FACTORS INFLUENCING THE PATHOGENICITY OF HELMINTHOSPORIUM SATIVUM A THESIS SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY LOUISE DOSDALL, M.A. IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY JUNE, 1922 FACTORS INFLUENCING. THE PATHOGENICITY OF HELMINTHOSPORIUM SATIVUM en STS SUP Mi TED TO) THe FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY E@QUISE DOSDALE, M.A. IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY JUNE, 1922 + | CONTENTS eee Page Imtroductionmand historical review emer eerie eerie neers 3 | 2XR(0| 04) 01 en ne ene yee oe Rm Sc. Uno OO.cr Bodo o-005b ood 4 Miethod’s) chess cid cas casey os deen eee Wl Be eee ae SEI 5 Source Of pathogens... pees u see oer ears «een on ee eee 5 Selection of host: varieties... ac. + tras 5,8 525 ae 4+. ose Oe ee 6 Ghrecle plants: “es he siecle ha ne cece oro Senter 6 Specitiemid entityvom:thempatho gence mere: ern enn eee 7 Temperature nrelattons wc scs clic coeieeieis nice te eine on ee ene eee 16 Gurowithoteiuncissonmspotatondextrosesacaineee ann eererten eter teen 16 Sporea germina bon ccs. saneiec, atere ease nes tote sree ene eee 18 Effect of hydrogen-ion concentration and temperature on spore germination 21 Tin RE CHO ti hone os tectnsisc ie he i eae teagan: RE 6 25 Influence ‘on typeorssoils 245% aceltead eee ee ae eee ae 27 Isainleresnves Ont Gorill smVeWiNMbIKS. 5c 55oncscdgndnocencodooee eicdNS ain tine ee 31 Influences ot Soil fertility:. ..).4ccrs ae see ee es ene 390 Comparison of séveral reot-rot causine ofpanisms. o.0......662- 2 See 40 Stimmernys and sicon clusions acces. cer ooo teee REO OE eee 44 eiterature Gcited!) . caress de aiceesesein Seiten Oe ee eee eee 47 ILLUSTRATIONS Fig.1. Types of curves obtained from measuring length of spores of Helminthosporium sativum produced on potato dextrose........... Il Fig.2. Length of spores produced on potato dextrose agar at various LEMPETALUGES. se j..c.5 sc qereracstione ousrstwhoveus mona Giese cist SOIC SOC Ee eee 15 Fig. 3. Length of spores produced on different substrata at 24° C............ 16 Fig. 4. Growth of H. sativum on potato dextrose agar in Petri dishes....... 17 Fig.5. Daily rate of growth of H. sativwm on potato dextrose agar in Petri GISHES Ais MN ee eter ae eos ah Lica AU arr 18 Fig.6. Percentage germination of spores in phosphoric acid-potassium hydroxide solutions of various hydrogen-ion concentrations....... 23 Fig.7. Percentage germination of spores in Czapek’s solution minus the sugar at various hydrogen-ion concentrations...............+.+00 24 Plate I. Helminthosporium sativum P.K.B. grown on potato dextrose agar at “different” temperatures... ojos: «eenvaceu seen eee 49 Plate Il. Helminthosporium sativum P.K.B. grown on potato dextrose agar at sdifterent temperatures. « occas doce aes ee eee eee 50 Plate III. Marquis wheat showing effect of Helminthosporium root-rot in different: SOUS. sis8. 65 ha atom occas Be eee ee St Plate IV. Lion barley plants, 3 weeks old, growing on soils inoculated with VAKIOUS) OF PatiSms) -.4 ss Sts Saree ree On an eee 52 Plate V. Lion barley plants 3 weeks old showing effect of soil organisms OnudevelopiientwomenOo EES Sietn See eee eres ee ers oo 53 Plate VI. Lion barley plents, 3% weeks old, showing effect of root infection by. Hl. sation PUK Bas se foc nee eee ee eee eee 54 LIBRARY OF CONGRESS os REQKIVYED « Gite fee “MAY 2 4 1924 DOCUMENTS DIVISION _ FACTORS INFLUENCING THE PATHOGENICITY OF HELMINTHOSPORIUM SATIVUM By Louise DosDALL, DVLRODUCTION AND HISTORICAL REV IEW In rg10 Pammel, King and Bakke (g) described a new Helmun- thosporium disease of barley which they called “late blight.” The causal organism was named Helminthosporium sativum n. sp. Pammel, et al., had observed the disease in Iowa in 1907 and 1908. In 1909 it was very serious. In the same year, they report that it was also found in South Dakota, Minnesota, and Saskatchewan. ‘These authors de- scribe the disease as follows: “Brown spots of irregular outline occur upon the leaves causing them to turn brown. The leaves are easily broken up, and in some cases completely destroyed. The disease also occurs upon the glumes, spikelets and seed. The straw at harvest is dull brown, and instead of standing erect becomes a tangled mass. The date of ripening of the grain corresponds with the time of full development of the late blight.” They observed that there was con- siderable difference in varietal susceptibility, the degree of infection ranging from o to 100 per cent. Late blight was considered the most serious disease of barley in Iowa. In 1913 A. G. Johnson (7) differentiated clearly the three Helmin- thosporium diseases of barley in Wisconsin, and he designated the one caused by H. sativum P.K.B., the “American blotch disease.”’ Louise Stakman (11), in 1920, showed that a Helminthosporium similar to the organism described as H. sativum by Pammel, King and Bakke, but isolated from various parts of diseased wheat and rye plants, was capable of causing a serious seedling blight of these hosts, and could also attack the older parts of the plants, namely, the leaves, nodes, culms, roots, glumes, and grains. In addition to wheat and rye, successful infections were obtained on barley and a number of grasses. In the spring and early summer of 1919, serious attacks of seedling blight caused by Helminthosporium occurred in practically all the wheat-growing regions of Minnesota. eels Stevens: (12), also: im) 1920) reported that a species. of Helminthosporium was constantly associated with foot rot disease of wheat in Madison County, Ill. Inoculations with the organism gave positive results. He concluded that Helminthosporium was the cause of the disease. 1The writer wishes to express her appreciation to Dr. E. C. Stakman, under whom the work was done, for advice and criticism, and to Mr. M. N. Levine for his helpful criticism in the presentation of the biometrical studies. : 3: a TECHNICAL BULLETIN 17 In January, 1922, Hamblin (5) reported a Helminthosporium foot- rot disease of wheat in New South Wales, Australia. The disease symptoms are very similar to those of the true take-all caused by Ophiobolus graminis Saccardo, but there are distinguishing characters. Hamblin’s description of the foot-rot in Australia corresponds very. closely with that of Mrs. Stakman and of F. L. Stevens. His descrip- tion of the poorly developed root system with an abnormal develop- ment of root hairs close to the culm, giving the dead or dying root a “fuzzy” appearance, and the frequent growth of secondary roots above the first node of the affected straws, applies equally well to conditions observed in Minnesota during 1921. In Hamblin’s opinion, the Hel- minthosporium disease was responsible for far more damage in 1921 in Australia than was the better known take-all. In recent years, a foot-rot disease of cereals, particularly wheat, rye, and barley, has been destructive in certain localities in Minnesota. This was especially true on certain peat lands in Anoka and St. Louis counties and on some of the sandy soils in Anoka, Nicollet, and Mahnomen counties. A Helminthosporiuin of the sativum type has been consistently isolated from the diseased plants. This organism is very widely distributed throughout the cereal growing region. The severity of its attack apparently must be greatly influenced by eco- logical conditions. In order to obtain more detailed and accurate in- formation concerning these conditions, a study of the physiology of the fungus, to the extent of its possible correlation with the pathogenicity under given conditions, was undertaken. PROBLEM In this study attention was directed primarily to the root- and foot- rots caused by H sativum. Little attention was given to secondary in- fections on leaves and heads. The soil environment was, therefore, of chief concern. In analyzing the factors which might influence the development of a disease of this type, temperature, moisture, and acidity would affect the growth of both the pathogene and the host,, and possibly also the reaction between the two. The vigor of the host conceivably might greatly influence the development of a disease caused by a facultative parasite, such as H. satiwum. ‘The type of soil in which they grew and the available nutriment might, therefore, change the balance between host and pathogene. It is difficult to separate and analyze the individual factors, because certain combinations intro- duce various complexities which are difficult to interpret. The following phases of the problem were investigated especially : 1. Relation of temperature to the growth of the fungus, to spore germination, to infection, and to the development of the disease. PATHOGENICITY OF H. SATIVUM 5 Relation of hydrogen-ion concentration and temperature to spore to germination. Development of the disease in various types of soil. Influence of soil moisture on the development of the disease. Influence of soil fertilization on the development of the disease. Comparison of several root-rotting organisms. Morphological variation in the fungus