight of one leaf 0.78 0.552 0.46 0.381 Ratio of dry matter in one leaf 100.0 70.8 100.0 82.8 Total nitrogen 4.64 4.92 4.04 4.39 Total nitrogen in one leaf 0.0362 0.02706 0.01858 0.01678 Ratio of total nitrogen in one leaf 100.0 Hs! 100.0 90.0 Decrease of nitrogen during 14 days; Healthy 100: Diseased 40.1. Nezumigayeshi (2). Kept in dark 11—25. 277 Healthy. Diseased, Oct. 11 Oct. 25 Oct. 11 Oct. 25 Nimbertotileaves\:cos.seseceradns 30 30 30 30 Dry weight of one leaf.........-.. 0.81 0.637 0.32 0.29 Ratio of dry matter in one leaf.) 100.0 78.7 100.0 90.5 sNotalimitnoxenieesseanesnee seston eee 4.53 4.78 3.61 3.76 Total nitrogen in one leaf,........ 0.036g 0.03044 0.01155 0.01090 Ratio of total nitrogenin one leaf} 100.0 83.0 1CO.0 94.3 Decrease of nitrogen during 14 days; Healthy 100: Diseased 83.5. c). Ogon, Kept normal, not in the dark. 11-25. Healthy. Diseased. Oct. 11 Oct. 25 Ocenia Oct. 25 Number of leaves ...........000000 30 30 30 30 Dry weight of one leaf,........... 0.83 0.60 0 30 0.26 Ratio of dry matter in one leaf.) 100.0 72.2 100.0 81.8 Mictalniinocenmy, ssc. sicsessnserse 403 4.05 | 332 3.86 Total nitrogen in one leaf......... 0.003345 0.00243 0.09106 0.COI004 Ratio of total nitrogen in one leaf 100,0 72.6 100.0 95.2 | Decrease of nitrogen during 14 days ; Healthy 100 : Diseased 27.6. 278 U. SUZUKI. These experiments clearly show that the migration of nitrogen compounds in the diseased leaves is very much slower than in the healthy ones.1_ These observations perfectly agree with the fact that the stems, roots and dormant buds in the diseased plants contain always very little reserve materials during the winter. On the existence of diastase tn mulberry leaves. On Oct. 5, leaves gathered at Komaba were dried at 30—40°C, pulverized and ground well in a mortar with some water, and then, after addition of some more water, filtered after 2—3 hours. The extract was devoid of reducing sub- stances. A few c.c. of this extract was added to a highly diluted starch paste and kept at 50—55°C for half an hour. A small amount of reducing sugar was now Clearly recognized in the extract of the diseased leaves, but only a trace in that of the healthy ones (The healthy leaves contained no starch, while the diseassd contained much.) Oct. 6. Jumonji, at Nishigahara, dired at room temperature and powdered. Here neither the healthy nor the diseased leaves showed any diastatic power (Here the healthy leaves contained no starch, while the diseased contained it moderately.) Oct. 6. Akagi at Nishigahara. Diastase. Starch content. Healthy. Diseased, Healthy. Diseased. Morning 6 o’cleck none much none much INGonwar2 es, none much very much much Evening 6 ,, somewhat somewhat somewhat much As the boiled extract in beth cases produced no sugar, the presence of diastatic ferment in the diseased leaves is most probable. Oct. 2. Roso at Komaba. 5 grams air dry sample were finely pulverized, ground with addition of water, diluted to 100 c.c., let stand for 24 hours and filtered. 2 c.c. of the filtrate was added to 1oc.c. of starch paste and kept for one and a half hour at 50°C. The extract of the diseased leaves reduced nearly 10 c.c. of Fehling’s solution, while that of the healthy leaves reduced only half as much. In the control test, where 1 I have not yet proved the existence of a proteolytic enzyme in the mulberry leaves, yet its presence is very probable and the oxidizing enzymes may probably inhibit also the action of it, as it is the case with diastatie ferments, MULBERRY-DWARF TROUBLES IN JAPAN. 279 the extract was boiled before the addition of starch paste no reducing sugar was produced. The presence of the diastatic ferment was beyond doubt. Oct. 12, Akagi (cut in autumn) at Nishigahara. The root bark of both, healthy and diseased plant, contained originally some reducing sugar. That of the healthy one however con- tained considerably more diastase than that of the diseased. Oct. 12. Takasuke at Nishigahara (cut twice in a year and deprived of the young leaves and hence diseased). The root bark, of both, the healthy and diseased plants contained very little diastase. Oct. 12. Akagi (cut in summer) at Nishigahara. The root bark, both of the healthy and diseased plants contained little diastase. We see from the above experiments that the diseased leaves contain generally more of diastatic ferment than the healthy ones, but no distinct relation was observed with the root bark.' Woods has observed that oxidase can inhibit the action of diastase; This depends of course much upon the relative quantities of oxidase and diastase. In the cases above described the oxidase was probably too diluted to prevent entirely the diastatic action. On the existence of catalase tn the mulberry leaves. Catalase was found by O. Loew in 1900 in the leaves of the tobacco plant; He first observed that the tobacco leaves contain an insoluble enzym which liberates oxygen gas from hy- drogen peroxid. Physiologists have until now attributed this action on hydrogen peroxid to every enzym. Loew confirmed by careful investigation the different and independent nature of the enzym and proved that diastase or any other enzym in their purest state never possess this property. He extended his observation to several other plants and at last reached the conclusion that the enzym is contained in every living cell of plants. It has decidedly the important function to destroy every trace of the 1 The apparent contradiction between the existence of diastase and that of much starch in the diseased leaves is easily explained on the ground that the sugar formed by diastatic action is not transported to the trunk and hence that it is always reconverted into starch by the chloroplasts. We further have to concede that there may be in the diseased leaves, as long ‘as they are fresh, only the zymogen of diastase, which in my experiments just mentioned, generally was transformed into diastase itself. 280 U. SUZUKI. poisonous hydrogen pcroxid that may be formed as a _ by- product in the process of respiration. I thought it of some interest to investigate whether there is an abnormal increase of this enzym in the diseased mulberry leaves. I took at first I gram of air dried and powdered leaves, mixed with 30c.c. water and added 5 c.c. of freshly prepared hydrogen peroxid of 2—3% strength. The development of oxygen gas was here so energetic that I used in the following tests only 0.1 gram of the dried leaves. Temperature and the reaction of the sample have much influence upon the development of oxygen, 30—40°C and neutral or slightly acid reaction being preferable.! Great care was taken to have exactly the same conditions in all tests. The following table shows the results. I. Akagi leaves gathered on Oct. 5, 0.1 g. air dry. Morning. Noon. Evening. 6 o'clock. 12 o'clock. 6 o’clock, Minutes. | Healthy. Diseased. | Healthy. Diseased. | Healthy. Diseased, (c.c. Oxygen.) (c.c. Oxygen.) (c.c. Oxygen.) 5 2.3 2.0 oy 3-7 2.5 4.0 fe) 3.4 42 40 i 6.8 4.7 6.6 20 5.0 9.0 8.0 11.5 9.2 11.2 30 6.8 11.2 10.7 14.5 Wt | 140 oe Roso leaves, fresh 0.5 grams. Oct. 21. Minutes. Healthy. Diseased. 5 HONG: De 10 10.0 4. 20 14.5 6.2 30 16.0 7-7 1 It is to be mentioned that by frequent shaking, the development of oxygen is very much accelerated. In this experiment, the mixture was shaken on the addition of hydrogen peroxid and afterwards left fo itself. In some cases the mixture was shaken at the last reading, MULBERRY-DWARF TROUBLES IN JAPAN. 281 a Shimanouchi, Oct. 27. Minutes, Healthy. 5 1.5 fe) BES 20 4.2 39 4-9 40 55 Ale Takasuke, Oct. 27. Minutes, 5 Io Healthy. 2.6 3:3 3.8 4.0 Diseased. 3.0 3-5 4.0 43 5.0 Diseased. 5. Jumonji, Oct. 27, air day 0.1 gram. Minutes. Healthy. 5 2.0 1 fo) 2.7 20 2.7 30 (2.8 After frequent shaking 50 6. Akagi, Oct. 27, air dry 0.1 gram. Minutes, Healthy. 5 4.4 10 4.5 20 45 After shaking, 4.3 1 The healthy leaves were here still very active, but the rather poor condition, Diseased. 3:3 1A 45 BED) We Diseased, 0.8 1.0 1.6 5:3 diseased ones were in a U. SUZUKI. No ioe) iS) 7s Roso, Oct. 27, air dry 0.1 gram. Minutes, Healthy. Diseased. 5 1.0 0.9 10 7 1.6 20 2.7 2.7 After shaking, 5-7 5-5 8. Tsuruta, Oct. 27, air dry 0.1 gram. Minutes. Healthy. Diseased. 5 0.5 1.3 10 1.5 2.7 20 2.8 aay) 3° 35 44 After shaking. 6.5 8.0 9. Yanagita, Oct. 27, air dry 0.1 gram. (Temp. 14°.) Minutes. Healthy. Diseased. 5 0.4 1.5 if) 1.0 2.6 20 nay 3.4 39 a ae 40 : 2.2 4.2 60 2.4 4-7 After shaking. 34 6.7 10. Nezumigayeshi, Oct. 27, air dry 0.1 gram. (Temp. 14°). Minutes. Healthy. Diseased. 5 3.7 1.0 10 4.3 1.8 After shaking. no more. no more. MULBERRY-DWARF TROUBLES IN JAPAN. 283 Ii. Yeji wase, Oct. 27eair dry 0.1 gram. Minutes. Tlealthy. Diseased. 5 1.0 6.3 10 1.6 6.7 After shaking. 2.3 9.7 12; ~ Ichihei, Oct. 27;amadry 0.1 gram. Minutes. Healthy. Difeased, 5) 6° 3-7 10 5.1 5:7 20 6.9 em 30 7.6 7.8 After shaking. 8.9 8.9 i355 Jumonji; Oct; 277 Minutes. Healthy. Diseased. 5 7.0 6.5 10 8.0 12,0 14. Akagi, root bark, Oct. 24, air dry 0.5 gram. (Teimip..21°). Minutes, Healthy. Diseased. 5 4.5 29 10 4.6 3:3 30 4.6 3-7 After shaking, eap Ten 15. Akagi, (cut in Autumn) root bark, Oct. 12, air dry 0.5 gram. Healthy. Diseased, After 2 hours. 1.2 6.0 284 U. SUZUKI. 16. Jumonji, root bark, air dry 0.5 gram. Healthy. Diseased. After 2 hours. 6.0 4.0 17. Akagi, (cut in summer) root bark, air dry 0.5 gram. Healthy. Diseased. After 2 hours. 1.0 4.0 We see from the above tables that the diseased leaves contain generally much catalase, but there are still some exceptions; and further the air dried leaves develop oxygen always less energetically than the fresh ones, hence I can not draw any safe conclusion until further experiments can be made next summer. According to Loew, there are two kinds of catalase, one soluble, and the other insoiuble in water, the latter being perhaps a kind of nucleo-proteid. In the leaves of the mulberry there exist also these two kinds. The soluble catalase develops oxygen very quickly but stops very soon. On the contrary, the insoluble catalase develops oxygen slowly but continues longer. The action of soluble catalase is almost destroyed by 5 minutes heating to 70°C, but after standing for 2-3 hours, the activity somewhat returns and from 0.1 gram., 5 cc. of oxygen were developed in two hours by frequent shaking. In another experiment the pulverized leaves were heated to 70°C for five minutes and filtered, the filtrate at that time had no activity, which however after standing for 48 hours developed about 5c.c. oxygen intwo hours. This was also the case when some chloroform was added as an antiseptic. Influence of cutting upon the decay of roots. The decaying of small rootlets and finally of the entire root system is one of the most remarkable phenomena of the mul- berry dwarf disease, and many have believed it to be due to parasites. But as I have already discussed in my former report, MULBERRY-DWARF TROUBLES IN JAPAN. 285 the principal cause of the disease is never of parasitic nature. It is exclusively the cutting in the growing season that causes the disease. Small rootlets which develop vigorously in the growing season can only support their activity by the supply of organic nutriments furnished by the leaves. Therefore the cutting off of the branches and leaves in the growing season stops the supply of food to the roots. At first the small rootlets starve and die off. New rootlets however come forth again after the ‘new shoots had reached a certain height and acquired power of assimilation. I have calculated that the new shoots coming from the cut stocks, rely upon the reserve material in the roots even as long as 4o days after cutting and until they have reached the height of 50—60cm, Therefore it is very evident that if the new shoots had used up all! the reserve materials in the roots before the development of new rootlets which can absorb the soil nutriments freely, then the new shoots must naturally suffer from starvation and disease must be the final result. I have proved this fact experimentally by examining the state of root development during the last summer. In the beginning of June, when the leaves were in full development, the growth of roots was very marked and small rootlets (so called white roots) were well developed forming a net work. After cutting in June (June 5—10) the growth of these rootlets was completely stopped and the white fresh appearance gra- dually turned yellow and reddish brown and finally the roots decayed. Even 30 days after cutting, no development of new rootlets was yet observed, while the development of the rootlets of the plants not cut was more and more advanced. In two weeks from cutting new shoots had developed and _ reached 50—60 centimeter in four or five weeks, but new rootlets were not yet visible, Later on they gradually appeared but were very scanty and until autumn they failed to achieve the nortnal development. These facts were observed with many varieties and under different conditions and agree perfectly well with my former assumption that the new shoots developing almost directly from the roots rely upon the reserve materials in the roots even after reaching the height of 50—60cm. Thus it is very evident that the exhaustion of reserve materials may very often take place before the new shoots can be provided with new mineral nutriments from the soil and this circumstance again retards 286 U. SUZUKI. the development of the entire root system, finally causing its starvation and death, the death being followed by decay which spreads*gradually into the larger roots. I have observed very remarkable cases which furnish a convincing proof. The rows ofa mulberry plantation were alternately cut at the beginning of June; a remarkable difference was here already observed at the end of July when the roots were examined. The normal plants had white rootlets like net works while the plants of the cut rows had no white rootlets whatever. Also the thickness of the root bark showed a considerable difference. Thus it is very evident that the power of developing new rootlets is more and more retarded in consequence of the successive cuttings in the growing season. On the quantity of reserve starch. I have already shown by careful analysis that the diseased leaves and stems contain always less nitrogen, and Miyoshi has also proved that the diseased stems and buds contain consider- ably less starch than the healthy ones. As the migration of starch and nitrogenous compounds from the diseased leaves is but very slow, these phenomena are naturally to be expected. But according to my assumption, the deficiency of reserve materials is not only the effect of the disease, but it must become the cause of its further development In other words, those plants which had used up the reserve materials must first show the disease. The following observations were made to test this view. 1). Jumonji, Sept. 30. The root bark of the healthy plants contained much starch but that of the diseased contained none. 2). Akagi, Sept. 30. The root bark of healthy plant contained some starch, but that of the diseased not. As this plant was cut at the end of August and the new shoots were still very young, no new rootlets had yet developed. Sufficient time has not yet elapsed for the new shoots to prepare the reserve starch, on the contrary they were still depending upon the reserve materials which had been prepared before cutting. We can say that the first appearance of the disease is caused by the exhaustion of the reserve materials in the roots. 3). Agaki(cut in autumn) was examined on Oct. 20, the MULBERRY-DWARF TROUBLES IN JAPAN. 287 disease had appeared shortly before the examination was com- menced. The healthy roots contained much more starch than the diseased ones and further there was a remarkable difference in the thickness of the rooot bark, that of the diseased one being only 1—4 as thick as that of the healthy one. 4). Takasuke. Some of the plants were subjected to cutting twice in summer and the new shoots coming were repeatedly deprived of the new leaves. Upon the appearance of the signs of a severe disease they were examined on Oct. 12. Those which remained still healthy under the same treatment, contained some starch, while the diseased ones were almost entirely deficient of it, a proof that the plants which used up the reserve materials first became also first diseased. We eLukagi ro) < plant ? (1) Etiolated seedlings. The seeds were first soaked in water for several days and kept in pure sea sand in perfect darkness at the temperature of 15-30°C. for several months. When the seedlings had reached 10-15 cm. and had two or three leaves, a portion (@) was analysed after washing ; the rest (4) was analysed after a further period of 17 days. eedlings (a) freed from cotyledons had small white leaves of 3-6 cm diameter, stems 6-10 cm, root 6-12 cm, dry weight=130 grams, Seedlings (0) 2-3 leaves opened, stems 13-16 cm, roots 6-12 cm, number of seedlings 228. dry weight=21.85 grams. (Dry weight of 100 seedlings=9.58 grams). In 100 parts of dry matter. (a) (2) Total nitrogen. 3.95 3-49 Albuminoid nitrogen. 1.54 1.33 Theine nitrogen. 0.127 0.127 Nitrogen in other forms. 2.28 2.03 (Theine) 0.48 0.48 Of too parts of total nitrogen. Total nitrogen. 1c0.0 100.0 Albuminoid nitrogen. 38.9 38.1 PHYSIOLOGICAL KNOWLEDGE OF THE TEA PLANT. 201 Theine nitrogen. 3.2 3.6 Nitrogen in other forms. 57.9 58.3 Every roo seedlings contain 0.046. g theine. (2) Seedlings grown in day light. Average leneth of stem 10 cm, average length of roots 10 cm, 3-4 leaves opened, the largest leaves 3 cm in length and 2.5 cm in width. Number of seedlings 235. Dry weight of seedlings (free from cotyledons)=20.44 g. (Dry weight of 100 seedlings=9.1 grams). Dry weight of cotyledons=71.2 (Dry weight of cotyledons of 100 seedlings=31.6 grams) (2) (2) Seedlings free from cotyledons. Cotyledons. In too parts of dry matter. Total nitrogen. 3.38 1.47 Albuminoid nitrogen. 1.78 0.79 Theine‘nitrogen. 0.174 0.013 Nitrogen in other forms. 1.43 0.67 (Theine). 0.66 0,05 In too parts of total nitrogen. Total nitrogen. 100.0 100.0 Albuminoid nitrogen. 52.7 53-7 Theine nitrogen. Bee 0.9 Nitrogen in other forms, 42.1 45.4 Every 100 seedlings contain. Total nitrogen. 0.307 0.465 Albuminoid nitrogen, 0.161 0.250 Theine nitrogen. 0.158 0.0041 Nitrogen in other forms. 0,130 O.2Tr(*) In another experiment the effect of sodium nitrate was tested. (3) Seedlings were kept in darkness until they reached 10-15 cm and afterwards exposed to day light for 16 days keeping them in half saturated gypsum solution. Control seedlings: (@) Number of seedlings 200. Dry weight of leaves 4.11 g. Dry weight of leaves of 100 seedlings= 2.00 g. Dry weight of stems and roots 23.5 g. Dry weight of stems and roots of 100 seedlings=11.8 g. Nitrate seedlings (0) were watered with a 0.2% sodium nitrate and half saturated with gypsum. Number of seedlings 340. Dry weight of leaves 6.50 ¢. (Dry weight of leaves of 100 shoots=1.gt g). Dry weight of (*) This agrees essentially with the observations of Clautriau; Nature et Signifi- cation des Alcaloides Végétaux Brussels 1900. p. 79- 292 U. SUZUKI. stems and roots= 36.5 ¢ (Dry weight of stems and roots of 100 seedlings=10.7 g). ; In 100 parts of dry matter. | (2) Watered with 0.29% (a) Watered with half Na NO, half saturated (c) sat. Ca SO, with Ca SO, Control Entire Entire eae seedlings) Stems | seedlings Stems an ae (free | Leaves.| and (free | Leaves.| and vee % from coty roots. from coty roots. gest ledons). ledons), Total nitrogen ...... 3.23 6.04 2.76 3.32 5-90 2.86 3.49 Albuminoid nitrogen 1.58 4.06 E15 1.61 3:83 1.22 1.33 Theine nitrogen...... 0.193 | 0.824] 0.082 0.17 0.752 | 0.066 | 0.127 i in other ey ea 1.48 1.16 1.53 1.54 1.32 sy 2.03 ((Pheine)iey.c-- Sponoidoc 0.73 eate 0.31 0.646 | 2.85 0.25 0.48 Total nitrogen ...... 100.0 100.0 100.0 | 100.0 100.0 100.0 100.0 Albuminoid nitrogen | 48.6 | 67.2 41.6 | 48.5 64.9 42.6 38.1 Nitrogen in other t LOLS eee 45-5 19.2 | | Theine nitrogen...... 5.93 | 13.6 | 3.0) meesen 12.7 23 3.6 55-4 | 464 | 22.4 | 55-1 | 58.3 Every 100 seedlings contain. Total nitrogen ...... 0.4484 | 0.1241 B15 | 0.4198 | 0.1128 0.307 | 0.3344 Albuminoid nitrogen | 0.2192 | 0.0840 | 0.1352 | 0.2043 | 0.0733 | 0.131 0.1274 Theine nitrogen...... 0.0266 | 0.0169 | 0.0097 | 0.0215 | 0.0144 | 0.0071 | 0.0121 Nitrogen in other ) | Bey Gos f |, 0.2032 | 0.0237 | 0.1795 | 0.194 tee: 0.169 | 0.1950 PHYSIOLOGICAL KNOWLEDGE OF THE TEA PLANT. 293 (3) Experiments with full grown tea plants. (2) On April 17. tea plants of the college farm about ten years old were deprived of all the old leaves, leaving only small buds. The leaves developed from the buds in full day light, were analyzed on May 16. In 100 parts of dry matter. Stem bark, Buds. Leaves. May 16 Brite old leaves| May 16 April 17. | April 17. | deprived normal Jekenge on April rowth | on April, oe 8 ; MotalimitOgen\y ss. ccc.-rs.cee 1.09 1.23 5.3 5:94 5-77 Albuminoid nitrogen......... 1.06 1.15 4.02 4.60 4.44 seme mitnos enh. eeeas.cecees 0.024 0.045 0.763 0.797 0.723 Nitrogen in other forms ... 0.01 0.03 0.53 0.54 0.61 (heme eres ne dias ocecse vos 0.09 0.17 2.89 3.02 2.74 In 100 parts of total nitrogen. Motalnitrogen!” 7 yeaess.s0--- 100.0 100.0 100.0 100.0 100.0 Albuminoid nitrogen ...... 97.2 93-5 75-7 77-4 77.0 Ane MA(S Tene feo ean osncegnaobee aD S7/ 14.4 13.4 12.5 Nitrogen in other forms ... 