BERKELEY LIBRARY UNIVERSITY OF CALIFORNIA c A COMPARISON OF A SCLKRCTINA FOUND ATTACKING APRICOT FRUITS IN CALIFORNIA ^ITH VARIOUS AMERICAN AND EUROPEAN FRUIT SCL^HOTINIXS. By Edith H. Phillips April, 1918 Berkeley, California Contents I. The Problem Page II. A. In Europe X B. In the United State* 11 C. In California 14 D. General Summary 14 III. Experimental 14 a. Introductory - Obtaining of the various strains used in culture and inoculation work 14 b. Apricot inoculations to obtain rate of growth, type of growth, color of conidia, and approximate sizes of conidia 18 c. Steamed rice cultures to obtain the color of the first trelve strains 25 d. Cherry inoculations to obtain color of conidia and general appearance of fungi on the fruits of all sixteen strains 27 e. Czapeck's agar cultures to obtain general types of growth of strains 1, 2, 3, 4 and 12 32 f. Inoculation of the five strains, 1, 2, 3, 4 and 12 into one apple to further test their individuality 33 g. Surnnfcry and conclusion* 35 IV* Literature cited 37 V. Additional bibliography 38 Illustration* I* Tli a Problem. During the past few years the brown rot fungus, a species of Sclerotinia, has grown increasingly important in the central coaet region* of California largely because of its parasitic attacks upon apricot trees and blossoms. The ripening fruit is likewise often attacked. This fungus occurring in California is locally called Sclerotinia fructigena, but should it be called by this name or by a different name? It was to find definitely if possible the ans- wer to the above question that I undertook the experimental work later described. a. In Europe. Persoon (1) in 1796 gives the following description of a fruit rot fungus: Torula fructigena: cinero-albida. eub rotunda, filorma articulis ova tie. Hab. in variis fructibus putridis Pruni domegticae Aaygd. Pereicae. autunmo praecipue frequens in piris putridis, cespitulos crassos sub- rotundus efficiens. Obs. Fila in novo hocce genere nee in capitula stipitata colliguntur, qualia axhibent Moniliae negus digitiforada trunco iaposita, uti in genere Aspergillo locum obtinet, and articulata; articulis deciduis, and glabra einrolicissiiaa sunt, quae in Dematio non observantur. The above may be translated: -2- Torula fructiKena; grtiyi eh -white, nearly round, egg-ehaped united in chains. Habitat: in various fruits of the domestic plum when decaying, peach, in autumn especially frequent in decaying pears, producing nearly round thick knobs. Observation. There are chains in this new kind and they are not collected into crowded heads in like manner to the Moniliae. and not like finger-like forms inposed on a trunk, as take* place in the Aspergillus form, and they art articulated: the articulations separate and are smooth and very simple, which are not observed in Dematium. His color term cinero-albida seems to refer to the color of the spores under the microscope, and not to the color of the fungus on the fruit as seen by the unaided eye. "Nearly round thick knobs" is his most definite morphological description. When he says, "There are chains in this new kind and they are not collected into crowded heads, e/r,." he seems to be referring again to the appearance of the fungus under the microscope. His description of his group Monilia is not very enlightening. Monilia. Erecta, fills moniliformibus capitulua constituentibus. His description of Torula is just as vague. Torula. Acaulis, fila moniliformia intricata, indeterminata effusa, Per so on changed the name of this fungus from Torula to Monilia and Monilia it remained for 100 years, or until the Monilia form was found to be an imperfect stage of a Sclerotinia. -3- Ehrenberg (2) in 1818 gives the following: Moniliae Oideura Lk. laxura mi hi 4} inpruno armeniaco putrido 4) CIDEDM laxum: floccie erectis divergentibus pallido cinereis; articulis rarius eonfluxis pellucidis magnis. Habitu et colore ab 0. fructigena Schmidt vtlde differt, cujus specimina, in herbsrio nostro servata, ab ipso arnico Dr. Schmidt examine ta eunt. Novter fungut Sporotrichum fare refert Ehrenborg'e description