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T)‘ I .127 -" I,’ .n , . “I I.‘.:.)- “ 3' ‘I f, “‘u-UI‘l | .'Ill ‘;";l<)‘ r . II‘) .I I; ' ..I, r I ,'.' ’ ”Eta-’2" 7 -45-- . of II‘I‘—;- I 43"». "3“ ‘1' I " I'M: ‘ . I II 2“ ’21 2”,." 2| H ' 1H,” [TI-'IIILI' firm), " OJ ‘ '5' . :I ' Til. 4-H W Illllllllllllllllllllllllllllllll 3 1293 00795 9202 \ - gun—...... -..y- -4.-. ./ l l ‘5'? T ; .2: I ‘.'" 3 ’"f . _._ .‘L .... i’t; ' ”saw: ‘ V' ‘~~ . s ‘ j ~ 2 «Wynn-*9 _ —-v-—-' / This is to certify that the thesis entitled FACTORS AFFECTING CARPOGENIC GERMINATION OF SCLEROTINIA SCLEROTIORUM (LIB.) DE BARY presented by William Lawrence Casale has been accepted towards fulfillment of the requirements for M.S. (bgeefil Botany and Plant Pathology \. 11L , Major professor Date 2/2 3/8Ll MSU is an Affirmative Action/Equal Opportunity Institution 0-7 639 Unwivmeruu r. a PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE Lu“; 1 6 3992 MSU Is An Affirmative ActiorVEqual Opportunity Institution own-Morn»: FACTORS AFFECTING CARPOGENIC GERMINATION OF SCLEROTINIA SCLEROTIORUM (Lib.) de Bary By William Lawrence Casale A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1984 ABSTRACT FACTORS AFFECTING CARPOGENIC GERMINATION CF SCLEROTINIA SCLEROTIORUM (Lib.) de Bary By William Lawrence Casale Isolate and size differences among sclerotia of Sclerotinia sclerotiorum affected carpogenic germination, whereas soil pH or splitting sclerotia in half did not. Carpogenic germination of sclerotia in PEG 8000 solutions was 100% from 0tx>-H bars osmotic potential. Germination in soil was 50% at -0.5 bars and (10% below -1 bar metric potential. Sclerotia imbibed similar amounts of water from - 0.5 to -10 bars soil metric potential. Carpogenic germination was stimulated by leaching sclerotia prior to incubation, and inhibited when material diffusing from sclerotia accumulated in, or was added to, the incubation medium. More sclerotia germinated in non-sterile soil than sterile soil. These data suggest a diffusible endogenous inhibitor of carpogenic germination. Sclerotia incubated in >u ug atrazine/g soil or a 10 UN atrazine solution formed only abnormal apothecia; percent germination was unaffected. Numerous stipes with abnormal apothecia grew from the aborted hymenia of immature apothecia soaked in 50 UN atrazine for 30 min. To my parents ii ACKNOWLEDGMENTS I wish to express my sincere appreciation to my major professor, Dr. L. Patrick Hart, for his guidance and support during the course of these studies. I would also like to thank Dr. John Lockwood and Dr. Gene Safir for their valuable suggestions and criticisms of this research while serving on my guidance committee. This research was supported, in part, by a grant from the Michigan Dry Bean Commission. iii TABLE OF CONTENTS LISTOFTABLES .......................................... LIST OF FIGURES ........................................ PART I: SOIL pH, SCLEROTIAL SIZE AND CONDITION AS INFLUENCES ON CARPOGENIC GERMINATION INTRODUCTION ............................................ MATERIALS AND METHODS .................................. Productionof sclerotia ............................... Comparison of carpogenic germination among six isolates ........................................... Incubation of several size classes of sclerotia ...... Incubation of intact and split sclerotia .... ......... Incubation of sclerotia at various soil pH ........... RESULTS ................................................ Production of sclerotia ........................ . ..... Germination rates of six isolates .................... Effect of splitting sclerotia and sclerotial size on carpogenic germination ..... . ...................... Influence of soil pH on carpogenic germination ....... DISCUSSION ............................................. LITERATURE CITED ....................................... iv PART II: THE INFLUENCE OF WATER POTENTIAL ON CARPOGENIC GERMINATION INTRODUCTION . .......................................... 2A MATERIALS AND METHODS .................................. 27 Water imbibition by sclerotia . ....................... 27 Incubation of sclerotia at various osmotic potentials ....... .............. ............. . 28 Incubation of sclerotia at various soil matric potentials ........................................ 30 RESULTS . ............................................... 30 Water imbibition by sclerotia ..... ...... . ........... 30 Influence of water potential on carpogenic germination ....................................... 32 DISCUSSION ............................................. 35 LITERATURE CITED ....................................... 38 PART III: EVIDENCE FOR A DIFFUSIBLE ENDOGENOUS INHIBITOR OF CARPOGENIC GERMINATION INTRODUCTION ............. . .................... . ........ Al MATERIALS AND METHODS .................................. A1 Electrolyte leakage by surface-sterilized sclerotia .. A1 Incubation of sclerotia in soil, soil filtrate or water ...................... . ...................... A2 Recovery of sclerotial leachate ..... ......... ........ AA Assay of sclerotial leachate for total carbohydrate .. AA Gas chromatography of sclerotial leachate ............ A5 Effect of sclerotial leachate on carpogenic germination ....................................... A6 Removal of diffusible material from sclerotia and its effect on carpogenic germination .................. A7 RESULTS .................... . ........................... A8 Electrolyte leakage by surface-sterilized sclerotia .. A8 Carpogenic germination of sclerotia incubated in soil, soil filtrate or water ... ................... A8 Recovery and assay of sclerotial leachate ............ 51 Germination of sclerotia incubated in sclerotial leachate .. ...................................... 5A Germination of leached and unleached sclerotia ....... 58 PART IV: EFFECTS OF HERBICIDES ON CARPOGENIC GERMINATION AND APOTHECIAL DEVELOPMENT INTRODUCTION ........................................... MATERIALS AND METHODS .................................. Mycelial growth on herbicide amended agar media ...... Effect of herbicide-amended soil on carpogenic germination ....................................... Effect of analytical-grade herbicides on carpogenic germination ................................... ... Effect of atrazine on apothecial disc development RESULTS ................................................ Mycelial growth on herbicide-amended media ........... Effect of herbicide-amended soil on carpogenic germination ....................................... Effect of analytical-grade herbicides on carpogenic germination ....................................... Effect of atrazine on apothecial disc development DISCUSSION ............................................. LITERATURE CITED ....................................... vi 8a 8b LIST OF TABLES Source of isolates of Sclerotinia sclerotiorum Number of carpogenically germinated sclerotia per 12 sclerotia for six isolates of Sclerotinia sclerotiorum with two conditioning tempEratures Number of carpogenically germinated sclerotia per 12 sclerotia of Sclerotinia sclerotiorum (H-isolate with 5 conditioning periods and 3 conditioning temperatures ............... . ............ . ......... Carpogenic germination of split (half) and intact (whole) sclerotia cd‘ Sclerotinia sclerotiorum incubated in soil at -0:§mbars metric—Eotential Number of carpogenically germinated sclerotia per 20 sclerotia of Sclerotinia sclerotiorum incubated at various soil pH's ............................... Carpogenic germination of sclerotia of Sclerotinia sclerotiorum after 50 days incubation at 15 C in sterile or non-sterile soil or soil filtrate, or in distilled water which was changed regularly or not changed ..................... .H.. ............ ..n. Total carbohydrate (detectable by phenol method) in sclerotial leachate samples from Sclerotinia sclerotiorum collected over 72 h .................. Carpogenic germination of sclerotia of Sclerotinia sclerotiorum (H-isolate) incubated i1) leachate solutions ......................................... Analyses of variances for carpogenic germination of sclerotia of Sclerotinia sclerotiorum incubated in leachate solutions from Table 8a .................. New stipe production by sclerotia of Sclerotinia sclerotiorum incubated if] sclerotial. Ieachate soIUtions ......................................... vii m DO (D O‘ 11 12 15 17 50 55 56 10 11 12 13 Growth of Sclerotinia sclerotiorum on herbicide amended 1% Bacto agar media after three days ...... Effect of herbicide-amended soil on carpogenic germination and apothecial development of Sclerotinia sclerotiorum. Sclerotia were removed Fomjierbicide-amended soil after 53 days and placed in distilled water ......... ....... .. ....... Effect of herbicide-amended soil on carpogenic germination and apothecial development of Sclerotinia sclerotiorum. Sclerotia were incubated for 28 days in the dark, then 18 days under fluorescent light, at 15 C ....................... . Effect of atrazine solutions on carpogenic germination EHHI apothecial development of Sclerotinia sclerotiorum .......................... viii 73 77 LIST OF FIGURES Figure 1 Soil moisture curve relating percent soil moisture to metric potental for Capac sandy clay loam soil Comparison of carpogenic germination among four size classes (1 = 81 mg, to A = 7 mg) of sclerotia of Sclerotinia sclerotiorum incubated at 15 C in water-saturated soiIE ...... H ...................... . Rate of water imbibition (weight increase expressed as a percentage of the initial dry weight) by sclerotia of Sclerotinia sclerotiorum incubated in distilled water .................................... Effect of soil metric potential on carpogenic germination of“ sclerotia cd‘ Sclerotinia sclerotiorum (ii-isolate) incubated for 3'0—days at 15 C, and on the amount of water imbibed by sclerotia after A8 h expressed as a percent of the initial fresh weight ............................... Effect of osmotic potential on carpogenic germination of unrotted sclerotia of Sclerotinia sclerotiorum in PEG 8000 solutions after 30 days at 15 C ...... ......................................... Electrolyte leakage from surface-sterilized and non-surface-sterilized sclerotia of Sclerotinia SClerotiorum 000.00.000.00... 000000000000000 0 000000 Recovery of leachate at 12 h intervals from sclerotia of Sclerotinia sclerotiorum incubated in sterile deionized water—for five days u.” ........ Effect of leaching on carpogenic germination of sclerotia of Sclerotinia sclerotiorum incubated on moist sand ............................... . ........ ix Page 1A 31 33 314 '49 52 1O 11 12 (A): Normal stipes and apothecia produced by sclerotia of Sclerotinia sclerotiorum incubated in water saturated soil at 15 C, under fluorescent light. (B): Abnormally formed, multiple-branched stipes of sclerotia incubated in atrazine amended soil at 15 C, under fluorescent light. (C): Abnormal apothecia produced by sclerotia incubated as in (B) for 53 days, then placed in distilled water at 15 C, under fluorescent light ........ ..u (A): Normal stipe (top) and apothecium produced by sclerotium of Sclerotinia sclerotiorum incubated in 1% methanol:water at 15 C, under fluorescent light. (B): Abnormal stipes produced by sclerotium incubated in 10 uM atrazine in 1% methanol:water at 15 (3 under fluorescent light. (C): Abnormal apothecia produced by sclerotium incubated as in (B) . ..... .........H”........HH.......HH........ (A): Normal (right) and abnormal apothecia produced by sclerotia of Sclerotinia sclerotiorum incubated in 0 and 10 UN atrazine, respectively, in 1% methanol:water at 15 C, under fluorescent light. (B)and (C):Efistorted and unexpanded apothecia of sclerotia incubated as it) (A) ..................... (A): Normally (left) and abnormally developed apothecia of Sclerotinia sclerotiorum soaked in 0 and 50 uM atrazine, respectively, in 1% methanol:water for 30 min prior to incubation in distilled water at 15(L under fluorescent light, for 10 days. (B): Darkened hymenia of apothecia shown in (A) with numerous stipes growing from their surface; early stage (left) and later stage with development of malformed apothecia. (C): Extensive branching and aborted apothecia produced by stipe soaked in 50 uM atrazine in 1% methanol:water as in (A) .......................... 75 80 82 PART I: SOIL pH, SCLEROTIAL SIZE AND CONDITION AS INFLUENCES ON CARPOGENIC GERMINATION INTRODUCTION Sclerotinia sclerotiorum (LibJ)de Bary [:Whetzelinia sclerotiorum (Lib.) Korf and Dumont] is an ascomycetous fungus, pathogenic to 361 species of plants in GA families (33). Economically important hosts include plants in the families Solanaceae, Brassicaceae, Umbelliferae, Compositae, Chenopodiaceae and Leguminosae (A7). Sclerotinia sclerotiorum is a discomycete of the family Sclerotiniaceae in the order Helotiales (A, 21). Libert gave the first Latin description of the present day S; sclerotiorum in 1837, naming it Peziza sclerotiorum (26). In 188A, deBary (11) changed the binomial 1x) Sclerotinia sclerotiorum. Comprehensive histories of the taxonomy and current status of the genus have been given by Kohn (20), Purdy (31), and Willetts and Wong (A7). Infection of host plants by §_._ sclerotiorum occurs either by means of ascospores or mycelium arising from sclerotia or neighboring plants (A7). No functional conidia are produced (microconidia have been observed, but their role is unknown). Ascospores are the primary inoculum for white mold of beans (1, 2, 7, 36, 37, A2) and lettuce drop (30), although mycelium from sclerotia has been reported to be infectious to been (29). Ascospores are forcibly ejected from apothecia (1A) and dispersed by wind as far as 25 m 1 2 (A2). The fungus infects healthy plants via germinated ascospores only if external nutrients are present (2, 12, 32). Senescent and injured organs or plant litter can provide this source of nutrients (A0). Soon after infection, a light-brown watery rot develops, followed by the appearance of cottony-white mycelium and collapse of non-woody tissue (27). After several days, small aggregates of mycelium develop, darken and become dry, hard survival structures called sclerotia. A mature sclerotium consists of aidarkly pigmented rind, 2—3 cells thick; just below this is a cortex of pseudoparenchymatous tissue, 2-A cells thick, and a central medulla of loosely arranged filamentous hyphae (22). Details of the formation and compositioncfi‘sclerotia have been reported ML 9, 1C, 25, A3). Sclerotia can remain viable in field soil for A-5 years ( LA) , 39, A9), and under some conditions for at least 10 years (5). The primary influence on survival appears to be biological, with normally occurring soil temperatures and pH being of minor importance (3). More than 30 species of fungi and bacteria have been reported as antagonistic or parasitic to Sclerotinia species, but only a few have been studied under field conditions (6, 16, 19, 28, AA, A5). Sclerotia of _S___._ sclerotiorum may germinate myceliogenically (production of mycelium) and/or carpogenically (production of apothecia). Myceliogenic germination occurs when sufficient external nutrients are 3 present [germination has not been observed on non-amended natural soil (2, 36)]. Secondary or daughter sclerotia may be formed in soil or culture, adjacent to or atzadistance from the mother sclerotia (A8). Apothecial initials evidently develop directly from medullary hyphae, usually just beneath the sclerotial rind [although microconidia are formed, they do not appear to function as spermatia] (22, 35). Saito (35) recognized four stages of apothecial initiation: 1) deeply staining areas of medullary tissue develop near the rind, and 2) become enclosed by thick-walled, darkly pigmented cells. These primordia 3) organize into clearly distinguished tissue, which A) ruptures the rind and grows as an apothecial stipe. The stipes are positively phototropic, and differentiation of apothecial discs occurs only in the presence of light (18, 25) [for S; trifoliorum, a closely related fungus, wavelengths greater than 390 nm were ineffective (15)]. Ultrastructural (19, 20) and histochemical (22) studies of the stipe and apothecium have been done. Several factors affecting carpogenic germination have been studied. Sclerotia grown on media containing vegetable extracts produce apothecia more frequently than those grown on potato-dextrose or synthetic media (3A,4A7L. Providing mature sclerotia with a conditioning or after-ripening period under moist conditions (A1) at high (13) or low (35) temperatures may enhance subsequent carpogenic germination. There is general agreement that 10-20 C is the optimum temperature range for the production of apothecia (36, A7). Cycling thermoperiods are not required for carpogenic germination of S: trifoliorum (38). The effect of light on apothecial disc differentiation has already been discussed, but Letham (23) suggested that light could affect the initiation of apothecia. The relationship between moisture and carpogenic germination is important and will be discussed in PART II. Sclerotia incubated in closed tubes produced stipes but disc formation was inhibited (2A). It is unclear, however, whether accumulation of inhibitory compounds or oxygen deficiency affected disc differentiation. There appear to be no reports of the influence of pH on carpogenic germination. Preliminary studies described herein yielded information for future experiments. A simple method of producing large quantities of readily germinable sclerotia was developed. Isolates of S; sclerotiorum were screened for Speed of germination and requirement for cold treatment prior to incubation, with selection of those isolates that facilitated experimentation. To insure reasonable uniformity of experimental units in future studies, the influence of sclerotial size»on carpogenic germinaton was examined. Soil pH as a possible influence on germination was also investigated, as was the ability of sclerotia to alter the pH of soil in their immediate vicinity. Split and intact sclerotia were compared to determine whether cutting affected carpogenic germination. MATERIALS AND METHODS Production of sclerotia Sclerotia of S. sclerotiorum were obtained in commercial fields in Michigan from infected dry bean plants (Phaseolus vulgarig in) (Table 1). Sclerotia were surface-sterilized by first quickly dipping them in 95% ethanol to reduce surface tension, then placing them IJJCL511 sodium hypochlorite (NaOCl) for 3 min followed by 2 min in sterile distilled water. Individual sclerotia were germinated myceliogenically on potato dextrose agar (PDA) and 5 mm discs were cut from the advancing colony margin after 3-A days. Autoclaved canned green beans were arranged in a single layer in plastic petri dishes, inoculated with inverted mycelial discs, sealed with Parafilm (American Can Company, Greenwich, CT‘ 06830) and stored at room temperature and light. Sclerotia were harvested after one month. This insured maximum yield, as new initials formed while others were maturing. Sclerotia were separated from macerated bean tissue in a metal strainer under a strong stream of tap water. Sclerotia were air-dried for 2A h, and stored at 5 C in plastic bags. Comparison of carpogenic germination among six isolates Sclerotia from six isolates of S; sclerotiorum (A, B, D, H, M and R) were stored in plastic bags at 5 C or room temperature (22 i 2 C) for 1, 2, 7.5 or 10.5 months. 5 Table 1. Source of isolates of Sclerotinia sclerotiorum. Isolate Source A Sclerotia collected from heavily infected dry bean plants in Bay Co., MI (8/19/81). Isolated from dry bean plants infected at soil line in Bay Co., MI (7/15/81). Isolated from heavily infected dry bean plants in Huron Co., MI (August, 1981). Isolated from infected dry bean plants in Huron Co., MI (7/16/81). Sclerotia separated from cull pile screenings from a central Michigan been elevator in 1980. Sclerotia obtained from R. Weinzerl, Department of Plant Pathology, Oregon State University, Corvallis 97331. Additional sclerotia from one isolate (H) were stored at 5 C for two months, then -5 C for five months. Following storage, six sclerotia from each isolate were incubated on water saturated Capac sandy clay loam soil in petri dishes sealed with Parafilm and incubated at 15 C. Each treatment was replicated twice. The stipes produced were counted daily starting 19 days after the beginning of incubation. A sclerotium was considered germinated if‘2M: least one stipe had broken through the rind. Incubation of several size classes of sclerotia Sclerotia from the B-isolate were separated into four size classes by length and mean fresh weight (n:15) of individual sclerotia: group 1, 11-2A mm, 81.17 mg; group 2, 7-13 mm, A6.78 mg; group 3, 5-6 mm, 19.83 mg; and group A, 2- 3 mm, 6.73 mg. Sclerotia were incubated at 15 C in water- saturated non-sterile Capac soil :hi Parafilm-sealed petri dishes. There were five sclerotia per dish and three dishes per size class. Incubation of intact and split sclerotia Twenty-five sclerotia (B-isolate) were cut in half and fifty sclerotia were left intact (large sclerotia were chosen for cutting so that the fifty sclerotia halves were similar in size to the intact sclerotia). After cutting, all sclerotia were incubated in plastic bags at 5 C for three days. Non-sterile Capac soil was adjusted to -0.5 bars metric potential (Figure 1) and 50 g placed in each of % SOIL MOISTURE 01 1 l i I I I I I i r 0 -é -1o MATRIC POTENTIAL Figure ‘L Soil moisture curve relating percent soil moisture to metric potential for Capac sandy clay loam soil. 20 petri dishes. Five intact sclerotia or sclerotia halves were lightly pushed into the soil in each dish. The dishes were sealed with Parafilm and stored at 15 C. Incubation of sclerotia at various soil pH Fifty milliliters of distilled water was added to 250 g of Capac soil (pH Si” in each of five beakers. The soil pH was adjusted with hydrochloric acid or potassium hydroxide and allowed to equilibrate for 8A.5 h. Five B-isolate sclerotia were placed on the surface of 50 g of equilibrated soil in each of four