0.6 2.8 9.9 2 ‘10.5 (6) On May 1. tea plants about fifteen years old were deprived of the old leaves. Some of these plants were covered with a box to shut out day light while others remained uncovered In 100 parts of dry matter. Leaves Leaves Buds May 16 May 16 May 1. day light, dark. Total nitrogen. 5-47 5-54 5-31 Albuminoid nitrogen. 4.42 4.18 4.01 - Theine nitrogen. 0.562 0.634 1,032 Nitrogen in other forms, 0.49 0.73 0.27 (Theine). 2.13 2,40 3.91 204 U. SUZUKI. In 100 parts of total nitrogen. & Total nitrogen. 100.0 100.0 100.0 Albuminoid nitrogen. 80.8 75.4 7555 ‘Theine nitrogen, 10.3 11.4 19.4 Nitrogen in other forms. 8.9 13.2 5.1 Leaves developed in the dark contain a greater percentage of theine than those grown in day light, but here it must be remarked that the former are very poor in other organic matters. Theine does not increase directly by the application of nitrates as above m= tioned, lence it is not a synthetical product analogous to asparagin; The following table shows the various amounts of theine-n’ ogen in % of total nitrogen in different parts of the tea riants: Bark of Coty- Stems and Leaves stems of ledonsof roots of Etiolated ! praia of full Seed. full grown young young seedlings, thet grown 5 home ae = seedlings, = plants. seedlings. seedlings. plants. none doubtful trace. 0.9 2.3-3.0 3.2-3.6 12.7-13.6 12.5-13.4 Further theine does not stand in any relation to protein production. An observation of Miyachi® also supports this view. He found that theine is a poor source of nitrogen for fungi and that in old leaves theine increases by the process of starvation, that is by keeping them in a dark place, thus :— Theine-nitrogen in % of total nitrogen : Original leaves. Starved leaves. 11.15 13.74 Here the processes of metabolism show more connection with theine production than those of assimilation and synthesis do. It remained however to be decided whether in the presence of a large amount of carbohydrates and in the absence of any other suitable source of nitrogen, theine may be used as a source of nitrogen for building up proteids in the tea leaves. Here the following points have to be mentioned. According to Kellner the quantity of theine in the leaves gradually decreases as they become older. G) Bull. College of Agr. Komaba. Tokyo. volII. No.7. p. 460. (2) Versuchs-Stationen 39. Bull. Coll. Agr. Tokyo, vol I. ~ PHYSIOLOGICAL KNOWLEDGE OF THE TEA PLANT. 295 Leaves gathered Theine nitrogen on in % of total nitrogen. Wary KGig sce PRM icceresycicto's 25's « geyuraqaree 16.5 ually seas SOME ojo Sloco w 0 sd eiiista’s -/etatyone 22.1 Novem berpgommeccasc-c. 10066: ses ccise 10.2 OldvleaVesi@ReWMey U5. s.cse.s se eccwen 8.6 Hence a gradual decrease of theine takes place from July to November not only relatively but absolutely. Since in autumn various products migrate toward the seeds or stems and roots, the decrease of total and theine nitrogen in the leaves in autumn might of course be expected ; and further, as we have found no theine in the seeds and only doubtful traces of it in the stems and roots of the tea plant, and as also there is only very little non-albuminoid nitrogen in the seeds as well as in the stems and roots, we may safely conclude that the theine once formed in the leaves may later again be transformed gradually into proteids. However it is not probable that it is directly used for this purpose, as we have already pointed out, but it seems that the process is an indirect one. Perhaps theine is firse destroyed and its nitrogen is liberated as ammonia, befort protein formation from that source can take place. My investigation permits the following conclusion :— 1) Seeds of the tea plant contain originally no theine. Neither do the proteids of the seeds yield theine by the action of hydrochloric acid. Hence the formation of theine during the germination process can not be due to a mere splitting off from the proteids, but must be due to a far reaching transformation of the products of metabolism. 2) Light seems to have no direct influence upon the forma- tion of theine. Since etiolated shoots as well as the shoots grown in day light, contain it in nearly the same quantities. 3) Cotyledons of germinating seedlings contain also some theine (though very little). 4) Stems and roots also contain a moderate amount of theine though its percentage is considerably lower than in the leaves. 5) Leaves contain the largest amount of theine, its quan- tity being nearly proportional to the development of the leaves. 6) No essential increase of theine was noticed by the ap- plication of sodium nitrate, making it also very probable that 206 U. SUZUKI. theine is not like asparagine a product of synthesis, but one of katabolism. 7) The bark of the trunk of the tea plant contains only doubtful traces of theine. But_the dormant buds are moderately rich in it. On the Localization of theine in the tea leaves. By U. Suzuki. Thus far nobody has succeeded in localizing the theine in the tea leaves, while in regard to many other alcaloids, the investigations of Errera,™ Maistriau and Clautriau, have shown that they are localized 1st. in the very active tissues: the vegetative tissues and the embryo. 2nd. Along the fibrovascular bundles (sourtout prés de la région libérienne et dans cette région méme ;) 3rd. in the epidermis: cortical layers and the shell of fruits etc. and generally all the outer tissues for pro- tection. 4th. in the special secretary organs. In regard to theine, Clautriau has not arrived at any satisfactory conclusion. This author says™ ‘‘J’ai en recours d’abord a la micro- chimie, et j’ai tenté de déterminer la localisation de la caféine dans le caféier ou le Thé. Malgré de nombreux essais sur un matériel tres abondant, je ne suis pas arrivé 4 mon but.” and he says again ‘‘11 n’en résulte pas que je considére la localisation microchimique de la caféine comme impossible, mais elle présente de grandes difficultés et demande encore de longues études préliminairés.” I have undertaken the same question as Clautriau and have solved it satisfactority in the following manner: At first I applied phosphotungstic acid upon sections of the leaves, but this produced more or less precipitate in all the cells, but decidely more in the epidermal cells. Since proteids are thus precipitated just as well as alkaloids, this reaction gave no decisive answer, I afterwards applied a concentrated solution of iodine in potassium iodide, but also in this case no decision could be reached. This reagent however acts decidely better (@) Errera; Akademie Royale de Belgique. Bull. No. 3. 1887. (2) Nature et signification des Alkaloids Végétaux Brussells 1900. p. 55 & 56. 298 U. SUZUKI. LOCALIZATION OF THEINE. on theine after the addition of a little hydrochloric or sulphuric acid and on application of this acidulated solution the epidermal cells gave decidedly a much more intensely brown color than the others which remained merely slightly yellowish. The most decisive answer however was reached by the following test. When a section of the leaf was placed in a 0.5% theine solution, the cells of the spongy and palisade tissues exhibited a very marked formation of proteosomes. This fact appeared rather strange to me, since the coffeine content of the fresh leaves is certainly more than 0.5% and thus the formation of proteosomes ought to take place in the fresh leaves without any application of coffeine.® Since however no proteosomes were observed in the epidermal cells (which are in other plants often very rich in the stored up active albumen) by the action of the 0,5% coffeine solution, there remain no other conclusion than this, that these cells contain no active albumin and at the same time contain all the theine of the leaves. A further test with tannin proved this conclusion to be correct. A section of the leaf was left for two days in a tannin solution of about 3-4% ; hereby a voluminous precipitate consisting of minute globules was produced in the epidermal cells, while the other tissues of the leaves showed only a slight turbidity. To prove that this precipitate consisted of tannate of theine was furnished by the application of highly diluted ammonia, which dissolved it at once. This affords an easy way of distinguishing the precipitate from minute proteo- somes, which latter solidify on the absorption of ammonia, and do not dissolve at all in it. Hence there can be no longer any doubt that the thecne in the tea leaves is localized in the epidermts. (1) Compare: IX and X of Chemische Energie der Lebenden Zellen by O. Loew Munich. 1899. @) This led me at first; to the supposition that the theine in the fresh leaves, was present not in the free state but in a loose combination with some other substance. Hence I extracted the green leaves, after crushing them, directly with chloroform, but I obtained about the same quantity of theine for the dry matter as with the tea of com- merce, The theine is therefore contained in the leaves in the free state. (3) It is well known that some authors made the great mistake of confounding tannate of theine with true proteosomes, hil ‘ar = EJ hal} By hl iy au xX f= & x ARETE SCRE | bot eS soR RGR] HS RMIRES VEE SCRMS | hILNeS ce ey ae Uber die Bekampfung der Mauseplage durch den Mereshkowsky’schen Mausetyphusbacillus. VON Y. Kozai. Eine der gréssten Plagen der Landwirthschaft ist bekannt- lich die Feldmaus. Dieselbe verrursacht wegen ihrer enormen Fortpflanzungsfahigkeit! und ihrer staunenswerthen Gefrassigkeit oft einen so grossen Schaden auf den Kulturfeldern, dass das Bebauen der letzteren iiberhaupt nicht méglich ist. Die Feld- miuse, die in diesem Frihling in der Provinz Ibaraki, nord- éstlich von Tokio, in ungezahlter Menge auftraten, zeigen in mancher Hinsicht Unterschiede von den in Europa verbreiteten (Arvicola arvalis) und gehoéren nach Herrn Prof. Sasaki? zu einer neuen Rasse, der dieselbe mit dem Namen ,, Arvicola hatanesumt”? belegt. Dieselbe ist nach dem genannten Autor* 10-13 cm. in Kérperlange und besitzt einen behaarten Schwanz von 3.2-4.2cm. Der Leib ist untersetzt, der Hals dick, der Kopf etwas rund, die hautigen Ohren sind kurz, die Augen klein und dunkelbraun, die Fiisse und Zehen fein, die Krallen kurz, der Schwanz ist ziemlich abgerundet. Die dicht anlie- gende und sanft anzuftiihlende Behaarung ist auf dem Oberk6érper grau bis rothbraun, auf der Unterseite dagegen weisslich. Der Bau, in welchem unsere Feldmaus ihr Leben fihrt, ist sehr 1 Nach Ritzema-Bos (Thierische Schiidlinge u. Niitzlinge, Berlin 1891.) kann ein Mausepaar unter giinstigsten Bedingungen in einem Jahre iiber 200, nach Brehm (Thierleben, Bd. II.) und Danysz (Maladies contagieuses des animaux nuisibles, Paris 1895.) tiber 350, und nach Ritdiger (Wien. Landwirthschaftliche Zeitung, 1892, S. 737.) sogar tiber 20000 Nachkommen haben, Dies bezieht sich natiirlich auf die europaische Feldmaus (Arvicola arvalis), 3 Hatanesumi bedeutet Feldmaus, 2 u, 4 Bericht der japanischen landwirthschaftlichen Gesellschaft rgo1. 300 Y. KOZAI. geschickt construirt. Das mit fein zerbissenen Pflanzentheilen sorgfaltig praparirte Nest liegt gewohnlich in den tieferen und sicheren Stellen des Feldes, besonders aber an Fusswegen und unter Gebiischen; von dort ab laufen Abzweigungen in allen Richtungen, so dass das Thierchen notigensfalls sich gut verber- gen kann. Diese Abzweigungen oder Seitenginge liegen ca. 20 cm. unter der Erde und stehen durch zahlreiche, schrag gegrabene Offnungen mit der Aussenwelt in Verbindung. Ge- wohnlich befindet sich in der Nahe des Nestes eine Kammer, in welcher Nahrungsvorrathe wie Getreideahren, Erbsen, kurz zerbissene Wurzelfriichte u. s. w. massenhaft aufgespeichert werden. In der oben erwahnten Provinz wurden nicht weniger als 5000 Hectar Felder von diesem Tierchen mehr oder weniger arg heimgesucht und alle Massregeln um dasselbe auszurotten, so z.B. Vergiftung u. Fangen, erwiesen sich als nicht geniigend wirksam. Bei dieser Sachlage wird man natiirlich auf den Gedanken kommen, ob nicht der seit Léffer mit Erfolg zur Mausevertil- gung beniitzte J/dusetyphusbacillus auch in unserem Fall mit Vortheil verwandt werden kénnte. In der That sind einige Laboratoriums- sowohl wie auch Feldversuche, angestellt von Herrn Dr. Onuwki und Herrn Sanuz1 mit dem J/dusetyphusbacil- Jus, als sehr gelungen zu bezeichnen, denn die im Kafig gehalte- nen Feldmause verendeten durch Jnfection per os in 5-14 Tagen und auch auf den inficirten Versuchsfeldern fand eine starke Abnahme der Mause nach einiger Zeit statt. Inzwischen erhielten wir von der Bezirksbehérde Ibaraki den Auftrag, die Wirksamkeit des Mausetyphusbacillus gegen unsere Feldmaus genau zu prifen und die praktische Verwendung desselben zur Mausevertilgung zu beaufsichtigen. Leider war der in unserem Besitz gewesene Léjjer'sche Bacillus abgestorben und eine frische Kultur konnten wir nicht aultreiben. So waren wir gezwungen, die von den oben erwahnten Herren beniitzte Kultur von unbekannter Herkunft in Anwendung zu bringen. Dieselbe zeigte aber bei naherer Priifung in ihren kulturellen und physiologischen Merkmalen cine gréssere Ahnlichkeit mit dem Mereshkowskyschen Bacillus als mit dem Léffer’schen. Spater theilte mir Herr Dr. Haschimoto in Sapporo mit, 1 Bericht der japanischen landwirthschaftlichen Gesellschaft 1900, BEKAMPFUNG DER MAUSEPLAGE. 301 dass die betreffende Kultur in der That von Jlereshkowsky selbst seinem Institut iiberwiesen worden war. Da dieser Bacillus im ‘Gegensatz zu dem Léffer’schen hauptsachlich zur von seinem Entdecker genau erforscht worden ist, so entschlossen wir uns, um seine Verwendbarkeit zu constatiren, denselben einer einge- henden Untersuchung zu unterwerfen. Zu diesem Zwecke haben wir nicht nur eine ganze Reihe von Infectionsversuchen sowohl im Laboratorium als auch auf freiem Feld ausgeftihrt, sondern auch den namlichen Bacillus in Bezug auf seine kulturellen und physiologischen Eigenschaften studirt. Die dabei gewonnenen Ergebnisse theilen wir im Folgenden mit. Dabei méchte ich nicht unterlassen, den Herren Dr. S. Kusuhara, K. Nakamura und G. Sasada in Ibaraki fiir die freundliche Unterstiitzung bei ‘der Ausfithrung der Feldversuche meinen aufrichtigen Dank aus- zusprechen. Ebenso bin ich Herrn ¥. Susuki, dem Assistenten am Institut, fir seine eifrige Hilfe zum grossen Dank verpflichtet. I. Kuiturelle und Physiologische Eigenschaften des Bacillus. Dieser Organismus stellt im hingenden Tropfen ein ziemlich plumpes Stdbchen mit lebhafter Eigenbewegung dar und lasst sich in dieser Bezichung von dem Léffer'schen Bacillus kaum unterscheiden. Doch ist der erstere in der Regel etwas danger in Form als der letztere und wachst haufiger zu Scheinfaden aus. Sein Verhalten den tiblichen basischen Anilinfarbstoffen gegen- uber bietet nichts Besonderes. Dagegen fallt die Farbung nach der Gram’schen Methode negaizv aus. In gewohulicher Boutllon ist das Wachsthum bei Briitwirme ein sehr kraftiges. Innerhalb der ersten 24 Stunden tritt eine starke Triibung ein und auf der QOberfliche der Fliissigkeit entwickelt sich eine zarte weisse Deckhaut, die beim Schiitteln in Fetzen zerfallt und leicht zu Boden sinkt. Nach 5-6 Wochen wird die Kulturfliissigkeit ganz klar. Die chemische Reaction wird durch das Bacterienwachsthum a/kalisch. In Alteren Bouillonkulturen zeigt sich eine schwache Indolreaction. Re- duction von Nitrat zu Nitrit tritt aber nicht ein. Ein eigen- thiimlicher Geruch, etwa an frisch gelassenen Pferdharn 302 Y. KOZAI. erinnernd, liasst sich bei der Offnung des Kulturkolbens deutlich wahrnehmen. In TZraubenzuckerboutllon ist die Entwickelung anfangs ebenso iippig wie in der gew6hnlichen, bald kommt sie aber zum Stillstand, ohne Zweifel weil die von dem Bacillus erzeugten Sauren das weitere Wachsthum verhindern. Die zuckerhaltige Bouillon schaumt beim Schiitteln, da unter andern etwas Kohlensaure gebildet wurde. In Zvraubenzsuckerbouillon im Gdarungskilbchen findet bei Briitwarme innerhalb 24 Stunden eine starke Triibung statt. Etwa die Halfte des geschlossenen Schenkels ist mit Gas gefiullt, das sich bei naherer Priifung grossentheils als CO, zu erkennen giebt! ; zugleich wird die Kulturfliissigkeit stark sauer. Bei Zimmerwarme geht die Entwickelung langsamer vor sich. Bei den Plattenkulturen in gewohnlicher Gelatine erscheinen am zweiten Tage feine Piinktchen, die nur sehr langsam an; Groésse zunnehmen. Bei schwacher Vergrésserung stellen sich die Oberflachencolonieen als rundliche, ziemlich glattrandige, hellgraue feinkérnige Scheiben dar. Die kleineren Tiefen- colonieen erscheinen bei schwacher Vergrdésserung als unregel- miassig runde, graue Scheiben. Der Nahrboden wird nicht verfliissigt. Bei Gelatinestichkulturen entsteht nach 24 Stunden dem Stichkanal entlang ein weisser diinner Streifen, der ziemlich gleichmissig bis zum Boden fortschreitet. An der Einstichstelle bildet sich eine weisse, diinne Auflagerung, die spater concen- frische Ringe erkennen lasst. Auf der gewihnlichen Gelatine erscheint am zweiten Tage langs des Impfstriches ein weisser, diinner Streifen, der sich langsam weiter entwickelt. Der Rand des Rasens buchtet sich mit der Zeit aus. Bei Agarplattenkulturen entwickeln sich bei Briitwarme innerhalb 24 Stunden ziemlich grosse Colonieen, die auf der Oberfliche der Verdiinnungsplatten einen Durchmesser von 2-3 mm. annehmen. Makroscopisch stellen sich die Oberflachen- colonieen als rundliche, hellgraue, das Licht stark brechende, etwas erhdhte Scheiben dar. Allmalich gewinnen dieselben ein charakteristisches Aussehen, indem um den dichten Kern sich * Nach Afereshkowsky soll sein Bacillus kein Gas entwickeln, (Centrabl, fiir Bakteriologie, Abth. I, Bd. XVIT. 1895. S, 742.) BEKAMPFUNG DER MAUSEPLAGE. 303 eine Anzahl concentrisch geschichteter Zonen lagert. Der Saum ist ziemlich regelmassig gelappt. Bei schwacher Vergrésserung ‘stellen sich die Oberflachencolonieen als runde, fein gelappte grobkérnige Scheiben dar. In den centralen Bezirken zeigen ‘sich oft faltige oder runzelige Figuren. Die Tiefencolonieen bieten mit unbewaffnetem Auge betrachtet, hellgraue Piinktchen dar: bei schwacher Vergrésserung erscheinen sie als unregel- miassig runde, grob kérnige Scheiben. Bei den 7raubenszuckeragarstichkulturen zeigt sich auf der Ejinstichstelle schon nach 24 Stunden bei Briitwirme eine weisse, diinne gelappte Auflagerung, die sich allmalich tiber die Agar- oberfliche hin ausbreitet. Langs des Stichkanals bildet sich ein feiner, weisser, bis zum Boden reichender Streifen. Stets findet ausgiebige Gasentwickelung statt. Auf Kartoffelstickchen ist das Wachsthum ein geringfiigiges. Nach 24 Stunden ist langs des Impfstriches ein kaum wahrnehm- barer Belag, der jedoch nach langer Zeit keine wesentlichen weiteren Fortschritte zeigt. In Mich wachst der Bacillus ziemlich gut. Keine Koagu- lation tritt ein, selbst nach mehreren Wochen bei Briitwarme nicht. Die Milch wird aber durch das Bakterienwachsthum deutlich sauer. Es findet auch eine schwache Gasentwickelung statt. Bei Adschluss des Sauerstoffs, d.h. in einer Wasserstoff-oder Stickstoffatmosphire sowohl auf den Agarplatten, als auch in Bouillon entwickelt sich der Bacillus viel dangsamer und gering- Jugiger als bei Zutritt des Sauerstoffes.1 Die entstandenen Colonieen resp. Bodensitze erreichen nie die Dimensionen der aéroben Vergleichsculturen. Gegen hihere Temperatur besitzt der Bacillus keine beson- dere Widerstandsfahigkeit. Dieselbe wurde in der Weise fest- gestellt, dass aus einer I-3 tagigen Bouillonkultur jedesmal 4 Oesen in frische, vorher im Wasserbade auf die gewiinschte Temperatur erhitzte Bouillon tibertragen wurden. Nach Ablauf von 5 Minuten wurden die R6hrchen unter dem Strahle der Wasserleitung rasch abgekthlt und dann in den Briitschrank gebracht. Es ergab sich dabei, dass eine Temperatur von 70° 1 Nach Angaben von MJereshkowsky (A.a.O.) soll der Bacillus bei Abschluss des Sauerstoffes sich gar nicht entwickeln., 304 Y. KOZAI. nach § Minuten den Organismus mit Sicherheit abtétete,. wahrend bei 65° C. der Erfolg kein gleichmissiger war. Gegen Licht ist der Bacillus ziemlich widerstandsfihig. Ganz diinne frisch ausgegossene Agarplatten, die zur Halfte mit undurchsichtigen schwarzen Karten bedeckt waren, wurden Ende Juni nachmittags dem Sonnenlicht ausgesetzt. Erst eine 8 Stunden dauernde Aussetzung tétete alle Organismen ab, wahrend nach 7 Stunden noch viele Colonieen nachtriglich zur Entwickelung gelangten. Die in diinner Schichte auf sterilen Deckglaschen tibertragenen Bacillen werden dagegen nach einer 30 Minuten dauernden Insolation in der Regel vernichtet. Die Austrocknung wird in diesem Falle dadurch ausgeschlossen, dass die Deckglaschen wahrend des Versuchs in einer mit feuch- tem Filtrirpapier versehenen Peérz’schen Schale gehalten wer- den. Gegen Austrocknung erweist sich der Bacillus ebenfalls ziemlich resistent. Bei einer Reihe von Versuchen, in denen mehrere, mit einer leicht triiben wiasserigen Bakterienauf- schwemmung beschickte Deckglischen im Exiccator bei Zim- merwirme aufbewahrt und nach verschiedenen Zeitraumen aut ihre Sterilitét gepriift wurden, stellte sich heraus, dass der Bacillus erst nach einem 30 Stunden langen Liegenlassen mit Sicherheit abgetétet war. Bei ahnlichen Versuchen mit einer hoch verdiinnten Aufschwemmung, in der sich keine sichtbare Triibung zeigte, erwiesen sich die inficirten Deckglaschen nach einem 26-stiindigen Aufenthalt im Exiccator vollstandig steril. In feuchtem Zustande vermag der Bacillus ferner lange Zeit seine Virulenz zu erhalten. Der aus trocknem Buchweizenmehl und einer 48-stiindigen Bouillonkultur angemachte Teig—die Form, in welcher der Bacillus zur praktischen Inficirung ver- wendet wird—zeigte nach einem 30-tagigen Aufenthalt in einem kalten feuchten Keller sich, wie Fiitterungsversuche mit Feld- miusen ergaben, noch vollig infectionsfahig. Endlich bildet der Bacillus in einer traubenzuckerhaltigen Nahrfliissigkeit ausser etwas Aethylalkohol nicht unbedeutende Mengen von £sstg- und Berusteinsdéure, ferner Spuren von Ametsen- und Jlilchsiure auch Ammontak besonders in alterer Kulturfliissigkeit. Hieriiber hoffen wir in Bailde mehr mittheilen zu konnen. BEKAMPFUNG DER MAUSEPLAGE. 