with regard to its specific ONS Graig N identity. METHODS SOURCE OF PATHOGENE During the spring and summer of 1920, tissue cultures were made from lesions caused by Helminthosporium on cereals and_ grasses. Twenty-two strains (isolations from various parts of different hosts or from different localities) of the sativum type were obtained from the roots, stems, nodes, leaves, and kernels of barley; from the roots, stems, leaves, and kernels of wheat; and from leaf spots of various grasses. Material was obtained from Anoka, Clay, Mahnomen, Nicol- let, Ramsey, and St. Louis counties in Minnesota; from Tennessee, and from Spruce Grove and Edmonton, Alberta. Seven of these strains were selected for preliminary inoculation experiments. As a virulent root-rotting organism was desired, only soil inoculations were made. Four-inch pots filled with soil were treated with live steam for two hours on each of three successive days. Six pots of such soil were inoculated with each of the various strains of Helminthosporum. For this purpose, spores were scraped from the surface of potato dextrose agar cultures and mixed with water. The suspension of spores was poured over the soil, and the pots were in- cubated for several days. Three pots which had been inoculated with each strain were then sowed with Marquis wheat and three with Manchuria barley (Minn. 105). Some infection was obtained in each case, on both the leaves and the roots. (The check plants were slightly infected, as the seed had not been treated.) The plants inoculated with strain 82a, however, were decidedly more heavily attacked than the others. This was especially true of the barley plants. A Helmin- thosporium of the sativum type was re-isolated from lesions on both the barley and the wheat. A single spore culture was then made from the original 82a culture, and all subsequent work was done with this single spore strain. Culture 82a was originally isolated from the darkened base of badly stunted barley plants sent to the laboratory from the peat plots on the Fens experimental field, St. Louis County, Minn., in the summer 6 TECHNICAL BULEEDRING 7 of 1920. A similar Helminthosporium was isolated from the nodes, sheaths, and blades of the same plants. In addition to H. sativum, Alternaria was frequently obtained from blackened kernels and nodes; a pink Fusarium was sometimes found on the base of the stem and roots; and Helminthosporium teres Sacc. was occasionally isolated from the leaves and stems. SELECTION OF HOST VARIEDIES In all experiments, the effect of the fungus on wheat and barley only was studied. In most cases where barley was tested, both Man- churia (Minn. 105) and Lion (Selection) were used. Manchuria is the barley most commonly grown in Minnesota. It is somewhat re- sistant to H. sativum, as shown by the work of Pammel, King and Bakke (9), of Hayes and Stakman (6), and of Christensen (3). For this reason it was used in the breeding work of Hayes and Stakman, It was crossed with the smooth-awned Lion, which is very susceptible to Helminthosporium, in an attempt to obtain a high yielding, smooth- awned, resistant variety. Marquis (Minn. 1239) was used in most of the experiments with wheat. In some cases Haynes Bluestem (Minn. 169) also was used. GHEE CK REANTS Since it is difficult to obtain seed entirely free from Helmintho- Sporium, especially in susceptible varieties, it was necessary to treat the seed in order to reduce infection in the check plants to a minimum. Silver nitrate was found to be the most useful disinfectant because the seed coats of both barley and wheat are impermeable to it (10), and the seed can be soaked for a long time in the solution without being injured. It also is more effective, especially against Hlelmintho- sporium, than mercuric bichloride. For experimental purposes, the method of seed treatment followed was essentially that recommended by Schroeder (10). The seed was dipped in 50 per cent alcohol to remove the air from the surface, soaked over night in N/too silver nitrate solution, dipped in a dilute sodium chloride solution to precipi- tate as insoluble silver chloride the silver nitrate remaining on the surface of the seed, washed thoroly in running tap water, and dried. Such treatment reduced the germination of Lion barley from 90 per cent to 78 per cent, and of Marquis wheat from 99 per cent to 97 per cent. PATAOGENICIEY OF Ho SATIVUM N SPECIFIC IDENTITY OF THE, PATHOGENE Three species of Helminthosporium are known to occur on barley in the United States. These are readily distinguished on the host by the symptoms. H. gramineum Rabh. causes the systemic stripe disease characterized by long, narrow, yellowish to brownish spots on the leaves and sheaths. Many spots often coalesce to form parallel stria- tions which run more or less the entire length of the blade and often down the sheath. Eventually the leaves may be reduced to shreds. H. teres. Sacc. and H. sativum P.K.B. both cause local lesions which are characterized by peculiar blotches on the leaves. HH. teres causes the European blotch or net blotch disease. The spots are yellowish brown in color, irregular in shape, and are scattered on the leaves. When held to the light, a characteristic net work is apparent. //. sativum causes the spot blotch disease characterized by irregular red- dish brown spots on the leaves. The spots are usually longer than they are broad, and, when abundant, may tend to form stripes. These three species also may be distinguished readily by their growth on potato dextrose agar. H. gramineum grows slowly, forms a fluffy, aerial mycelium which does not sporulate (at least not readily ), and usually gives the medium a reddish or purplish tinge. H. teres also grows rather slowly. The mycelium grows very close to the surface of the agar. The color of the reverse side of the colony is greenish black. Grayish white tufts of mycelium are formed irregu- larly on the surface of the colony. Cylindrical, thin-walled spores are formed, but usually they are not abundant. In contrast to both these species, H. sativum grows very readily and sporulates abundantly, forming a flat, black or greenish black colony on agar. The abundance of conidia gives the surface a powdery appearance. Organisms similar to the one isolated from typical barley spot blotch have been isolated hundreds of times by workers in this laboratory from various parts of barley, wheat, and rye plants, and from numerous grasses. Pammel, King and Bakke (9) described the spores as cylindric in shape, straight or curved, slender, widest at the middle, from 105 to 130 microns in length by 15 to 20 microns in width, pale greenish gray to dark brown in color, with 7 to 14 cells. Later workers have found much shorter spores, altho observations on shape agree fairly well. Johnson (7) states that the spores are narrowly spindle-shaped, usually more or less curved. Mrs. Stakman (11) describes the spores of the organism with which she worked as either straight or curved, dark blue-green to brown in color, averaging 41 by 20 microns in size, and containing from 3 to 8 septa. Two types were isolated from dis- eased wheat: one a fuscous type measuring 35 by 22 microns and containing from 3 to 4 septa; the other straw-colored to fuscous, 8 TECHNICAL BULLETIN 17 measuring 60 by 20 microns, and containing from 4 to 7 septa. Both of the latter are described as elliptical in shape. Stevens (12) makes the following statement regarding the form causing the foot-rot of wheat: “The spores, observed as grown on autoclaved wheat leaves or stems in humid air, are from 24 to 122 microns long, the majority of them falling within the limits 80 to 90 microns, with septa or pseudo-septa varying from o to 13, usually 5 to 10. The spores are usually typically thickest in the region about midway between the base and the middle point of the spore, approach- ing a narrow or broadly elliptical shape, tapering somewhat toward each end. They possess an outer dark wall that is thin and extremely fragile and an inner, colorless, thick wall that is frequently soft and gelatinous . . . The spores usually, perhaps always, germinate either from one or both ends, not laterally, and are functionally only one-celled.” After making a large number of isolations from Helminthosporium lesions on barley, wheat, and rye, great variations were found in the size of the spores of the various cultures, altho they resembled each other more or less in shape and color. In order to find out just what variations might be expected in one strain, as a guide to the interpreta- tion of the species, a single spore was again isolated from culture 82a and a biometric study was made of the spores produced under various conditions. The single spore was planted on a potato dextrose agar slant and incubated at 24° C. for ten days. Transfers were then made to potato dextrose agar and to ripe autoclaved barley heads. Agar cultures were incubated at the following temperatures: 5°, 14°, 18°, 24°, 28°, 32°, and 36° C. The cultures grown at 5° and 36° did not