petri dishes at each pH value (pH A.82, 5.28, 6.11, 6.A3 and 7.0A). The dishes were sealed with Parafilm and the sclerotia incubated at 15 C. The number of germinated sclerotia and the number of stipes produced were counted at regular intervals. After 33 days, the pH of soil immediately beneath each sclerotium and thelfliof‘soil in each dish as distant as possible from any sclerotium was determined with an electronic pH meter RESULTS Production of sclerotia White cottony mycelium grew throughout inoculated bean tissue after one week to produce sclerotial initials, tufts of white mycelium, L45 mm in length. Clear droplets developed on the surface of the initials two to three days later; these gradually disappeared as the sclerotia matured 10 and darkened. Sclerotia, dry and black, were apparently mature after another A-7 days. Sclerotia were irregular in shape and ranged from <1 to >20 mm in length, resembling sclerotia produced on infected plants. Larger sclerotia arose from the fusion of several neighboring initials. Sclerotia grown on PDA were generally smaller and more regular in shape (circular/concave-convex). Germination rates of six isolates With the exception of the R-isolate, all isolates had 83-100% carpogenic germination after seven months incubation in soil at 15 C. There were differences in the speed of germination among isolates (Table 2). Isolate-A sclerotia conditioned at 5 and 23 C prior to incubation had 50% germination after 22 and 37 days, respectively. Thus, a conditioning period at 5 C accelerated, but was not required for carpogenic germination. Isolate-Elbeheved similarly. Fifty percent of H-, B- and M-isolate sclerotia had germinated by 7 months, but not before A6 days, at both 5 and 23 C. Only two of the 2A R-isolate sclerotia germinated after seven months. The speed of carpogenic germination of H-isolate sclerotia conditioned at 5 or 23 C for 1, 2, or 7.5 months was similar, with the most rapid germination by sclerotia conditioned at 5 C for 10.5 months (50% by 22 days), and those conditioned at 5 C for two months then -5 C for 5 months (50% by 21 days) (Table 3). Thus, the effect on carpogenic germination of 5 C conditioning was apparently no 11 Table 2. Number of carpogenically germinated sclerotia per 12 sclerotia for six isolates of Sclerotinia sclerotiorum with two conditioning temperatures. Conditioninga Incubation time (days)a Isolate temperature (C) 19 22 26 29 37 A2 210 A 5 1 6 8 8 8 8 10 23 O 1 2 5 6 6 12 D 5 O 1 3 7 8 12 12 23 O 0 2 A A 11 11 H 5 O 1 1 2 2 2 10 23 O 1 2 2 A A 12 B 5 O O O 0 2 3 12 23 O O O O O O 12 M 5 O O 0 0 0 O 12 23 O O O 0 O O 12 R 5 O 0 O 0 0 O O 23 0 O O 0 O 0 2 a Sclerotia were conditioned in sealed plastic bags for 745 months at 5 or 23 C prior to incubation. b Sclerotia were incubated at 15 C in hater-saturated soil contained in Parafilm-sealed petri dishes. 12 Table 3. Number of carpogenically germinated sclerotia per 12 sclerotia of Sclerotinia sclerotiorum (H-isolate) with 5 conditioning perins and 3 conditioning temperatures. Conditioninga Conditioning Incubation time (days)b period (months) temperature (C) 19 21 22 26 37 210 1.0 5 0 1 1 2 9 12 23 0 0 0 0 A 11 2 0 5 0 0 0 2 10 12 23 0 0 1 2 6 6 7.5 5 0 0 1 1 2 10 23 0 0 1 2 A 12 10.5 5 0 5 9 12 12 12 2.0 at 5, then 5.0 at -5 3 10 12 12 12 12 a Sclerotia were conditioned in plastic bags prior to incubation. b Sclerotia were incubated 2M: 15 C in water-saturated soil contained in Parafilm-sealed petri dishes. 13 different than 23 C conditioning for 7.5 months or less, although conditioning for 1CL5 months or at -5 C accelerated germination. Effect _<_>__t: splitting sclerotia and sclerotial size on carpogenic germination The number of carpogenically germinated sclerotia was independent of size up to 21 days incubation, but dependent on size after 22 days (Figure 2). Germination at 21 and 22 days was statistically analyzed for independence of sclerotial size using chi-square (3:0.05) testing of A1<2 (size class x germination) contingency tables. Essentially 100% of the largest sclerotia (size classes 1 and 2), 50% of size class 3,2nul30% of size class A had germinated after 37 days. The mean number of stipes per germinated sclerotium after 23 days wasi3.6, 2.9,‘L1 and 1.1 for size classes 1, 2, 3 and A, respectively. In future experiments, sclerotia as nearly the same size as possible were selected. Germination of intact and split sclerotia was compared (Table A). When the number of germinated sclerotia after 2A and A6 days was analyzed using 2 x 2 (condition of sclerotia x germination) contingency tablesiand chi-square (3:0.05), germination was independent of the condition (intact or split) of the sclerotia . The mean number of stipes produced per germinated sclerotium was similar for intact and split sclerotia, but could not be statistically tested. The cut surfaces of the split sclerotia, initially white, turned off-white to black (similar to rind tissue) s ,1! P- """ e—o C) I: 2 Lu J! 0 w 1.0 v- , E; A “J 3 1.. ‘z‘ '2- <>— ----------- o --------- o o: 4 LLI (5 C5 2 2'0 215 1’70 315 INCUBATION TIME (days) Figure 2. Cbmparison of carpogenic germination among four size classes (1 = 81 mg, to A = 7 mg) of sclerotia of Sclerotinia sclerotiorum incubated at 15 (I in water- saturated soil. ' 15 Table A. Carpogenic germination of split (half) and intact (whole) sclerotia of Sclerotinia sclerotiorum incubated in soil at -0.5 bars metric potential. No. stipes/ No. germinated/50 germinated sclerotium Sclerotia 2A days A6 days 2A days A6 days Whole 238 A63 1.35b 1.511b Half 1A AA 1.07 1.39 a Values in a column were not significantly different, based on chi-square (3:0.05). b Differences could not be statistically tested. 16 after 2A days of incubation. Influence of soil pH_gn carpogenic germination After 8A.5 h equilibration, the soil pH's used were A.82, 5.28, 6.11, 6.A3 and 7.0A. After incubation of sclerotia for 33 days, the soil immediately beneath sclerotia in treatments two to five was significantly more acidic than the surrounding soil (Table 5). The number of sclerotia that germinated after 2A days was tested using a 5 x 2 (soil pH x germination) contingency table and chi-square (§=0.05): germination was independent of soil pH for the range of pH examined (Table 5). Contingency tables could not be used to analyze germination data at 192nml33 days due to the low number of germinated sclerotia at 19 days and ungerminated sclerotia at 33 days. DISCUSSION Sclerotia collected from infected plants or plant debris in the field were difficult to free of bacterial and fungal contaminants. Sclerotia grown on green beans in petri dishes and harvested by washing under tap water were easily surface sterilized in sodium hypochlorite. The green been substratelmay provide sclerotia with nutrients similar to those obtained from infected bean plants in the field. There was a large variance in the time required for germination among sclerotia of the same isolate grown and 17 Table 5. Number of carpogenically germinated sclerotia per 20 sclerotia of Sclerotinia sclerotiorum incubated at various soil pH's. Initial soil pH Incubation time A.8 5.3 6.1 6.A 7.0 19 days 0x 1 5 A 1 2A days 15 10 1A 8 11 33 days 18 20 18 20 20 Final soil pHY Beneath sclerotia A.2aZ A.2a 5.1c 5.7d 6.2e Surrounding soil A.Sab A.9 be 6.0 de 6.6 f 7.0 g Differences in the numbers of sclerotia germinated at 2A days were not significant, based on chi-square (3:0.05). Differences at 19 and 33 days could not be statistically tested. y Final soil pH was measured after 33 days immediately beneath each sclerotium, and also at a point as distant as possible from any sclerotium (surrounding soil). Differences between final pH values with a common letter are not significant according to Tukey‘s test (P;0.05). Analysis of variance for final soil pH Source df Mean square F Treatment 9 5.1290 131 ** Error A0 0.0391 in Differences among treatments are significant at P=0.01. 18 harvested at the same time. This may be due to natural variation among individual sclerotia or to sensitivity to slight differences in environmental factors. The variance may, however, result from the method of sclerotial production, since new sclerotia are being initiated and formed throughout the 30 day incubation on green been tissue and sclerotia are harvested at the same time. This results in a difference of several weeks maturation time among the sclerotia. Although sclerotia appear to be morphologically mature, differences in physiological maturity may occur among sclerotia in a petri dish. Variance may be reduced by harvesting the sclerotia as soon as the first sclerotia apparently mature. This would sacrifice yield in favor of reducing variability. Cutting sclerotia in half‘hadru1effectcn1the percent germination (Table A). This finding was supported by Saito (35), who studied the initiation and development of apothecia from small cubes of sclerotial medullary tissue. Variability in experiments due to differences among individual sclerotia may, therefore, be reduced by applying different treatments to alternate halves of the same sclerotium. There were slightly more stipes produced per germinated intact sclerotium than from half sclerotia, but this difference could not be statistically tested due to the experimental design and limitations of contigency tables. The regeneration of the dark pigmented layer in cut sclerotia has been reported (35). 19 The size of sclerotia was positively correlated with the percent germination after 20 days incubation, but not before 20 days. The largest sclerotia produced the most stipes, with fewer stipes formed as sclerotial size decreased. Large sclerotia contain greater amounts of medullary tissue from which apothecial initials may arise (therefore, potentially more stipes)anulmore reserves to support apothecial development than smaller sclerotia. The results indicate that selecting sclerotia of similar size increases uniformity of germination and stipe production. Conditioning at 5 C was not required for carpogenic germination of the isolates tested (Tables 2, 3). The conditioning period is not well defined, and little or nothing is known of the processes associated with it. Saito (35) believes it is the time of formation of stipe initials within the medullary tissue. There were considerable differences among isolates in the incubation time prior to germination (specifically, stipe emergence). Variation among isolates with respect to conditioning and germination rate may have resulted in some of the disagreement found in the literature. Carpogenic germination of sclerotia was unaffected by differences in the soil reaction between pH A.82 and 7.0A. Since the unadjusted pH of the Capac soil used in future experiments (pH 51” was within this range, no attempt was made to alter its pH. The pH of the soil immediately below the sclerotia decreased. This change was particularly 20 pronounced intfluemore alkaline soils. Culture filtrates and cellular extracts of S; sclerotiorum contain high concentrations of organic acids (A6), and sclerotia have been shown to leak amino acids (17),vflflxfl1may account for the observed drop in soil pH. The lowering of soil pH in the immediate sclerotial environment may have resulted in the apparent independence of carpogenic germination and soil pH. A lower soil pH might also inhibit bacteria in the vicinity of the sclerotia, thereby reducing rotting and enhancing survival. The change of pH in the immediate sclerotial environment must, therefore, be considered when studying the effect of pH on S; sclerotiorum. LITERATURE CITED 1. Abawi, 0.5., and R.G. Grogan. 