305 ll. Infectionsversuche im Laboratorium. Die simmtlichen zu diesen Versuchen beniitzten Feldmiuse stammten von der oben erwihnten Provinz. Sie wurden jedes- mal nach dem Einfangen eine Zeit lang in grossen aus Zink- blech und Drahtnetz bestehenden, mit trockener Erde und sauberem Stroh versehenen Kisten unter scharfer Beobachtung aufbewahrt. Nach Ablauf einer 14-21 Tage dauernden Quaran- taine! wurden die Thierchen aus dem Behialter herausgenom- men, in anhliche aber kleinere Kasten gebracht und der In- fection unterzogen. Als Infectionsmaterial diente, wo nicht speciell anderes bemerkt wird, eine 24.-stiéindige Bouillonkultur.* Mit 5-6 c.c. dieser Kulturfliissigkeit wurde ein Teig aus trockenem Buch- weizenmehl angemacht und fiir 24 Stunden jenen Miusen, jedes- mal 4 Stiick betragend, bei denen eine Jnfection per os eintreten sollte, als alleinige Nahrung gereicht. Nach dieser Frist, binnen welcher die inficirte Speise in den meisten Fillen voll- standig verzehrt worden war, erhielten die Thierchen gewohn- liche, d. h. aus Reis oder Weizen und etwas Rettig bestehende Nahrung. Subcutan behandelte Thierchen bekamen 0.1-0.5 c.c. derselben Kultur und wurden in einem besonderen Kasten mit normalem, nicht inficirtem Futter ernahrt. Auf diese Weise haben wir eine grosse Anzahl von Feldmiusen inficirt, wobei gleich bemerkt sei, dass alle der Infection erlagen. Von dem allgemeinen Verhalten der erkrankten Thierchen ist zunachst zu bemerken, dass friiher oder spiter sich eine immer zunelimende Apathie -bemerklich macht. Die schwer Kranken kauern namlich mit halbgedffneten Augen und ge- straubtem Haare zusammen und geben auf mechanische Stésse nur eine schwache Reaction. Ferner lasst sich, worauf schon Mereshkowsky* aufmerksam gemacht hat, an den _hinteren Extremitaiten eine immer zunehmende Parese wahrnehmen, so 1 Wahrend dieser Zeit bekamen die Thierchen in der Regel abgerundete Figuren und glanzendes Fell. 2 Die urspriingliche Kultur war vor dem Versuche durch Verimpfen von Maus zu Maus in ihrer Virulenz gesteigert worden. So Aah EOP 306 Y. KOZAI. dass die inficirten Thierchen vor Todeseintritt nur noch auf den Vorderfiissen sich langsam fortschleppen kénnen. Bei der Obduction der verendeten Thierchen und zwar der per os inficirten lassen sich in den inneren Organen dieselben Verinderungen wahrnehmen, wie sie schon von Mereshkowsky beschrieben worden sind.! Am auffallendsten ist die Ver- grésserung der Leber und Milz. Die letztere zeichnet sich ferner besonders durch dunkelrothe Farbung und derbe Be- schaffenheit aus. Die Leber ist zumeist parenchymatés getriibt und weist mehrere kleine gelbe Flecken auf. Auch der Ver- dauungskanal lasst mehr oder weniger wesentliche Verander- ungen wahrnehmen und ist nicht selten stark mit Gas gefillt. Der Inhalt der Harnblase ist etwas dunkel und zeigt zuweilen eine miassige Triibung. Was nun die bakteriologischen Untersuchungen der ver- endeten Thiere anbetrifft, so haben wir in der Regel einige Blutstropfen aus dem Herzen ferner Milz- und Leberstiickchen unter den nétigen Cautelen in Bouillonréhrchen iibertragen und diese im Briitschrank gehalten. Ausserdem haben wir in 6 Fallen auch den Inhalt sowohl der Harnblase wie auch des Darmkanals bakteriologisch untersucht. Die hierbei erzielten Ergebnisse lassen sich in folgenden Satzen zusammenfassen. Die mit Leber Milz und Harn beschickte Bouillon zeigte jedes- mal eine massige Tvibung innerhalb 24 Stunden und cie Kulturen im hangenden Tropfen sowohl wie auch Plattenkulturen wiesen stets Reinkulturen des namlichen Bacillus auf. Auch der Darminhalt enthielt in sammtlichen von uns_ untersuchten Fallen den namlichen Bacillus. Der Beweis dafir konnte aber wegen der Anwesenheit anderer Bakterienarten nur durch die subcutane Injection der Bouillonkultur mit Sicherheit erbracht werden. Was nun die /etale Wirkung des Bacillus betrifft, so trat dieselbe in unserem Falle, wie aus nachfolgender Aufstellung ersichtlich, in folgenden Zeitfristen ein : 1 A.as0: BEKAMPFUNG DER MAUSEPLAGE. 307 5 Tage nach der Inficirung starben 4 Feldmause 6 9 a” ” ” 9? 4. ” 7 ” ” ” ” ” 6 ”” 8 ” ” ” ” 99 6 be) 9 ” ” ” ” ” 7 ” 10 9 95 7 A0 9 5 ” II 2 i 3 - Hy 7 a 12 99 %? ” ” ” 8 ” 13 $ a x 3 a IO 14 is 5. ¥ a rr LE am 15 9 ed 9 ” ” 14 ” 16 a 5s 5 a " 8 nS 18 Lb) ” >” ” my) 4 yy 20 ” ” ” ” 9 3 ” 21 - 3 3 A 5 Zz a 22 - Pr i s is I _ 23 ” ” ” ” ”” 2 3) 25 7 ” bad 9 ” I ” 27 ) a” ” ”” ” 2 ’” 30 y ” ” ” I ” 35 a9 ” ” ” ” I ” 38 a i ts i! i I ay Im Ganzen 108 Feldmiause Die Sterblichkeit tritt also schon am 5. Tage der Inficirung auf, steigt dann rasch und erreicht das Maximum am 15. Tage, um wieder allmdlich herunterzugehen. Auffallend ist die That- sache, dass in 2 Fallen die Krankheit erst nach 35 resp. 38 Tagen ablief. Jedenfalls ist es sicher, dass der Mereshkowsky’- sche Bacillus auf unsere Feldmaus hoch virulent wirkt. Dieselbe scheint aber xzzcht so empfanglich zu sein, wie die in Europa verbreitete Art (Arvicola arvalis), denn die letztere soll nach Mereshkowsky binnen 1-9 Tage nach der Infection sterben. Die oben erwahnte Krankheitsdauer bezieht sich nur auf diejenigen Miause, die eine geniigende Menge der inficirten Speise verzehrt haben. Bei den Thierchen dagegen, die nur eine ganz geringe Menge des Infectionsstoffs aufgenommen haben, verlauft die Krankheit in der Regel noch viel langsamer. LAC. as O:; 308 Y. KOZAL Von den 2 Feldmiausen, denen versuchsweise von der inficirten Speise ganz wenig gegeben war, starb die eine nach 40, die andere erst nach 75 Tagen. Um dieses experimentell zu priifen, haben wir eine Anzahl von Miausen mit verschiedenen Mengen einer 24- stiindigen Bouilionkultur sowohl durch Darminfection wie auch durch sudbcutane Einspritzung inficirt und unter scharfer Be- obachtung im Aufbewahrungskasten gehalten. Die dabei ge- wonnenen Ergebnisse lassen sich aus folgender Tabelle ersehen. Darminfection. Subcutane Injection. y E : : ish ‘Il o. Bouillon- a Bouillon- a kultor | 22h! Verendet e kale eo Verendet & angewandt |, ,., nach, =. flangewandt.}, ,.. innerhalb. =. eel Mause. Ss Re Mause, + Tao. l2nk2alentes 1.0 9 14 z 4 24. Stunden 14., 16., 30. Tagen Bhp Wk, lt, lr, 3 6 17,7 3 5 24.) 20., 35. Tagen Ac LSS lises 10 5 16,8 b 5 4 » 20., 30. Tagen San om 205 l fey ep» eye hy 20 5 17,8 20 6 5 254030: m agen! j 9., Io. Tagen LO: 20.1205 } Sul Ze emlas 46 5 - 2.454 40 } 11,5 32., 40, Tagen \ 14. Tagen IO, 20., 40., Rhy Wp M2 1 o 5 32.4 #0 4 12 oh 40., 52. Tagen 14. Tagen Es unterliegt also keinem Zweifel mehr, dass die Krank- heitsdauer bis zum letalen Ausgange gewissermassen von der Menge der in den Kérper gelangten Microbien abhangig ist. Zuptk? kam zu einem &ahnlichen Resultat mit dem Ldffler schen Bacillus. Der Einfluss der individuellen Disposition des 7 1 In allen Fiillen liess sich der Bacillus in den ianeren Organen der Leichen nach- weisen, j 2 Centrabl. f{ Bakteriologie, Abth. I. Bd. XXI. 1897. S. 446. BEKAMPFUNG DER MAUSEPLAGE. 309. Thierchens ist in dieser Beziehung auch nicht zuverkennen. Ob beim Einfithren von ganz geringen Mengen des Infections- materials in den Organismus jegliche Wirkung ausbleibt oder demselben gar Immunitat ertheilt wiirde, konnten wir bis jetzt nicht entscheiden. Ganz in derselben Weise wie das Hinfiihren von geringem Infectionsmaterial verhalt sich die Infection mit der durch lang fortwahrende Ziichtung auf kiinstlichem Nahrboden abge- schwiachten Kultur. 4 Feldmiuse, die mit einer 4 Monate alten Agarkultur fer os inficirt waren, blieben nach Verlauf von 40 Tagen noch am Leben. Eine dieser Mause wurde mittelst Chloroform getétet und die Organe, die eine charakteristische Veranderung zeigten, bakteriologisch untersucht, wobei sich der Bacillus aus Milz Leber und Herzblut in Reinkultur gewinnen liess. Ferner zeigte das Blutserum desselben Thierchens eine sehr starke agglutinierende Wirkung auf den Bacillus. Nach Ablauf weiterer 15 Tage wurde die eine der tiberlebenden Miuse zwecks bakteriologischer Untersuchung ebenfalls mittelst Chloroform getotet, wiaihrend eine andere (a) fer os und die letzte (b) durch subcutane Lnjection mit einer hoch virulenten 24 stiindigen Bouillonkultur inficirt wurde.t Die durch Chloroform getotete Maus liess den Laczllus in Milz Leber und Herzblut nachweisen. Auch das Serum gab eine starke Widal’sche Reaction. Die Anwesenhett des Bacillus tm Blut bet Lebzetten der Méuse wurde noch bei speciellen diesbeziiglichen Priifungen constatirt. Dieselbe Beobachtung hat Mereshkowsky? mit dem Liffler'schen Bacillus gemacht. Von den zum zweitenmale in- ficirten Mausen starb die eine (a) nach 14, die andere (b) nach 3 Yagen, wahrend von den in derselben Weise behandelten Controllthierchen die Zer os inficirte nach 5 Tagen die subcutan injicirte nach 86 Stunden zu Grunde ging. Das Einfiihren des abgeschwichten Bacillus in den Organismus scheint also die Wirkung des hoch virulenten Bacillus einigermassen zu paraly- steven, ohne aber in unserem Falle dieselbe aufzuheben. Nach Mereshkowsky* soll die Infection mit einer 7 monatlichen 1 Zur Darminfection wurde 1 c.c. und zur subcutanen Einspritzung 0.3 c.c. der Kultur beniitzt. 2 u. 3 Centralbl. fiir Bakteriologie, Abth, I. Bd, XVI. 1894. S. 612. 310 y. KOZAI. Agarkultur des Léffer’schen Bacillus europadische Feldmiuse (Arvicola arvalis) tmmun machen. Von grossem Interesse ist noch, die Frage nach der Wir- kungsweise des Bacillus zu entscheiden. Es wurden zu diesem Zwecke sowohl aérobe, als auch avaérobe Bouillonkulturen des Bacillus hergestellt und nach einen 14 tagigen Aufenthalt im Briitschranke folgenderweise behandelt. a) Filtration durch ein Pukal'sches Filter, b) Eine 5 Minuten lange Erhitzung auf 70° C., c) Zusatz von 0.3% Trikresol. Nachdem die so behandelten Kulturen durch specielle Priifungen sich als steril erwiesen hatten, wurden je 2 Feldmiause mit je 2 c.c. der Kultur swdcutan inficirt. Ausserdem wurden je 2 Mausen 0.2 c.c. der aéroben sowohl wie auch der anaéroben Kultur ofue jegliche Behandlung unter die Haut eingespritzt. Nach 2 Zagen starben die mit uwzbehandelter Kultur inficirten Controllthierchen und zwar ohne wesentlichen Unterschied in der Krankheitsdauer, wahrend alle iibrigen dauernd gesund blieben. Daraus kann geschlossen werden, dass unter den bei unseren Versuchen obwaltenden Verhiltnissen der Bacillus kecnerlet hildung von Toxin veranlasst und dass die pathogene Wirkung des Bacillus nur der /zvaszou an sich zuzuschreiben ist. Es sei schliesslich noch die Thatsache erwahnt, dass die De- jectionen der kranken Individuen als Verbreiter der Krankheit in hohem Maasse fungieren kénnen. 3 gesunde Feldmiause, die wir versuchsweise in einem mit Dejectionen erkrankter Thier- chen besudelten Kafig aufbewahrt hatten, starben sammtlich nach 25-35 Tagen und zwar von der Wirkung des Bacillus. In einem anderen Fall erlagen 2 von 6 im besudelten Kafig ge- haltenen Individuen binnen 35 Tagen. Zuzpzk* fand in ahnlichen Fallen, aber mit dem ZLdéffer’schen Bacillus, nur selten eine Infection. Ill. Infectionsversuche auf freiem Felde und Praktische Inficirung. Hatten die vorerwahnten Laboratoriumsversuche die Em- pfanglichkeit unserer Feldmause gegen den Mereshkowsky’schen sh i cy, (0) BEKAMPFUNG DER MAUSEPLAGE. 31k Bacillus sichergestellt, so bleibt noch iibrig, den praktischen Werth des Infectionsstoffs d. h. die Wirksamkeit desselben auf freiem Felde zu erproben. Bei den ersten diesbeziig- lichen Versuchen wahlten wir ein Feldstiick aus, auf dem Tabakstengel zu Haufen aufgeschichtet waren. Es wurden 10 solche Haufen mit dem namlichen Bacillus inficirt, indem man je Io aus Buchweizenmehl und einer 24-stiindigen Bouillonkultur angemachten Portionen von Teig in der Grésse von etwa einem Cubikcentimeter in dem Innern eines jeden Haufens vertheilte. Am folgenden Morgen waren die meisten Teigstiickchen ver- schwunden und die wenigen zuriickgebliebenen wiesen frische Bissspuren auf. Nach 5 Tagen nahmen wir die inficirten Haufen auseinander und gruben zahlreiche darunter befindliche Mause- lécher sorgfaltig aus.1_ Hierbei trafen wir aber im Ganzen nur 4 kranke Miuse,? wahrend nach den anderwirts gemachten Erfahrungen eine weit gréssere Anzah] zu erwarten gewesen ware. Wahrscheinlich hatten viele Thierchen wahrend der Versuchszeit im kranken Zustande die Flucht ergriffen. Aus diesem Grunde wiahlten wir beim zweiten Versuche auf breiten Flussdiammen und im freien Felde (im Dorf Oba) mehere, passende, zahlreiche Mauselécher enthaltende Parzellen aus, welche, um das Entrinnen kranker und Hereinkommen frischer Miause zu verhindern, mit starken tief in die Erde einge- schlagenen Bambuspfahlen dicht umgeben wurden. Ausserdem wurden diese Umziunungen, um Raubvégel abzuhalteu, mit Schniiren tiberspannt. Die so vorbereiteten Parzellen wurden sodannam 24. Januar dieses Jahres sorgfaltig inficirt, indem je ein Teigstiickchen, etwa 1 c.c. Bouillonkultur enthaltend, in die Mauselécher geschoben wurde. Bei der am folgenden Morgen vorgenommenen Besichtigung waren die gelegten Teigstiickchen meist bereits verschwunden und die noch zuriickgebliebenen liessen oft Spuren von Scheidezihnen der Mause erkennen. Am 29. Januar, also am 5 Tage nach der Inficirung schritten wir zur sorgfailtigen Ausgrabung der Versuchsparzellen. Die dabei angetroffenen Miause, die lebenden sowohl wie auch die toten 1 Um ein Entrinnen von Miusen durch die Seitenginge zu verhindern, wurde zuerst jedesmal in tiefer Graben um den Haufen hergestellt, 2 Specielle bakteriologische Untersuchungen ergaben, dass diese Thierchen in der That von dem Bacillus angegriffen waren, 312 Y. KOZAI. wurden der bakteriologischen Untersuchung unterworfen. Die so erzielten Ergebnisse sind in der folgenden Tabelle zusammen- gestellt. Nr, der Nr. d Der Bacillus? Mause in aay in avant ee fortlaufender oa <1 oan] aera e merkung. Reihenfolge. | P@t°"- | Milz, | Herzblut.| Leber. | Bt + + + Lebendig eingefangen. 2 a - + I 3 + + + 4 + + + ps Shone SERS. lla 5 ey a 5 a> 6 - 7 + 8 = 2 9 I. + + 10 + a 11 + + 12 — =k 13 - + 14 + + 15 + 16 - + Tot im Nest gefunden. 17 URE = - - Lebendig eingefangen. 18 = + = ” 19 = = ag 2 20 ~ + + ” Von den 20 untersuchten Mausen waren also 17 Individuen von dem Bacillus angegriffen, so dass der Prozentsatz der in- 1_Jn dieser und folgenden Tabellen wird die Anwesenheit des Bacillus durch (+) die Abwesenheit dagegen durch (—) bezeichnet. BEKAMPFUNG DER MAUSEPLAGE. 113 ficirten 85 betrug. Auch die drei gesunden Nager wiren schwerlich dem Eindringen des Bacillus entgangen, wenn die Miauselécher nicht so zeitig ausgegraben worden wiren, da mehere infectionsfihige Teigstiickchen! noch darin vorhanden waren. Der dritte Versuch wurde in der Nachbarschaft dcs zweiten Versuchsfeldes vorgenommen und zwar in ahnlicher Weise wie vorher. Die Parzellen I und II wurden nimlich mit dem ublichen Infectionsmaterial beschickt, wahrend wir auf der Parzelle III 2 schwer erkrankte Mause fretlaufen liessen. Die am folgenden Morgen vorgenommenen Besichtigungen ergaben, dass die erkrankten Miause sowohl wie auch die meisten Teig- stiickchen verschwunden waren. Vier Tage nach der Inficirung d. h. am g. Februar wurden die Versuchsparzellen sorgfaltig ausgegraben, die angetroffenen Miause gefangen, in den schon erwihnten Kasten unter scharfer Beobachtung aufbewahrt und zwar so lange bis die meisten Thierchen der Wirkung des Bacillus erlagen. Die dabei gewonnenen Ergebnisse bringt die folgende Tabelle zur Veranschaulichung. 1 2 Miuse, denen diese Stiickchen zum Fressen gegeben waren, starben binnen 12 Tagen und zwar von der Wirkung des Bacillus. 314 Y. KOZAI. Nr, der Der Bacillus in Miuse in Nr. der | Infections- B k fortlaufender | Parzellen.} material, ANSE NO NES Reihenfolge, Milz. Herzblut. Verendet nach : 21 + + 5 Tagen der Inficirung, 22 + ae 6 ” ” ” 23 at ar 7 ” »” ”» 24 =f oF 8 ” » » ie 25 + + 12 ” ” ” 26 + + 16°55) a 27 + + 16 ” %” »” Inficirte 28 Teig- — — Getotet nach 24 Tagen. stiickchen. 29 re. aan ” ” 30 ” 30 + + Tot gefunden, 31 + + Verendet nach 12 Tagen. 32 16 + + ” ” 12 ” 33 ar + ” ” 14 ” 34 te ae ” ” 15 ” 35 + + Verendet nach 10 Tagen 36 + 3 ”» a 12 45 37 Ill. rae + 1 ” ” 13 ” 38 ae + Be ” ” 15 ” 39 = - Getitet nach 30 Tagen. 40 = = ” » 30 » Das Resultat des dritten Versuches bestatigt also das beim zweiten erzielte und liefert einen recht bedeutenden Prozentsatz der inficirten Mause (80). Ferner geht daraus hervor, dass der Ablauf der Krankheit im Freien ebenso schnell stattfindet wie beim Laboratoriumsversuche. Hier wie dort fangt am 5 Tage nach der Inficirung das Absterben an, das am 10-16 Tage am starksten auftritt. Endlich sei noch hinzugefiigt, dass Feld- miause in der That auch auf frezem Feld wie in der Gefangen- BEKAMPFUNG DER MAUSEPLAGE. 315 schaft ihre kranken Genossen auffressen und so den tétlichen Bacillus in sich aufnehmen. Setzten also die oben geschilderten Versuchsergebnisse die Wirksamkeit des Bacillus als Mausevertilgungsmittel ausser Zweifel, so blieb doch noch festzustellen, ob die Infection in ebenso giinstiger Weise stattfindet, wenn das Inficirmaterial in geringerer Menge in Verwendung kommt. Demzufolge wurden 3 Parzellen auf cinem alten breiten Flussdamme im Dorf Oba in schon erwahnter Weise umzaunt und itiberspannt. Dieselben wurden dann mit Buchweizenteigstiickchen beschickt, welche die Agarkultur des Bacillus in 3 verschiedenen Verdiinnungen enthielten. Es wurden namlich in einem Liter einer $%%-igen Kochsalzlésung gleichalterige und gleich gut entwickelte Bak- terienrasen aus I, resp. 3 und § Agarrdhrchen auf das Feinste vertheilt und mit jeder so gewonnenen Aufschwemmung Teig bereitet. Die meisten der gelegten Stiickchen waren am folgenden Morgen verschwunden. Am 7. Tage der Inficirung (am 4. Marz) wurden die Mauselécher ausgegraben und die gefangenen Thierchen der _ bakteriologischen Untersuchung unterworfen. Die folgende Tabelle zeigt die so erzielten Resultate. 316 VY. KOZAI. Zahl der Der Bacillus Nr, der = . | Agarkultur - G Nr. der cae Miuse in Versuchs-| i. einem B k fortlaufender Parzelle Liter der eMersungs Reihenfolge. ; ' | Kochsalz- ljsung. 41 | 4 ele as Tot gefunden. 42 = = = Lebendig eingefangen,. 43 zi, i rt ” 44 oF Pr z ” 45 + sF + “5 46 + ee tieys Tot gefunden. 47 | (a —- | = L.ebendig eingefangen. 48 + ar af » 49 I I — = = ” 50 } = a = ” 51 ~ - ~ » Lebendig eingefangen,schwer 2 eis |) gets a krank u. starb nach ; a einigen Stunden. 53 a; # Lebendig eingefangen. 54 | met he 5 55 | at = = ” 56 + + + 55 57 = = ia ” 58 or = co ’ Lebendig eingefangen, sehr 59 | | 25 ot Sis | krank u. starb am | folgenden Mergen. Go | 4 £ a | Tot gefunden. 61 | + + SF Lebendig eingefangen. 62 = = > ” 63 - + AF =F ” 2 10, 3 64 et = A ” 65 + + + Tot gefunden, 66 = - = Lebendig eingefangen. 67 + oF ot: ” > Lebendig eingefangen, starb 68 at aE ca | am folgenden Morgen. | . . 69 + | + + Lebendig eingefangen, 70 + + ae Tot gefunden. ir + + st ” 72 - - = Lebendig eingefangen. 73 Ill. 5 + + ae ” 74 + + + Tot gefunden. 75 + + + ( Lebendig eingefangen, sehr schwer krank und starb 76 + se + nach einigen Stunden. 77 - = - Lebendig eingefangen. 87 | sh ote + Tot gefunden. BEKAMPFUNG DER MAUSEPLAGE. 317 Betrachten wir vorliegende Tabelle, so werden wir gewahr, dass die Verbreitung der Krankheit gewissermassen von der Menge der inficirenden Bakterienkultur abhangig ist. So betrug die Prozentzahl der inficirten Individuen in der Parzelle III, wo die stiirkste Bakterienemulsion beniitzt worden war, 82 (9 inficirte u. 2 gesunde Miause), also fast gleich viel wie die beim zweiten und dritten Versuche erzielten (85 resp. 80), bei denen Bouillonkultur resp. kranke Mause in Verwendung kamen. Dagegen belief sich dieselbe in der Parzelle II auf 74 (6 inficirte u. 3 gesunde Miuse), und in der Parzelle III, wo die geringste Bakterienkultur zur Inficirung verwendet worden war, erreichte dieselbe kaum 45 (7 inficirte u. 11 gesunde Miause). Es sei gleich bemerkt, dass die hier beniitzte Kultur nicht die geringste Abschwiachung erfahren hatte. Denn 2 Miuse, denen 0.3 c.c. einer 24-stiindigen Bouillonkultur desselben Bacillus subcutan injicirt wurden, starben binnen 48 Stunden. Ausser diesen Versuchen haben wir noch eine Anzahl von Feldexperimenten ausgefithrt, aus denen ein abschliessendes Urtheil tiber die dabei erzielten Resultate gezogen werden konnte. Bei diesen Versuchen wurden solche Felder ausgewiahlt, die von allen Seiten mit irgend einer natiirlichen Grenze, d. h. Bachufer, Fusswegen, Sumpffeld u. derg]. mehr, umgeben waren. Es wurden zuerst alle Mauselécher zugetreten und die nach 3 Tagen aufgefundenen, frisch aufgegrabenen Lécher mit dem Teig in tiblicher Weise beschickt, der die Bakterienkultur in verschtedener Starke enthielt. Nach 5-8 Tagen wurden auch diese Locher gezahlt uud wieder zugetreten. Dieselbe Opera- tion wurde alle 7-10 Tage wiederholt und zwar so lange bis keine resp. sehr wenige Locher auf’s Neue aufgegraben worden waren. In einigen Fallen wurden die neuen Locher zum zweiten Male mit dem Inficirmaterial behandelt und weiterer Beobachtung unterworfen. Die Daten dieser Versuche sind in folgender Tabelle zu- sammengestellt. 