produce spores, The barley head cultures were incubated at 24° C. Fresh barley leaves were taken from the greenhouse, placed in moist chambers, inoculated with spores of the same culture, and incubated at 24°. The length of time required for the cultures to sporulate at the different temperatures varied considerably; those at 24°, 28°, and 32° were ready for measurement in 16 days, while those at 14° required 37 days. 6£°0- zz°O> If*o- | 09 | ooo! 9 €O1 | goz | 192 |161|¥%6 | Sz |62 |€ asorjxep Le-ve bE-v1 | Z0°6S | | 0}e}0g | ze — ee ee en ee eee a a gS °0o- TSO== chose 09 oos | I v GS | o&r | 6yr | EZ | ay | Sr | Qe | asoljxop zg be Ig‘t1 99°6S | | 0}8}0g ze a. = — — ——s oe — — — —— — ——— | —— -— SACO z= ob oF ESO == 09 oof 5 |ee 122 les les We IQ ee Ie asoryxop LS°Sz 69°F | by Ls | [ 01e}0g | ze —— — —— — —_—_—_ ——s — ——— te ——t )— ee Ql rs 090+ Sg°0= 09 | Oo1 I Ay C2 || EAS | ml is | aso1}xop gi‘oz gS "er of*z9 | 0ye}0g | ae | =| a — = =) = = |e | | | ozr|orr|oor|06 | 0g |oZ |o09 |o OF Oe oz or *"d seetsap AyIqersiea JO uoeiAap ue apo | paansevow oe - aUInIpayy ‘91ny JUsTOYyo0d) piepurys | | Satods JO ‘ON (SUOIOIUL) Sasse[o UT UOTINAIA4SICG | -viodwa yp | | peonpoid a1aM sa1ods YoryM Japun suonipuody) AZIS LNaNAIAIq] AO SNOILVINdOG ONINASVAJY WOT GANIVIEQ MnatywS wmAodsoyUuUujaF] JO SAXOdS JO HLONAT YO SLNVISNOD GNV SNOILVIUVA I W1advL 10 TECHNICAE, BU LEE IN Wz Spores from these various sources were then measured for length. In all cases, measurements were made with a Bausch and Lomb micro- scope, using the 4 mm. objective and an eyepiece micrometer calibrated so that one space was equal to 3.4 microns. It was observed that at an extreme temperature, such as 32° C., there was a great deal of variation in length and a large number of measurements would be re- quired to obtain a normal curve. Data were therefore recorded for the measurements of 100, 300, 500, and 1000 spores. From these data the mean was calculated for each group and the differences were com- pared in relation to the probable errors, according to the methods given by Babcock and Clausen (2). These data are summarized in Tables I and II. For 100 spores the mean was found to be 62.30 + 0.85 microns ; for 300 spores 57.44 + 0.57 microns; for 500 spores 59.66 = 0.45 microns; and for 1000 spores 59.07 + 0.31 microns. Their accuracy can be seen by-comparing these results with their probable errors. In Table II the comparison of the means for 100 and 300 spores with the means of each of the other three groups, shows that the difference between any two is from 3 to 5 times the probable errors of the differ- ence. This borders on the verge of a significant difference, so that 100 or perhaps even 300 spores are scarcely enough to use as a basis for drawing conclusions. When the mean for 500 spores is compared with that for tooo, the ratio is 1:1. The results obtained by measuring 1000 spores are only very slightly more accurate than those obtained by measuring 500 spores. The difference is certainly not great enough to necessitate the measurement of the second 500 spores. TABLE II SumMMaARY OF CoMPaRISONS BETWEEN MEANS AND COEFFICIENTS OF VARIABILITY FOR LENGTH OF Spores or Helminthosporium sativum OBTAINED FROM MEASURING POPULATIONS OF DIFFERENT SIZE (From Data SUMMARIZED IN TABLE I) Means Coefficients of variability Conditions es Difference divided by Difference divided by compared Difference the probable error Difference the probable error ——___—_ of the difference of the difference No. of spores too and 300 4.86+1.02 5 5-41-1.73 3 tooand 500 | 2.64+0.06 3 4.66+1.31 | 4 100 and 1000 3.230.901 4 ip Wit ae) 3 300 and 500 2.22 One 3 0.75 +0.93 I 300 and 1000 1.63+0.65 3 I.300.85 2. sooand 1000 | 0.590.585 I 0.55+0.68 | I PATHOGENICITY. OF Eo SATIVUM IL These comparisons are perhaps brought out more clearly by the curves in Figure 1, in which the data obtained from measuring the different lots of spores have been plotted after grouping the measure- ments into 10 micron classes. The lowest curve, representing 100 spores, very clearly does not give a true index of the lower extreme of the total population. This explains why the mean obtained from 100 spores is too high. The three succeeding curves show that, as the number of individuals increases, the curve gradually approaches GET EET ETE Be SEE Eseecbesasee eis Fig. 1. Types of Curves Obtained from Measuring the Length of 100, 300, 500, and 1000 Spores of Helminthosporium sativum Produced on Potato Dextrose ANgarnatusen "Cs 12 TECHNICAL BULLETIN 17 a normal one. In general contour the 300-spore curve and the 500- spore curve approach the 1000-spore curve, altho the first 1s somewhat more irregular. The slight rise at the lower extreme indicates that the short spores tend to group themselves about a mode of their own, It is possible that improvement in the method of sampling might 1n- crease the accuracy of the results obtained from a smaller population. In the present study about 100 spores were measured from one mount. The spores were distributed as evenly as possible in the drop of water and each spore was measured in passing systematically over the slide from the upper left to the lower right hand corner. An attempt was made to make the mount so that two or three spores would come into the field at once. For all other conditions, 500 spores were measured. Results obtained in the study of the morphology of spores de- veloped on potato dextrose agar at various temperatures are interesting. Table III shows very little difference in the means of spores de- veloped at 18° and 24°. From the comparisons in Table IV it is seen that these differences are insignificant. If, however, we examine the coefficients of variability, we find that there is a significant difference in the amount of relative variation in the length of spores. This fact is very clearly brought out in the curves in Figure 2. The degree of variation is not increased by a temperature 4 degrees lower (14° C.), but the mean length of the spores is slightly increased. This may be due to the fact that at a lower temperature the black outer wall on the spores and mycelium is laid down much more slowly, so that the spores have a longer time in which to form. This is further sub- stantiated by the fact that at 32° the spores are very much shorter. The amount of relative variation is practically the same as at the lower extreme. These differences in length of spores produced at various temperatures are graphically represented by the curves in Figure 2. The most striking difference in spore morphology was obtained by comparing the spores produced on different media. As the fresh leaf and the autoclaved head cultures were incubated at 24° C, we may compare these results with those obtained from the agar culture at 24° C. Comparing first the spores from the head and from the agar, we find that the former are slightly longer. The amount of relative variation in the two is practically the same. On the fresh leaves, however, the spores are very much longer and decidedly more uniform. 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(Ce 17.39=0.47 37 7.530.48 | 16 Leaves and agar ASO (Ce 27.16+0.45 60 9.34+0.52 : 18 These facts show that in a single spore strain of a Helmintho- sporium, of this type, marked variations may be found in the length of spores developed under various conditions. Differences in spore measurements by various authors are therefore to be expected, and very fine specific or varietal differences can not be drawn on, the basis of spore size unless a large number of carefully controlled compara- tive studies have been made. Seemingly, the original spore meas- urements given by Pammel, King and Bakke are rather large (105 to 130 microns). Stevens has come nearest to approaching this length with a maximum of 122 microns. The same author states that the majority of his spores fall within 80 to 90 microns. By examining Table III it will be seen that the majority of the spores developed upon fresh barley leaves in a moist atmosphere fall within the 80 and go classes, or within 75.0 to 94.9 microns. Out of 4000 spores meas- ured, only 43 were longer than 100 microns. On the fresh leaves the longest spore measured was only 115.5 microns. However, the opti- mum conditions for maximum and minimum spore length have not PATHOGENICITY OF H. SATIVUM 15 necessarily been obtained in these studies. On the basis of spore shape and similarity with the organism obtained from typical spot blotch lesions and the ability to produce spot blotch symptoms on barley, the organism undoubtedly should be included in the species Helmuin- thosporium sativum P.IK.B. 240. 0° 60 20 5) % 80 ww Q i) SS 9 40 ie % 8 S g oO Oo 20 FO 60 40) /00 120 Length of spores (microns) Fig. 2. Length of Spores of Helminthosporium sativum Produced on Potato Dextrose Agar at Various Temperatures The shape of the spores was found to be more or less the same under various conditions. At 24° and 28°, the spores tended to be fat, spindle-shaped to broadly elliptical, sometimes slightly curved. At 32° the thickening in the middle was less evident, and they tended to be more uniform in diameter. The small spores were globose to ovate. At 14° the longest spores were mostly narrowly cylindrical. There was a marked tendency for the thickened portion to occur nearer the base than the apex, giving the spore the shape of a slender flask. 