1975. Source of primary inoculum and effects of temperature and moisture on infection of beans by Whetzelinia sclerotiorum. Phytopathology 65:300—309. -' '"—— 2. Abawi, G.S., F.J. Polach, and W.T. Molin. 1975. Infection of been by ascospores of Whetzelinia sclerotiorum. Phytopathology 65:673-678. ——————————— 3.Adams, P.B., and W.A. Ayers. 1979. Ecology of Sclerotinia species. Phytopathology 69:896-899. A. Alexopoulos, C.J., and C.W. Mims. Introductor Mycology, 3rd ed. John Wiley and Sons, New York. 63 PD- 5. Brown, JJL, and K.D. Butler. 1936. Sclerotiniose of lettuce in Arizona. Ariz. Agric. Exp. Stn. Tech. Bull. 63:A75-506. 6. Cambpell, W.A. 19A7. A new species of Coniothyrium parasitic on sclerotia. Mycologia 39:190-195. 7. Cooke, G.Eq .LR. Steadman, and M.G. Boosalis. 1975. Survival of Whetzelinia sclerotiorum and initial infection of dry edible bEEfis in western Nebraska. Phytopathology 65:250-255. Y 2 10. 11. 12. 1A. 15. 16. 17. 18. 19. 20. 21. 22. 21 Cooke, R.C. 1969. Changes in soluble carbohydrates during sclerotium formation by Sclerotinia sclerotiorum and S; trifoliorum. Tradngfi:_MyEblT Soc. 53:77-86. Cooke, R.C. 1970. Physiological aspects of sclerotium growth in Sclerotinia sclerotiorum. Trans. Br. Mycol. Soc. 5A:3‘61’-365.-—- Cooke, R.C. 1971. Physiology of sclerotia of Sclerotinia sclerotiorum during growth and maturation. Trans. Br. Mycol. Soc. 56:51-59. de Bary, IL 188A. Vergleichende Morphologie und Biologie der Pilze Mycetozoen und Bacterien. Leipzig. 558 pp. (Comparative morphology and biology of the fungi, mycetozoa and bacteria. Translated by H.EJR Garnsey, revised by 1.8. Balfour, Claredon Press, Oxford. 1887). de Bary, A. 1886. Ueber einige Sclerotinen und Sclerotien—krankheiten.Bot.Z.AA:377-A7A. Hareda, Y., H. Tanba, and H. Watanabe. 197A. Cultural studies on apothecial formation in Sclerotinia sclerotiorum: part 1. Germination tests-ET—EEIEFBTIE produced at different temperatures and aged for various periods. Bull. Fac. Agric. Hirosaki Univ. 22:37-A3 [in Japanese with English summary]. Hartill, W.FJL, and AJK Underhill. 1976. "Puffing" in Sclerotinia sclerotiorum and 8. minor. New Zealand J. BEE. 1Az355-358. —— Honda, Y., and T. Yunoki. 1975. On spectral dependence for maturation of apothecia in Sclerotinia trifoliorum Erik. Ann. Phytop. Soc. Japan A1:383-389. Huang, H.C. 1976. Biological control of Sclerotinia wilt in sunflowers. Pages 69-72 in Rep.—Annu. Conf. Manit.Agron.1976. Huang, H.C. 1983. Histologyy amino acid leakage, and chemical composition of normal and abnormal sclerotia of Sclerotinia sclerotiorum. Can. J. Bot. 61:1AA3- 1AA??— Ikegame, H. 1959. Effect of light on maturation of apothecia of Sclerotinia trifoliorum. Ann. Phytop. Soc. Japan. 2Az273-280 [in Japanese with English summary]. Jones, D” and EL Watson. 1969. Parasitism and lysis by soil fungi of Sclerotinia sclerotiorum (Lib.) deBary, a phytopathogenic fungus. Nature 22A:287-288. Kohn, L.M. 1979. Delimitation of the economically important plant pathogenic Sclerotinia species. Phytopathology 69:881-886. ——-' Korf, R.P. 1973. Discomycetes and tuberales. Pages 2A9-319 in (LC. Ainsworth, F.K. Sparrow, and ILS. Sussman,'§ds. The fungi: an advanced treatise, vol. IV A. Academic Press, New York. Kosasih, BJL, and HHL Willetts. 1975. Cmtogenic and histochemical studies of the apothecium of Sclerotinia sclerotiorum. Ann. Bot. 39:185-191. 230 2A. 25. 26. 27. 28. 29. 31. 32. 3A. 35. 36. 38. 22 Letham, ILB. 1975. Stimulation by light of apothecial initial development of Sclerotinia sclerotiorum. Trans. Br. Mycol. Soc. 65:3'3—3-335. — Lethmn,ELB. 1976. Productioncfl‘apothecial initials by New South Wales isolates of Sclerotinia sclerotiorum. Aust. Plant Pathol. Soc. NEWEIT-5YA:5T' Le Tourneau, D. 1979. Morphology, cytology, and physiology of Sclerotinia species in culture. Phytopathology 693887-890. Libert, M.A. 1837. Plante crytogamicae arduennae (Exsiccati) No. 326. Published by iLA. Libert. Lumsden, R.D. 1979. Histology and physiology of pathogenesis in plant diseases caused by Sclerotinia species. Phytopathology 69:890-896. Makkonen, R., and 0. Pohjakallio. 1960. On the parasites attacking the sclerotia of some fungi pathogenic to higher plants and on the resistance of these sclerotia tx> their parasites. Acta Agric. Scand. 10:105-126. Natti, J.J. 1971. Epidemiology and control of been white mold. Phytopathology 61:669-67A. Newton,iLC.,and L.Sequeira. 1972. Ascospores astlm [wimary infective propagules of Sclerotinia Seglerotiorum in Wisconsin. Plant Dis.—R€p—.-5677—98: 0 . Purdy, L.H. 1955. A broader concept of the species Sclerotinia sclerotiorum based on variability. PfiyfaiaffiaTBEy'A5EA21—A27. Purdy, L.H. 1958. Some factors affecting penetration and infection by Sclerotinia sclerotiorum. Phytopathology A8:605-609: ———————————————————— Purdy, LJL 1979. Sclerotinia sclerotiorum: History, diseases and sympTBmatology, host range, geographic distribution, and impact. Phytopathology 69:875-880. Saito, I. 1969. Effect of some nutritional conditions on the formation and germinability of sclerotia of Sclerotinia sclerotiorum (Lib.) deBary. Bull. HERE—a-fd-b_Pr§fe—c—t': Agric. Exp. Stn. 19:1-7. Saito, I. 1973. Initiation and development of apothecial stipe primordia in sclerotia of Sclerotinia sclerotiorum. Trans. Mycol. Soc. Jpn. 1A:3A3-351. Saito, I. 1977. Studies on the maturation and germination of sclerotia of Sclerotinia sclerotiorum (Lib.) deBary, a causal fungus of bean stem rot. Rep. Hokkaido Prefect. Agric. Exp. Stn.lkL 26. 106 pp. Schwartz, H.F., and J.R. Steadman. 1978. Factors affecting sclerotium populations of, and apothecium production by, §2163221212 eslergtiozgm- PhytOpathology 68:383-388. — '——-' Sproston, T” and DAL Pease. 1957. Thermoperiods and production of apothecial initials :ni the fungus Sclerotinia trifoliorum. Science 125:599-600. 39. 210. A1. A2. A3. uu. A5. A6. A7. A8. A9. 23 Starr, (LC., H.J. Walters, and G.H. Bridgmon. 1953. White mold (Sclerotinia) of beans. Wyo. Agric. Exp. Stn.Bull. 322. Steadman, J.R. 1983. White mold--a serious yield- limiting disease of been. Phytopathology 67:3A6-350. Steadman, .LR., and KfW. Nickerson. 1975. Differential inhibitioncfl‘sclerotial germination:U1Whetzelinia sclerotiorum. Mycopathologia 57:165-170. — Suzui, T., and T. Kobayashi. 1972. Dispersal of ascospores of Sclerotinia sclerotiorwn(LibJ deBary on kidney bean plants: part 1. dispersal of ascospores from a point source of apothecia. Hokkaido Nat. Agric. Exp. Stn. Res. Bull. 101:137-151 [in Japanese with English summary]. Trevethick, J” and RJL Cooke. 1973. Non-nutritional factors influencing sclerotium formation 2h) some Sclerotinia and Sclerotium species. Trans. Br. Mycol. Soc.60:559-566. Tribe, H.T. 1957. On the parasitism of Sclerotinia trifoliorum by Coniothyrium minitans. —Trans. Br. ‘MycoI. sac. A0:A894A99. Uecker, F.A., W.A. Ayers, and P.B. Adams. 1978. A new hyphomycete on sclerotia of Sclerotinia sclerotiorum. Mycotaxon 7:275-282. Vega, R.R., D. Corsini, and D. Le Tourneau. 1970. Non- volatile organic acids produced by Sclerotinia sclerotiorum in synthetic liquid mediaT"My33lngE 57:332-338. Willettsw H.J.and J.Au4“ Wong. 1980. The biology of Sclerotinia sclerotiorum, S. trifoliorum, and S. minor with emphasis on specifiE—nomenclature. B68. Rev. A6:101-165. Williams, GJL, and J.H. Western. 1965. The biology of Sclerotinia trifoliorum Erikss. and other species of sclerotium-forming fungi: II. the survival of sclerotia in soil. Ann. Appl. Biol. 56:261—268. Young, P.A., and H.E. Morris. 1927. Sclerotinia wilt of sunflowers. Montana Agric. Exp. sen. Bull. 208. PART II: THE INFLUENCE OF WATER POTENTIAL ON CARPOGENIC GERMINATION INTRODUCTION Diseases caused by Sclerotinia sclerotiorum are most severe under wet conditions (20,‘HD. Cultural practices and modifications of plant architecture that reduce moisture in the soil or canopy have had some success in reducing disease incidence and severity (A, 6, 10, 2A, 26, 28, 29). Wet conditions might affect inoculum production, infection of the host plant and lesion development. S; sclerotiorum does not infect unless there is a film of water on the plant surface, although growth of the fungus from ascospores, mycelial disks or sclerotia is not adversely affected by osmotic potentals of -A0 bars and higher (9). Lesion expansion on bean leaves was stimulated by lowering osmotic potential from -1 to -28 bars, and‘wasrunzreduced until -6A bars, but expansion of established lesions was stopped when leaf surfaces were allowed to dry. There have been few reports on the relationship between water potential.(WM) and carpogenic germination. Bedi (3) reported that sclerotia did not germinate carpogenically at 100% relative humidity,but requirmdfree water. Morrall (21, 22) compared carpogenic germination oT‘ sclerotia incubated in soil cc nanocmyw .nwnsflocfi mew: mwuoemaom no moHQEmm on» no some so: mmumowfiaoe we cow .Som .mwcflcmmeom Loewe/6H6 ammo so: omuoojnoo mum: mfiuoewaom mumaomfirz ”pcmeflgwnxc one on LOHLQ mhmo mm Lou Aemv menumLOQEOO Boon Lo 0 m pm pqox one: mwuoecaom mpmaomfiim czocwrzcoumeonmq .Loumz omHHHumfio 2H omamnzocfl ashoHuocmHom mwcfiuoeoaom mo mfiuocmaom >2 Apsmfioz zap HmeHCH esp mo mmmpcooeoa m mm omnmocnxm unmoeocw ucmwozv :OHOHOHQEH Loam: mo comm .m oesmwm A9505 m2; 0 m N P O O. _ _ _ _ O mum—02-5— ... 0-11.0 Hm \BBOme To l U m \ 320$ -m c': 6 a 6 6 g 9 co _¢o e asvauONI lI-IOIEIM % ICE. 32 1.32%, 5.0 i 1.A9%, and 1A.5 i 1.AA% (mean of four replicates the standard deviation of the mean) of their initial dry weight after 118 h, respectively. Consequently, the actual percent moisture during the course of the experiment was slightly higher than indicated. At 118 h the weight of sclerotia was no greater than it had been at 27 h. Similar amounts of water were imbibed in A8 h by sclerotia held at metric potentials from -0.5 to -10 bars (Figure A). Weight increase was measured as a percentage of the initial fresh weight. In the previous experiment, R- isolate sclerotia stored at 5 C had an increase Of 97% of the initial fresh weight after A8 h in distilled water (Ww=0 bars). Influence of water potential on carpogenic germination In the first experiment, the number of germinated sclerotia in PEG 8000 solutions was highest at -1.0 bar, and approximately 30% germinated at -8.8 bars (Figure 5). Sclerotia rotted at the higher osmotic potentials, which affected the germination. In tn”: second experiment, antibiotics were included; germination was 100% between 0 and -A.0 bars, and nearly 30% at -8.0 bars (Figure 5); there was no germination at -15.0 or -20.0 bars. Evaporative water loss during the experiments, which would have altered osmotic potential, was negligable. In soil, 50% of the sclerotia germinated at -0£5bars, and less than 10% germinated at metric potentials below -1 bar (Figure A). 