318 Y. KOZAT. (ee Tage an welchen Miuse- al der ec lécher inficirt, resp. | Nr. und Grésse des | >. . gezahlt und zugetreten | Versuchsfeldes. fic RU never mee wurden, | eas 2. Februar gezahlt und . 130 inficirt. Sn, gezahlt und Aus Versehen nicht zugetreten. gezihlt, 14. ” ” ” 64 17. ” ” ” 26 I; Eine 24-stiindige ARs sp » 3 Weizenfeld. Bouillonkultur, 18 (0.4 Hectar) | 28. ” ” » 6 4. Marz f . 3 13 ” ” ” I 23 ” 3 ” oO ; (a) (b) 18, Februar gezihlt und 37 42 inficirt. 2s gezahlt und 39 45 zugetreten. 28, 5 » » | II. a. ‘In 1 Liter einer 0.59¢-| 13 13 Weizenfeld | igenKochsalzlésung 3. Marz 3 os (0,4 Hectar), | wurden 4 Agarkul-| 9g 8 ,turen beim Versuchs- 9. 1s » » | feld (a) und 3 Agar-| 7 3 II. b. | kulturen beim Ver- 14, 5 FA * Weizenfeld suchsfeld (b) gut} 7 6 Nochmals inficirt (0,6 Hectar). vertheilt. 17. 4 mit entsprechenden 7 6 Teigstiickchen. 20. 4, gezahit und fo) 2 zugetreten. 27. ” ” ” 12) 3 Ate: ; (a) {b) 3. April gezahlt und IIT. a. 85 72 inficirt. IIT. a. 5 Agarkulturen in 9. 5 gezahit und Weizenfeld 1 Liter einer 0.5%- | 191 183 zugetreten. (1,3 Hectar). igen Kochsalzlésung. 12, ” 3 ” 49 57 2A 5 7 III. b. III, b. 19 26 Weizenfeld 1 Agarkultur in 3. Mai + = (1,4 Hectar). derselben Menge Io 20 der Lésung, 13. ” ” ” £ ms BEKAMPFUNG DER MAUSEPLAGE. 319 Zur besseren Veranschaulichung dieser Ergebnisse wurde in folgender Tabelle die Prozentzahl der frisch aufgegrabenen Lécher in gewissen Zeitraumen ausgerechnet, wobei die am Tage der Inficirung aufgefundenen Mauselécher als 100 ange- nommen werden. Nr. der Versuches. 1G ie Ill. Zahl der Agarkultur. Inficirmaterial. Bouillonkultur. (a) 4 (b) 1 (a) 5 (b) 1 Am Tage der Inficirung. 100 100 1co 109 100 Nach 3-7 Tagen. — 105 107 225 254 pu 20 24 19 58 79 1 21 a 14 19 7 22 36 » 39 ” 2 19 14 12 28 » 40 ” ° fe) 7 fo) 21 Eine plétzliche Steigerung der Prozentzahl der frisch auf- gegrabenen Mauselécher am Anfange des Versuches ist auf den Umstand zuriickzuftihren, dass die Aufzahlung und die Inficir- ung derselben 3 Tage nach dem ersten Zutreten, d. h. zur Zeit, wo die Mause ihre Locher noch nicht genug aufgegraben hatten, stattfand. Ferner scheint nach dem Zahlenverhiltnisse der frisch aufgegrabenen Lécher zu urtheilen, die Sterblichkeit der Mause binnen 7-15 Tagen nach der Inficirung am intensivesten aufgetreten zu sein, eine Erscheinung, die mit den Ergebnissen der schon erwahnten Laboratoriums- sowohl wie auch der Feld- versuche in gutem Einklange steht. In weiteren 15 Tagen liess sich noch eine Abnahme der frisch aufgegrabenen Lécher beobachten, viele Mause schienen wahrend dieser Zeit erst zu Grunde gegangen zu sein. Ob diese Miause, bei denens die Krankheit sich lange hingezogen hatte, zu wenig von dem inficirten Teig aufgefressen hatten oder durch Benagen der Kadaver erst spater inficirt worden sind, muss natiirlich dahin- gestellt bleiben. Endlich sehen wir aus der Tabelle, dass in den Parzellen, die entweder von Anfang an stark oder zweimal inficirt worden waren, wie Versuche I. II. (a) u. III. (a), keine 320 Y. KOZAI. frischen Locher nach 40 Tagen mehr beobachtet wurden. Da- gegen wurden beim Versuche II. (b) u. III. (b), frische Lécher, obgleich nicht zahlreich, doch nach dieser Zeitfrist immer noch beobachtet. Diese geringere Infection muss sicher durch die Anwendung der verhialtnissmassig verdiinnten Bakterienemul- sion bedingt worden sein. Dies Resultat stimmt mit dem beim Laboratoriumsversuche erzielten und bestatigt gleichzeitig in vollem Umfange die von Mereshkowsky1 gemachte Beobach- tung, dass die Kultur seines Bacillus erst bei geniigend intenstver Inficirung sich zwecks Mausebekampfung verwenden lasst. Abgesehen von der Krankheitsverbreitung auf dem inficirten Feld wird die Infection auf die Nachbarschaft tibertra- gen, worauf Mereshkowsky? schon aufmerksam gemacht hat. Wir trafen namlich bei der Besichtigung der Versuchsfelder einige kranke, sich langsam fortschleppende Mause auf den benachbarten Feldern. Dieselben wurden in der That, wie specielle bakteriologische Untersuchung zeigte, von dem nam- lichen Bacillus angegriffen. Ferner fanden wir in einem auf etwa 200 Schritte von dem Versuchsfeld II. (a) entferntem Felde gelegten Strohhaufen 2 am Hals und Bauch etwas abgenagte frische Leichen von Mausen auf, die, wie bakteriologische Priifung erwies, in der That der Wirkung des Bacillus erlegen waren. Endlich starben von 20 lebendig eingefangenen, zum Versuchs- zwecke aufbewahrten Mausen nach 4 Tagen 4 Individuen ab und zwar von der Invasion des Bacillus. Da diese Thierchen von einem nicht inficirten Flussdamme (im Dorf Oba) eingefangen worden waren, sO muss man annehmen, dass die Krankheits- keime von dem etwa 500 Schritte entfernten Versuchsfeld (Nr. I) fortgeschleppt worden waren. Nachdem die oben erwahnten Versuche den Mereshkowsky- schen Bacillus als Bekampfungsmittel gegen unsere Feldmause bei richtiger Anwendung als sehr geeignet erwiesen hatten, schritten wir dazu, die geschadigten Felder der genannten Provinz zu inficiren. Zu diesem Zwecke wurde zuerst eine Strohabkochung mit Zusatz von etwas Kochsalz, Pferdfleisch- auszug und Natto, einem aus Sojabohnen hergestellten, an Peptonen reichen Nahrungsmittel, in grossen Mengen verfertigt. Die so bereitete, mit kohlensaurem Natron neutralisirte Flussig- iu, 2 A. a. O; BEKAMPFUNG DER MAUSEPLAGE. 321 keit, in der, wie Vorversuche geigten, der Bacillus sich gut entwickelt, wurde in meheren, etwa 10 Liter enthaltenden, aus Weissblech hergestellten T6pfen vertheilt und durch dreistiind- iges Erhitzen im Dampftopf sterilisirt. Nachdem die Kultur- fliissigkeit auf 30-40° C. abgekiihlt worden war, wurde sie mit einer Reinkultur des Bacillus inficirt und im Briitschrank bei 25-37° C. 2 Tage lang aufbewahrt. Nach dieser Zeit zeigte die Flissigkeit in der Regel eine starke Triibung und war fertig zum Versenden. Dazu wurde der Wattepfropf in kurzen Stutzen des Topfes glatt geschnitten, eine passende Blechkappe darauf gestilpt und die Fuge mit schmelzendem Siegellack dicht verstrichen. Beim Gebrauch wurde die Flissigkeit theils ohne Verdiinnung, theils aber mit der gleichen Menge einer sterilen ca. 0.5 %-igen Kochsalzlésung verdiinnt, mit Buchweizen- mehl unter Zusatz von etwas Weizenmehl! zu einem dicken Teig geknetet. Derselbe wurde nun an die Arbeiter vertheilt, die sodann in Reihen tiber die Felder gingen und ein Teigstiick- chen in der Grésse von etwa einem Kubikcentimeter in jedes Mauseloch hineinschoben. Ausserdem wurden benachbarte Walder, Feldraine Fluss- u. Bachdi’mme, Graben u.s. w. wo Mause besonderes gern ihr Nest bauen, ebenfalls sorgfaltig infi- cirt. Auf diese Weise haben wir bis Ende Juni ca. 3000 Hectar bebauter Felder behandelt, die von Miausen stark gefahrdet waren. Bald liefen zahlreiche Berichte bei uns ein, welche einen glanzenden Erfolg der Inficirung mittheilten. In der That boten die Felder bei der Ende October von uns vorgenommenen Besichtigung ein ganz anderes Bild dar wie friiher. In diesem Frithling als wir die Inficirung in grossem Maassstab vornahmen, zeichneten sich die Felder durch zahlreiche Mauselécher aus und Kulturgewachse jeder Art liessen das Zerst6rungswerk des Nagers erkennen. Und jetzt fand man nur ausnahmsweise hie und da ein paar Mauselocher, und junge Pflanzen, wie Bohnen und Erbsen wuchsen tippig weiter. Die im Friihling in staunens- werthen Mengen auftretenden Feldmiause sind wie durch Zauber verschwunden. Wie glanzend der Erfolg des Mittels war, zeigt auch die folgende Thatsache. Im Friihling pflegten die Bauern im Dorf Oba gegen kleine Zahlung die zum Versuche beniitzten Feldmause jeder Zeit in jeder Zahl uns zu liefern. Jetzt aber 4 Dies geschah um die Zihigkeit des Teiges zu vermehren. 322 Y. KOZAI. liefern sie keine Mause mehr, selbst wenn wir den Preis hoch ansetzten. Auf Grund der oben erwahnten Ergebnisse nehmen wir keinen Anstand mehr, den Mereshkowsky’schen Bacillus an die Seite des vielfach erprobten Léffer’schen zu stellen und ihn ebenfalls als ein sicher wirkendes Mittel gegen die Mauseplage zu erklaren. Ob derselbe Bacillus eine mit dem Léffer’schen oder dem WDanysz’schen gleiche. Virulenz besitzt, wird den Gegenstand einer weiteren Mittheilung machen. Uber die Bildung des Pyocyanolysins unter verschiedenen Bedingungen, VON O. Loew und Y. Kozai. Vor Kurzem haben Bulloch und Hunter beobachtet, dass Culturen des Bac. pyocyaneus einen Korper enthalten, das Pyocy- anolysin, welcher die Blutkorperchen des Ochsen, des Schafes, des Kaninchens und anderer Thiere hamolysirt.1 Die Menge dieses KGrpers variirte in verschiedenen Fallen, altere Culturen sind reicher daran als junge. Von Interesse ist ferner, dass 15 Minuten langes Erwarmen auf 100° die hamolytischen Eigen- schaften der Culturen noch nicht aufhebt, und dass dieser K6rper vorzugsweise in den Zellen bleibt, so dass Filtrate der Culturen weit schwacher wirken als sterilisirte, unfiltrirte Culturen. Von einigem Interesse schien uns die Frage, ob das Pyocy- anolysin in verschiedenen Nahrloesungen gleich stark auftritt und ob das Maas des Luftzutritts einen Einfluss aussert, ferner ob jener Korper in massigen Mengen totlich auf Thiere wirkt. Die oben genannten Autoren verwendeten lediglich Boutllonculturen, von denen (nach Entfernung oder Tétung der Bacillen durch 15 Minuten Erwarmen auf 60°?) 0,05 bis 2 c.c. mit 2 c.c. einer 59% Aufschwemmung von Blutkérperchen 18-20 Stunden lang bei 37° im Incubator gehalten wurde. Bei den starkeren Graden der Hamolyse waren die Blutkérperchen vollig gelost. 1 Centralbl. f. Bakt., Band 28, S. 866. Wie dem Einen von uns von Prof. JZ, Nenchki mitgetheilt wurde, haben Vencki und Steber schon vor einiger Zeit die gleiche Beobachtung gemacht. 2 Unter gewissen Verhaltnissen vertragt #2. Pyocyaneus jedoch weit hohere Tem- peraturen. Die Totung bei 60° wurde jedenfalls durch das bacteriolytische Enzym in der Cultur beschleunigt. ©. LOEW UND V. KOZAT. Uo ey aN Wir verwendeten zu unsern Culturen drei Nahrloesungen : I. Bouillon II. Pepton 199§+ Glycerin 0.1% III. Asparagin 0,5% + Glycose 0,5 % Jede dieser Loesungen wurde auf zwei je 200 c.c. fassende Erlenmeyerkolben vertheilt, der eine enthielt nur zu 2 cm. Hohe Nahrloesung, was ausgiebigen Luftzutritt erméglichte, der andere war bis zur Verengerung am Kolbenhalse gefiillt, der Luftzutritt war hier sehr beschrankt und anaérobes Leben in den unteren Schichten begiinstigt. Loesungen II. und III. erhielten noch 0,29 secundares Kaliumphosphat und 0.019% Magnesium- sul:.t (letztres in sterilisirter Loesung erst bei der Infection zugegeben). Die am 20. Marz inficirten Loesungen zeigten (bei 36° gehalten) am 4. April folgende Erscheinungen : Bet veichlichem Luftsutritt: Schleimige Loesung und schlei- miger Bodensatz, braune Farbung. ‘| Bee geringem Luftzutritt: Sehr schleimige Beschaffenheit von Loesung und Bodensatz, schwach braunlich. Let reichlichem Luftsutritt: Dunkel braungriin, Vegetation abgelaufen, die anfanglich bedeutenden Bacterienmassen I1J Wieder gelost. Bodensatz gering, zum Theil krystall- inisch. Loesung kaum merklich schleimig. Bet geringem Luftsutritt: Noch viele Bacterienballen am Boden. Loesung hellgriin, tribe. Lei reichlichem Luftsutritt: Schwach braunlich. Die vor- her geringen Vegetationsmassen waren fast vollig wieder verschwunden. Geringer, zum Theil krystallini- scher Bodensatz. Schwach braunliche Farbung. Bet geringem Luftzutritt: Bacterienballen am Boden. Schwach griinliche Farbung. Es schien demnach, dass bei reichlichem Luftzutritt mehr Pyocyanase gebildet wird, als bei geringem, was mit Erfahrungen an anderen Enzymen iibereinstimmt. Bei II. war das ganz ausser Zweifel. Die Loesungen blieben bei alltaglich fortgesetz- tem einmaligen Umschwenken bis zum g. April bei gewohnlicher Temperatur stehen und dienten dann zu folgenden Versuchen mit Rinderblut, welche im Wesentlichen wie von 4Azlloch und Hunter ausgefihrt wurden. Blutkérperchen aus frischem Ochsen- blut wurden in 5% tiger Aufschwemmung in physiologischer Kochsalzloesung verwendet und zwar wurden stets 2 c.c. dieser 1618 BILDUNG DES PYOCYANOLYSINS. 325 Suspension mit 0.5, resp 2 c.c. der Culturen vermischt und bei 37° gehalten. Die abgemessenen Culturfliissigkeiten wurden vorher 15 Minuten auf 60° erhitzt. Das Resultat war nach 20 Stunden folgendes : Himolyse. Gace. Gering. 2c.c. Miassig stark. Omc.c. Spur. ec, .Gering. 0.5 c.c. Ziemlich stark. 2 c.c. Sehr stark. Dunkelrote Loesung. Oi c.c, Keine: 2c.c. Schwach. qyy_{ Cultur mit: viel Luftemorrec. «Stark. (Cultur mit wenig Luft; 0.5 c.c. Stark. Es ergab sich also, dass in Bouillon weniger Pyocyanolysin entwickelt wurde, als in den andern beiden Loesungen, dass ferner Luftzutritt die Bildung des Pyocyanolysins forderte bei Pepton und Bouillon als Nahrstoff. Auffallend war das Resultat bei Asparagin und Glucose als Nahrstoff, bei II]. Zucker setzt in der Regel die Enzymbildung bei Bacterien herab, hier aber finden wir, dass nicht nur mehr gebildet wird als in Bouillon, sondern auch, dass der reichlichere Luftzutritt keinen Unter- schied von dem geringeren Luftzutritt ergab. Um nun zu beobachten, ob der Hamolysingehalt obiger Pyocyancusculturen Thieren schadlich sein wiirde, wurden die oben erwahnten Culturen zunachst mit 0.39% Tricresol versetzt und nach 1 Tag 0.5 c.c. von jeder Cultur weissen Mausen injicirt. Bemerkt sei, dass bei der zwei Jahre lang fortgesetzten Cultur dieses Microben auf Agar die Virulenz verloren gegangen war. Das Resultat war ; tee mit viel Luft; Maus lebt noch munter nach 10 Tagen. ‘(Cultur mit wenig Luft: Maus tot nach 3 Tagen. | eae mit viel Luft: Maus munter nach 10 Tagen. ‘(Cultur mit wenig Luft: Maus munter nach 10 Tagen. ci eed mit viel Luft: Maus tot nach 15 Stunden. Cultur mit wenig Luft: Maus tot nach 4 Tagen. Das Resultat steht somit in gar keinem Zusammenhang mit dem Pyocyanolysingehalt. Gerade die Loesung, welche am meisten Pyocyanolysin enthielt, die Peptoncultur bei reich- (Cultur mit viel Luft. Ig Cultur mit wenig Luft. | oe mit viel Luft. { II. Cultur mit wenig Luft. 326 O. LOEW UND yY. KOZAI. lichem Luftzutritt, war ganz harmlos, wahrend die Bouillon- cultur bei geringem Luftzutritt, in der sich nur geringe Mengen Pyocyanolysin fanden, t6otlich wirkte.t Loesung III., welche gleichstarken Haimolysingehalt zeigte, bei reichlichem sowohl wie bei geringem Luftzutritt, ergab einen sehr bedeutenden Unterschied in der totlichen Wirkung in beiden Fallen. Es war aus dem Asparagin etwas kohlensaures Ammoniak entstanden, wie das Wessler'sche Reagens ergab und zwar mehr beim reich- lichen Luftzutritt. Es ware nicht unmdglich, dass dieser Ammoniakgehalt den Tod der Thiere bedingte. Es ergibt sich hieraus : 1. Bei reichlichem Luftzutritt wird mehr Pyocyanase ge- bildet als bei geringem. In Bouillon und in Pepton-Nahrloesung bedingt reich- licher Luftzutritt auch eine Vermehrung des Pyocy- anolysins. Bei Anwesenheit von Zucker (bei III.) war dieser Einfluss reichlichen Luftzutritts nicht zu be- merken. er Pyocyanolysingehalt obiger Culturen ist bei Injec- tion von 0.5 c.c. der abgetéteten Culturen weissen Miausen nicht schadlich. Nachschrift: Nachdem diese Versuche abgeschlossen waren, ersahen wir aus einem kiirzlich erschienenen Artikel von Weingeroff,? (welcher die Beobachtungen von JSzlloch und Flunter bestatigte), dass dieser ebenfalls zum Schlusse kam, dass das Toxin virulenter Pyocyaneusculturen nicht identisch mit dem Pyocyanolysin ist. Dieser Autor verwendete Culturen von Pyocyaneus, dessen Virulenz durch 6fteres Passiren durch Kaninchen so gesteigert war, dass 10 c.c. des Filtrats ein Kaninchen binnen drei Tagen tétete. Ferner ist hervorzuheben, dass Wetngeroff ausschliesslich Bouillonculturen verwendete. No Lo) 1 Vielleicht wird das Pyocyanolysin leicht im Thierkérper zerstért. Die Hamolysine anderer Bacterienarten (Erysipel) scheinen durchaus nicht so harmlos zu sein, 2 Centralbl, Bakt. Bd. 29, S. 777 (1901). 3 Pepsin und Trypsin zerstéren in 24 Stunden das Haemolysin, aber nicht das Toxin ; letzteres wird bei 100° selbst nach 30 Minuten noch nicht zerstort. Ueber die coagulirende Wirkung des Chloroforms. VON O. Loew und K. Aso. Vor Kurzem ver6ffentlichte 4. Sa/kowski! einige inter- essante Beobachtungen iiber eine coagulirende Wirkung des Chloroforms auf Blut und Milch. Auch Eigelb wird allmalig coagulirt, aber nicht das Weisse von Eiern, ebenso wenig das Lactalbumin in der Milch. Der eine von uns (L) wurde dadurch an eine schon vor langerer Zeit gemachte Beobachtung erinnert. Stiicke von der Pancreasdriise des Schweines nehmen namlich in Chloroformwasser aufbewahrt bald ein anderes Aussehen an als beim Aufbewahren in verdiinntem Alkohol von 15% ; in jenem Fall wird sie heller als in letzterem und die Flissigkeit giebt dort weniger Gerinnsel wie hier. Dieses veranlasste uns zu einigen Versuchen mit Leber und Muskeln vom Rind. 59g. frischer feinzertheilter Leber wurde einerseits mit etwas Chloro- form,” andrerseits mit etwa dem doppelten Gewicht verdtinnten Alkohols von 15% zwei Tage bei Zimmertemperatur stehen gelassen, hierauf mit 200 c.c. Wasser eine Stunde lang bei 50° extrahirt, die Filtrate mit Zusatz von etwas Salpetersdure zum Kochen erhitzt und das Gerinnsel nach dem Waschen und Trocknen bei 100° gewogen. Der mit der einen Leberprobe in Contact gewesene Alkohol von 15% wurde ebenfalls zum Kochen erhitzt, mit etwas Salpetersdure angesduert und das Gerinnsel ebenfalls gewogen. Das Resultat war folgendes: Es war gelést worden nach der Behandluug mit : 4 Z. physiol. Chem, Bad. 31, S. 320. 2 Das Chloroform reagirte vollstandig neutral. 328 O. LOEW UND Kk. ASO. Chloroform. Alkohol von 15% 0.574 g. 1.899 g. +0.476 g. (gelést im 15% igen Alkohol.) 2.375 $- Es war somtt bet Abwesenhett des Chloroforms etwas iber viermal so viel Proteinstoff gelist worden. Dieses ist bei manchen physiologischen Arbeiten wohl zu beachten. Bemerkenswert ist noch, dass das Wasserextract nach der Behandlung mit Chloroform nur schwach gelblich war, wahrend nach der mit verdiinntem Alkohol rot von geléstem Haemoglobin. Der in analoger Weise mit Muskelfleisch! ausgefiihrte Versuch ergab Ber: Chloroform. Alkohol von 15% 0.938 g. 1.055 g. +0.446 g. 1.501 g. Hs ist also in beiden Fallen eine coagulirende Wirkung des Chloroforms ersichtlich. Es erinnert dieses an die katalytische Wirkung mancher Aetherarten, wobei wahrscheinlich eine Uebertragung gewisser Schwingungszustande (chemische Ener- gie) anzunehmen ist. Nachschrift. Wie wir soeben aus einem der neuesten Hefte der Zeitschr. Biol. ersehen, hat auch Aviiger denselben Gegen- stand bearbeitet und schon vor elf Jahren einen Niederschlag durch Chloroform in wasserigen Extracten verschiedener thieri- scher Organe beobachtet. Er fand auch, dass Haemoglobin vollstandig dadurch gefallt wird. 1 Die nachherige Extraction geschah hier nicht mit blosem Wasser sondern mit finfprocentiger Loesung von Magnesiumsulfat. On Kaki-Shibu, a Fruit Juice in Technical Application in Japan. BY M. Tsukamoto. The name “ Kaki-shibu,” or more briefly ‘“‘Shibu,” is given in Japan to the juice of the unripe fruit of the Kaki tree (Diospyros Kaki, L.). This juice serves for the preservation of fish-nets and fish-lines which are soaked in the liquid and left to dry wellin the sun. After this treatment the nets or lines are much more durable. It further serves as an application to packing papers,’ especially such as are used for packing tea. Since such paper is less penetrable by moisture, the danger of mould development in the tea in warm and moist climates is diminished. Also tubs and other wooden vessels are treated with the juice to render them more durable. Further the juice is frequently mixed with India ink and the mixture is then used as a paint for the outer walls of wooden buildings and also for wooden fences. Considering that the Kaki-shibu has such important techni- cal applications it appeared desirable to subject the juice to an investigation. First of all it should be stated that not every kind of Kaki fruit yields suitable juice. There are found two varieties of the Kaki in Japan: one becomes very sweet when ripened, while the other remains astringent and is very rich in tannin,” yielding in great quantity the juice desirable for making Kaki-shibu. Such fruit when full grown is only 3-4 cm. in diameter. 1200 kg. of the fresh fruit when well crushed and mixed with about 2 hl. of water will yield nearly 7 hl. of Kaki-shibu. The mixture remains three or four days in 1 Also other kinds of paper are thus treated and used for various household and commercial purposes. 