16 TECHNICAL BULLETING 7 Throughout the culture work, bizarre forms frequently appeared, especially forked spores which were sometimes almost stellate. Seven or eight such single spores were isolated and planted on agar slants. In each case normal spores were produced and the bizarre type occurred so rarely that it was quite impossible to isolate another single spore of the same shape from the progeny. 240 200 ---- Agar ns Ss LYING /20 ¢ 80 & Q 2) SS .) 40 \ v Q & S S O O 20 4O 60 80 /00 /20 Length of spores (microns) Fig. 3. Length of Spores of Helminthosporium sativum Produced on Different Substrata at 24° C. The solid line represents spores produced on autoclaved ripe barley heads; the line with short dashes, spores produced on potato dextrose agar; the line with alternate long and short dashes, spores produced on fresh barley leaves in a moist chamber. TEMPERATURE, RAO S GROWTH OF FUNGUS ON POTATO DEXTROSE AGAR In determining the temperature relations of H. sativum, the first problem studied was the growth of the fungus in pure culture. In all, four series were run to determine the range of growth, the same gen- eral method being used in each. Thirty cubic centimeters of potato dextrose agar were poured into petri dishes ro centimeters in diameter, PATHOGENICITY OF H. SATIVUM 17 The plates were inoculated in the center and incubated at the various temperatures. Each series was run in triplicate. The diameter of the colony was taken as the index of growth. In some cases it was impos- sible to control the temperature within several degrees, so that one series can not be checked quantitatively against the other. Different lots of potato dextrose also were used in the different series. The results of two series are given in Table V. In each case, the size of the colony represents the average of three plates. TABLE V EFFECT OF TEMPERATURE ON GROWTH OF Helminthosporium sativum on Potato Dextrose Agar First Series Second Series (Feb. 28—March 3, 1921) (Dec. 15-22, 1921) Temperature, | Diameter of colony | Temperature, Diameter of colony degrees C. (after 9 days) degrees C. (after 7 days) mm. mm. o- 2 4 3510 9 6-8 | 14 12-13 18 13-15 28 15-18 30 17-22 2 20-23 77 21-24 51 27-28 &y 30-32 36 31-33 35 34-35 13 | 35-39 4 40-42 (o} hee ee ol exavetrere 10 60 60 40 > a ee Temperature (°Centigrade) 0 5; /0 Vi 20 25 gO a5 40 20 Fig. 4. Growth of Helminthosporium sativum on Potato Dextrose Agar in Petri Dishes The curve represents the diameter of the colonies at the end of seven days. Data obtained in the second series are shown graphically in the curves of Figures 4 and 5. Figure 4 shows the relative growth of the fungus at the various temperatures after seven days. Figure 5 18 TECHNICAL BULLE TN s17 shows the daily rate of growth at each of the temperatures tested. Plate I shows the final appearance of the colonies in the first series, Plate il in the second: From these results we may conclude that the minimum temperature for the growth of Helminthosporium sativum lies near o-2° C., the maximum temperature between 35° and 39° C. and the optimum be- tween 24° and 28° C. | 90 80 70 60 50 x 3 WwW G S ‘ 40 : 3. Ss 32°C S ANT EE 30 17°C 9 lL : / eos aN 9 20 g ZA) = A : et a) Pe. os ~ jet RMN ee py ernie | ee as 5°C Time (days) oO / 2 3 + a 6 7 Fig. 5. Daily Rate of Growth of Helminthosporium sativum on Potato Dextrose Agar in Petri Dishes PATHOGENICITY OF H. SATIVUM 1g SPORE GERMINATION In the first series of studies to determine the effect of temperature on spore germination, hanging drop cultures were made on the covers of petri dishes, using distilled water and Czapek’s solution, minus the sugar, as media. The spores were taken from a six-days-old bean agar culture. Germination counts were made after 48 hours. The results are given in Table VI. TABLE VI SporE GERMINATION OF H. sativum in DisTILLED WATER AND IN CZAPEK’S SOLUTION AT Various TEMPERATURES Tempera- Distilled water Pu 6.7 Czapek’s solution—sugar PH 6.0 ture, de- —— -—-——-- —- ——- = —|- - = grees C. 1st end arden) (edt TS ence ah gna 4th drop drop drop drop Av. drop | drop drop drop Av. se Eira) er % % ao \) Ya % qe % % % To I I— | I I I I— 20 10 15 10 14 zl : =} z: = es) y 6 10 8 20 | 13 30 50 40 59 42 14 10 20 15 55 50 2 40 52 18 40) | 40 30 37 66 50 60 59 lea 5 24 68 41 44 58 85 86 75 75 80 aos eat =| = 30 AS eS 48 | ~41 50 77 70 59 80 72 = | | x = 34 50 60 | 55 36 50 25 42 38 42 oO oO oO oO ia) oO (3) (9) io) oO With the exception of the results obtained at 34° C., a higher percentage of germination was obtained in Czapek’s solution than in distilled water. In both cases the optimum occurred at 24°. At a temperature of 1° C. 14 per cent of the spores germinated in Czapek’s solution, while less than 1 per cent germinated in distilled water. At ‘42° no germination occurred in either case, while at 34° the germina- tion in distilled water was practically the same as at 30°, but in Czapek’s solution a marked inhibition occurred at the higher temperature. After trying various methods for germinating spores, including hanging drops in petri dishes, in van Tieghem cells, films on slides in moist chambers and in moist atmosphere, the most satisfactory method proved to be floating the spores on the surface of a thin layer of a liquid medium in Syracuse watch glasses. In such cultures the spores can be counted directly on the surface under the low power of the microscope. In several series, through the different temperature ranges, con- sistently high percentages of germination were obtained at ithe extreme temperatures when water which had been redistilled over glass was 20 TECHNICAL BU DEE TIN 17 used. Fluctuations occurred in the different cups at any one tempera- ture. The results given in Table VII are typical. The percentages given in the table represent the average of several counts. TABLE VII Spore GERMINATION OF H. sativum IN REDISTILLED WATER AT VARIOUS TEMPERATURES Average percentage of germination Sames floating on Sass ins ae Temperature, degrees C. surface of water bottom of cup 55 1025 i eu ae 7 76 ‘ ar 7) 93 ‘ ; "es 17.0-19.0 j 73 37 21 ene 62 i ee ‘ 28 she 70 / 66 30 532.0 : : 2 83 46 34 035.0 ea 65 : 63 38.0-39.0 7 zi 2 4 e 65 In a second series, using hanging drops in petri dishes, 67 per cent of the spores germinated at 6°, 54 per cent at 12°, 79 per cent at 1674 OI per cent at 22°, 72 per cent at 28°, O1 per cent at 20-307) Somper Cent at 82 2°82 per cent at 35>..and 87 per Cental sou In a third and fourth series in watch crystals, the spores were not counted but the germination was indicated as poor, moderate, and good. After 24 hours incubation, in these series, the germination was poor at 6°, moderate to good at 12°, 18°, and 22°, and good at the higher temperatures. By count, 89 per cent of the spores germinated at 39°. At the end of 48 hours the germination was good at 6°. From these results with redistilled water it is difficult to detect any quantitative effect of temperature on the number of spores which ~ germinate. Even at 1° C. a small number of spores will germinate. This, however, is probably very near the lower limit. At the lower temperatures, 1°, 6°, and 12°, pieces of mycelium in the cultures always germinated much more readily and sent out longer tubes than did the spores. At 40-42° no germination occurred. in the first series for which the results are given. In later series, however, high germi- nation sometimes occurred at 38-39°. Comparing these results with the data presented in Table V, we find 35-39° to be the maximum temperature for the growth of the mycelium on potato dextrose. At temperatures as high as 35° and 39° the germ tubes appeared very quickly, but were always short and did not increase much in length atter two or three days. On the othes handivat, 22728 oy amcmezer PAMAOGENICILTY OF A. SATIVUM 21 the tubes formed such a mat of mycelium by the end of 24 hours that it was often difficult to determine the percentage of germination. At the lower temperatures a longer time was required for the germ tubes to appear, and they increased in length very slowly. In redistilled water, therefore, spores of H. sativum germinate about equally well at temperatures from 6° to 39° C. No very definite optimum temperature for germination is apparent. The char- acter of the germ tubes and the length of time in which they appear, however, would indicate that an optimum temperature lies between e228@ and 32°C. Prom these results it would seem. that for the above-ground parts of the host, temperature is not a limiting factor in infection so far as spore germination 1s concerned. From the data in Table VII it will be seen that in most cases the percentage of germination of the submerged spores is only