33 100 1 ~90 60'1 WATER meIeI'rIou A 430$ Z 2 ~70 g I- -l - <40 1; z ‘2 2 _ m m E m _ ‘5 20‘ '1 g i'.‘ GERMINATION < A B o —2 -¢l -é -A 30 SOIL MATRIC POTENTIAL (be rs) Figure A. Effect of soil metric potential on carpogenic germination of sclerotia of Sclerotinia sclerotiorum (H-isolete) incubated for 30 days at 15”C, endcnithe amount of water imbibed by sclerotia after A8 h expressed as a percent of the initial fresh weight. .cmme on» mo :oHumH>oo ccmocmum on» oofizu Hmsom memo Hmowpeo> .mpwfiomw -z u Ad; “33034 n loo .0 m; an mama om Lain 28338 ooom 0mm 5 ESLOHOOLmHom mficfluocwflom mo manoeoaom oouuoecs mo coHumcfisLom Oficmwoqcmo co Hmwpcouoo oHuosmo mo pommmm .m meswwm Time: ...<_._.Zm._.0m o.._.O_ZmO 0N1 mrI 07. ml w1 v: NI ‘0 0 0 I ‘ o o v m % 6 0 NOIIVNIWHSO 4 1 O (D O O DISCUSSION The rate of water imbibition and dry weight lost upon soaking was similar for sclerotia (R-isolate) conditioned at 5 C or room temperature (Figure 3), suggesting that the 5 C treatment did not increase either the amount of soluble compounds able to diffuse out of sclerotia or the permeability of the membranes to such compounds. The M- isolate sclerotia gained weight at a rate similar to the R- isolate sclerotia. There was, however, three times the initial dry weight lost from M-isolate sclerotia compared to the R-isolete. Their final percent moisture was therefore higher than the R-isolate. Huang (11) reported greater leakage of amino acids from what he referred to as abnormal sclerotia: those having a severely fractured rind and a brown medullary region. The M-isolate sclerotia used in the present EXperiment had ten to greyInedullee and were more brittle than the laboratory produced R-isolete sclerotia which had white medullae. The condition of the M-isolate sclerotia may have been due1x>the method of their storage at a bean elevator. Since the maximum amount of water was imbibed by sclerotia after'7tiincubation in distilled water (Figure 3), a soaking period of 7 h was used for subsequent experiments to obtain a maximum amount of water imbibition with a limited amount of leakage of solutes, particularly in the water potential experiments, where imbibition of water 35 36 by sclerotia would have lowered the water potential of the incubation medium. The different patterns of germination exhibited by sclerotia incubated in soil adjusted to specific metric potentials (Figure A) and by sclerotia incubated in PEG 8000 solutions(Figure 5)cfl‘similar water potentials indicate that all properties of a system for maintaining specific water potentials must be considered. Aside from water potential, ion effects and characteristics such as diffusibility of active compounds within the system may have significant effects on the behavior of the organism under study. Phytophthora cinnamomi had reduced growth rate and increased susceptibility to water stress on matric- controlled as compared to osmotic-controlled agar media, and it was suggested that this response reflected differences between the tn“) systems ix: solute transport (27). Phytophthora cinnamomi and Alterneria tenuis were less tolerent of soil metric potentials than of equivalent osmotic potentials of agar media controlled osmotically with KCl or sucrose (2). Growth of Fuserium roseum f. sp. cerealis was stimulated as the osmotic potential was lowered over the range, -1.5 to -8“2 bars; growth was not stimulated when soil metric potential was lowered over the same range (5). The hypothesis that carpogenic germination of S; sclerotiorum was stimulated by the loss of inhibitory compounds through diffusion was supported by sclerotial 37 germination occurring at nnxfli lower solute potentials than soil metric potentials. The amount of water imbibed by sclerotia, however, decreased only slightly at the lower soil metric potentials (Figure A), suggesting that the dry weight lost consisted, in part, of compounds which inhibited carpogenic germination. Although water imbibition was only slightly affected, the volume of soil water present at lower metric potentials may have been insufficient for the diffusion of inhibitory compounds away from sclerotia. Polyethylene glycol 8000 solutions provided a medium through which inhibitory compounds could diffuse, even at relatively low osmotic potentials. "Regermination" of sclerotia of S; sclerotiorum at -15 bars (21) indicates that sclerotia can imbibe enough water to support germination at this low water potential, provided germination has been initiated. Morrall (22) reported 28% germination of sclerotia of S; sclerotiorum at a soil metric potential of -7.3 bars, while in the present study only A% of sclerotia germinated at -7 bars metric potential (Figure A). The higher germination rate reported by Morrall may have been due to saturating soil in dialysis bags prior to immersion in PEG 20,000 solutions of appropriate osmotic potentials. Three days were required for equilibration and the process was repeated during the experiment due to microbial decomposition of the bags. Loss of inhibitory compounds may have occurred during exposure to very high water potentials for several days during the incubation period. Sclerotia in the present study 38 germinated at osmotic potentials lower than those reported by Grogan and Abawi (9) or Morrall (21). This may be due to different types or volumes of osmotica used, or to fungal isolate differences. (A) 10. LITERATURE CITED Abawi, 0.8., and R.G. Grogan. 1979. Epidemiology of diseases caused by Sclerotinia species. Phytopathology 69:899-90A. ——————————— Adebayo, AJA., and R.F. Harris. 1971. Fungal growth responses to osmotic as conpared to metric water potential. Soil Sci. Am. Proc. 35:A65-A69. Bedi, KA& 1962. Light, air and moisture in relation to the formation of apothecia of Sclerotinia sclerotiorum (Lib.) deBary. Proc. Indiafi—A'c—a'af_S€i—.T SEEtu’B 55:213-223. Blad, B” .LR. Steadman, and A. Weiss. 1978. Canopy structure and irrigation influence white mold disease and microclimate of dry edible beans. Phytopathology 68:1A31-1A37. ‘ Cooke, RHL, R.I. Papendick, and D.M. Griffin. 1972. Growth of two root-rot fungi as affected by osmotic and metric water potentials. Soil Sci. Soc. Amer. Proc.36:78-82. Coyne, D.P., J.R. Steadman, and FUN. Anderson. 197A. Effect of modified plant architecture of Great Northern dry been varieties (Phaseolus vulgaris) on Dis. Rep. 58:379-382. Duniway, J.M., R.G. Grogan, and J.R. Steadman. 1977. Influence of soil moisture on the production of apothecia by sclerotia of Whetzelinia sclerotiorum. (Abstract) Proc. Am. Phytop. Soc. A:1T5. Griffin, DAL. 1972. Ecology of soil fungi. Syracuse Univ. Press, Syracuse, New York. 193 DP. Grogan, R.G., and G.S. Abawi. 1975. Influence of water potential on growth and survival (If Whetzelinia sclerotiorum. Phytopathology 65:122-128. — Haas, J.H., and IL Bolwyn. 1972. Ecology and epidemiology of sclerotinia wilt of white beans in Ontario. Cen..L Pl. Sci. 52:525-533. 11. 12. 13. 1A. 15. 16. 17. 18. 19. 20. 21. 22. 2A. 25. 39 Huang, H.C. 1983. Histology, amino acid leakage, and chemical compostion of normal and abnormal sclerotia of Scerotinia sclerotiorum. Can. J. Bot. 61:1AA3- 1AA7I Jaffee, B.A., and E.I. Zehr. 1983. Effects of certain solutes, osmotic potential, and soil solutions on parasitism of Circonemella xenoplax by Hirsutelle rhossiliensis. PhytopathoIOgy'73:5AA:5A6. -— Janes, B.E. 1966. Adjustment mechanisms of plants subjected to varied osmotic pressures of nutrient solution. Soil Sci. 101:180. Jarvis, P.G., and M.S. Jarvis. 1965. The water relations of tree seedlings. 1L growth and root respiration in relation to osmotic potential of the root medium. In B. Slavik, ed. Water Stress in Plants. The Hagfie. Kosasih, B.D., and H.J. Willetts. 1975. Ontogenic and histochemical studies of the apothecium of Sclerotinia sclerotiorum. Ann. Bot. 39:185-191. Lagerwerff, J.V., G. Ogata, and H.E. Eagle. 1961. Control of osmotic pressure of culture solutions with polyethylene glycol. Science 133:1A86-1A87. Lewlor, ILW. 1970. Absorption of polyethylene glycols by plants and thier effects on plant growth. New Phytol. 69:501-513. Lesham, B. 1966. Toxic effect of ,carbowaxes (Polyethylene glycols) on Pinus halipensis seedlings. P1. Soil 2A:322. Michel, ELE., and M.R. Kaufman. 1973. The osmotic potential of polyethylene glycol 6000. Plant Physiol. 51:91A-916. Moore, W.D. 1955. Relation of rainfall and temperatures to the incidence of Sclerotinia sclerotiorum in vegetables in south FloriEE'durIng the years 19AA to 195A. Plant Dis. Rep. 39:A70-A72. Morrall, ILA.A. 1975. Water potential and the germination of sclerotia of Sclerotinia sclerotiorum (Lib.) de Bary. (Abstract) Proc. Can. PhytOpath. Soc. A2:17. Morrall, ILA.A. 1977. A preliminary study of the influence of water potential on sclerotium germination in Sclerotinia sclerotiorum. Can. J. Bot. 55:8-11. Painter, L.I. 1966. Method cd‘ subjecting growing plants to a continuous soil moisture stress. Agronomy J. 58:A59—A60. Partyka, B.Eq and W.F. Mai. 1962. Effects of environment and some chemicals on Sclerotinia sclerotiorum in laboratory and pot'Et'cT-Ti-el-cf Phytopathology 52:766-770. Saito, I. 1973. Initiation and development of apothecial stipe primordia in sclerotia of Sclerotinia sclerotiorum. Trans. Mycol. Soc. Jpn. 1A:3A3;351. 26. 27. 28. 29. 30. A0 Schwartz, iLF., J.R. Steadman, and D.P. Coyne. 1978. Influence of Phaseglus vulgaris blossoming Sclerotinia sclerotiorum. Phytopathology 68:A65-A70. Sommers, IHE., R.F. Harris, F.N. Dalton, and W.R. Gardner. 1970. Water potential relations of three root-infecting Phytophthora species. Phytopathology 60:932-93A. Steadman, J.R. 1979. Control of plant diseases caused by Sclerotinia species. Phytopathology 69:90A-907. Steadman, J.R., D.P. Coyne, and G.E. Cook. 1973. Reduction of severity of white mold disease on Great Northern beans by wider row spacing and determinate plant growth habit. Plant Dis. Rep. 57:1070-1071. Steadman, .LR., and KLW. Nickerson. 1975. Differential inhibition of sclerotial germination in Whetzelinia sclerotiorum. Mycopathologia 57:165-170. . Williams, J., and C.F. Shaykewich. 