2 In order to make the fruit of some of this variety palatable, it is necessary either to treat it when well-ripened with a very diluted lye, or to dry it in the sun after taking off the skin, 330 M. TSUKAMOTO. large tubs when a certain kind of fermentation sets in, which is recognizable by the development of gas. The Kaki-shibu is prepared principally in the southern parts of Japan and the best quality of it comes from Kyoto, Osaka and Nara. The largest quantity of it comes from the vicinity of Hiro- shima. The juice is generally prepared in August! and may be applied fresh or after standing for two or three years. Some persons claim that the juice is improved by long standing in a cool place. In contact with the air an amorphous film is formed on the surface, probably by oxidation, while on the bottom of the vessels some bacteria and ‘yeast-like cells are deposited causing the characteristic smell of butyric acid. This development of microbes proves that the preserving properties of the Kaki-shibu do not consist in any peculiar antiseptic action, and therefore the beneficial effects have to be traced to another source. The Kaki-shibu leaves on evaporation a film insoluble in water, and this substance fills up the pores of fibres and wood, thus diminishing the water-holding capacity and the chances for destructive fungi. It is further very evident that the fibres of lines and papers are more closely united to each other so that they closely cohere. Thus the deterioration by mechanical wear and tear is considerably lessened.?, One can easily convince himself of this peculiarity by dipping filter paper in Kaki-shibu and letting it dry. The difference in behaviour of this paper and ordinary filter paper when rubbed in the moist state with the fingers, is quite remarkable. The former offers considerable resistance to the separation of fibres, while the latter does not. Chemical Examination. The most characteristic constituent seems to be a kind ot 1 Jt is said by the practical Kaki-shibu-makers that the season for the preparation of the juice is not of long duration, being only about 10 days ; both before and after that season the quality and quantity of the product is inferior. 2 The view expressed by Prof, I, Ishikawa (Journ. of Tokyo Chem. Soc, Vol, IIT. (1882) Transactions p. 19.) on this same subject is a little different from that stated here : namely, that the effective power of the juice is due to the formation of the film, which is formed on the surface of the Shibu when allowed to stand for some length of time. It seems to me, however, that the film formed on standing is different in its nature from that formed on evaporation, and that the former can hardly be supposed to be formed so soon, ON KAKI-SHIBU, A FRUIT JUICE IN JAPAN. 331 tannin; but this has some abnormal properties, as will be seen later on. When water is mixed slowly with old Kaki-shibu it can be observed that the latter remains for sometime suspended in the water in the form of a reddish and slimy liquid and only on shaking well does a complete distribution result, but this solution remains opalescent. Fresh Kaki-shibu is whitish and nearly odourless ; old Kaki-shibu is more or less reddish, brown, and gives off the odour of butyric acid. Old commercial Kaki-shibu in the undiluted state will yield when exactly neutralized with sodium carbonate a flocculent precipitate, and soon afterwards a jelly which is almost insoluble in boiling water and alcohol. Boiling alcohol does not extract a tannin-like compound while the insoluble part turns black at once in contact with ferric chloride, which shows that the tannin compound has _ here exceptional properties. The jelly mentioned is, however, soluble in dilute acids, but it is again precipitated by the addition of an excess of strong acids, as whitish floccules. The acid solution also gives precipitates by ferric chloride and basic lead acetate, but not by phosphotungstic acid. The jelly is soluble in dilute sodium carbonate, which latter solution becomes very dark on contact with the air. The fresh juice shows only a moderate acid reaction but this reaction increases on standing,’ which is no doubt due to the moderate development of microbes. On distillation in a current of steam a considerable portion of the acid distills over.2. I have determined by titration the total acidity, and further the amount of non-volatile acid after evaporation of 10 c.c. to dryness. It was thus found that the amount of volatile acid is almost equal to that of the non-volatile acid in new Kaki-shibu, while it is more than twice as great as the amount of non-volatile acid in the old Shibu.* 1 For my observation for the acidity served two kinds of Kaki-shibu prepared in the same place, one of which was made this year (1901) and the other last year, The total acidity per litre of the new and of the old corresponded respectively to 0.978 g. and 1.948 g. of SO,. 2 The nature of this volatile acid was investigated about twenty years ago by Prof, I, Ishikawa (I. c. p. 19-20) who found butyric acid in it. * The samples that served for my analysis came from Awotani-mura, Tsuzuki county near Kyoto, The new Kaki-shibu was prepared this year (tgo1), and the old in the last year (1900). M. TSUKAMOTO. Us Ww tN Commercial Kaki-shibu even in high dilution gives precipi- tates with basic lead acetate, mercuric chloride, mercuric nitrate and copper acetate. Since dilute phosphotungstic acid yields no precipitate, proteins seem to be absent to a notable degree. One of the most striking properties of the Kaki-shibu is the production of an insoluble film on evaporation to dryness. Whether this film is exactly the same as the film formed on the surface of the liquid on long exposure to the air may be doubted, since there evidently takes place a considerable oxidation in the latter case. In the former case, however, the loss of solubility in water is only due to the loss of the greater part of the acid by volatilization.2 Furthermore, these two films show the following differences in their behaviour: The former film swells somewhat when moistened, and is very easily broken. The latter film is elastic and more coherent. When treated with dilute acetic acid the former first swells up and gradually dissolves on standing, but the latter is almost insoluble and shows no perceptible change after long standing. On addition of concentrated sulphuric acid the former dissolves at once, while the latter only very slowly. On boiling with dilute nitric acid the former dissolves very easily giving a fine yellow solution, but the latter is dissolved only with difficulty. When boiled with caustic potash the former dissolves also much more easily than the latter. In order to determine the quantity of the film formed by evaporation, 10 c.c. of Kaki-shibu was evaporated to dryness and this residue was treated repeatedly with cold water until no acid reaction could be percieved in the washing. The insoluble film was dried and weighed, giving 0.351 g. or 3.420% for a sample of the new Kaki-shibu and 0.360 g. or 3.512% for a sample of the old ;3 they contained respectively 0.349 g. or 99.5% 1 In the new Kaki-shibu a little turbidity is observed by phosphotungstic acid. 2 Fresh juice is not a homogeneous solution but contains much fine suspended matter, This gradually goes in solution as the amount of acid is increased by bacterial action, since the suspended matter of the fresh juice gives a much stronger tannin reaction than the filtered liquid does. 3 It appears that the film formed on evaporation from the new Kaki-shibu is more soluble than that of the old juice. ON KAKI-SHIBU, A FRUIT JUICE IN JAPAN. 333 and 0.358 g. or 99.3% of organic matter. This film turns black with ferric chloride. When Kaki-shibu is mixed with an equal volume of alcohol no precipitate is observed, but on a further addition of alcohol and some ether an almost white flocculent precipitate was formed and this on standing gradually turned reddish brown. Very remarkable is the behaviour towards acids. A moderate quantity of mineral acids will give no precipitate; while on further addition much precipitate is produced,? which is again soluble in pure water and alcohol, and very easily in dilute acids. Of organic acids a larger quantity is required than of the mineral acids. 10 c.c. of old Kaki-shibu requires nearly 15 c.c. of concentrated sulphuric acid to produce complete precipitation. In this behaviour towards acids the tannin compound is like ordinary tannins, although in some other respects its behaviour is quite different. I have carried out the tannin determination by the Lcewen- thal method as improved by von Schréder, and have found the results calculated as both gallotannic acid and quercitannic acid; they were respectively 4.883 g. and 5.041 g. in 100C.c. of the new Kaki-shibu ; and 3.535 g. and 3.649 g. in 100 c.c. of the old. The former correspond respectively to 74.7% and 77.1% of its total solid matter ; while the latter had 58.79% and 60.6%. The commercial Kaki-shibu neither contains sugar, nor does it yield sugar on decomposition with sulphuric acid. The Kak tannin, therefore, is no glucoside. These reactions for reducing sugar were always made after the tannin had been removed with basic lead acetate. In one experiment 500 c.c. of commer- cial Kaki-shibu were boiled on the addition of 15 cc. of concen- trated sulphuric acid for three hours, and thereby a red precipitate was separated on cooling in the form of little globules. This precipitate, which increases on standing for twenty-four hours, was then collected on a filter and washed with water for a short time, since prolonged washing dissolved gradually a portion of it. This precipitate resembling oak-red (Eichenroth), was colored 1 It may be mentioned that the freshly cut surface of the unripe Kaki fruit when moistened with ferric chloride gives no uniform black reaction, but merely in numerous isolated points, and it seems therefore that the tannin is limited to certain cells, 2 The fresh Kaki juice behaves differently, since it gives no precipitate with an excess of acids. 334 M. TSUKAMOTO. violet black by ferric chloride ; in cold alcohol dissolved easily: with a fine red color; in caustic soda, with a brown red color. The aqueous solution is precipitated ona moderate addition of sulphuric acid. From genuine oak-red, however, our substance is distinguished by its solubility in cold water and cold alcohol. The liquid from which the red precipitate was separated was of a dark reddish brown color, and contained evidently more of the red substance in solution, The sulphuric acid was at first removed by barium carbonate and then the rest of the tannin by basic lead acetate. The filtrate now obtained was treated with sulphuric acid to remove the lead, and the filtrate evaporated on the water bath with the addition of barium carbonate. The search for sugar or chitosamine was in vain. I intend to repeat this experiment with the insoluble film obtain- ed by evaporation of Kaki-shibu. Finally for analytical results we have :— New Kaki-shibu. Old Kaki-shibu, Specific gravity at 15°c 1.0230 1,0250 Total solid Matter. 6.391% 5-874% Total fixed organic Matter. 5-993 », 5.566,, Ash. 0.398 ,, 0.308 ,, Insoluble film. B-A20),, SiSizee : ‘ as gallotannic acid. 4. Re a etnies | as raercieniie acid. pa Ses #3 Volatile acid (as acetic acid.) 0.087,, O2174- Fixed acid (as lactic acid.) 0.085 ,, O,102. >, Nitrogen ! 0.030 ,, General Conclusion. The industrial value of Kaki-shibu is due to its containing a peculiar tannin which in some respects differs from all other kinds of tannin known, since it is insoluble in alcohol and water, and soluble in dilute acids. This tannin becomes insoluble when the volatile acid of the Kaki-shibu evaporates, and the insoluble film thus formed protects fibrous objects against mechanical wear and tear. A partial oxidation in contact with 1 This nitrogen is probably due to an admixture of small quantities of protein. The amount varies greatly. ON KAKI-SHIBU, A FRUIT JUICE IN JAPAN. 335 air still improves the qualities of the film. This film also dimin- ishes the water absorbing capacity of such fibrous materials as paper and strings, and thus diminishes the chances of attacks from fungi. In conclusion I must express my thanks to Prof. O. Loew for his kindness in giving me the direction of this work, and to Messrs. T. Ono and K. Onodera for their assistance in the investigations, Investigations on the Digestive Enzymes of Some Lepidoptera. BY S. Sawamura. The digestive organs of Vertebrata consist of the mouth, stomach, and intestines. The saliva secreted in the mouth is alkaline; the gastric juice of the stomach, acid; and the pancreatic juice and bile secreted into the intestines, alkaline: thus the reaction of the fluids in the alimentary canal from mouth to rectum changes several times. But, as to Lepidoptera, the reaction of the fluids is alkaline throughout the digestive canal. Hence, there arises the question as to whether the digestive enzymes of these animals are different from those of the Vertebrata. As the silk-worm (Lombyx mort.) is the most useful insect in this country, it has been much studied, and thus the mor- phological character of Lepidoptera has become comparatively well known; but our knowledge of its digestive process is still very scanty. O. Kellner! made an investigation on the digestibility of mulberry-leaves by silk-worms, and found that they could digest albumin, fat and carbohydrates except cellulose; but his investigation did not extend to the process of digestion itself. Not only with Lepidoptera, but also in the whole division of Zusecta, the digestive enzymes are very imperfectly under- stood. The first investigators on this subject were Plateau and Fousset.2 The former concluded, from his investigations, that the fluid in the fore-intestine (now commonly called the fore-stomach) being neutral or alkaline, contained diastatic enzyme, which the investigator thought to be secreted by 1 Landw. Versuchsstationen. 1886. 2 Jahresbericht iiber die Fortschritte dec Tierchemie, 1877. 338 S. SAWAMURA. salivary glands ; and that in the middle intestine (now generally called the stomach) the fluid being neutral or alkaline, had the power of decomposing albumen and saponifying fat (with Hydrophilinen and Scarbaeiden diastatic enzyme was also present); and finally that in the end-intestine (now generally called the intestine) there existed no digestive ferment. The conclusion obtained by Yousset, who studied Blatta ortentalts, was nearly the same as that of Plateau, the only difference being that in Alatte ortentalzs, the fluid in the middle intestine was acid, and contained no diastatic enzyme. The investigations made by Krukenberg} were much more extensive than those above mentioned, but as unfortunately the author of this paper has had no opportunity of reading his original reports, the details of his investigations can not here be mentioned. The chief points of the summary of his experiments are, however, as follows :— I, In Zzsecta and in other Arthropoda trypsin predominates over pepsin. II. The property of the tryptic enzyme of insects is differ- ent from that of Vertebrata, other Arthropoda, and Mollusca. III. In /uvertebrata there being no division of the intestines which is specialized as stomach, corresponding to the stomach of Vertebrata, a certain part of the intestines is commonly regarded as a stomach. Krukenberg called the tryptic enzyme of insects “‘/sotrypsin,” but in what respect it differs from the known trypsin is not ex- plained. Biedermann? studied the digestion of TZenebrio molitor (Coleoptera), and found that the secretion in the upper part of the middle intestine had an acid reaction, while in the lower part it was always alkaline; and further that it acted upon starch, disaccharides, fat, and albumen, but not upon cellulose ; and finally that the albumen was split up into amido-compounds as in the case of tryptic digestion. Besides these investigations, the discovery of invertin in the head of honey-bees by Er/en- meyer and Planta* may be mentioned. 1 Fortschritte der Tierchemie. 1880. 2 Arendt, Chemisches Central-Blatt. 1893. i 2 Biedermanns Central-Blatt fiir Agrikulturchemie. 187¢. THE DIGESTIVE ENZYMES OF LEPIDOPTERA. 339 Some general knowledge of the digestive enzymes of insects may be obtained from the investigations mentioned, but as there exist among insects both herbivora and carnivora, the character of the digestive enzymes may also be different. Further, since the investigations of Krukenberg and others hitherto made have related chiefly to proteolytic enzymes and not to amy- lolytic and lipatic enzymes, some further questions remain to be solved. The author has, therefore, undertaken to investigate the digestive enzymes of Lepidoptera, especially those of the sz/f- worm. The insects used in the first experiment were the living larvae of Dasychira lumulata Butl. They were dissected, and the expanded part of the digestive canal (stomach) was taken out, and freed from its contents by washing it in water, and 3 ers. of these stomachs were triturated in a mortar with the addition of powdered glass. The crushed mass was divided into two parts. One part was digested with 15 grms. of glycerin containing 10% of water, while the other part was made faintly acid with dilute acetic acid, according to the trypsin-extraction method of He¢denhain,1 and exposed to the air for a short time, whereupon it was digested with glycerin like the other part. To furnish a control case for the method of extracting enzymes, the stomach and intestines of a snake were treated in the same manner as those of the insects, After three days the glycerin extract prepared without the acid treatment was filtered. To the filtrate strong alcohol was added till a precipitate was produced. The precipitate was collected on a filter, dissolved in water, filtered and again precipitated. But this precipitate was too insignificant to be separated again from the filter. It was, therefore, exposed to the air on the filter, and after the evaporation of the alcohol, the whole was divided into two parts. (ne part was dissolved in about 20 c.c. of water containing 0.196 of HCl, and the other in 20c.c. of 0.3% solution of sodium carbonate, since, according to Hammarsten,? trypsin acts best in a fluid containing 0.3—0.4 99 of sodium carbonate. The extract of the stomach of the snake was treated 1 Hammarsten, Lehrbuch der Physiologischen Chemie. S. 171. 2 Ibid, S. 172. 340 S. SAWAMURA. in quite the same manner. The enzyme-solution thus prepared, served for the following experiments. The first experiment ,was made to ascertain the influence of the reaction on the behavior of the enzymes. The materials tested with the additicn of some thymol at room-temperature, or at 36° C., were sharply cut slices of coagulated egg-albumen, fibrin and starch-solution. The results were as follows :— Materials Solutions. ace Temperature, Intervals. | Results. - . room- not of 0.1 9 HCl. oo temperature. (ar dissolved. Acid-solution of the lightly enzymes of snake- - % | i slightly Stomach: dissolved. | ” egg- ” ” alsaaen } corroded, ” ” + not | starch, | 3 hours. Gane | Acid-solution of the | a i t- ibri 2G | 2 days. 4 Sy ee ee pet ae Fie dissolved. j + 5 room- F not temperature, ei dissolved. ” egg- * 5s not albumen. corroded. ” ” a not starch. 3, hours, changed. 0.3 % solution of - 4 y not sodium carbonate. ae = he dissolved. Alkaline solution of the enzymes of snake- stomach. THE DIGESTIVE ENZYMES OF LEPIDOPTERA. 341 Solutions. saute Temperature. Intervals, Results. ; | Alkaline solution of dissolved, and the enzymes of the 3 43 = produced biuret insect-stomach. | } reaction. ! Ss | | wee 72 | | 207.C. | 2 days. _ | | | oun | starch starch, a 3 hours, completely | temperature. | disappeared, The acid extract of the insect-stomach and that of the snake-intestines were examined in just the same way as the former. The results were the same as those obtained with the insect-stomach. I therefore infer: I. That the enzymes of Lepzdoptera act only in an alkaline, and not in an acid solution. II. That in the digestive organs of Lepidoptera tryptic enzyme is present, while pepsin is absent. Since, however, it might be objected that there was present pepsinogen, which failed to be converted by the highly diluted acetic acid into the active state, a second experiment was made, and this time with old silk-worms. 16 grs of stomachs and 3 grs of intestines were obtained from silk-worms in the same manner as in the former case, and these were divided into two portions. After adding to one part some HCI of 0.19% (according to Podwysozki'! pepsinogen is easily transformed into pepsin by HCl), both portions were crushed as above and digested with glycerin as above. After three days it was filtered, and to the filtrate strong alcohol was added. The precipitate was collected on a filter, dried and divided into two parts. Since according to K/ug,? pepsin acts best in a solution containing 0.5—0,.69¢ of HCl, and according to Ewald ° trypsin acts also in a solution containing 0.39% of HCl, one part was dis- solved in about 30 c.c, of 0.49 HCI, and the other in about’ 40 c.c. of 0.3% solution of sodium carbonate. Both solutions were t Neumeister, Lehrbuch der Physiologischen Chemie. S. 178. 2 Oppenheimer, Yermente und ihre Wirkungen. S. roo. 2 Thid. S. 113. 