slightly less than that of those on the surface. In all cases, however, the germ tubes produced under water were very short and abnormally branched in cgmparison with the long straight tubes produced on the surface. The germ tube first appears as a hyaline tip at the apex of the spore. It is difficult to determine whether the tube breaks through the wall or emerges through a pore. After the tube has increased in size, the delicate exospore is split, sometimes for a third of the length of the spore. A second tube soon appears at the base of the spore, just to one side of the scar where the spore was attached to the sporo- phore. The connection between the two tubes is continuous through the spore, showing the false nature of the septation in the endospore. The endospore is frequently drawn away from the exospore and forms a constricted tube through the latter. Two germ tubes are not always formed from each spore. In one lot of spores germinated in redis- tilled water at 22° C., it was found that 52 per cent of the spores pos- sessed germ tubes at both ends and 39 per cent at only one end. Nine per cent of the spores did not germinate. Very rarely lateral tubes are found. One spore was observed with a lateral germ tube from each of five adjacent cells at one end of a ten-celled spore. In a few other cases, one or two lateral tubes were observed, usually arising from central cells. Fusions between germ tubes are very common. EFFECT OF HYDROGEN-ION CONCENTRATION AND TEMPERA- TURE ON SPORE GERMINATION As so many spores always germinated in redistilled water at the various temperatures which permitted germination at all, the effect of hydrogen-ion concentration on germination was studied. Culture solu- tions, based on Clark and Lub’s (4) titration curve for ortho-phosphoric acid, were made by adding varying quantities of n/5 KOH to 50 cc. 22 TECHNICAL BULLETIN 17 n/s H, PO, to give a series of hydrogen-ion concentrations ranging from pH 2.4 to pH 12. The pH value of the solutions was deter- mined colorimetrically, except for the highest three alkaline solutions for which the theoretical value according to the curve is given. Spores from a seven-weeks-old barley head culture were dusted over the surface of the solutions in Syracuse watch glasses. The percentage of germination was determined after 18 hours and after 36 hours. Similar series were run in triplicate at 19°, 24°, and 32° C. .The results are given in Table VIII. TABLE VIII SporE GERMINATION OF H. sativum In Hs POs—KOH Soxuutions or Vartous HyDROGEN-ION CoNCENTRATIONS AT DIFFERENT TEMPERATURES Germination pH Hours ie) (Op 2a (Cs Rian (Ce I 2 3 | Ay I 2 3 Avy. I 2 | es iealeass = — | — = —_ | | | 9% Ge | %o % %o % To To % % % 2.4 | 18 l= @e |. (0) o o o O Ce) o to) o 36 eel ect I 3 oO () oO 2 oO 3-4 18 2. ry x 2 5 6 o 4 8 10 10 9 36 2 4 7 4 io) 2 2 10 13 7 10 | - 4.4 18 15 | 27 || 24 | a5 |) 40 || 36°) 28 9) "35 | 44) aes 36 | | as) 14 18 41 33 u(y" || EX) 31 en | 27 28 es ls a 5.2 | 18 27 | 28) 25 27 55 38) || 46 |, 46" || dow | azn es sHea eae: 36 28 33 25 29 52 39 52 | 48 | 44 | 4 52 44 6.4 18 31 25 || 40. | 933 50 | 59 Ge | Sa ty 70.) |) S2aieas 36 32 RG. || gy 33 56 649) 62) || or | 4am 66 | 55 54 : Meal! ZeO 06 ij ABS AOe Ica AN CO areal 7m lea Nea eal ee | 76.5) 72s | aa 36 | a6 | 45 | 53 | 45 || 68% | 65 | 84.) (72° | 65 1 Si7aan eames E | | \ ee. | | eee | | eae 7.4 | 18 | 30 | 39 68 | 46 8r 75 72 7 78 | 80 | 84 Sr 36 345 OO. 67 54 85 83 73 80 72 83 | 87 81 7.8 18 [55 allie #35 36 42 8s 278 80 80 88 | 90 | 94 gt 36 | 30 34 SB ae | Be | toe ie 22 88 | 97 94 92 |, 94 8.0 18 35 24 20 20 | 72 62 | 68 67 | 72 65 74 70 36 35 22 38 2276 68 | 82 75 75 79 78 Wi 8.2 18 | 21 18 18 19 | 60 | 43 | Sor )) Se | 70 80 | 90 8o 36 TOM 24: oy || 2 72 ||| 6m P65 66. | 75, || “Sci eSaanieen 9.2 18 25 40 34 cep || es EDL | (53 (aS |) Ge 87 84 36 AD | 7 | 39 85 y Aon Xe) 2) ||| 89) | (90) | ones oz TTA 18 22 22 20 21 33 40 ZY 35 84 83 82 83 36 40 35 36 37 73 TA \s2 76 86 | 93 90 | 89 11.8* 18 12 18 12 14 | to} 8 3 40 26 39 35 36 26 34 37 2255 eco ° 20 10 30 69 48 39 T2108 18 ° to) I to) o to) ° fo) ) I to) 36 2 14 I 6 fo) 22 to) 7 9 35 O Deen * Theoretical value according to Clark and Lub’s titration curve for ortho-phosphoric acid. PARAOGENICILY OF TH. SATTVUM 23 After an incubation of 18 hours at 19° C. no germination was obtained at a hydrogen-ion concentration of pH 2.4. Very slight germination occurred at pH 3.4; while at pH 4.4 the germination showed a marked increase, rising steadily until a hydrogen-ion concen- tration of pH 7 was reached. From this point a gradual decrease oc- curred, reaching the lowest point at pH 8.2. At pH 9.2 there was a second rise followed by a gradual falling off, until at pH 12 no germi- nation occurred. After incubating for 18 hours longer there was scarcely any change in the amount of germination on the acid side. There was a slight increase on the alkaline side. At the higher temperatures the results were very much the same except that the percentage of germination was increased and the point of maximum germination was shifted slightly to the alkaline side. At both 24° and 32° the optimum germination occurred on the alkaline side of neutrality at a hydrogen-ion concentration of pH 7.8. A much greater increase in germination occurred at the higher temperatures on the alkaline side than on the acid. The average germination after 18 hours incubation at the different temperatures is represented by the curves in Figure 6. O00 80 < 9 “ Ww S 60 —S § QQ G D 40 —y SN 3 KK iS v 20 —¥ % Q i Ae Hydrogen ion concentration - pt 3 4 5 ° 7 8 9 /0 7, 12 Fig. 6. Percentage Germination of Spores of Helminthosporium sativum in Phosphoric Acid— Potassium Hydroxide Solutions of Various Hydrogen-Ion Concentrations Webb (14) germinated spores of Aspergillus niger, Penicillium cyclopium, Fusarium sp., Botrytis cinerea, and Lenzites saepiaria in n/5 mannite solutions in which the hydrogen-ion concentrations were adjusted by the use of H, PO, and NaOH according to Clark and Lub’s titration curve for ortho-phosphoric acid. The results obtained with Fusarium sp. are the only ones comparable with those obtained with H. sativum in the wideness of the range of hydrogen-ion con- 24 TECHNICAIES Oizo TaN est 7. centration which permits spore germination. It may be pointed out that both Fusarium and Helminthosporium are chiefly soil organisms. Among the organisms that Webb studied, only Fusarium responded favorably to an alkaline medium. Maximum germination occurred at hydrogen-ion concentrations of pH 2.8 and pH 7.4. From pH 6.2 a steady increase in germination occurred with the increase in hydrogen- ion concentration up to pH 2.8. from the same point, a steady increase in germination also occurred with the decrease in hydrogen-ion con- centration and practically the same maximum was reached at a con- centation of pH 7.4. Examining the data of H. sativum again, there is a steady decrease in germination from the neutral point with the increase in hydrogen ions up to a concentration of pH 2.4, where no germination occurred during 18 hours and only very slight germina- tion during 36 hours. However, the usual bimodal curve is obtained, but, in this case, both maxima occur on the alkaline side at hydrogen-ion concentrations of pH 7.8 and pH 9.2. With H. sativum, germination occurred chiefly in the alkaline solutions. A series of spore germination tests was also made in Czapek’s solution minus the sugar, with various hydrogen-ion concentrations ranging from pH 2.6 to pH 9.8. The results are represented by the curve in Figure 7. In this. case also the bimodal curve was obtained. The first maximum, however, occurred on the acid side of neutrality at a hydrogen-ion concentration of pH 6. The second was on the alkaline side at a concentration of pH 8. 