1969. An evaluation of polyethylene glycol (P.E.G.) 6000 and P.E.G. 20,000 in the osmotic control of soil water metric potential. Can..L Soil Sci. A9:397—A01. Zur, B. 1966. Osmotic control of the metric soil—water potential: I. soil-water system. Soil Science 102(6):39A-398. Zur, B. 1967. Osmotic control of the metric soil-water potential: II. soil-plant system. Soil Science 103(1):30-38. PART III: EVIDENCE FOR A DIFFUSIBLE ENDOGENOUS INHIBITOR OF CARPOGENIC GERMINATION INTRODUCTION Results of experiments reported in PART II suggest that carpogenic germination cu‘ Sclerotinia sclerotiorum is stimulated by diffusion of some compound(s) out of and away from sclerotia (see PART II, DISCUSSION). The composition of sclerotia (8, 9, 16, 17, 18) and amino acids leaked from sclerotia (7) have been examined, but there have been no reports of endogenous compounds influencing germination in this manner. The present study was undertaken to determine: a) whether leaching sclerotia stimulated germination, and b) if incubating previously leached sclerotia in sclerotial leachate inhibited germination. MATERIALS AND METHODS Electrolyte leakage by surface-sterilized sclerotia Loss of electrolytes was used to determine if changes in permeability of sclerotial membranes resulted from surface sterilization. Sclerotia were rinsed under running deionized water for 30 sec to remove surface material, air— dried for 2 h, then divided into six subsamples (approximately 730 mg fresh weight each) and weighed. A1 A2 "Surface-sterilized" sclerotia were placed in a metal sieve and rinsed with 10 ml 95% ethanol, then 50 ml deionized water. Rinsed sclerotia were placed in A0 ml 0.5% NaOCl for 3 min, A0 ml sterile deionized water for 2 min, blotted dry and transfered to 15 ml fresh deionized water. Conductivity was measured with an electronic conductivity meter (Markson Science Inc», Del Mar, CA 9201A) immediately and at 30 min intervals for 2.51%. "Non-surface-sterilized" sclerotia were treated similarly, but deionized water was substituted for 95% ethanol and 0.5% NaOCl. Three replicates were included. The experiment was repeated with sclerotia air-dried for 60 min after blotting and before soaking in 15 ml deionized water; conductivity was measured for three hours. In a third experiment, conductivity was measured over A3 h, and 100 ug/ml each of penicillin-G, streptomycin sulfate and neomycin sulfate was added to eliminate bacterial growth. Incubation of sclerotia in soil, soil filtrate or water Material diffusing from sclerotia was removed from, and a carbon source added to, several incubation media to determine the effect on carpogenic germination. Sclerotia were incubated in sterile or non-sterile soil or soil filtrate, each with or without glucose amendment. Sterile soil was obtained by placing Capac sandy clay loam in glass beakers covered with aluminum foil and autocleving at 121 C for 30 min, 3 times. Five surface-sterilized sclerotia (H-isolate) were placed on 50 g of sterile or non- 43 sterile soil in each of five replicate 90 x 15 mm plastic petri dishes and 2OInl sterile distilled water or sterile distilled water plus 1% glucose was added to each dish. The dishes were sealed with parafilm and incubated at 15 C. To remove contaminants from send to be-used in other treatments, the sand was rinsed well with water and placed in an enamel bucket with enough hydrochloric acid (37%) added to just cover the send. After 2A h of frequent mixing, the acid was poured off; The sand was then stirred under running distilled water until therfliof'the runoff water was equal to the pH of the distilled water. Soil filtrate was obtained by filtering five parts Capac soil mixed with two parts distilled water (w/v) through #1 Whatman paper in :3 Buchner funnel. Twenty milliliters of sterile or non-sterile soil filtrate or soil filtrate plus 1% glucose was added to each of several petri dishes containing 50 g acid washed, autoclaved sand. Five sclerotia were placed in each petri dish, and there were five replicates per treatment. The dishes were sealed and incubated as described previously. Sclerotia were also incubated in water which was changed at regular intervals or not changed. Five sclerotia, 50 mg acid washed sand, and 20 ml sterile distilled water (with or without 1% glucose amendment) were added to each dish. Sclerotia in half of the dishes were kept in the same solution during the entire incubation period, and the remaining sclerotia were aseptically removed every 2A-A8 h AA and replaced in fresh sterile distilled water or sterile distilled water plus 1% glucose. Changing of the solutions prevented the accumulation of any compounds that leaked from sclerotia. Five replicate dishes were incubated as described above. Recovery of sclerotial leachate Two grams of sclerotia (H-isolate) was rinsed with deionized water and placed in 75 ml sterile deionized water (two replicates).The solution was collected and replaced with fresh sterile deionized water each hour for the first 12 h, then every 12 h for five days. The samples were concentrated separately in a rotary exeporator at A5 C, dried under nitrogen and stored over calcium chloride, in vague, fer A8TL Assay of sclerotial leachate for total carbohydrate Because of similarities in the effects of glucose and sclerotial leachate in the incubation medium, sclerotial leachate was assayed for the presence of carbohydrate. Three grams (fresh weight) of sclerotia (H-isolate) in eacklof three 150 ml flasks was rinsed by shaking in 50 ml sterile deionized water for 15 sec; the water was poured off and replaced with 501nl fresh sterile deionized water. At 1, 3, 7, 12, 2A, A8 and 72 h, the solution was poured off, collected, frozen at -5(3until needed, and replaced with fresh sterile deionized water. After thawing, each leachate sample was filtered, reduced to dryness in a rotary L15 evaporator at A5 C and resuspended in A0 ml deionized water (the 6-7 h leachate sample had to be diluted A times for the assay). The leachate was assayed for total carbohydrate by the phenol method (A, 5). The absorbency at A88 nm of the samples was measured (H) a Varian Series 63A spectro- photometer (Varian Aerograph, 2700 Mitchell Drive, Walnut Creek, CA 9A598l. Deionized water (for reagent blank) and glucose standards (25, 50, 75 and 100 ug/ml) were prepared and absorbency measured in the same manner as the leachate samples. Samples were replicated three times. Gas chromatography of sclerotial leachate Alditol acetate derivatives of the sugars and polyols in the sclerotial leachate were prepared from the samples collected above. Aliquots containing;20 ug leachate were dried in 1 ml reaction vials under nitrogen. Twenty-five milliliters of freshly made sodium borohydride (20 mg/ml in 3 M ammonium hydroxide) was added to each vial and incubated for 1k1et 23(L Glacial acetic acid(A-3 drops) was then added until bubbling ceased. After the addition of 100 ul methanol, the solution in each vial was carefully mixed on a vortex mixer and dried under nitrogen. This step was repeated. To each vial was then added 100 ul methanol:water (1:1); the solutions were mixed and dried under nitrogen. Another 100 ulInethanol was added to each vial, the solutions mixed and dried. This last step was repeated twice. After the final drying, 50 LU. acetic A6 anhydride was added to each vial and the vials were heated at 121 C for 1 h. Two-microliter samples of derivatized leachate and standards were injected into a 6 ft x 2 mm glass column packed with 3% SP-23A0 on 100/120 Supelcoport (Supelco, Inc., Bellefonte, PA 16823) in a Varian Aerograph Series 1200 gas chromatograph. A detector temperature of 300 C and inlet temperature of 230 C were used with a column temperature programmed from 180-275 C at A C/min. Nitrogen carrier gas was adjusted to a flow rate of 25 ml/min. Effect of sclerotial leachate on carpogenic germination Crude leachate was obtained by soaking sclerotia in distilled water for 82 h and filtering the resulting yellow solution through #1 Whatman paper. Sclerotia that had been leached for 13 days under running tap water were incubated on 25 g of washed and ignited sand in a 90 x 15 mm petri dish containing 10 m1 Of 58, 580 or 5800 ug leachate/ ml aqueous solution. Five sclerotia in each of 20 replicate disheswereincubeted et15 C. Germinated sclerotia with short stipes were incubated in leachate solutions to determine if crude leachate had an effect on continued stipe elongation and continued stipe production in addition 1x) its effect on initiation of carpogenic germination. Sclerotia (H-isolate) were germinated on moist soil, in the dark, at 15 C. Most sclerotia had germinated after A8 days and produced several stipes each. The number of stipes on germinated sclerotia 47 was counted and the sclerotia placed on a 3 mm bed of washed and ignited sand in six petri dishes, four sclerotia per dish. Ten milliliters of sclerotial leachate was added to each dish at the rate of 0, 0.29 or 2.9 mg leachate/ml. Included in sill treatments were 100 ug/ml each of penicillin-G, streptomycin sulfate and neomycin sulfate. Sclerotia in 2 replicate sets of dishes were incubated at 15 C. Removal of diffusible material from sclerotia and its effect on carpogenic germination Sclerotia were leached on a layer of glass wool in a glass funnel under running tap water. Other sclerotia were spread in the bottomsIof glass petri dishes and maintained at 100% relative humidity HUD by positioning them above deionized water in sealed Mason jars. Paper towels lining the walls of the jars extended into the water to increase the surface area of the water. A third group of sclerotia was exposed to ambient relative humidity (:eir-dry). Sclerotia (H-isolete) were treated as above for 30 days. Leeched, 100% RH and air-dry sclerotia were placed on a 3 mm bed of washed and ignited sand moistened with 6 ml distilled water in 100 x 15 mm plastic petri dishes, 10 sclerotia per dish, and A replicate dishes per treatment. The dishes were sealed with Parafilm and incubated at 15 C. The experiment was repéated using sclerotia (A and D-isolete) which were leached or held at 100% RH for 23Idays prior to incubation on moist sand, with nine replicates per treatment. RESULTS Electrolyte leakage by surface-sterilized sclerotia Surface-sterilized sclerotia leaked significantly more electrolytes in the first 30 min then non-surface-sterilized sclerotia (Figure 6). After the first 30 min, however, the differences were not significant. Results were similar when sclerotia were air-dried for 60 min prior to incubation and when conductivity was measured for A3 h. Carpogenic germination of sclerotia incubated in soil, soil filtrate or water Seventy—two percent of sclerotia incubated in non- sterile soil germinated carpogenically, while of the sclerotia incubated in non-sterile soil + glucose and sterile soil, only 12 and 16% germinated, respectively (Table 6). Four and 32% of the sclerotia rotted in the non- sterile soil and non-sterile + glucose, respectively. Of all the soil filtrate treatments, only the non-sterile soil filtrate treatment had any germination (8%) after 50 days, although 8% of the sclerotia rotted. Fifty-two percent of the sclerotia which were incubated in regularly changed sterile distilled water germinated carpogenically. There was no carpogenic germination in the regularly changed sterile distilled water + glucose or either of the unchanged sterile distilled water treatments. Some sclerotia in all treatments except sterile soil, A8 A9 o: o 9 SURFACE /' STERILIZE/:/ I NON- 4004 0 SURFACE- STERILIZED A) O 9 I pmhos leaked/g fr wt sclerotia 0 3'0 6'0 9'0 120 150 TIME (min) Figure 6. Electrolyte leakage from surface- sterilized and non-surface-sterilized sclerotia of Sclerotinia sclerotiorum. Surface- sterilized scIEFOEI5_WEFe rinsed briefIy in 95% ethanol, placed in 0. 