342 S. SAWAMURA. used for experiments as in the former case. To test the digestion of fat, olive oil was used. For this purpose a little soda solution was added to the olive oil, and after the mixture was well shaken it was dissolved in ether. The ethereal solution was separated, washed with water, and the ether left to spontaneous evaporation. The neutral olive oil thus obtained was mixed with some alkaline enzyme-solution in a small glass tube, a little azolithmin solution being added to indicate the change of the reaction of the solution. After 8 days the results observed were as follows :— Material Solutions, a: Temperature Results Bie ae 0.4% HCl. fibrin, | 36°C. | not dissolved. Acid solution of | + the enzymes. 4 z = EEE room- 5 | - temperature. 3 starch. | 5 not changed. | . { 207 t = - . 0.370 solution of fibrin. | 260 not dissolved. sodium carbonate. 1 SONS SVT a | | ay | Alkaline solution of d > dissolved. the enzymes. é | = foe | } i | room- | sh temperature. x = | a : . a starch | starch. |} 36° C. disappeared. . aa » fat. | acid set free. | Gelatine was liquefied on bringing it in contact with some of the alkaline solution of the enzymes in the presence of some thymol. Also freshly precipitated casein was easily dissolved by it. THE DIGESTIVE ENZYMES OF LEPIDOPTERA. 343 From the results of the experiments above mentioned, we may conclude I. That in the stomach of silk-worms ¢ryptic, diastatic and lipatic enzymes are present, but that pepszz and pepsino- gen are quite absent. If. That the digestion of fat is effected by decomposition into fatty acids, as in the case of Vertebrata. The third experiment was made to ascertain the presence of digestive enzymes in the intestine of insects. 3 grs. of the intestines of silk-worms, which had been carefully separated from the stomach and freed from their contents by washing in water and then crushed in a mortar together with glass powder, were digested with glycerin as in the former case. After three days the mixture was further treated as above. One of the portions was dissolved in about 20 c.c. of 0.4% HCl, and the other in 20 c.c. of 0.3% solution of sodium carbonate. With both of these solutions the experiment was performed as before, and after six days the results observed were as follows :— Solutions. | Mate 5 | Temperature. Results. Acid solution of | fibrin. go. GC: not dissolved. the enzymes. | | es > ca ae os ss room- BS temperature, starch. gon C: not changed. x 5 room- | temperature. Alkaline solution of fhm |} 30°C. | dissolved. the enzymes, as | 7 room- | ae temperature, | | — — | SS _. —— a | eestareunes|) 3071C., i) not changed, | | | | | 3 room- , temperature. 30° C. acid was not set free. 344 S. SAWAMURA. From these results it may be concluded, that there exists a difference between the enzyme production of the intestines and that of the stomach, no diastase and lipase being present in the former. Another experiment was made with the intestines of Caligula japonica Moor. 6 grs. of the intestines were washed in water, crushed in a mortar together with glass powder, and digested with 409 alcohol. After seven days it was filtered, and to the filtrate ether-alcohol (1 part of ether and 3 parts of alcohol) was added. The precipitate collected on a filter was exposed to the air for a short time to evaporate the alcohol, and then divided into two parts. One part was dissolved in 20 c.c. of 0.1%: HCl, and the other in 20 ¢:c. of ao )fecoluman of sodium carbonate. The tests were made as in the former experiments, and the mixture kept in a thermostat for six days. The results observed were as follows :— Solutions. odes Results, | used. 0.1% HCl. coagulated | not corroded. egg-albumen, | Acid solution of *. a the enzymes. | de fibrin, not dissolved. es starch. not changed. 0.3% solution of coagulated not corroded. sodium carbonate, egg-albumen, Alkaline solution of | =“ corroded and biuret the enzymes. | reaction produced. - fibrin. dissolved and biuret reaction produced. FA starch. | not changed. fat. acid was not set free, THE DIGESTIVE ENZYMES OF LEPIDOPTERA. 345 We may therefore infer, that there exist enzymes in the intestines which act upon albumin, but that amylolytic and lipatic enzymes are completely absent. /P/ateau’s opinion that there are no enzymes in the intestines, seems to be erroneous. As regards the products of digestion I have made tests with the enzyme precipitates obtained from the stomach of the silk-worm and of Caligula japonica. ‘These precipitates were dissolved in a 1% solution of sodium carbonate (cryst). Some fresh egg-albumen served for the test with the enzyme from the silk-worm (a), while coagulated albumen was used with that from Caligula japonica (6). After the addition of some crystals of thymol the mixtures were kept at 36° C. for eighteen days. Putrefaction had been successfully prevented by the presence of the thymol. There was still some unchanged albumen present, as the test with nitric acid showed. The undigested albumen was removed from (a) by lead acetate according to H/ammersten’s method,! and after freeing the filtrate from lead by H,S, it was neutralized with NH, and evaporated. The residue thus obtained was extracted with boiling alcohol, but no crystals of leucin or tyrosin could be observed on slow evaporation of this extract. The part insoluble in alcohol gave no indication of tyrosin crystals on slow evaporation of its solution ; it consisted of a syrupy mass that behaved towards the usual reagents in every respect like peptone. The test for tryptophan was, like that for leucin and tyrosin, ineffectual ; a solution of chloride of lime failed to give the purple-red color.2, Tryptophan is a characteristic product of the tryptic digestion and according to Neumeister the formation of leucin and tyrosin can directly be inferred, when the reaction for tryptophan gives a positive result. We observe therefore that the end-products of digestion were peptones, but no further decomposition products. Hence, the proteolytic enzyme of the stomach of Lepidoptera is different from the pepsin as well as from the trypsin of Vertebrata ; it agrees with the pepsin in so far as it produces as end-products of digestion peptones, but differs from it as to the reaction of the active solution ; and on the other hand, while it agrees with the trypsin 1 Physio, Chem, S. 175. * The same results were obtained from (4) after it had stood seven days at 36° C, 346 -- S, SAWAMURA. as to the alkaline reaction of the active solution, it differs from it in so far as it does not produce any amido-acids and tryptophan, since the action stops with the production of peptones. Similar tests with starch solution were made to determine the end-products of the action of the diastase of Lepidoptera. After six days at 36° C, the mixtures still contained some starch. After evaporation to dryness, and extracting the residue with water, a solution was obtained that gave a ved reaction with iodine, showing the presence of erythrodextrin. The chief sugar formed was maltose, to judge from the properties of the osazone obtained. ‘To decide whether traces of dextrose were present some Saccharomyces apiculatus Reess, which has the peculiarity of fermenting dextrose only, but not maltose, was added to the solution in a fermenting tube. After standing for a few days, the evolution of some carbon dioxide was observed, which shows that some dextrose must have been present. There was probably present also some maltase-like enzyme, that further transformed the maltose produced by the diastase, into dextrose. Hence, the following conclusions may be drawn. I. All the enzymes secreted in the digestive canal of Lepi- doptera act in an alkaline solution, the action ceasing completely in an acid solution. II. The proteolytic enzyme of Lepidoptera decomposes albumen into peptones but does not further decompose peptone into leucin and tyrosin. Therefore, this enzyme resembles trypsin only in the reaction of its active solution, while it resembles pepszz in regard to the end- product of digestion. III. The amylolytic enzyme liquefies starch, forming dextrine and maltose. Possibly there is also present some maltase-like enzyme. IV. The lipatic enzyme of Lepidoptera like that of Vertebrata decomposes fat into fatty acids. V. While in the stomach these enzymes are present ; in the intestines proper only proteolytic enzyme is found, the two other enzymes being absent. VI. Though the expanded part of the intestine of Lepidoptera is commonly called the stomach, its physiological THE DIGESTIVE ENZYMES OF LEPIDOPTERA. 347 function resembles rather that of the intestines of Vertebrata. Krukenberg’s view that in /nvertebrata there is no part of the intestines that is comparable with the stomach of Vertebrata, is true at least for Lepidoptera, since there exists no genuine acid gastric juice in them. “ eas | i ap eel, bE rh maa } Jiitit” VES ee Ki «vis sh Ay ee (403 ay — . a Vite se aw heat fA rou 3 ee vm oon 1. We 4 was ry te ve tan? 4 j a _ nh &s : Cm AG is ur ‘ 4 é i i ‘ On the Occurrence of Cane Sugar in the Seeds of Gingko biloba and Camellia theifera. BY U. Suzuki. Recent investigations have shown that cane sugar occurs very frequently in the vegetable kingdom, having been observed not only in seeds but also in stems, roots and leaves. It not only plays an important role as reserve material during the germination process, but it is also the principal form in which starch is transported, and therefore germinating shoots may contain it, even when the original seeds do not. Inthe following lines I shall describe its occurrence in the resting seed. I. Seeds of Gingko biloba. The hard shells of the seeds of the Gingko were removed and the content dried and finely powdered—630 grams—was at first freed from fat and then repeatedly extracted with hot alcohol of 90%. This extract yielded according to the method of Z. Schulze for the isolation of cane sugar, nearly 10 grams of crystals which on minute examination proved to be identical with cane sugar. ' The aqueous solution gave a strong red coloration with resorcin and hydrochloric acid ; reduced Feh/éng’s solution, not directly but after inversion, became brown and finally black by the action of concentrated sulphuric acid ; and the optical rotatory power calculated from observation on Solezl-Ventzkes sacchari- meter was found to be [a]p= + 66.5.° A quantitative determination showed that the seeds of Gingko contained nearly 6% of soluble sugars of which $ reduce Fehling’s solution only after inversion. Further tests left no doubt that there were present also other sugars in small quanti- ties, which have less rotatory power and are soluble in alcohol with greater difficulty than cane sugar. 350 U. SUZUKI. CANE SUGAR IN SEEDS. Il. Seeds of Camellia theifera. The seeds were at first deprived of their shells. 600 grams of these dried and powdered seeds were, after extraction with petroleum ether, extracted with hot 90% alcohol. The alcoholic extract yielded on cooling a yellowish white precipitate which was removed by filtration. The filtrate was now evaporated to a syrupy consistency and then warmed with the addition of just as much 90% alcohol as was necessary to dissolve the whole mass. After standing a few hours, crystals of cane sugar were abundantly produced, which were recrystallized several times from 90% alcohol. Thus nearly 30 grams, or 5% of the dry matter of the seeds, were obtained. As far as my knowledge goes, this is the first time that such a high percentage of cane sugar has been found in seeds. Thus FE. Schulze found in those of : Oats } gram from 3 kilo Earth nut 8.0 gram from 1 kilo. Rye 0.15 Pe BS Yellow Lupine 4.0 Prine sie ay Buckwheat 3.5 5 oy Sun flower 0.7 5 500 gram. Coffee 2.5 7 BX 55 A high percentage, however, is also found in pollen, viz. 11-14% in that of the pine and of the hazel nut (P/anta). On the Formation of Asparagin in the Metabolism of Shoots. BY U. Suzuki. It is a fact that while the various amido-compounds formed from protein during the germination process disappear gradually with the further development of the shoots, the asparagin increases to a certain phase whereupon also this disappears. The more carbohydrate is present in seeds, the quicker will the asparagin disappear in the following period. But with seeds rich in protein the gradual transformations of the nitrogen compounds can be much better traced; the accumulation of asparagin is more considerable, and it is here that the sugar is finally formed by assimilation in the young leaves which causes the consumption of the asparagin in the process of protein formation. We owe to Prof. £. Schulze and his students many very valuable investigations in this line. But thus far it is not entirely clear how the primary amido-compounds such as leucin, tyrosin, arginin, etc. formed by the action of a proteolytic enzyme upon the reserve protein of the seed, are gradually transformed into asparagin. It seems to me that most probably the primary amido-compounds are destroyed more or less completely by an, oxidation process, and that the nitrogen liberated thereby as ammonia serves for the synthesis of aspara- gin, since ammonia would act noxiously by its accumulation.! Besides, as I proved some years ago, ammonia offered to the roots in larger doses than is needed for the immediate protein formation, is transformed into asparagin and is stored up as such 4 Compare also the theory of protein formation of O, Loew, in Chapt..8 of: Die Chemische Energie der Lebenden Zellen. 352 U. SUZUKI. for further use.! The carbon for the asparagin is furnished either by the products of partial oxidation of the amido-com- pounds or by sugar. Now if this view is correct, then the partial destruction of the primary amido-compounds, and hence also the formation of asparagin, would be stopped when air is withheld and oxidation is prevented. I therefore tested the behaviour of shoots in the absence of oxidation, which deprivation they can endure for a certain time. Such an experiment had already been made by Palladin,? who observed that in the absence of oxygen asparagin formation stops, which fact he, however, tried to explain in a manner quite different from that which we can accept.? Clausen repeated these experiments with less decisive results. My experiments, however, with barley and soy bean shoots confirm the observation of Pal/adin, and I infer therefore that asparagin must be considered as a synthetic product formed with the atid of an oxidation process, after partial destruction of the primary amido-compounds. In my experiment, the etiolated shoots were kept in an atomosphere free of oxygen ® for 45—52 hours at a temperature of 15—20°. I then compared the amount of ammonia and of asparagin with that of the control plants. In my first experiment with barley I had withheld the oxygen for too long a time and some shoots had died off; hence this experiment with barley shoots had to be repeated. Experiment with barley. Etiolated shoots of barley of an average length of 24 cm. were carefully deprived of their endosperm ® and divided into three portions : 1 Bull. College cf Agr., Imp. Univ. Tokyo. Vol. II. No.7. (1897). Ber. D. Bost. Ges., vol. 6. (1888). 3 He believed that the protein yields asparagin by direct oxydation. + Landw. Jahrbicher, Vol. 19. n 5 The oxygen was removed by caustic soda and pyrogallol in the usual way. By measuring the decrease of the volume of the air in the bell jar, the test was made for the complete absorption of the oxygen. 6 The presence Of the endosperm would have rendered the result much less decisive. It was to be assumed from analogy that the young plant had absorbed a sufficient amount of the primary amido-compounds, formed previously in the endosperm from protein, to assure a proper answer to our question. ASPARAGIN IN THE METABOLISM OF SHOOTS. 353 (a) Original, dried at the beginning of the experiment. Number of shoots 250, average length 24 cm., dry weight= 5.4126 ¢. (6) Control. Kept 45 hours in distilled water in darkness Number of shoots 250, average length 24 cm., dry weight= 5-1355 g- (c) Test shoots. Kept 45 hours in darkness, deprived of. oxygen. Number of shoots 250, average length 24 cm., dry weight =4.678 g. The analysis gave the following results : Original. | Control. Total nitrogen ...... 5.00 4.90 Albuminoid | nitrogen ... 2.46 | 1.99 Asparagin | nitrogen ...| 1.40 1.88 Ammonia nitrogen ... 0.20 0.17 Amido nitrogeny...| 0.94 0.86 In 100 parts of dry matter Test shoots. || Original. 5-45 100,0 49.20 27.96 4.00 18.84 Control, 100.0 40.61 38.12 3:55 17.72 For 1co parts of total nitrogen Test shoots, 100.0 41.65 27.90 4.04 26.42 a EE EE SR SR EE SS ES NS In 100 parts of dry matter Gonitroll esses esc 8.85 In absence of air... 7.16 Original cscs. , 6.59 Asparagin. 0.1818 0.1336 0.1427 Every 100 shoots contain 1 The shoots were nearly dead, but the roots were still fresh. + Amido-nitrogen means here nitrogen in the form of the primary amido-compounds formed from protein. U. SUZUKI. Experiment with the Soy Bean. Since an experiment with shoots of the soy bean had convinced me that very small shoots of g—10cm. height do not give a decisive answer, larger shoots were selected for the chief test. The shoots had an average length of 18 cm. (2) Original, dried at the beginning of the experiment. Number of shoots 51, dry weight=5.197 g. (6) Control. Kept 52 hours in distilled water in darkness. Number of shoots 51, dry weight=4.804 g. (c) Test shoots. Kept 52 hours in darkness, deprived of oxygen. Number of shoots 50, dry weight=4.592 g. Analysis : _ OO In roo parts of dry matter For 100 parts of total nitrogen Original. | Control. | Test shoots. |} Original. | Control. | Test shoots, Total nitrogen .,.... 9.69 9.83 16.39 100.0 100.0 100.0 Protein nitrogen ... 2.49 2.44 2.50 25.70 24.93 24.07 Asparagin nitrogen 5.80 6.20 5.90 59.70| 62.95| 57.41 Ammonia nitrogen 0.23 0.26 0:31 2.35 2.65 3.01 Amido-nitrogen ...| 1,17 0.93 1.68 12.25 9.51 15.51 Asparagin. In absence of oxygen ... In roo parts of dry matter 29.20 27.79 Every 100 shoots contain In addition we place here the amount of the primary amido- compounds in the presence, and in the absence of oxygen, showing that their decrease in the presence of oxygen is inti- mately connected with the increase of the asparagin. ASPARAGIN IN THE METABOLISM OF SHOOTS. 355 Nitrogen in form of the primary amido-compounds. Barley. Soy bean. In 100 parts of | In 100 parts of In 100 parts of | In 100 parts of dry matter | total nitrogen dry matter | total nitrogen \IMUR CSS 2 SonpopsonoRe 0.86 G2 0.93 9.51 Without oxygen......... 1.44 26.42 1.68 15.51 The following conclusion may be drawn from these results : In etiolated shoots, the decomposition of protein continues in the absence of oxygen as well as inits presence. This is in accordance with what we know of the action of enzymes. The shoots had remained apparently healthy for 45—52 hours when deprived of oxygen. Only some shoots had lost turgor, but these were quite free from any bacterial attack. The roots were in contact with but little water and retained their normal appearance, A very decisive difference in the production of asparagin was however noticeable : No increase in the absence of oxygen, but an tnerease tn tts presence. Just the reverse is observed with the primary amido-com- pounds, which decrease by the production of asparagin from them. Analytical Data. Total nitrogen Protein nitrogen Asparagin nitrogen | Animonia nitrogen Seclon | 2 lane = leks o8| = |nBulos z awe | © Aare os ls 2 ol ae o. ae a) sc 3 iene = tos) | eal See ae) ee aE al ae SP) gee BIS 'ts es i) 2 7, 5 S| eZ a Sl 4 z £ ZA | 0.465] 9.1 |0.02325| 0.930] 9.2 |0.02288] 0.465] 1.3 0.00325] 0.465] 0.37] 0.00093 (1) | “a ” 9.5 | ” 9.1 % 1.3 ” 0.37 3S | °o | 7,2 - Fa { 0.461) 9.1 |0.02263]| 0.922] 7.4 |0.01838] 0.461] 1.7 |0.00431] 0.461/0.37| 0.00080 2 m1 ”) ; , ” 9.0 ” i 9) ” 1.75 ” ie] 27 3S = | ( 0.465|10.2 |0.02534] 0.930] 8.7 |0.02106) 0.465] 1.4 |0.00353] 0.465] 0.37/0.000988 (3) | Wiles stents 5) Sas jy (aay Bowe 356 U.. SUZUKI. ASPARAGIN IN SHOOTS. Analytical Data. Total nitrogen Protein nitrogen | Asparagin nitrogen} Ammonia nitrogen Dry matter used BaO replaced replaced. N. found Dry matter used BaO replaced. N. found. Dry matter used BaO replaced. N. found | 0.460|17.8 |0.04457] 0.920] 9.6 |0.02291| 0.460] 5.3 [0.01332] 0.460) 0.42/0.00105 (1) wn > . > ” s ” 6 ( 0.456)18.0 |0.04482] 0.912) 9.0 |0.02229] 0.456] 5.6 [0.01407] 0.456) 0.47|\c.01175 ai { 0.453|19.3 |0.047C6} 0.906) 8.9 |0.02266] 0.453] 5.4 [0.01345] 0.453] 0.57|0.001425 (3 » |0.48 wn au Soy bean shoots. l ° ” « ” 5:45 ” 0.59 (1) Original shoots. (2) Control shoots. (3) Shoots in absence of oxygen. 10oc.c. H,SO,=0.303 g BaSO,. toc.c, H,SO,=14.6c.c. Ba(OH),. Ic.c, Ba(OH), =0.00249 g. Nitrogen. For the determination of ammonia in the shoots, the air dried, fine powder was well mixed with some water and then put in a flask, 100 c.c. water and 1c.c. of 1% MgO suspension was added, and then subjectcd to distillation, the escaping ammonia was absorbed by standard sulphuric acid and titrated as usual. The control test with pure asparagin solution shows that no noticeable amount of ammonia is developed by the above treatment. It therefore can safely be assumed that the developed ammonia is not derived from a decomposition of asparagin (or other amido-compounds) in the shoots, but that it was present as such in the shoots. Asparagin nitrogen was determined by Sacchsse’s method. The Composition of the Nuts of Gingko biloba. BY U. Suzuki. The analysis of the nuts of Gingko biloba, a tree of frequent occurrence in Japan, is naturally of some interest since they are extensively consumed as food. Freed from the shells, the nuts composition ; showed the following In 100 parts of dry matter, Ciiide- proteitt 4...) oe. et ee yes Dies Grude fat .....: 2 ee 2.0 Sleecithitr 2...” .. AMM Sc ccslowues, patie oan O07 SrudeGbre *: 5 7 ee oo) ako we eevee 12 Ash (free from carbon and insoluble matter).. 