700 80 60 20 £ 9 ~ as S i § Xe G ® v S NN S 8 Ne % Hydrogen ton concentration - p /7 Fig. 7. Percentage Germination of Spores of Helminthosporium sativum in Czapek’s Solution Minus the Sugar at Various Hydrogen-Ion Concentrations to UL PATHOGENICITY OF H. SATIVUM While the germination of H. sativum spores in these solutions is not necessarily the same as in a soil solution, certain general relation- ships may be pointed out. The spores will germinate through a wide range of hydrogen-ion concentration. Optimum germination occurs near the neutral point or on the alkaline side. The spores will tolerate high degrees of alkalinity. Germination studies in solutions more nearly approximating soil soiutions are still desirable from the stand- point of a closer analysis of the development of the disease. INFECTION Marquis wheat and Lion barley were grown under sterile conditions in test tubes containing white sand. When the seedlings were about an inch high, the coleoptile was inoculated with a suspension of spores and incubated at various temperatures. At 22°, 25°, and 30° C. char- acteristic minute brown lesions were visible after 18 hours. At the end of five days no infection had occurred at 6° on the barley; very light infection was evident on the wheat. Light infection also occurred on both wheat and barley at 14° and 34°, and on wheat at 30°. Mod- erate to heavy infection occurred on both hosts at 22° and 25°, and also on the barley at 30°. In these cases, the typical basal browning characteristic of the seedling blight occurred. This was as far as it was possible to follow the disease under these conditions. The results indicate that infection will take place to some extent through a rather wide range of temperature from 6° to 34° C. but that for the severe development of the disease the range is narrower, probably 22° to 30°. To some extent moisture, as well as temperature, was the limit- ing factor at the extremes. F. L. Stevens (13) reports that, “In an adaptation of the rag-doll seed tester, which allows the use of seedlings under aseptic conditions and variations of moisture and temperature as desired, inoculation by spores of Helminthosporium upon the uninjured sheath was followed within 24 hours by entrance of the mycelium into the host cells, and within 48 hours by a browned, diseased spot visible to the naked eye. Subsequently, when conditions favored, the mycelium invaded the inner- most leaves and caused general rotting and death. When inoculated upon the roots, there was general invasion of the cortex with very slight discoloration.” Stevens does not report under what conditions of tem- perature and moisture the disease developed best. An attempt was made to arrive at the temperature relations gov- erning leaf infection by inoculating fresh excised leaves with spores of H. sativum, placing them in moist chambers and incubating them at various temperatures. After incubating for 72 hours at 6° C., both 26 TECHNICAL BULEE TIN 17 inoculated and uninoculated check leaves were dark green, turgid, and normal in appearance. No signs of infection were apparent. Micro- scopic examination showed that many spores had germinated but so far as could be detected from free hand sections, the germ tubes had not penetrated. After the same incubation period, at 12° C., very small blue-green water-soaked areas were visible at the points of inoculation. The remainder of the leaf tissue and the uninoculated check leaves were still green and normal in appearance. These water-soaked areas were not yet visible at the end of 48 hours’ incubation. At 18° C., by the end of the third day, there were green water-soaked areas on which conidiophores were beginning to appear on the inoculated leaves. The tissue of the leaves was still firm and the cells were turgid. While the infected areas retained a dark blue-green color, the rest of. the leaf was yellow. The uninoculated check leaves were light green to yellow in color. After 72 hours’ incubation at 23°, 27-5 andy20u1e= there were large dark green blotches of infected tissue covered by a velvety mass of conidiophores. The leaf tissue was beginning to soften and the check leaves and non-infected areas were yellow. In the in- fected areas the cells were beginning to disintegrate, but the chloroplasts were still green. At 34° C. small water-soaked areas, 3 or 4 mm. in diameter, were apparent after 36 hours’ incubation. At this time the border was beginning to turn brown. By the third day, there were small, brown, definite leaf spots, similar to the normal lesions produced on leaves in the greenhouse and in the field. The remainder of the leaf tissue and the check leaves were yellow. Under the conditions just described, there was always an abundance of moisture, so that the difference in reaction must have been due to the influence of temperature on host and fungus. During the first 36 hours the results were probably more or less comparable to results obtained in growing leaves attached to the plant; during the second 36 hours, at some temperatures at least, the relationship was probably saprophytic. The most that can be claimed for results obtained in this way is that they are only indicative of what may happen on growing plants. The results obtained from these experiments would indicate that at temperatures of from 18° to 30° C., penetration into the leaf will take place about equally well in the presence of sufficient moisture. Below 24° the spots increase in size more slowly, above 24° more rapidly. At 12° a much longer incubation period is necessary for the development of water-soaked areas than at higher temperatures. At 6° no visible infection was obtained. At a temperature as high as 34°, on the other hand, the development of the spots and the browning of PALHOGENICLLY OF Hi SATIVUM 27 the host tissue occurred so rapidly that further development of the fungus was checked. While no control experiments were made with soil or leaf infection on growing plants, results obtained in the greenhouse agreed in general with those obtained on the temperature relations of the fungus. When the average temperature was between 75° and 85° F., much better infec- tion was obtained than when the average was lower. Better results were obtained on an inner bench over the steam pipes than on an outer bench next the outside wall on the west end of the house where it was always cool, and vigorous plants developed in spite of heavy soil inoculation. These results also agree with those reported by McKinney (8). He says, “Controlled soil temperature experiments, conducted in the ‘Wisconsin temperature tanks,’ and field experiments show that seedling infection in both spring and winter wheat and in spring barley is great- est at relatively high temperatures. The optimum temperature appar- ently lies between 26° and 28° C. This is very near the optimum rate of growth of H. sativum in pure culture.” PNB UENCE OF IPE OF SO The statement has already been made that particularly severe infec- tions of Helminthosporium foot- and root-rots were observed during the summer of 1920 on sandy soils and on peat soils in certain localities in Minnesota. Consequently one of the first tests undertaken was a study of the development of the disease in different types of inoculated soil in order to gain, if possible, an insight into the individual factors which might be influencing the situation. A heavy loam, a sandy loam, a sand, and a peat soil were selected for use. The heavy loam was a black dirt used without modification ; the sandy loam was obtained by mixing two parts of the heavy loam with one part of quartz sand; and the sandy soil by mixing one part of the heavy loam with two parts of coarse sand. All this soil was passed through a 5-millimeter mesh screen before being packed into the pots. The peat was a high-lime peat obtained from Anoka County through the Division of Soils, and fertilized according to directions with acid phosphate and potassium chloride to secure maximum yield from this particular type of soil (1). Small pots of steam-sterilized soil were planted with Marquis wheat and Lion barley. After the seeds were planted, the soil was watered several times with a heavy suspension of Helminthosporium spores. 28 TECHNICAL BiG TIE DIN si When the plants became crowded in the small pots, they were trans- planted to larger pots containing sterilized soil which had been inocu- lated in the same way. In these pots, the plants were grown to maturity. There was no very serious seedling blight in any of the pots. The coleoptiles of most of the plants were darkened, and lesions were formed on the first leaves. The seedlings in the inoculated soils were not noticably smaller than those in the uninoculated, sterilized, check soils. When about six weeks old the height of the plants was meas- ured in order to determine the effect of the disease on growth. The results are given in Table IX. In each case the table gives the average height of 30 to 4o plants. Any differences in height of the plants in the different soils in either the uninoculated or inoculated series may be considered due to the influence of the soil in which they grew. As will be seen from Table IX, the differences between the plants in the different types of soil in the inoculated series, altho small, agree fairly well with similar differences in the uninoculated series. The differences between check plants and inoculated plants in the same type of soil may be considered to be the result of the disease. A comparison of the dif- ferences between plants in inoculated and uninoculated soils of the different types will give an index of the influence of the soil type on the development of the disease. TABLE IX AVERAGE HEIGHT OF WHEAT AND BARLEY PLANTS GROWN IN INOCULATED AND UNINOCULATED Sorts or Various Types | Marquis wheat Lion barley Type of soil ae a f Inoculated Check | Jnoculated Check cm. cm. cm. cm. Heavy loam 22.8 30.2 24.5 | 27.6 Sandy loam 21.0 B58) 29.0 | Brat : = pe SS ss A Ives Sand DzOyr | 23:6" 2553 | 28.3 | Peat 29.4* Antagin | 20.4 33-6 * Plants were measured two days later than those in the heavy loam and in the sandy loam, and so can not be compared with these. Judging by the height of the plants at this stage, the barley de- veloped about equally well in the heavy loam and