may be translated: Moniliae Oideum Lk. laxum mine 4, in decaying apricots 4) f.IDEUH laxum: tufts erect diverging pale ashen; spores large, pellucid, rarely holding together. Habit and color irihich differs exceedingly from 0. fructigena. Schmidt, whose specimens, which have been preserved in our herbarium, have been examined by Dr. Schmidt himself. Our fungus is closely related to Sporotrichum. The first part of this description seems to be the only part of much value. Ehrenberg seems to describe a fungus different from the one Persoon describes, the difference apparently lying in the morphological appearance of the conidial pustules. According to Aderhold and Ruhland (3) the name Oideum laxum was changed to Qospora laxa by Wall roth in 1833, and was later determined by Saccardo and Voglina as Monilia. Bonorden (4) in 1851 claims to have found another fruit rot fungus which he calls Monilia cinorea and describee it as follows: Mo nil i a cinerea rt; Komrat auf faulenden Fruchten vor und hat graue Hyphen und unregelmtssig - elliptische Sporen. Bildet kleine graua, etwas briunliche BtJschel oder RHufchen deren Mycelium in den Fruchten (Kirschen) eehr leicht beobachtet werden Kann, wenn man seine perpendi- kulflre Schnitte da von unter das Mikroskop bringt. Das Mycelium becteht aus artikulirten Fa* den, welche eich fie tig in den Zellen der Frucht ver- breiten und mit epitxen, nicht septirten, im Inneren KBrnigen, frei in die Zellen hineinragenden Faden endigen. The above may be translated as follows: Monilia cinerea Mine; habitat on decaying fruits. Hyphae gray; spores irregularly elliptical; eporodochia small, gray, somewhat brownish; mycelium from which sporodochia are produced is readily seen in vertical sections of the fruit (cherries). Mycelium of septate filaments which spread, branching in the cells of the fruit, and end in free filaments containing granular contents within the cells. Saccardo (5) in 1886 mentions the three species of Monilia. fructigena. laxa and cineroa and besides these gives two varieties of M. fructigfma. var. gyconophila and var. Candida. ?:'onilia fructipena Pers. Oidium fructipenum Link, Torula fructigena Pers. caespitulis compaetiusculis, pulvinatis, saepe circinantibus confluentibusque, tomentosis, albidis dein carneo^ochraceis, hyphis fasciculatis breve ramosis; conidiis longe ramoso-catenulatis, ovoideis v. oblongis 25 10-2 e hyalino carneolis. -5- Hab. in fructibus Piri, Kali, Persicae. Arrneniacae in Germania, Gtdlia, Italia, Britannia, Belgio, Austria, America bor. Var. syconophila. Cfs. Rabenh. Flora 1850 p. 632 • conidiie ecutionibue; caeepitulis subaurantiacis. In fructibus Fici siccii Casamiceiola Ital. auetri. Var. cendida Walk (sub Cospora) : caespitulis cendidis. Ad poma sub dio putrescentia in Thuringia. An haec var. raera typi forma junior? Sporotrichum fructigenum Link, videtur quoque statue infane. Monilia cinerea Bon. caespitulis minutis, cinereis compaetiusculis; hyphis cinereis ramuloeis septatie; conidiis irregulars ellipsoideis v. sfaepius liraoniiformibus, 15-17- 10-12, e conereo hyelinis, longt concatenatis. Hab« in fructibus putrsscentibus Pruni Ceraei in Germania et Italia. I'onilia lexa ?»alk Sacc. et Vogl., Cospora laxa Walk.vOidium laxua g^hrcnb. .Acrosporiun laxum ^erg... conidiis catemiletis, erectinsculis, divergenti-nr aosic, dense aggregatis, grieeis in articulos (conidia) singulbs, oval es secedentibus. Hab. in fructibus putridis Pruni Araeniacte in Geraania (Ehrenberg) *An diverse a !