3.0 Starch Vc... > | See ee Ce RS. 62.4 Canesugar |: . Ae. os ew 2 INCAUCINE SUS aT — em. yo ales Sa ether s fal Hotal nitrogen “Paws... 228.2. ee ee. 1.8 Albuminoid nitrogemmeern..+.ee.csce2 cack. - 1.4 Nuclein and other non-digestible nitrogen 0.26 Non albuminoid nitrogen (mostly basic) .... 0.4 In 100 parts of pure ash (including CO,). TO. pe RUMMMMIEE Sc -tascp ao odees three 47.3 NacO™ . Number of young plants appearing. Cabbage. l : CaO:MgO | CaO:MgO} CaO:MgO CaO:MgO No: L.(3 = x)eel| Nos tk (2 : 1) ie SIL (x = 3) [Nos iV (eee) | | April 24, | i: I. 2: o. 25. 2 2 5 I | » 26. 2 3 6 I 20:95 || 2. 4. | 6. I. | Since some of the seeds failed to germinate, a number of young plants taken from the field served on May 3rd. to increase in each pot the number of shoots to seven. On May 3oth.,a TO WHAT EXTENT SHOULD A SOIL BE LIMED, 375 photograph was taken, see Plate XLV. which shows that the development of pot No. II., was the best, next came No. IIL, while that of No. IV. was exceedingly poor. On June 18th., the plants were cut and weighed in the fresh state with the following results :— Galhase Rate of Total weight of Weight of the Average a CaO : MgO. seven plants. largest plant. weight. Nore B Oe | 375 gi 130 gr. 54 gi No. II. B38 Si 475 gv. | 140 gr. 68 gr. No, III Test 390 gr = 100 gr 56 er, No. IV. | tee E 75 or | 45 gr. II gr It follows therefore that for cabbage the ratio of 2: 1 between the assimilable lime and the magnesia present in Pot No. IIL, was far superior to the other ratios. Experiment with Buckwheat. On April 1oth., buckwheat was sown, there being two series of four pots each in eight of the pots, each pot receiving fifteen seeds. On May 3rd., the number of equally well developed shoots in both series, A and B, was reduced to eight. On May 5th., the flowers began to develop, but with different degrees of rapidity as is seen from the following table : — | Number of plants bearing flowers. le Wot No: Ty | iRotNo: IT. Pot No. IIT. Pot No. IV. Buckwheat. | CaO:MgO | CaO: MgO CaO : MgO CaO : MgO | Ses Dees I fe Cees ee ee) oo — a! — — | —. ee ae A | \ Fs | d I \ d Z f I i [@) a) | LB fo) | 3 I B fo) Lb fo) | A 2 A 5 A I EN I Beeat: B 3 B 4 B 2 3 376 T. FURUTA. On May 12th., flowers were open on all the plants. It will be seen that the flowers in Pot No. II. opened sooner than those in the other three pots. On May 15th. a photograph was taken (see Plate XLVI.) which shows that pot No. I. had the most leaves, and that No. II. showed the greatest height of plants, while No. IV. was backward in development. On May 30th., the seeds approached their ripening stage. On June 12th., white spots of a fungus appeared on several leaves, especially on leaves of No. I]. A. On June 19th. the plants in the series A of these pots were cut and weighed with the following results : Buckwheat. | No. I. | No. IL | No. III. | No. IV. 8 plants ) CaO : MgO | CaO: MgO | CaO: MgO | CaO: MeO in each ;ot. eyes. tis 2 2 85 Fee 1 22 Total weight. 382 er. | 220 gr. 190 gr. | 106 gr. Av eight of we ppc z 35.3 s- | 27.5 gt. | 238 gr. 13.3. gr. Average height. | 72 cm. | 68.3 cm 57-4 gt. | 48.6 cm ee Number of leaves. | 199. 155 31° | 118, | | Number of (ripened. | 112. 06 75 62 the bundles of seeds, mise 18. 10 64. 19 Weight aieaes | 55- gr. 45- 2F 34. gF. 20. gf. se unripened. 5-5 gr. 5-5 er. 14.5 gr 5-5 oF. | | = aa Weight of 100 os Sees = bi illo o ripened seeds. | 453 8% | 435 &f- | 3-92 §T. 3-86 gr. Conclusion :—On glancing over this table it will at once be seen, that the yield from the pot No. I., was in every respect the richest. Tolerably close follows the yield of pot No. IIL, but with pot No. III, a very considerable falling off will be noticed 1 Pot No. III., was rich in leaves, but many of these leaves were very small, TO WHAT EXTENT SHOULD -A SOIL BE LIMED. Vik and still greater is the decrease from pot No. III., to No. 1V. There can therefore be no doubt that the ratio of 3 parts of CaO to 1 part of MgO in the soil is the most favorable for buckwheat. A further increase of lime, to judge from observations with other crops, would in all probability depress the yield. The second series, B, of buckwheat plants, (8 plants in each pot) was cut on June 28th, with the following results : Nowe Noy 10 No, III. No. IV. Buckwheat. CaO : MgO | CaO: MgO | CaO: MgO | CaO: MgO 3) cael 2 AD pia ee! nog @ Total weight. 340 gr. 275. gr. 281. gr. 240. gr. Average weight. 42.5 gr. 34.4 gr. Rept Ash 30.0 gr. Average height. 75.1 cm. 67.1 cm. 67.3 cm. 56.3 cm. Number of leaves. | 267. 182. 226. 173. Weight of {ripened., 64.5 gr. 58.0 gr. 58.5 gr. 56.0 g) the seeds. | unripened. 6.5 gr II.O gr 8.5 gr. 8.0 gr Weight of Ico meee : Jor ; ripened seeds, 4.30 (ote 5-70 gr. 4.12} gy. 4.64 gr. We observe from this comparison that also here the richest yield as to straw and seeds was obtained with the ratio 3 CaO to 1 MgO while on the other hand the single seeds were best formed with the ratio 2 parts CaO: 1 part MgO, since 100 seeds weighed here considerably more than in the other cases, Experiment with Oats. On April toth, four pots were sown with oats, each receiv- ing 15 seeds. The rate of germination was about equal in the four pots. On April 28th, the young plants were thinned out to eleven in each pot. On May 12th, it was noticed that in pot No. IV., some leaves were withering and becoming brown, which phenomenon had made considerable advance on May 3oth. 378 T. FURUTA. On June 8th, buds began to appear on the plants except in No. IV., where they appeared later, as will be seen from the following table ; Number of stalks bearing flowers. Oats. | ; = —— — | No; I} @GiieaeNo: 'I1.\ * |. iNest teh eeomenn z | er | cae eS June 10. | 8 8. 4 (a) Pham (72 | Io 8. 7 fe) — — — | — ————— ~— | Rela: 12 TO. 10 I | = ms | | | | = : ra ire sel 13. 13. 12. 4. | | = eae | | Zee TL lg | 18 21 21 fe) I On June 1oth, black Aphzdes appeared on the leaves and stalks of the plants except on those of No. 1V. On June 17th, a photograph was taken which is reproduced in plate No. XLVIII. At the same time the length of the tallest stalks was measured . with the following result : No “i 140 cm. Now Tic 145 cm. Now il 145 cm. No. IV. 108 cm. The plants in pot No. II., were richest in leaves and stalks ; then followed those in No. III., while those in No. 1V., showed a rather poor development. The results obtained with oats differed from the results obtained with buckwheat. The oats had ripened on July 22nd, and yielded on harvesting the following data : TO WHAT EXTENT SHOULD A SOIL BE LIMED. 379 CaO Ratio of ———. Natl MgO | | il) = Nowe ie No. II | No, III No, IV. 3 I 2 I | I I iB 2 | a Average height ......... 113,cm, | 116cm. | 20cm, | gt cm. Motaliweight ...,2-s.c0. 260 g. 280 g. | 297 g. 219 g. Fresh weight of | a | | g Ss grains with uae 46 g. | SS DES ae: Fresh weight of straw...| ONG (5 229 g. | 247 g. 203 g. As a general result we observe that No. I., and No. IV., gave a smaller yield than No. IJ., and No. III. An increase of ; ; 2 CaO lime beyond the proportion of 7 MgO brought on a moderate decrease, while an increase of magnesia in No. IV., caused a considerable decrease in the yield. : CaQ - : The most favorable ratios WeO in my experiments were therefore : Buckwheat = 3: I Cabbacemwe = 2 : I Oats = Ne | In turning back now to our original question : “ To what extent should a soil be limed?”, we would have to answer: After the amount of casily asstmilable lime and magnesia has been determined in the way above indicated by me, the ratio should be corrected by adding lime to such an extent that tt becomes 3: 1 toa given depth, when crops rich tn foliage are to be grown, while the ratio 1:1 has to be prepared when oats and simtlar cereals are to be grown. > ‘ © * ee eo = = + On the Lime-factor for Different Crops, BY O. Loew. Remarks on the foregoing communications of Mtr. Aso and Mr. Furuta. On reviewing the results of Mr. Aso and Mr. Furuta the fact that the greatest yield of a certain crop depends—other things being equal—upon a distinct ratio between lime and magnesia cannot be denied. Mr. Aso has operated with water cultures and restricted) his observations to the period before flowering and fruiting, while Mr. /wruta has operated with soil- cultures and directed his observations to the ripened harvest. On comparing Aso’s results with barley with Furuta’s results with oats, the conclusion may be drawn that cereals before the fruiting stage require more lime relatively to magnesia than in the fruiting stage. This does not surprise us, since on the one hand the formation of the leaves requires much lime, while on the other hand the formation of grains, much magnesia. It is a fact that the grains of cereals contain more magnesiumphos- phate than calciumphosphate. The greater the leaf surface to be developed in a given time the greater will be the amount of lime required, hence we observe also in the above experiments that soy beans (Aso) and buckwheat (/uruta) require more lime relatively to magnesia a, than oats (furuta). The best ratio of eo is according to 382 O. LOEW. these experiments ! for oats (ripened harvest) 1: 1 for barley, before flowering, 2: 1 cabbage 2: I buckwheat 3: 1 If we take the amount of magnesia as the unit we may call the relative amount of lime present the “4mefactor and express the result thus: The limefactor for buckwheat is 3; for cabbage, 2; for oats 1. If we find the limefactor in a soil 1, the soil does not need liming for a crop of oats, but will need it when buckwheat or onions are to be grown. The beneficial influence of the rotation of crops is partly due to the variations of the limefactor in the soil caused by the growth of different crops. Since the limefactor of cereals before flowering is larger than later on after the flowering stage, great benefit might accrue from the application of a highly diluted solution of calcium nitrate to the young plants. The limefactor for various crops may be calculated from the average of many ash analyses ; and thus we find the limefactor by calculation from MWol//’s tables of ash analyses, for Wheat in the flowering stage.... 1.4 2 Clover ” ” ” ” Oy" 3: Lettuce: 5: see: «ss ee 2.8 Sugar icaneaeeemen..: . 2 bersemers 1.05 But it is safer to determine the limefactor by actual test, since the analysed plants may have taken up more lime than necessary. Many plants will render this surplus partly or wholly innocuous by precipitating it as oxalate. Should plants, however, take up more magnesia than is required from a soil rich in magnesia but poor in lime, the injury cannot be repaired, the harvest will be very unsatisfactory. Such a soil is devoid of natural fertility, even if it be rich in potassa, phosphoric acid and nitrogen compounds. One of the beneficial effects of liming is believed to consist in, ‘unlocking such potassa, as is at the time unavailable for 1 With onions in water cultures the results were best with the ratio 2: rf, but the results with the ratio 1 : 1 were nearly the same, These experiments have to be repeated with soilcultures as must also those with soybeans, since Aso’s tests relate only to a very early stage. These figures relate as usual in agricultural relations to absolute quantities, not to the number of molecules, ON THE LIME-FACTOR FOR DIFFERENT CROPS, 383 plants”; but it may be objected that such potassa as can be unlocked in the soil by slaked lime or carbonate of lime, can surely also be unlocked by the rootlets themselves. The writer showed years ago that ove of the effects of tncreasing the lime consists in the rich development of roothairs) and this explains satisfactorily the fact that on liming the soil, the plants become capable of absorbing an increased amount of potassa. A second very important physiological effect of lime is the rich production of dark green and normal chlorophyllbodies. The retarding effect of an abnormal excess of lime over magnesia in the soil, or of an excessive liming, may be overcome by an application of powdered magnesite. Burnt magnesia or precipitated carbonate of magnesia should be avoided, being too finely divided and hence much more easily absorbed than the lime compounds. It was in the year 1892 that I first called attention to the importance of a proper ratio between lime and magnesia in the soils, but nobody took notice of my deductions. I had said ?: ‘“ Aus unseren Studien ergibt sich also, dass, ein so notwendiger Bestandteil der Pflanzennahrung auch Magnesia salze sind, sie doch bei gewissem Ueberschuss schadlich wirken, wie kein anderes Nahrsalz. Ist zu viel Magnesia im Verhialtniss zum Kalk vorhanden, so ist eine pathologische Wurzelentwickelung oder baldiger Tod der Wurzeln die Folge, ist aber zu wenig vor- handen, so wird die Entwickelung der Pflanzen verzégert. Dort treten Gift-, hier Hungersymptome auf. Es ware nun von Interesse, festzustellen, welches Minimum von Magnesia im Boden bei gegebenem Kalkgehalt noch eine annehmbare Ernte zulasst, und andererseits wie viel Kalk bei gegebener Magnesia menge nothig ist, pathologische Erscheinungen zu verhindern.” It was Mr. A/ay in Washington D.C., who at my suggestion first took this problem up last year and who obtained very decisive results.* Among other things he found that in certain cases gypsum can better overcome the injurious action of an excess of magnesia in the soil than carbonate of lime can. The writer’s theory of the physiological functions of lime and magnesia was first published in Flora (1892) and later on 1 Flora 1892, p. 384. 2 Landw, Versuchsstationen, vol. 41, p. 474. ® Bulletin No. 1 of the Bureau of Plant Industry, Washington, rgor. 384 O. LOEW. in English in Bulletin No. 18 of the Division of Vegetable Physiology and Pathology, U. S. Department of Agriculture 1899, under the title: The Physiological Réle of Mineral Nutrients.1.| From this a few lines containing the main points may be extracted. The lime is according to this theory necessary for the formation of certain calcium compounds of nucleoproteids required in the organized structures of nuclei and chlorophyllbodies, while the magnesia serves for the assimi- lation of phosphoric acid, since magnesium phosphate can give up its phosphoric acid more easily than any other phosphate that occurs in plant juices. While calcium is fired in the organized structure, magnesium is wovable, since one and the same atom can in the form of secondary phosphate serve repeatedly the same purpose as a Carrier of assimilable phos- phoric acid in the formation of nucleoproteids and lecithin. It follows therefore that in the case of an excess of lime being absorbed, the assimilation of phosphoric acid will be rendered more difficult, since this acid will then chiefly combine with the lime whereby the chances for the formation of magne- sium phosphate will be diminished. The effect will then be the sane as if the amount of available phosphoric acid in the soil were lessened, i. e., the growth of the plant will be retarded and even starvation phenomena will set in. The effect of this excess of lime will be still more marked with the decrease of the phosphoric acid present. If on the other hand an excess of magnesia is entering the cells, the calcium nuclein compounds of the organized structures can not be formed or when previously formed, will be changed into the respective magnesium compounds, which are not suited for the same function, perhaps on account of a very different capacity of imbibition. Hence nuclei and chlorophyllbodies will first suffer from the excess of magnesia, and this can be traced under the microscope on filaments of Spirogyra. Even ina 0,1 per cent. solution of magnesium nitrate these cells will die within five days, while on the addition of 0,3 per cent. calcium nitrate they will remain alive for a number of weeks, although on account of the absence 1 Comp. especially pp. 28 ;37 3 42 ; 47 ; 60. and Bul, No. 1. Bureau of Plant Industry. That Bul. will be sent free of charge to any one who applies to the U.S. Dept. of Agriculture, Washington, D. C. ON THE LIMEFACTOR FOR DIFFERENT CROPS. 335 of other mineral nutrients the multiplication of cells will cease. The functions of lime and magnesia are intimately connected with each other, since the nuclei require lime and phosphoric acid as separate constituents. These inferences also hold good for the animal organism and the law inferred by the writer,— the greater the nucler mass of an organ the greater ts also tts lime content has been confirmed in a number of cases,! 1 Cf the above cited Bul. No. 18, p. §7. & zed _ ‘ety: J . Fo hs ar 7 é 4 = Ba * ® rE ee ? rr ive walks SURES BILE ated tam | i oF Vee va i \ «ane eke » ALA , . al ie s ioe s nh ; a a - fa 1] o> ie , Y cy o ,!@a 1 ae On the Lime Content of Phanerogamic Parasites. BY Of phanerogamic parasites, only one, Cuscuta europea, has thus far been examined with regard to the composition of the ash. When compared with other phanerogamic plants, the striking fact was revealed that this ash is exceedingly poor in lime, containing only about 29; while its host, the clover, is rich in lime, yielding an ash containing about 30—36%. The fact that parasites being devoid of chlorophyll require less lime than green plants, is of much interest. Seedlings require less lime as long as they have no chlorophyll. Further, etiolated leaves of Vicia faba contain less lime than the green leaves.! According to Church,? also less lime (and more potassa) is present in albino leaves than in normal ones. These facts agree well with the inference that not only the nuclei but also the chlorophyll-bodies, require lime. I had therefore believed it of interest to investigate in this respect another phanerogamic plant without chlorophyll, and selected Gastrodia elata Bl., an orchid. This plant is characteristic of Asia and is not found in Europe. It occurs frequently on the main island of Japan.* The stemlike, brownish peduncle which has some scales comes forth from the ground in the spring and reaches 60—7o0 cm. in height; in June appear some yellowish brown flowers which yield capsules with numerous very small seeds. The under- ground part consists chiefly of a rhizome covered with scales 1 Palladin, Ber. d. Deut. Bot, Ges., Vol, X., p. 179. 2 Jour. Chem. Soc., 1878 and 1886, 3 Near Vikko it is very frequent. 388 k. Asé. and is very rich in starch.t Whether this plant is parasitic or saprophytic is not yet quite decided. I collected the plant in the beginning of June and separated the rhizomes from the other parts.* The ash 6f each part was separately analyzed with the following result : In 100 parts of dry matter. Above-ground part .......... 5.25 % Under-ground part.......... 3.904 % In 100 parts of dry matter. Above-ground part. Under-ground part. SO, +... +2 eee G.100. . Ao eae 0.073 P.O; ...2. see 500. . <2 eae 0.679 KO: 2.2, 23a P2322 . 4 ae eae 2.009 Na,O |. ss. eee 9370. 46E bee 0.541 CaO... 22s ae BISA sie eee 0.200 Ma... eee TE tv Ae soe 0.251 Re,0.,.. <>. ee ES ae pee oe 0.102 In 100: parts of ash. Above-ground part. Under-ground part. SU. > 3+... eee BETS peers 1.85 PGS gn sss 64 eee 257 . . eee 17.24 KO... eee HAL? S . . eee 50.99 NatO ... Zee 22. eee 13.73 01 6 pea. - B31... cee 5.08 MgO. 7.25 -<2 ape BOO. <2 sche eee 6.37 FeO. 2... ee Rol . Se Saeko e eee SiO] atid lossaeee- 8? =... aera 245 When we compare these results, with the ash constituents of green plants, a striking fact is noticed in regard to the ratio of lime and magnesia. In the chlorophyll-bearing parts, the content of magnesia is always surpassed by that of lime; for instance, with cereals in the blossoming stage the ratio of CaO : MgO is 2: 1; in lucerne before flowering 8: 1 ; and in rape before flowering 5.5: 1. Church compared the ash of albino leaves with that of green leaves of Quercus rubra and found. in regard to lime and magnesia the following data : 1 After fruiting, the plant dies having used up all the nutrients in the rhizome. 2 According to Mr. S#idata, botanist in the College of Science, Imperial University, Tokio, this plant is probably a saprophyte, because no one has observed any connection between its rhizome and the root of other plants. 3 TI found only traces of calcium oxalate in this plant. ON THE LIME CONTENT OF PHANEROGAMIC PARASITES. 