in the sand, the difference in the average height being 8 millimeters in the inoculated series and 7 millimeters in the check series. The increases over this amount were about equal in the sandy loam and in the peat, the advan- tage being slightly in favor of the latter. The difference between the height of plants in inoculated and uninoculated soils was practically the PATHOGENICITY OF H. SATIVUM 29 same in the heavy loam and in the sand. This would indicate that these two types of soil had practically an equal influence on the development of the disease. The difference was less pronounced in the sandy loam, showing that here the disease had least influence on the size of the plants. The great- est difference in height was in the peat soil, indicating that here the disease had most influence on the growth of the plant. From these results it is apparent that root-rot of barley produced the greatest effect on the host in the peat, a less marked effect in the sand and heavy loam, and the least effect in the sandy loam. On the whole, the differences were very small. The further development of the disease on the barley plants was not followed. The conclusions to be drawn from the height of the wheat plants at this stage must be derived from comparisons between the differences in the height of diseased and check plants in the same type of soil. It is obvious that the effect of the disease is much more marked on the wheat than on the barley. The least effect of the disease on the growth of the plants was obtained in the heavy loam. There was practically an equal increase in effect in the other three types of soil. The wheat was then transplanted to larger pots of inoculated soil. In each case, the most severely diseased plants were transferred. After transplanting, the check plants grew much faster than the diseased g, plants, and headed several days earlier. Plate III shows the compara- tive vigor and size of the plants in the different types of soil at maturity. Final observations were made on the Marquis wheat just before the heads began to turn yellow. The plants were removed from the soil, carefully washed, and examined for foot- and root-rot. In the heavy loam soil, both diseased and check plants averaged 3.5 culms per plant. While the severity of infection, measured by the degree of browning at the base of the plant, was moderate, there was very little difference in the extent of the root systems. The check plants headed four days earlier than the diseased plants and were con- siderably more vigorous. A slight browning occurred at the base of most of the mature check plants which resembled slightly a light infec- tion by Helminthosporium. The lesions, however, were less definite and no organism was obtained from tissue cultures. HA. sativum was isolated from the base of diseased plants. . In the sandy loam, the average number of culms on each diseased plant was 3, on each check plant 2.5. The basal infection was moderate, There was ‘little difference in the root systems. In sand the average number of culms on each diseased plant was 3, on each check plant 2. The infection at the base of the diseased plants 30 TECHNICAL BULLETIN a7, ranged from moderate to heavy minus. The root systems of the dis- eased plants were considerably less extensive, brown lesions were numerous, and the roots were very easily broken. The contrast between diseased and check plants was greatest in this type of soil. In the peat soil the average number of culms on inoculated plants was 2.7, on uninoculated 2.6. Basal infection was light to moderate. There was very little difference in general appearance of the plants grown in inoculated soil and in uninoculated soil. The best plants in both series were obtained in the peat soil. Under the conditions studied, the root-rot inhibited the growth of Lion barley most, during the first six weeks, in the peat soil. The effect of the disease was less evident in the heavy loam and the sand, and least evident in the sandy loam. During the same period, the growth of Marquis wheat was least inhibited in the heavy loam. The effect of the disease on the growth of the plants was markedly in- creased, and to practically the same extent, in the other three types of soil. By the time of maturity, however, the disease had developed much more severely in the sand, as evidenced by the smaller size of the plants, their decreased vigor, the amount of basal browning, and the breaking down of the root system. The effect of the disease was almost as severe in the heavy loam. In both the sandy loam and the peat there was only a very slight difference between the plants grown in inoculated and uninoculated soil. In analyzing the factors involved in these various soils, it may be pointed out that in the loam soils, in addition to the change in physical texture brought about by adding increasing quantities of sand to the original heavy loam, there has been a dilution of the mineral nutrients of the host, a decrease in the water-holding capacity, a decrease in the amount of organic matter in the soil, and an increase in the amount of soil aeration. All these factors may be assumed to have an influence on both the host and the pathogene. On the other hand, in the peat soil, we have a high organic content, a high water-holding capacity, and an optimum of mineral nutrients for the host. The abundant moisture and high organic content of the peat soil should seemingly be conducive to extensive saprophytic growth of Helminthosporium, thus greatly 1n- creasing the amount of inoculum and the chance for infection of the growing host. This tendency, however, seems to be counterbalanced by the optimum conditions offered for the growth of the host. On the other hand, the greater severity of the disease in sand and heavy loam suggests a possible influence of the soil water. These results led to a further study of the influence of the soil moisture and of soil fertility on the development of the disease. PATHOGENICITY OF H.uSATIVUM 31 INPEWUENCE OF SOM MOISLURE Preliminary series of experiments were carried out in the green- house in the following manner in order to determine the effect of soil moisture on the development of H. sativum on Lion barley. Light loam soil was sifted through a 5 millimeter screen, packed into jars, and sterilized. The sterilized soil was mixed: with a culture of H. sativum grown on sterilized oats seed. Five degrees of soil moisture were main- tained more or less uniformly by adding definite amounts of water each day. Inthe fifth series the soil was kept saturated by standing the porous pots in jars of water. In the other four series the soil was in glazed jars and the soil moisture was regulated by adding different amounts of water. Each moisture series was carried out in triplicate, in both inocu- lated and uninoculated soil. The seed was sterilized with silver nitrate before planting. Comparative results on the infection above ground at the end of three weeks and below ground at the end of four weeks are summarized in Table X. In this table the infection is designated by fractions; the denominator represents the number of plants in one pot, the numerator the number that were infected. On examining the data, it is seen that, as far as the above ground parts of the plants are concerned, the per- centage of infection, as well as the severity, is increased as the amount of soil moisture is increased. Comparatively few infections occurred on the check plants. The relation of soil moisture to root infection is a little more difficult to see, as here the development of the roots in inoculated and uninocu- lated soils with the same moisture content must be compared, and then these differences compared for the various series. The roots were most severely rotted in the saturated inoculated soil, and the difference in the extent of the root systems of diseased and check plants was greatest here. The next greatest difference was in the first series, with a soil moisture content averaging 9 per cent, while the least difference was found in the third and fourth series. In these two series the plants grew best of all, in both the inoculated and uninoculated soils. Injury to the roots is brought about by rather limited local lesions which kill the root tips or cut off portions of the roots when the lesions occur back from the tips. Very often the roots are rotted off near the seed. These results would indicate that plants suffer most from root infec- tion by H. sativum in soils containing both maximum and minimum extremes of moisture. 