£. cinerea Bon?" "She foregoing descriptions by Saccardo may be translated thus: Monilia fructiRena Pers. Oidium fructiRenua Link, Torula fructigena Pers. spore masses more or less compact, cushion- shaped, often rounding end confluent, hairy, whitish then flesh color with ochre yellow, hyphae bunched, with short branches; conidia in long branching chains, egg-ehaped or oblong 25 x 10 -12 microns, from transparent to flesh color (carneolis). Habitat, in the fruit of pears, apples, peaches and apricots in -6- Gerraany, France, Itely, England, Belgium, Austria and North America. Var« gyconcphila. Cfr. Rabenh. Flora 1850 p. 632: ith rather pointed conidiaj epore maeeee shading into orange. On the dried fruits of the fig in CaBf«aicciola, southern Italy. Var. Candida ?7alk (sub Ooepora): ppore zaaeses white. In fruit (tipple) rotting in the open in Thuringia. "10 thit veriety an early form of the genus! - one will recall that Sporatrichum fructigenum Link seems ai»o an undeveloped form.1* Bonilia cinerea Bon, minute tufts, ashy (colored) more or less contacted; hyphee aehy (colored), branching, septate; conidia irregularly ellipsoidal or more frequently lemon-shaped, 15-17 x 10-12 microns, from achy to transparent, in long connected chains. Hab. in rotting cherry fruit* in Germany and Italy. Monilia laxa (ffalk) SRCC. et Vogl., OoBpora laxa Walk. Oidium laxum Shrenb. Acroeporiua l^jrum Pers. Fith conidia in chains, more or less erect, branching divergently, thickly clustered, ashy (colored) separating into single oval spores. Hab. in rotten fruit of the apricot in Oerajany (Ehrenberg) "Is this Uonilia different from M. cinerea?" In general Saccardo's descriptions, with their names, agree with the d-ecriptioa» of the three fonoa alraady given. Woronin (6) in 1900 definitely establishe* t*o species of Sclerotinia, which he call* Sclerotinia fructipana and- Sclerotinia cinerea. At that time none of the apothecia of any of the fruit rot Monilias had been found and described, but Woronin was so sure that the f orme he was -7- working with were really the imperfect stages of a Sclerotinia that he called the two forms Sclerotinia fructigena and Sclerotinia cinerea instead of Monilia fructigena and Monilia c in area. He gives for the average site of the conidia of Sclerotinia f ructigena taken from tha surface of naturally infected pears and apples, which have fallen to ihe ground, as 20.9 x 12.1 microns. Tha maximum measurements for conidia produced in the open he gives as 24.5 x 13.2 microns. In cultures on different favorable media the site of the conidia increases still more, reaching 20.7 - 30.8 x 14.9 » 16.5 microns. Spores of Sclerotinia cinerea taken from leafy shoots of cherry and peduncles of cherry blossoms and from the surfaces of different stone fruits average 12.1 x 8.8 microns.* Ha says however, that tha et«e is not constant. The largest spores of Sclerotinia cinerea col- lected by him in the open raeaeura 13.2 x 9.9 microns. Spores in cultures on different favorable madia are larger, and measure usually 17,5 x 11.2 microns. Single spores he adds in the favorable culture media msy reach even 24*2 x 13.2 microns. Woronin found the most striking difference between thase two forms to lie in the general form and color of the conidial pustules. Sclerotinia f ructigena produced the compact knob-like yellondsh pustules, wkil« Sclerotinia cinerea produced the grayish more powdery pustules. See Plate 28 for copies of the color blocks appearing with Woronin* s work. Aderhold and Huhland (3) in 1905 claim the distinction of rediscovering the species laxa. In their work they compare the apothecial stage of the species they call lexa with the apothecial stags -8- of jS. f ructigena. and compare the conidial stage of their species with the conidial stage they call cinerea. The epecies they call Isxa is quite distinct from J5. fructigena. but in the final analysis about the only distinction between their J5. cinerea and j>. laxa ie the fact that £. laxa occurs naturally on apricots, and S. cinerea on other stone fruits, eepecially cherries, although B. laxa is supposed to have spiaewhat larger conidia than their S. cineree. They also decide that the form attacking peaches and plums in North America is S. cinerea t»nd not S». fructiRena. as Norton calls it, for the following reasons: 1. Preserved asei and aecopores they obtained from Norton are somewhat stoallar than the asei and escospores of their above Sclerotinia laxa. 2. Because Smith, 2.F. describes conidim occurring on the peach in the Eastern United States as ashen-gray. The first reason they think bars Norton's species from lax*, the second reason from fructigena. Following are the principal distinguishing characteristics of Sclerotinia fructigena as given by Aderhold and Ruhland (3): -9- Table I. Shoving principal distinguishing characteristic* °* tclerotinia, fructigena. Sclerotinia laxa, and Sclerotinia cineroa t>s given by Aderhold and Ruhland. Specie* Ascoepores (micron*) Conidial tuft* Aeci (microns) Habitat S. laxa 11-12.5 x 5.6 -6.8 yellow, pointed, larger without oil drop* 11.5-13.5 x 5.2-6.9 gray Hunt, often smaller with small oil drops 120-180 x 9x12 pome* 121« 5-149. 