5% NaOCl for 3 min, then placed in deionized water for 2ImhL. Non-surface-sterilized sclerotia were treated similarly tn) surface-sterilized sclerotia, but with deionized water replacing 95% ethanol and 0.5% NaOCl. After treatment, each sample (AGO-750 mg) of sclerotia (3 replicates) was immediately incubated in 15 ml deionized water at 23 C. 50 Table 6. Carpogenic germination of sclerotia of Sclerotinia sclerotiorum after 50 days incubation at 15 C in sterile or non-sterile soil or soil filtrate, or in distilled water which was changed regularly or not changed. One percent glucose was added to a similar set of treatments. Percent germinationx Treatmenty - 1% glucose + 1% glucose Soil + H2O: Sterile 16 cdz 36 bc Non-sterile 72 a 12 00 Sand + soil filtrate: Sterile 0 d 0 d Non-sterile 8 cd 0 0 Sand + sterile H20: Changed 52 ab 0 0 Not changed 0 d I 0 d Means of 5 replicates. y Five sclerotia were incubated on 50 g soil or sand plus 20 ml distilled water or soil filtrate in each petri dish (90 x 15 mm). Differences between entries with a common letter are not significantly different according to Tukey's test (P=0.05). Analysis of variance Source df Mean square F Treatment 11 2930.36 12.38 ** Error A8 236.67 ax Differences among treatments are significant at P=OJTL 51 non-sterile soil and regularly changed sterile distilled water germinated myceliogenically. Recovery and assay of sclerotial leachate There was A6 mg dry material/g dry wt sclerotia recovered from sclerotia soaked in sterile deionized water for 5 days; most of this material (35 mg/g dry wt) was recovered in the first 12 h, with A3 mg/g dry wt recovered in the initial 72tl(Figure 7). For the total carbohydrate assay, the concentration of carbohydrates was expressed in ug glucose equivalents (ug glc eq), where no. ug glc eq/g sclerotia : (no. glucosyl units) x volume of leachate (ml) (3) mass of sclerotia (g) where 1 glucosyl unit equals the absorbency of 1 ug D- glucose/ml prepared by the phenol method. The total detectable carbohydrate recovered in the first 12 h was 69% of that recovered in the first 72 h (A031 ug glc eq/g sclerotia) (Table 7). Therefore, detectable carbohydrate accounted for 8 and 9% of the total mass of leachate recovered after 12 and 72 h, respectively. These values are approximate since the phenol method will detect pentoses, hexoses, heptoses and amino sugars. Of the carbohydrates detected by gas chromatography, glucose and mannose (or their polyol forms) were found in greatest abundance. Lesser concentrations of 52 0) 0| co 35$ h) mg LEACHATE/g DRY WT. SCLEROTIA 1 2 3 4 TIME (days) Figure 7. Recovery of leachate at 12 h intervals from sclerotia of Sclerotinia sclerotiorum incubated {K sterile Hgfo'n-i'i'é'd—w'éfgr for five days. Vertical lines equal twice the standard deviation of the mean. Bars without vertical lines were not replicated. 53 Table 7. Total carbohydrates (detectable by phenol method) in sclerotial leachate samples8 of Sclerotinia sclerotiorum collected over 72 h. Sampling ug glucose equivalents/ ug glucose equivalents/ time (h) g sclerotia g sclerotia/ hour 0 - 1 3A2 a 35.8b 3A2 i 35.8 1 - 3 A8A t 10.3 2A2 t 5.3 3 - 7 1233 * 17.8 309 t 19.1 7 - 12 726 t 1A.A 1A5 t 3.2 12 - 2A 611 t 1A.3 51 t A.1 2A - A8 A73 2 5.9 20 t 0.3 A8 - 72 162 t 5.6 7 t 0.3 a Leachate was obtained by soaking each of 3 replicate 257 g samples of sclerotia in 50 ml sterile deionized water. The leachate solutions were collected and replaced with fresh sterile deionized water at 1, 3, 7, 12, 2A, A8 and 72 h. Means of 3 replicates t the standard deviation of the mean. 5A glyceraldehyde, deoxyribose, arabinose, galactose and glucosamine (or their polyol forms) were also detected. Germination of sclerotia incubated in sclerotial leachate Leeched sclerotia were incubated in 0, 58, 580 or 5800 ug leachate/ml. The lowest concentration used (58 ug/ml) was about 16% of that recovered in a previous experiment after 5 days (Figure 7), based on the mass of sclerotia incubated in the leachate solution. Germination in 580 ug leachate/ml was lower than that in controls, and only 1% germinated in 5800 ug leachate/ml after 35 days (Tables 8a, b). Increasing amounts of mycelium were produced in the leachate solutions as the concentration of leachate increased. Secondary sclerotia developed from sclerotia that germinated myceliogenically in the 5800 ug leachate/ml solution. In a similEW' experiment, germinated sclerotia (H- isolate) with short stipes, but no apothecia, were incubated for 21 days in 0, 0.29 or 2.9 mg leachate/ml. Significantly fewer new stipes were produced after 1A days in 0.29 mg leachate/ml than in either water controls or 2.9 mg leachate/ml treatments (Table 9). By 21 days, however, differences were not significant. Stipes formed prior to incubation continued to elongate in crude leachate solutions as well as in controls. 55 Table 8a. Carpogenic germination of sclerotia of Sclerotinia sclerotiorum incubated in leachate solutions. The test sclerotia were leached under running tap water for 13 days prior to incubation. Leachate for the treatment was collected during 72 h from other sclerotia. Percent of sclerotia germinating Leachate concentration carpogenically after:x (ug/ ml) 1A days 23 days 3A days 0 18 ay 55 a 83 a 58 11 ab 39 ab 79 ab 580 O b 21 be 71 b 5800 0 b 0 c 1 c x Means of A replicates. Each replicate consisted of 5 dishes, each dish containing 5 sclerotia, 25 g sand and 10 ml leachate solution. y Per column, entries with a common letter are not significantly different according to Duncan's Multiple Range test (P:0.05). 56 Table 8b. Analyses of variances for carpogenic germination of sclerotia of Sclerotinia sclerotiorum incubated in leachate solutions—from TabIe 8. Analysis of variance for 1A day data Source df Mean square F Treatment 3 313.0 6.75 * Error 12 A7.67 Analysis of variance for 23 day data Source df Mean square F Treatment 3 22u1.o 1o.u9 '* Error 12 213.67 Analysis of variance for 3A day data Source df Mean square F Treatment 3 5977.3 19u.91 ** Source 12 30.67 2 Differences among an , Differences among treatments are significant at 2:0.05. treatments are significant at P=0.01. 57 Table 9. New stipe production by sclerotia of Sclerotinia sclerotiorum incubated in sclerotial leachate solutions. The sclerotia were first germinated in soil. Leachate concentration No. new stipes/ dish after:x (mg/ ml) 1A days 21 days 0.0 uu.0 by 75.0 a 0.29 2A.5 c 77.0 a 2.9 71.0 a 108.0 a x Means based on 2 replicates, each consisting of A germinated sclerotia, 25 g sand and 10 ml sclerotial leachate solution in a 90 x 15 mm petri dish. y Per column, entries with a common letter are not significantly different according to Duncan' 3 Multiple Range test (P: 0. 01) Analysis of variance for 1A day data Source Of Mean square F Treatment 2 1090.5 107.26 ** Error 3 10.17 Analysis of variance for 21 day data Source df Mean square F Treatment 2 68A.7 <1 ”'5' Error 3 880.0 *2 Differences among treatments are significant at P=0.01. ”'5' Differences among treatments are not significant. 58 Germination of leached and unleached sclerotia Leaching sclerotia under running tap water prior to incubation on moist sand enhanced carpogenic germination of all three isolates of S; sclerotiorum (Figure 8). After 20 days, 5, A0 amd 80 percent of the leached sclerotia of isolates D,lland.A,respectively,innlgerminated, whereas less than 5% of each isolate had germinated when held at 100% RH or ambient RH prior to incubation. Forty-nine, 87 and 100 percent of the leached D-, H- and A-isolate sclerotia, respectively, had germinated after 60 days. Of the sclerotia held at 100% RH prior to incubation, only isolate A had greater than 50% germination DISCUSSION It was hypothesized in PART II that the leakage of inhibitory compounds from sclerotia stimulated carpogenic germination. When material diffusing from sclerotia was removed from the incubation medium, either directly or via possible microbial sinks in non-sterile soil, carpogenic germination was higher than if this material was allowed to accumulate (Table 6). The addition of glucose also suppressed carpogenic germination in all treatments. Although glucose, as well as other sugars end/or polyols, was detected in sclerotial leachate, inhibition by other leachate components cannot be ruled out. Phenol-detectable 59 Figure 8. Effect of leaching on carpogenic germination of sclerotia of Sclerotinia sclerotiorum incubated on moist send. (a), H-_i_solate scleFOTia were jeeched under running tap water (0), held at 100% relative humidity (D), or held at ambient relative humidity (A) for 30 days prior to incubation. (b), A-isolate( ) or D-isolate (----) were leached (O) or held at 100% relative humidity (Cl) for 23 days prior to incubation. Vertical lines represent twice the standard deviation of the mean. 60 SCLEROTIA GERMINATED/1O NO. 2'1 /; SCLEROTIA GERMINATEo/m b 00 I l I NC. i 10‘ 30 50 INCUBATION TIME (days) 61 carbohydrate comprised only 9% of the total leachate (Table 7; Figure 7), although large amounts of trehalose and mannitol were found in ground and extracted sclerotia, with lesser amounts of arabitol, glycerol, glucose, galactose and traces of fructose, galactitol and arabinose (8, 17). Huang (7) detected seven amino acids in sclerotial leachate. Carpogenic germination of sclerotia was almost completely inhibited by incubation in soil filtrate (Table 6). An exchange of ions between the soil solution and the colloidal fraction or organic matter in the soil may have removed inhibitory compounds from or altered the osmotic potential of the soil solution during incubation, accounting for the near absence of germination in soil filtrate. The addition of leachate to the incubation solution inhibited carpogenic germination of sclerotia previously leached to remove readily diffusible compounds (Table 82). It is unlikely that the inhibition of carpogenic germination was due to the lowering of osmotic potential by the leachate. Although the precise composition of the sclerotial leachate is unknown, limits can be set on the probable osmotic potential of the leachate solutions by the formula, I'l: iCRT (A) where Tlis the osmotic pressure in bars, 1 is the average number of particles per molecule, 0 is the concentration of the solute in moles/liter, R is the gas constant “L0832 bar 62 liter"1 mol'1 K’I), and T is the absolute temperature in degreesl((15). An upper limit can be set by assuming the leachate to be composed entirely of hexose, in which case the osmotic potential of the 5800 ug leachate/ml solution would be -0.77 bars. As an extreme lower limit, a 5800 ug NaCl/ml solution would have an osmotic potential of-JL76 bars. The actual osmotic potential of the 5800 ug/ml solution would lie between these limits, which is higher than the lowest osmotic potentiad. at which carpogenic germination occurred (Figure 5). Surface sterilization of sclerotia was necessary to prevent microbial degradation of sclerotial leachate. The effect of surface sterilization