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At the end of two months, the cultures were practically masses of mycelium and spores. These masses were passed through a meat grinder and the pulp was thoroly mixed with sterilized soil in which the wheat and barley were then planted. The observations on seedling injury at the end of twenty days are sum- marized in Table XIV. TABLE XIII SumMary oF Data In TasBLe XII Infection classes oe 1.0 to I.3 1.4 to 1.8 1.9 to 222 Yield - — | ~ -—- K N 12 | K N 12) K N 12 300 100 600 oe) & | as: 200 300 RS HW 2 a 7 1: a 300 200 | No fertilizer ao B + faa} eqe No fertilizer 600 100 300 200 600 200 oO On 300 600 200 | ns ie oo - | ro tons manure a 3 jen] | | ee 20 tons manure | o a5 600 600 200 600 wn Ss Ow oO gS 600 100 oe 300 300 100 300 100 The results on the Lion barley were very sharp. Unfortunately, rats molested some of the pots. In the barley series, however, only the check plants were injured. Three plants were left in each of the three pots. 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The amount of injury caused by foot- and root-rot was very much less than on the barley. The Illinois strain of Helminthosporium seemed to cause slightly more injury than the Minnesota strain. This series was started in the greenhouse during warm weather early in October when the temperature in the house was very high. The sub- sequent development of the disease was most itteresting. After three weeks, the plants were thinned so that only three remained in each pot except for those inoculated with H. sativum, culture 82a, of which only three plants in each pot survived. These were badly stunted and 1n- fected at the time. The pots were kept next to the outer west wall of the greenhouse, where the temperature was always low during winter. The position of the pots was changed periodically so that all the plants would have more or less equal advantages as to sunlight. The plants grew remarkably well, and after a few weeks scarcely any differences could be detected between the different series. The barley stooled ex- cessively and did not head well. The wheat was very good. At heading time, late in April, there was practically no difference between either the wheat or the barley plants grown in the clean soil and in the soil inoculated with the Minnesota strain of H. sativum or the Fusarium culmorum. The wheat in the soil inoculated with the Hlinois strain of Helminthosporium was very bushy and developed only one or two heads per pot, while the other wheats developed from four to eight. The Lion barley was also slightly poorer in the soil inoculated with the Illinois strain than in the others. The barley did not head well, how- ever, in any case. Under the conditions of this experiment, then, the Helminthosporium caused more injury to Marquis wheat and Lion barley than the Fusa- rium, both in the seeding and the mature stages. While the Minnesota strain of Helminthosporiuan caused decidedly more seedling blight on the barley, the Ilinois strain caused slightly more stunting of the mature plants. The Illinois strain caused more injury to the wheat at both stages. SUMMARY AND CONCLUSIONS In recent years a foot- and root-rot of wheat, rye, and barley has been serious in certain localities in Minnesota. A Helminthosporium of the sativum type has been constantly isolated from the diseased plants. In addition to causing a foot- and root-rot, the same type of organism attacks the leaves and stems and especially the nodes, glumes, and kernels of cereals and a large number of wild grasses. A strain of the organism was isolated from a foot-rot of barley. A pure culture was secured by isolating a single spore. The morphology of the organ- PAI OGENICIRY «OR. EH. SARTV OM 45 ism was studied under various conditions with regard to its specific identity. The physiology and pathogenesis were studied with special reference to environmental conditions most favorable to the develop- ment of foot- and root-rot. The organism is capable of causing disease symptoms similar to those described by Pammel, King and Bakke in Ig1o0. Discrepancies are found between the spore measurements of this organism and that described by Pammel, King and Bakke, but since wide variations oc- curred under different conditions in a single-spore culture of the organism studied, the similarity of disease symptoms is considered sufficient justification for considering the organism to be Helminthos- porium sativum P, Kk. B. Variations in the morphology of the spores were found to occur under different conditions of growth. For spores as variable in length as those of H/. sativum, it was found necessary to measure 500 spores in order to obtain accurate results. On potato dextrose agar, significant differences in mean length of the spores occur when the organism is grown at different temperatures. The shortest spores with a mean length of 55.98+-0.35 microns were produced at 28° C. The longest spores, with a mean length of 67.32+0.55 microns, were produced at 14° C. The difference between the two means is 14 times the probable error of the difference. The greatest differences in length were found between spores pro- duced on different substrata. At 24° C. the mean length of the spores produced on potato dextrose was 65.75-+0.37 microns, on autoclaved ripe barley heads 67.74--0.38 microns, and on green barley leaves 83.140.29 microns. The difference between the means of the spores produced on the agar and on the leaves is 37 times as great as the probable error of the difference. The temperature relations of the fungus were studied and it was found that the mycelium will grow at from 1° C. to 37° C., the optimum lying near 28°. The spores germinated in redistilled water about equally well at temperatures ranging from 6° to 39°, but the length of the germ tubes indicated that the optimum temperature is between 22° and 32°. Germ tubes penetrated the tissue of both the coleoptile and the leaf at from 12° to 34°, but severe infection occurred through a narrower range, from 22° to 30°, the disease developing faster at the higher temperatures. Above 30°, however, the development of the lesions seemed to be checked, altho they appeared very soon after inoculation. In general, we may say that rather high temperatures are most favor- able to the growth of the fungus, to spore germination, to infection, and to the development of the disease. 46 RECHNICALS UR Tine: In phosphoric acid—potassium hydroxide solutions, the spores germinated through a wide range of hydrogen-ion concentrations. A double optimum occurred, both maxima falling on the alkaline side of neutrality at pH 8.2 and pH 9.2. In Czapek’s solution minus the sugar, the maximum germination occurred at pH 6 and pH 8. In general, the spores germinate better in alkaline solutions than in acid solutions, The spores will tolerate high degrees of alkalinity. Leaf infection increases directly with the amount of moisture present. Greenhouse experiments indicate that the effects of root and foot infections are more severe in extremely dry and extremely wet soils than in soils containing an optimum amount of moisture for the growth of the host plant. During one year’s field experimentation, no correlation was found between the fertility of the soil and the development of foot- and root-rot. The pathogenic effect of H. sativum isolated from barley plants in Minnesota was compared with that of a Helminthosporium isolated from stunted wheat in Illinois and with Fusarium culmorum isolated from scabby wheat. Experiments were made to determine the ability of these organisms to cause root- and foot-rot of Marquis wheat and Lion barley. Under the conditions of the experiment, the Helmin- thosporiums caused more injury than the Fusarium. The Minnesota strain of Helminthosporium caused the greater amount of seedling injury on the Lion barley, while the Illinois strain caused the greater dwarfing of the mature plants on both wheat and barley. As a result of these studies, the wide-spread occurrence of H. sativum may be explained by the fact that the fungus responds sapro- phytically to such a wide range of environmental conditions. Neither the effect of temperature nor acidity seems to be a limiting factor in the development of the disease so far as spore germination is concerned, As a parasite, the fungus causes rather limited local infections. The amount of injury is determined largely by the number and size of the lesions. A direct correlation exists between the amount of moisture present and the number of lesions. The severity of the infection 1s ereater at rather high temperatures than at low temperatures. The disease may be expected to develop most severely, therefore, at high temperatures in the presence of sufficient moisture. Root and foot infections are more severe in certain soils than in others. This is probably largely due to differences in soil moisture and temperature. In general, the disease causes the greatest injury under conditions unfavorable to the growth of the host. Factors, such as soil fertility, which might then be expected to influence the disease, apparently have little effect. iS Io. PATHOGENICITY OF H. SATIVUM 47 IAT RON URE, (CMI) . Alway, F. J. Agricultural value and reclamation of Minnesota peat soils. Minn. Agr. Exp. Sta. Bul. 188. 1920. (Out of print.) Babcock, E. B. and Clausen, R. E. Genetics in relation to agriculture. New York, 1018. Christensen, J. J. Studies on the parasitism of Helminthosporium sativum EAC Bee VMiasters) dhhesisau Vitnne Nore Ee x