9x8.5- 11.8 apricot S. cinerew 6.2 -9.3 x 3.1 - 4.6 blunt gray email er 8.9-107.6 x 5.9- 6.8 * tone fruiti The foregoing measurement* for Sclerotiaia cinerea were made from upoth*cia on a peach nuaay *ent to Aderhold and Ruhland by Norton, the m«a*ur9:aent* for Sclerotinia fructigania and Sclerotinia laxa were made from material obtained in their own country. Maseee (7) 1910 give* only one name, and that Sclerotinia fructigena. to the fora that attack* apple*, cherries, plum* and peache* in England. He *t*y* that the fungu* fruit* are grayish-white or whitish. He describe* the Monilia stage as foil owe: "tuft* consisting of simple or branched chain* of ovoid or lemon- shaped hyaline spores, 21-25 x 10-12 micron*. The size* of the spore* here given correspond more nearly to the -10- siies given by Saccardo for fructigena than for cinerea. but the color of the sporee corresponds moro nearly to cinerea than fructigena. Worrauld (8) 1917 in studying the classification of Monilias distinguishes four types of lionilia as found on cultivated fruit trees of the genera Fyrus and Frunue. He used prune juice &gar plate cultures, and cultures on steamed potato in Roux* tubes as a basis for his classification, which ie given below. Prune juice agar plate cultures Cultures on steamed potato in Eoux tubes (1) A: occurring com- monly on apples and pluias and frequently on sweet cherries (2) Bloesoa wilt Monilia of the Margin almost entire or lucin.it. ta i no known color* ation; conidia absent Margin with deltoid or flaballi form lobes, growth usually arrested about aid- Conidial tufts yellow, well -developed at upper end of potato, forming raised zones apple, also occurring occasion- way between canter and side ally on plums of plate, and new outgrowths as flabelli-fora lobes develop tsually from the tdnuses; olive green to brown 20113 s appear, the first usually at 0.5 to 1 cm. from the center; conidia absent (3) A grey Monilia fraqusnt oa plums and sweet ehorrios (4). American form of As above but no brown zones appear Margin entire or crenate; canidial tufts numerous, usually in concentric circles; brown colora- tion of the agar absent or appeare as a peripheral band near the edge of the pl&te; growth generally more rapid than in (2) tufts grey •ore numerous than in (2) often appearing in concentric circles t» round point of inocu- lation. Conidial tufts grey, almost covering the whol« surface in a continuous layer. -11- Sclerotina fructigena is the only form to which he gives a specific name. In hie summary horevor he says in part: "The causal organism is a grey Monilia. easily distinguished from &[. fructigena; at present it is to be referred to konilia cine^ea Bon. On culture media the habit of the fungus is different from that of the grey Monilia (also referred to ]£. cinerea by American vorkert) which is coomonly found in North America," b. In the United States Smith (9) in 1889 states that Mpniliu fructigena causes a destructive rot of peach fruit and blight of young p^ech branches in the orchards of Delaware and Maryland. He says, "In the fungus the corranon node of propagation from peach to peach, and the only knom one, is by aeant of ash-gray ccnidis, which are pro- duced in greet numbers on ih« brown surface of the effected parts* These spores generally occur in little hemispherical tufts or confluent masses on bundle* of hyphcd threads which have burst through the skin of the peach.1* Smith si>ys that he firet discovered the blight in the summer of 1887 in Delaware* Ha also says that this fungus occurs destructively on peaches, apricots, plums and