on permeability of sclerotial membranes was, therefore, of concern. Although surface-sterilized sclerotia initially leaked a greater amount of electrolytes than non-surface-sterilized sclerotia, there was no further difference after the first 30 min (Figure 6). This likely reflected changes in membrane permeability of only the outer layer of tissue from which solutes would diffuse most rapidly. Since stipe initiation occursiJImedullary'tissue (1A),it was assumed that surface sterilization would not unduly affect the response of sclerotia to compounds inhibitory to carpogenic germination. Sclerotial leachate did not appear to inhibit continued stipe production and elongation by previously germinated 63 sclerotia, suggesting that leachate may effect only the formation of apothecial initials and not subsequent events. Leaching sclerotia prior to incubation (Figure 8) may enhance carpogenic germination by removing a specific inhibitory compound(s)mxzh on mcfintooom ucmofimflcwfim yo: mew Louumfi coaeoo m nufiz moweuco comzuon mwocmtmwwwo > .coHOSHow ocHoHans HE or can :om m om wcficwmucoo swan Heuoa as m— x om comm sun acumen—02H mew: meOLOHom cos x o m or o o o o o o.oF o o o Fm o o o o m.o cflusnweuo: o— F: ooF o o n m: ooP 0.0? oz oz N22 0 0 pm mm om m.o mcflwmefim w mm so, 0 o a am 002 .o.oa om :0 cow 0 0 pm mm cor m.o wcflumeu¢ 111 III 111 o o as P: mm 111 _ Hoepcoo mwomsuoa< moafium cofiumcfi manoewfiom mfloonuoa< moafium :OHOmcH Aawom w onwofinem: 18200 a acmocoowm IELOU « \.H.w wsv cofiumeucoocoo mHUOLQHOm OF \.02 name ¢m Loam: eefiflaamae amen .mame mm Hwom poocosm mowowneo: :H maaoeeaan em \.02 mzmo mm Hflom cmncoEmrmoonneon :H oopsoEmimEoHnLo: so: om>oswe 6.83 mHOOLOHom Hmfloonpoqm new :oHumcwEme owcwwoaemo co xHHon nonsmEmIOOAOASLo: mo uoommm .LOpmz nwaamumfio cm omomaq new .mzmo mm Lopmm Hwom .ESLOHOOLOHom mficwuoeoaom mo acoEQOHo>mn .FP OHan 7A soils germinated carpogenically, but at 0.5 and 1.0 mg/g soil several germinated with mycelium that grew across the soil surface to produce secondary sclerotia. Although many stipes were produced by sclerotia in the atrazine and simazine treatments, mature apothecia developed only in the unamended control after 53 days. Sclerotia removed from the atrazine and simazine amended soil after 53 days, produced only abnormally formed apothecia (Figure 9C). In the second experiment, all of the stipes produced by sclerotia incubated in 20-500 ug atrazine/g soil were darkened, abnormally formed and produced no apothecia when exposed to light for 18 days (Table 12; Figure 9B). Nearly all stipes produced by sclerotia in >0.8 ug atrazine/g soil were abnormal, and only abnormal apothecia developed. There was no carpogenic germination of sclerotia in 500 ug metribuzin/g soil, and normal stipes but no apothecia were produced by sclerotia in 100 ug metribuzin/g soil. Sclerotia in all other treatments produced normal stipes and apothecia (Table 12; Figure 9A). Effect of analytical-grade herbicides on carpogenic germination Sclerotia incubated in 1% methanol:water for 17 days germinated at a slightly lower rate (75 t 5%) than those incubated in water (95 t 5%), but produced normal apothecia. Rates of carpogenic germination for sclerotia incubated in 0, 2, 10 and 50 uM atrazine in 1% methanol:water were not Significantly different after 17 days (Table 13). Stipes 75 Figure 9. (A): Normal stipes and apothecia produced by sclerotia of Sclerotinia sclerotiorum incubated in water saturated SOiT-at 15 C, fider fluorescent light. (B): Abnormally'formed, multiple-branched stipes of sclerotia incubated in atrazine amended soil at 15 C, under fluorescent light. (C): Abnormal apothecia produced by sclerotia incubated as in (B)tkn'53 days, then placed in distilled water at 15 C, under fluorescent light. 76 Figure 9. A) B) 102 DAYS IN ejeéimiglflliizllE/EBEASOILI. C) { 0.5 mg ATRAZINE/g son A 77 Table 12. Effect of herbicide amended 3011 on carpogenic germination and apothecial development of Sclerotinia sclerotiorum. Sclerotia were incubated for 28 days in the dark, then 18 days under flourescent light, at 15 C. Total no./ 20 sclerotia Percent Stipes Apothecia Conc. germination (ug a.i./ of unrotted Nor- Ab- Nor- Ab- Herbicidex g soil sclerotiay mal normal mal normal Control --- 50 abcz 16 0 1 0 Atrezine 0.8 35 abcde 9 0 2 0 A.O 65 a 2 19 0 A 20.0 5 de 0 1 O 0 100.0 55 ab 0 18 0 0 500.0 22 bcde 0 6 0 0 Metribuzin 0.8 A0 abcd 11 0 6 1 A.0 50 abc 18 0 13 0 20.0 A8 abc 17 0 1 0 100.0 16 cde A . 0 0 0 500.0 0 e 0 0 0 0 Diuron 0.8 17 bcde A 0 3 0 A.O 20 bcde A 0 3 0 20.0 A5 abc 12 0 3 0 100.0 28 bcde 10 0 3 0 500.0 20 bcde 5 0 2 0 x Ten sclerotia were incubated in each 90 x 15 mm petri dish containing 50 g soil and 10 m1 herbicide solution. y Means of two replicates. Differences between entries with a common letter are not significant according to Duncan's Multiple Range test (P=0.05). Analysis of variance for % germination Source df Mean square F Treatment 15 920.33 3.u9 * Error 16 263.AA * Differences among treatments are significant at 2:0.05. 78 Table 13. Effect of atrazine on carpogenic germination and apothecial development of Sclerotinia sclerotiorum. Ten sclerotia were incubated in each petri di§h containing 10 ml atrazine in 1% methanol:water, under fluorescent light at 15 C, for 17 days. Atrezine Total no./ 20 sclerotia concentration Percent Stipes Apothecia (uM) germination Normal Abnormala Normal Abnormalb 0 75C A1 0 33 0 2 70 AA 0 21 7 1O 70 0 6A 0 26 50 95 0 118 0 A a Abnormal stipes were darkened and misshapen (Figure 108). b Abnormal apothecia were malformed and sterile (Figures 10C, 11). C Mean of 2 replicates. Analysis of variance for percent germination Source df Mean square F Treatment 3 - 283.33 1.03 n.s. Error A 275.00 “'5‘ Differences among treatments are not significant at P=0.05. 79 produced by sclerotia in 1% methanol controls and 2 uM atrazine were light colored and shaped normally (Figure 10A); stipes of sclerotia in 10 and 50 uM atrazine were darkened, and abnormally shaped (Figure 108). While all of the apothecia produced by sclerotia in the 1% methanol control were normal, 25, 100 and 100% of apothecia produced by sclerotia in 2, 10 and 50 uM atrazine, respectively, were malformed and sterile (Table 13; Figures 10, 11). Results were similar when~sclerotia were incubated in 2, A, 6,8 and1C)uM atrazine(nearlsrall apothecia produced in >A uM atrazine were abnormal), while sclerotia in 10, 50 and 100LM4metribuzin(N'diuron produced all normal apothecia (data not Shown). Effect of atrazine on apothecial disc development The hymenium of apothecia soaked in 50 uM atrazine for 30 min darkened after 2 days, although stipes appeared normal. Numerous stipes grew from the hymenial surface of these apothecia after 10 days, and each developed a malformed apothecium (Figure 12A, B). Stipes soaked in atrazine appeared normal after 2 days, but developed highly distorted apothecia by 10 days (Figure 12C). Sclerotia soaked in 1% methanol control developed normal apothecia (Figure 12A). 80 Figure 10. (A): Normal stipe (top) and apothecium produced by sclerotium of Sclerotinia sclerotiorum incubated in 1% methanol:water at 15 C, under fluorescent light. (B): Abnormal stipes produced by sclerotium incubated in 10 uM atrazine in 1% methanol:water at 15 C, under fluorescent light. (C): Abnormal apothecia produced by sclerotium incubated as in (B). 81 Figure 10. ) A B) C) 82 Figure 11. (A): Normal (right) and abnormal apothecia produced by sclerotia of Sclerotinia sclerotiorum incubated in 0 and 10 uM atrazine, respectively, in 1%Imethanol:water at 15 C, under fluorescent light. (B) and (C): Distorted and unexpanded apothecia of sclerotia incubated as in (A). 83 Figure 11. A) B) C) 8A Figure 12. (A): Normally (left) and abnormally developed apothecia of Sclerotinia sclerotiorum soaked in 0 and 50 UN atrazine, respectively,—In 1% methanol:water for 30 min prior to incubation in distilled water at 15 C, under fluorescent light, for 10 days. (ED: Darkened hymenia of apothecia shown in (A), with numerous stipes growing from their surface; early stage (left) and later stage with development of malformed apothecia. (C): Extensive branching and aborted apothecia produced by stipe soaked in 50 UN atrazine in 1% methanol:water as in (A). 86 DISCUSSION Studies on the effect of herbicides on soil-borne plant pathogens have been reviewed by Kaiser at al (11) and Katan and Eshel (12). Atrezine stimulated growth (17, 18, 20, 2A), respiration (6, 23) or glucose catabolism (26) Of some fungi at low concentrations (about 10 ug/ml), but was often inhibitory at higher concentrations (20, 21, 22, 23). Some fungi have been found to utilize simazine as a nutrient (13). None of the herbicides tested in the present study stimulated mycelial growth on agar. Metribuzin, a triazine with an asymmetric ring, had a different effect on germination of sclerotia than did atrazine or simazine (symmetric triazines). Metribuzin prevented carpogenic germination, but there was considerable mycelial growth across the soil :nul secondary sclerotia were produced. It does not seem likely that metribuzin directly stimulated mycelial growth, since mycelial growth was not stimulated on metribuzin—amended agar. It is possible that metribuzin acted indirectly through suppression of antagonistic or competitive microorganisms, or by the microbial breakdown of metribuzin into compounds that could beInetabolized by S. sclerotiorum. Diuron (a substituted urea) inhibited mycelial growth and carpogenic germination. Based on my definition of carpogenic germination as a Single stipe breaking through the rind, atrazine and 8? Simazine did not affect carpogenic germination. The stipes produced, however, by sclerotia incubated in atrazine- or simazine-amended soil were distorted, highly branched, and apothecial discs were difficult to distinquish or were absent (Figure 9B). Removal of sclerotia from the atrazine- and simazine-amended soils or incubation in low concentrations of atrazine in 1% methanol diminished the effect of the herbicides and differentiation of apothecial discs occurred, Inn; these discs were morphologically abnormal and sterile (Figures 9C, 10, 11). Direct application of atrazine to apothecia resulted in abortion of the hymenium and the growth of numerous stipes £71m: the hymenium; stipes soaked :NT atrazine branched repeatedly and developed aborted apothecia (Figure 12). All of the herbicides used in this study inhibit photosynthetic electron transport in plants (1), probably by affecting a quinone step. Atrezine and simazine were the only compounds that differentially! allowed carpogenic germination while inhibiting normal apothecial development. Apothecial disc expansion is light dependent, and atrazine may affect the photosensing mechanism. This could not be the only site of action, however, since stipes grown in the dark were deformed. Carlile (3) suggested that the activity of respiratory enzymes may be indirectly affected by light. If apothecial disc expansion is associated with increased respiration (and the energy required for cell.