380 Albino leaves. Green leaves. CaO) ” F, 235—250°C | slightly acid | G. 250—260°c acid és H. 260—270°C neutral | 4g. I 270—275°c | = 32. it 275—280°c ” | 3 &- K, 280—300°C | —— | very little es Residue. | ees 2.4 g. Since the fraction H, which distilled between 260-270°c, and and most of it more exactly at 264,° as a second trial showed, exceeded all the other fractions in quantity and showed the very agreable odour of the wood ina high degree, I subjected it to an elementary analysis with the following result : I. 0.1740 g. yielded o.1940 g. Hz, O, and 0.5376 ¢. CO, corresponding to 12.38% H, and 84.43 of C. II. 0.2414 g. yielded 0.2453 g. H.O, and 0.7542 CO, corres- ponding 11.3 % H, and 85.2 % C. This would correspond tolerably well to the formula CrgilasO: Theory Experiment I. II, Ge Seu 84.9 84.43 85.2 Ee eee es 12.38 19-3 6 A ot eta Re 4.0 3.5 The supposition might be entertained that the new com- pound is a camphor-like substance related to triterpene, or VOLATILE OIL IN CRYPTOMERIA WOOD. 405 to the cholesterins. Indeed the solution in chloroform produces on the addition of some concentrated sulphuric acid a dark red color. I propose to call it Swgzol, from sugi, the Japanese name for Cryptomeria Faponica. Sugiol is an oil of neutral reaction, almost completely in- soluble in water, but easily soluble in alcohol, ether and chloroform. Its boiling point is 264°, spec. gravity 0.935. It yields no crystalline acetyl and hydrazon compound, but reduces alkaline silver solution very slowly in darkness. hee Mand, oh! iP a 2 oe a O ¢, ‘te of a > Pa ee aid - «] : Ca i. 7 ifs 7 ae +f tal a a ba <4 Pa , : on eee ian cehtben 7 io ‘lee +) ue On the Poisonous Action of Quinone. BY T. Furuta. ae Numerous phenol derivatives have been tested for poisonous properties, but the ordinary quinone has been almost wholly neglected in this regard, although it is of some physiological interest. Beserinck has recently shown that Streptothrix chromogena, a soil fungus, has the remarkable property of producing quinone from proteids.t This author is even inclined to ascribe the process of the formation of humus in the soil largely to the quinone produced by this fungus. Quite recently Phisalix has observed a peculiar poison in the secretion of a certain myriapod which he and Séhal after- wards identified as quinone. Since quinone is a labile di-ketone it appeared to me of particular interest to ascertain whether it is a general poison, and to compare it with related compounds. As subjects, served shoots, twigs, isolated leaves, algae, mould fungi, bacteria, insects, tadpoles and mice. Experiments with Shoots. 1. Young plants of the soy bean 12—14 cm. high were placed in 1% solutions of quinone, hydroquinone, resorcin, pyrogallol and phloroglucin. The roots in quinone showed first, after a few hours, a decided injury and discoloration and it took only four days to kill the entire shoots, while it took eight days with hydroquinone, seventeen with pyrogallol, and twenty with phloroglucin. The control plants were still alive. Phloro- glucin is trioxybenzene, but it is considered by some authors as a tri-ketone. Nevertheless one would expect that such a tri- ketone would prove about as strong a poison as quinone. 1 Central-Blatt f. Bakt. (II, Abt.) VI. 1900. 408 T. FURUTA. 2. Wheat. Young wheat plants of 15 cm. length kept in the same solutions were first injured by quinone, then followed hydroquinone and the other compounds in the same order as observed with the soy shoots. A second experiment with 0.5 per mille solutions yielded essentially the same results, Experiments with Twigs. Twigs of plum trees of about 20cm. length and bearing 20—22 flower buds were placed in the same solutions. In quinone the buds withered and gradually dropped off; not one had opened. In hydroquinone, only four buds opened within twenty-one days; the others had withered, In resorcin, two buds opened within eleven days ; later on all the buds developed into flowers. In pyrogallol seventeen buds opened within twenty-one days. A second test with bud bearing peach twigs 15 cm. long yielded similar results —Vapours of quinone seem to be much more poisonous than the aqueous solution. A young cabbage branch placed in a flask covered with a glass plate, was exposed to the vapours which one gram of quinone developed from the bottom of the vessel at the ordinary temperature. This branch died within twenty-two hours ; the turgor was lost and the normal color was changed. Young leaves of Zyifolium, Photinia, and Rhododendron died under these conditions within twenty-five hours. Leaves with a thick cuticle, as those of Camellia, succumbed more slowly. Isolated leaves placed on a 1 per mille solution of quinone died within two days ; while it took 4—5 days with hydroquinone, and ten days with resorcin and pyrogallol. Experiments with radish seeds left no doubt, that the germinating power was greatly injured by three days soaking in a I per mille quinone solution, Of 50 seeds only 8 germi- nated, as compared with 25 in the control case. Experiments with Algae. Diatoms and filaments of J/esocarpus and Spirogyra were placed in 1 per mille solutions of quinone, and observed every thirty minutes under the microscope to ascertain whether the protoplasm was contracted and the contents of the cells would ON THE POISONOUS ACTION OF QUINONE. 409 stain quickly by highly diluted methylene blue. It was thus found that diatoms and the cells of MZesocarpus were killed by quinone in three hours and forty minutes ; soon afterwards also Spirogyra. Yowards algae hydroquinone also proved very poisonous ; indeed with diatoms hydroquinone acted nearly as powerfully as quinone. With resorcin, pyrogallol, and phloro- glucin, death was observed more than a day later. Experiments with Mould Fungi and Bacteria. Penicillium glaucum developed on beerwort to which 1 per mille quinone had been added, while Asfergzllus oryzae did not. Bacteria were inoculated in bouillon to which after steri- lization 1 per miile quinone had been added. The bacillus of typhoid fever of mice could not develop in this solution, while Bac. pyocyaneus gave a meager growth on the surface. Experiment with a Mouse :— The vapour which quinone emits at the ordinary temperature exerts a highly poisonous action on warm blooded animals. About } gram of quinone was placed in a deep beaker glass (1 liter capacity) and upon this at a height of 3 cm. above the quinone a wire gauze was placed. A small white mouse was then put in the beaker glass and this covered witha glass plate, which was often lifted to admit fresh air. The animal became at once very much agitated, tried to escape and soon, gave evidence of irritation of the eyes and nose. After one hour all the motions became sluggish, and after two hours more the animal was dead. Under the same conditions horse-flies died within three hours. Experiments with Tadpoles. Tadpoles were placed in the o.1 per mille solutions of quinone, and the time required to kill the animals was : (1) with quinone : 33 minutes. (2) ,, hydroquinone: 58 i (3) ,, pyrocatechin: 22 hours and 23 minutes. (4) ,, pyrogallol: Cy eee Any el 56 (5) ge SESOneIne cy 5S 9 (6) ,, phloroglucin and control (plain water) : they were still actively moving after six days, 410 T. FURUTA. POISONOUS ACTION OF QUINONE. The highly. poisonous character of quinone became still more evident by the observation that even in 0.005 per mille solution it killed tadpoles within one hour and thirty-five minutes. Other aquatic animals (Ase/lus, Copepoda) were killed by this highly diluted solution within eight hours. Conclusion: We can infer from all these results that quinone is a very strong poison,—a much stronger poison indeed than the other closely related benzene derivatives. In the face of these facts does there exist any probable basis for the hypothesis of Bezerinck, that the relatively large amount of humus in the soil is due to the action of quinone? It seems to me that another hypothesis would be more natural than this namely, that the quinone produced by Streptothrix chromogena must be changed almost as soon as formed, for otherwise animal and vegetable life in the soil becomes impossible ! Are Coffeine and Antipyrin in High Dilutions Poisonous to Plants ? BY S. Sawa. Many poisons show a stimulating action of some kind or other when they are so highly diluted that any further injurious action can not take place. Gamaleta ' has observed that coffeine exerts a stimulating action on the growth of yeast and certain bacteria. Since coffeine is trimethylxanthine and xanthine is a constituent of the nucleoproteids of the protoplasm, I thought it of interest to observe the action of coffeine in high dilution on phaenogams. The first experiment was carried out with onion plants which were placed ina solution of 1 p. mille of coffeine and antipy- rine with and without the addition of nutrients. It was observed that the plants remained alive for nearly four weeks. In both solutions some new branches started but these developed much better with antipyrine than with coffeine. Coffeine proved gradually to be a much more noxious compound than antipyrine, and the primary leaves withered much sooner in the former than in the latter case. The development after twenty-three days in the presence of all mineral nutrients is seen in the following table giving the measurements of the still living parts. 1 Jahresbericht fiir Thierchemie, 1896 p. 923. 412 S. SAWA. COFFEINE AND ANTIPYRINN. Increase in length of all Length of principal shoot || shoots combined, | at the beginning. | | absolute relative cm. cm. % jae Ee | ie A 26.0 3.0 11.5 Antipyrin | B 21. 3.5 | 16.3 So | = = —_ Coffeine J a 108 | e | Pp ( B 21.5 0.2 | 0.9 (A 38.8 26.7 | 688 Control 1B 40.5 | 26.5 | 65.4 In the second experiment the amount of these bases was reduced to 0.1% and 0.25%. ‘This time young celery plants of about 15cm. height served for the test. After about two weeks some injurious action was observed, commencing with the wilting of the rim of the leaves. The injurious effect of coffeine in this high dilution appears very remarkable since the epidermis of the tea leaves is compara- tively rich in it and nevertheless remains uninjured. The coffeine is here probably shut up inthe vacuole surrounded by a tonoplast of such density that the cytoplasm and nuclei are sufficiently protected against the intrusion of the coffeine. A stimulating effect of some kind or other can only be expected in still higher dilutions of coffeine and antipyrine. Has Urea any Poisonous Action on Phaenogams ? BY S. Sawa. Sometimes urine is applied as manure before its urea is completely split up by bacterial action into ammonia and carbonic acid. But while the ammonia can be absorbed in the soil and thus becomes only gradually accessible to the roots, a poisonous action by a too high concentration of ammonia being thus avoided,’ the urea is not absorbed in the soil as Kellner has shown. It might be supposed, however, that urea could act injuriously and I have therefore made an experiment with young onion plants in order to test this point. Thus far it has been generally assumed that urea would be a good source of nitrogen for the phaenogams, but some objections may be raised against the tests thus far made.? On April 22nd two plants were placed in Axof’s solution with an addition of 0.5 p.m. urea and.also two control plants without this addition. These plants were kept over five weeks, in a room near the window at 15—I8°C. The solutions were twice renewed. A start towards the formation of flowers was made by each plant, but only in the one to which urea had been applied was a full flower developed, in the other three plants there being no further development. Gradually new branches developed with the four plants, but much better with the control plants than with those in the urea solution; soon afterwards, however, the old leaves com- menced to die off from the tip downward. Finally on the 30th of May, the parts still living were measured. The results may be seen in the following table : 1 On the poisonous action of ammonium carbonate in water culture, see Bul. of the Coll, of Agr., Tokyo, Vol. IIL, No, 3. 2 Comp. Hampe, Landw, Versuchs, St. Vol. 10. Also Thompson, Centr, f. Agr. Chem, Aug. Igor ; the urea solution being permitted to act for only 3—5 hours each day. 414 S. SAWA. POISONOUS ACTION OF UREA. On April 22. | On May 30. cm. cm. A 85.0 67.0 (decrease 18) Urea. eae 81.0 52.0( » 29) A 68.0 127.0 (increase 59) Control, B 69.0 12.01) eso) The injurious action of urea even in the high dilution of 0.5 per mille is therefore quite evident. This recalls another observation of Loew and Bokorny on algae.1 Spirogyra was much injured within five days in a solution of 0.2 per cent. urea, and microscopic observation showed that it was especially the chlorophyll bodies that were attacked.? Probably in these organoids, the urea is too readily split up into ammonia and carbonic acid, and the nascent ammonia killed the chlorophyll bodies. The fact that urea in such dilutions does not act poison- ously on fungi and bacteria, and is even a good source of nitrogen for them, would be in accord with this explanation. 1 Journ. f. prakt. Chemie, Vol. 36 (1887) p. 379. 2 Thio-urea acted still more poisonously. On the Poisonous Action of Potassium Persulphate on Plants. The salts of persulphuric acid exert a moderate oxidizing action on certain compounds. Thus Jlorre// and Crofts observed a slow action on glucose in the presence of ferrous sulphate whereby glucoson is produced.t AHugounencg observed an oxidation of uric acid, haematin, and bilirubin by these salts ;? uric acid in alkaline solution yields thus allanturic acid. Préscher found that while methyl green is quickly oxidized, other color bases are not attacked as e.g. methylen blue. He further observed that apomorphine is oxidized by it. The oxidizing character of these salts makes it very probable that they may act poisonously also on living cells ina way similar to the action of hydrogen peroxid. Indeed it was observed by Wacker,* and further by Bérard and Nicolas,* and finally by Friedlinder,® that these salts easily kill bacteria. The last named author states that $% sodium persulphate in aqueous solution prevents the growth of pathogenic bacteria, and a 5% solution kills them; and further that one gram of sodium persulphate suffices to kill a rabbit of 2 kilo weight. Mcolas® observed that an intraveinous injection of 0.04 gram of sodium persulphate per kilo body weight kills a rabbit ; and per os 0.3 gram per kilo, a guinea pig. Loew observed that infusoria died in a 0.59% solution of the potassium persulphate in about thirty minutes. I thought it of some interest to extend such observa- tions also to chlorophyll-bearing plants. 1 Journ, Chem, Soc. 77, 1219. (1900). 2 Chem, News, 1gor. 3 Merck's Report 1894, 35. Semaine Medicale 1899, No. 43, p. 342. 4 5 Therep. Monatschefte 1899, Feb, 6 La Semaine Médicale, Vol. 20 (1900), 416 S. SAWA. For the experiment with algae, as Spzrogyra, Mesocarpus and diatoms, a 0.5% solution of potassium persulphate was applied.1 All these organisms died within one hour. Branches of the rape plant about 17cm. long with numerous buds showed in the 0.5% solution of the same salt an injurious effect after twenty-four hours: the leaves became curled, the tips of the branches withered after three days and the lower part of the branches bleached out. One day later the plants had entirely died off. In the control cases with potassium sulphate and with distilled water, the branches had remained healthy and many of their buds had developed into flowers. The poisonous action of the potassium persulphate on chlorophyl-bearing plants is therefore plainly seen, but it is surprising to see that the poison- ous action becomes very weak on a further dilution of the 0.5% solution to 0.19%. In such a solution plum branches as well as onion plants remained alive for a considerable time, although some injurious action could be noticed. Thus only a few buds of the plum branches developed within the next five days, while in the control case with distilled water all the buds had developed into flowers. After about ten days the branches exposed to the persulphate seemed to be entirely killed. The onion plants remained alive considerably longer in the 0.19 solution ; even after ten days only the tips of the leaves showed signs of withering. Further growth seemed to stop; while in the control cases with potassium sulphate and with distilled water alone it was not inconsiderable, as may be seen from the following table. After the ten days of observation, all the necessary mineral nutrients were added in the form of Kznop’s solution, and while now the growth in the control cases was very marked, the plants in the persulphate were very backward. One of these plants showed a further drying up from the tip downward, while with the other only a very insignificant increase was noticed (see Table). 1 In this case common well water was applied in preparing these solutions, to avoid the poisonous action of the very minute trace of copper sometimes contained in distilled water, 417 POISONOUS ACTION OF POTASSIUM PERSULPHATE. Length of Plants. Increase, Length of Plants, Increase. At the beginning After 10 days hc 23 days after full om (March 16) ae i ue Absolute | Relative HOGRcHaReRy (April 18) Absolute | Relative individual leaves (hela a o/ individual leaves om 07 cm. Summed up j Ze cm, : & ae: fo) 10.0 73 fo) 20. - o’ = dead (4) A o! ea (1) 37.4 38.0 0.6 1.6 a 23.6 416 3.6 9.5 n/ 8. Potassium persulphate se Z bs =< 5 19.1 fo) dead f U B of 14.2 42.6 42.0 —0.6 —1.4 on doa 36.5 —5.5 igh © 9.3 (2) 4 n 13.5 ty ‘lets 7. Oo 17.0 fo) 24.4 A o! 34.2 ° A Bt 10.9 t 35:3 45.4 10.1 28.6 m 26.5 87.2 41.8 92.1 Potassium rat 9.5 (5) sulphate . — = pas fo) dead fo) 20.1 o! 30.6 6 8, ; j 80. 32.6 67. Control e o! 17.5 37 a Mods 27-4 n 41.5 0.6 3 7.1 n 8.5 (6) = 0 14.9 (3) o dead - A caf 13.5 28.4 38.8 10.4 36.6 fo) 30.0 65.5 26.7 68.8 \Watere ce ; n 35-5 ie - fo) dead B o ae 31.7 40.5 8.8 27.8 of 30.0 | 67.0 26.5 65.4 : n 37.0 \ i EE a ee Foot notes :— (1) 1.6cm. on the tip had died off. (A) BOOM oy ty hm (3) 3-3 cm, ” ” ” ” ” ” ” ” (4) Osold leaves ; N=new leaves. (5) A bud was formed on April 18, (6) ” ” ” ”» ” ” 418 S, SAWA. POISONOUS ACTION OF POTAS. PERSULPHATE. It seemed to me of interest to test the effect of a still higher. dilution. Accordingly young Cucurbita plants of about 6 cm. length “with 3 leaves were placed April 22nd in a o.1 per mille solution of potassium persulphate containing all the mineral nutrients in the form of KAwop’s solution; but even in this case after two weeks some leaves had become curled and yellowish. We must infer therefore that potassium persulphate shows considerable poisonous effects upon phaenogams. 1 Compounds which may not be stimulants in a certain dilution tor phaenogams may however be stimulants for fungi. Thus A, Hattori (Journ. College of Science, Tokyo, 1901) observed that copper sulphate injures the growth of roots of the pea and maize even at 0.000001 % while at 0.004% it stimulates the growth of fungi. Note on Hamananatto, a Kind of Vegetable Cheese. BY This peculiar product is prepared from soy-beans, as are also two other kinds of vegetable cheese manufactured in Japan, the WZso and the Natto ;1 but it has a different flavour and taste, and lacks: the slimy character of the common /Vatto. It is manufactured only in the central provinces of Japan—especially in those of Mikawa and Totomi, from which it finds its. way all over the country. It has an agreeable salty taste and a peculiar odor somewhat resembling that of the fresh crust of brown bread. There is not any mycelium discernible with the naked eye. The soy-beans composing it form no compact mass, and are of a brown colour with a thin layer of a salty taste and a somewhat sticky consistency. In preparing this product, the soy-beans are well washed, boiled to softness, spread on straw mats, and mixed with wheat flour (6 liters flour to 10 liters soy-beans). Moldfungi will now develop, but soon afterwards this mixture is exposed to the direct sunlight for three days, probably to kill the fungi, and is then put into flat tubs, After 12—13 days some common salt and ginger are added. The entire mass is then kept in tubs under pressure for about thirty days, A portion, carefully freed from the pieces of ginger and particles of straw mats used in its manufacture, was dried, pulverized and sifted through a 0.5mm. sieve. I found the chemical composition of the dry matter to be as follows :— Albuminoid! nitromentter)..:..-. <2. <.45. nae G7 UG, Crude. fats 5 qe Peet: © <<< sckea tatters 3.44% Crude fibre NI os 0:0 0p Se a or (6.87% Total carbohydrate excluding cellulose.... . 8.40% Total ash including the salt added ......... ap CSA! by 1 The composition of Af:so was studied by VU. Kel/mer and AZ. Nagaoka. Vsul. Col. of Agr. Tokyo, Vol. I., No. 6 ; and that of Watto by K. Yade, ibid, Vol. 11, No. 2. 420 S. SAWA. NOTE ON HAMANANATTO. The fresh sample contained 44.73% water and 55.23% dry matter. There exist at least three different kinds of bacteria in this product. The most numerous colonies on agar are of two kinds. I, The largest colonies on bouillon agar show under the microscope very large chain-like forms. It hardly liquifies gelatin. Surface culture on agar: white, without lustre. Stab culture in agar: much development on the surface, but very little along the needle track; no gas bubbles visible. On potato : light brown, with minute foldings. In bouillon: kahm- haut white and lustreless, breaking easily on shaking II. The second microbe forms small colonies on bouillon agar. It readily liquifies gelatin producing thereby an acid reaction. Stab culture in agar: developing along the track, with chalk-like white colour and showing formations like the Bac. mycoides. On potato: greyish, with irregular foldings. In bouillon: developing on surface white and lustreless. Solution remains clear. 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