cherries, and to scot extent kltso on apples, « pears , and quinces. Cordiey (10) in 1899 gives the name Monilia fructJEena to the form occurring on prunes, cherries, peaches, apples, pears and quinces in Oregon. As to color ho says: "In passing through almost any of our prune orchards when the green fruit is being picked, or even earlier, one cauy see here and there a -12- prune that IB pertly or wholly covered with this ash-grey or blue-gray 'mold1 On cherries and peaches At the disease spreads the surface of the diseased tissues becomes covered with the characteristic ash-grey conidial tufts. In apples, pears, and quinces the disease spreads in much the sane way but more slowly, and usually with a less abundant spore formation.1* He says that the name of this fungus was determined in 1895 by means of prune specimens. Norton (11) in 1902 changes the name of the form found in Maryland s peach and plum orchards from Monilia fructigena to Sclerotinia fructigena. after definitely connecting the Monilia stags with a perfect stage. Norton says that the asci and paraphyses are of the usual form, of the Pezitaceae and of the genus, and -that the asci are 45-60 microns long and 3-4 microns wide with 8 spores in the apical half. He also say* that cultures obtained from the ascoapores produced the characteristic yellowish gray conidia of Monilia fructigena. The question of the identification of species has interested several workers in this country, they no doubt being stimulated by the work of Woroain, Aderhold and Ftuhland and other European workers. But our workers seem to have given little consideration to the possible Sclerotinia laaa. or, disregarding the specific names, to the fact that there may be more than two specific, economic fruit rot Sclerotinias. It will be remembered that Norton called the form he studied fructigena. Read (12) in 1908 calls the form he has collected on various hosts fructigena. The measurements of the asci he gives as 125-215 x 7-10 microns. -13- The spores he says are ellipsoid, ends round or less pointed, hyaline, often containing refractive granules, and measure 10-15 x 5-8 microns. The measurements of chlamydoepores, conidia, he gives as 10-28 x 7-17 microns, mostly 7 x 11 microns. He gives the color of the pustules as at first cinereous, later on cherries and plums becoming ochraceous-buff to Isabella color (R), on peaches even drab to Isabella color (H), on pears and apples acquiring a blackish tinge. He eays also they are lemon-shaped, continuous, hyaline, inarticulate di - or trichotomously branched chain* without dis junctors. Reads* s description seems to fit better into Aderhold and Ruhland's description of £. laxa than their description of J>. fnactigana. S« fructigena is supposed to have ascospore* pointed at both ends, which do not contain granules or oil drop. Mathney (13) in 1913 decides that the American brown-rot fungus is Sclerotinia cineroa and not Sclerotinia fructigena. He knows of *derhold and Ruhland's work dealing with the three species, fructigena. laxa and cinerea but he evidently does not consider laxa very seriously. Mathney*s general averages of asco spore measurement* taken from Connecticut, Massachusetts, Maryland, New York, Indiana, and Wisconsin follow: Host Asci Aecoepores Microns Microns Peach 135-190 x 6.9-10.5 10.5 -14.5 x 5.2-7.5 mostly 163 x 8.9 mostly 12.5 x 6 Plum 135-173 x 6.8-10.8 9.3-14.2 x 5-7.4 mostly 151x9.4 mostly 11.8 x 6.3 The average measurements for the American brown-rot conidia he hives as 14.7 x 9.9 microns. Conidia from California, Indiana, and New Hampshire and local conidia were used in getting these measurements. These measurements -14- might find a place either under Scleritinia cinerea or Sclerotinia laxa as given by Aderhold and Ruhland. Mathney gives the color of the conidia of Sclerotinia fructigena as yellowish; of Sclerctinia cinerea as ash-gray. c. In California In California the brown rot fungus is called Sclerotinia f rue tiff ena. A strain, which ie apparently typical of the California form ie discussed more in detail under Experimental, d. General Summary I. There are three early virginal descriptions of fungi, all of v-hich have fim lly been put into the genus Sclerotinia. which attack the common domesticated fruits. Vague though these descriptions be, Per soon, describing Torula fructir.era in 1796, Ehrenberg describing Gideua Itocua in 1518, and Bonorden describing Monilia cinerea in 1851 all seen to be describing different fungi* II. That there are more than theoo forms of Sclerotinia. or Monilia as he calls them, that attack the comon domesticated fruits is demonstrated by vcrntid. He distinguishes four kinds. The only form he calls definitely by name is Sclerotinia fructigena* • III. There is greet need for exact systematic work witfc standardized methods in order to determine the number of species of fruit rot Sclerotinias. III. Experimental a. Introductory - Obtaining of the various strains used in culture and inoculation work. -15- As apothecia1 have never bean reported in California and as they are often difficult to get in other places, the cultural work has been confined to the Monilia stage. 1. I searched in the spring of 1917 during the blossoming period of the apricot trees for apothecia of our fruit rot, Sclerotinia. and for apothecia °* Sclerotinia libertiana. I brought to the laboratory from one orchard several of what I supposed to be Sclerotinia libertiana apothecia. These were all taken from the soil and none had any evident connection with a "mummy ". Miss Smith (assistant professor of Plant Pathology at the University of California) placed these apothecia in a moist chamber with the adhering soil and considerable water. In a short time three apothecia showed to be slightly different from the typical Sclerotinia libertiana apothecia. They were somewhat darker and redder, a little coarser and tougher and more cup-shaped. Miss Smith used two of these apothecia to make rough cultures, first rinsing off the apothecium with distilled water and then planting a piece of it in a flask of bread and prune juice. From four flaeks three apparently pure culture* of I'onilia were obtained* Examination of the asci and ascospores of the other apotheciua revealed a great » c»iuj, similarity to Sclerotinia libertiana asci and ascospores. The spores were somewhat larger than the spores of Sclerotinia libertiana and were inclined to be egg shaped, and posse sued a ready ability to form a cross wal] , and produce sterigmata and gonidia without first producing mycelium* Gonidia were sometimes produced directly by an ascospore without the interposition of a typical sterigma. The spores ,r i*ight Inoculated November 22, 1914 Photograph Ji-nuury 21, 1916 Slightly reduced. Pt 32 P?.ate 33, Photograph Phovirtg croft eec lions of canker* and chscke thovm in plates 31 end 3?. Fetch t.t left, epricot ct right. Upper; chaekt lover: inoculation. Photograph JiinuaxY 28, 1916 (x PI. 33 Plate 34. A. Shovdng variation in size and shape of conidia of strain 1 when grown on sterilized plum wood in a test tuba* Culture 15 days old. (xlOOO) B. Showing variation in site and shape of conidia of strain 1 when grown on standard nutrient gelatine in a test tube* Culture 15 days old* (X 1000} C. 1 } These numbers 1, 3, 4, 12 3 ) correspond to the strains of 4 ) the eaiae nuaber, and show the 12 ) appearance of separating conidia Taken froa Royal Atone cherries July, 1917. (x 1000) PI B 12 Plate 35. A) Conidia from the surface of an apricot B) naturally infected with the California apricot rot Sclerotinia (x 1000) C. 3 mature conidia from a culture of strain 1 on Solan urn tuberogum 14 daye old (x 1000) and ger Minuting conidiuai of California apricot rot Sclerotinia (2 day» in tap wbtfciO (X 1000} " D. Spore-like bodie* from culture on Diamond plum ( x 1000) September 19, 1914. See pi -to £7, uppar photogr&ph, E. Gancint-ting gonidiua of the California apricot rot Sclarotinia in 100^ peach juice, the acidity of which wee +10,4. Drop culture 8 days old. (x 2000). F. Gonidia of the California apricot rot Sclarctlnia prooucwd ia a tap water drop culture of contdia from e noture-lly infected apricot. 49 d&ye cl