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LIBRARY BlN IE HS Jun 2, 12353 Ell: yul All 1' ABSTRACT FACTORS AFFECTING SAPROPHYTIC SURVIVAL OF SELECTED ISOLATES OF RHIZOCTONIA SOLANI KUHN IN MUCK SOIL BY Keh-Ming Pan The organic soils on Michigan State University's Muck Experimental Farm appear to be essentially devoid of the fungus Rhizoctonia solani Kuhn. Because of this, investigations on the factors influencing sur- vival of isolates of the fungus in muck soil were made. Beet seedball colonization and soil segment assays were employed to assay 3. solani population changes in soil. Eighteen out of 19 isolates survived poorly in muck soil held in closed plastic bags for 30 days; only isolate R54 survived well in the same soil under the same conditions. Soil aeration influenced the survival of certain isolates in muck soil but not others. When five isolates were compared for their survival capability in open and closed storage conditions, three isolates R47, R54, and PR56, were benefited by aeration while two other poor survivors, R51 and R57, were not. Keh-Ming Pan Isolate R54 survived well in muck soil under open storage condi- tions for as long as 150 days, but the same isolate survived poorly in mineral soil in open storage. There was no evidence of naturally occurring inhibitory sub- stances that would account for inhibition of growth of poorly surviving isolates in the muck soil. Soil moisture, soil temeprature, soil reaction, growth rate of the mycelium on either muck soil or on culture media, or ability to pro- duce sclerotia or monilioid cells was unimportant in differential sur— vival. Isolate R51 had a high capability for producing sclerotia, and the sclerotia survived well in muck soil for as long as 26 weeks. Never— theless when sclerotia of R51 were incubated on the surface of muck soil for various times from O to 6 weeks, there was a gradual reduction in growth rate of mycelium from the sclerotia transferred to agar and the sclerotia had a high bacterial contamination. In contrast, the growth rate of isolate R54 remained relatively constant and the sclerotia had a very low bacterial contamination. High bacterial contamination of R51 sclerotia may influence the inoculum density of this isolate in muck soil. Maintaining the population of 3, solani in soil was not solely dependent on sclerotia. A positive relationship was found between Keh-Ming Pan populations of Rhizoctonia isolates and their ability to produce mycelium resistant to lysis. Hyphal lysis of isolate R54 on muck soil was 32% after 35 days incubation, but other isolates had lysed 70-lOO% after the same time. Meanwhile the viability of R54 mycelium in colonized muck soil remained high whereas other isolates died out after 2 weeks. The successful survival of certain B, solani isolates appears to be inversely related to populations of other soil microorganisms occurring naturally in the muck soil. Isolate R57 survived well and re- sisted lysis only when the soil had been freed of competitors by sterili- zation. Use of p-dimethyl aminobenzenediazo sodium sulfonate (DASS) in the soil to eliminate possible competition from Pythium spp. or from other DASS sensitive microorganisms did not benefit R57. The effect of soil microorganisms on growth on agar of relatively poor and relatively good survivors was not differential. In addition to the soil microorganisms occurring naturally in muck soil, some other factor or factors may also determine Rhizoctonia survival because isolate R51 survived poorly and lysed completely in either the presence or absence of other organisms. Apparently the absence of 5, solani in muck soil is due to the absence of resistant isolates which have not yet been introduced into the soil or which have not yet arisen as mutants. FACTORS AFFECTING SAPROPHYTIC SURVIVAL OF SELECTED ISOLATES OF RHIZOCTONIA SOLANI KUHN IN MUCK SOIL BY Keh-Ming Pan A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1976 To my wife and parents ii ACKNOWLEDGMENTS I wish to express my sincerest gratitude to Dr. Donald J. deZeeuw, committee chairman and advisor, for his guidance, encouragement, and many helpful comments throughout the course of this study. I also thank him for arranging financial support and for reviewing and criticizing the manuscript. I wish to extend my sincerest gratitude to the other members of the committee: Dr. John L. Lockwood, Dr. Melvyn L. Lacy, Dr. William J. Hooker, and Dr. Harry H. Murakishi, for guidance; for critical reviewing and for valuable suggestions on the manuscript. I would like to make special mention of my appreciation to the late Dr. Takashi Matsumoto and to Dr. Hong-Ji Su for their kind recom- mendations. They helped me to continue my education in graduate school at Michigan State University. The long term love, patience and encouragement given by my wife and parents, really sustained me during the completion of this work. iii TABLE OF CONTENTS LIST OF TABLES O O O O O O O O O O O 0 O O O O O 0 I O O O 0 LIST OF FIGURES O O O O O O O O I 0 0 O O O O O O O O O O 0 INTRODUCTION 0 O O I O O O O O I O O O O O O O O O O O O O 0 LITERATURE REVIEW 0 O O O O O O O O O O O O O O O O O O I 0 MATERIALS AND METHODS O O O O 0 I O O O O O O O O O O O O 0 Sources of Rhizoctonia solani . . . . . . . . . . . . . Culture and assay media . . . . . . . . . . . . . . . . Preparation of Rhizoctonia—vermiculite inoculum . . . . Preparation of uniform propagules from mycelial mat and sclerotia . . . . . . . . . . . . . . . . . . . Experimental soils. . . . . . . . . . . . . . . . . . . Assaying populations and survival capability of Rhizoctonia solani. . . . . . . . . . . . . . . . . Beet seedball colonization assay° . . . . . . . . . Soil segment assay. . . . . . . . . . . . . . . . . Comparative survival ability of isolates of Rhizoctonia solani in muck soil under closed and open storage conditions 0 O 0 O O O O I O O O O O O O O O Q 0 O 0 iv Page viii 18 18 l9 19 20 21 22 22 23 23 TABLE OF CONTENTS (cont'd.) Comparative survival ability of isolates of Rhizoctonia solani in mineral soil under open storage conditions . . Effect of volatile substances on growth of Rhizoctonia solani . . . . . . . . . . . . . . . . . . . Soil moisture and temperature effects on survival of Rhizoctonia solani . . . . . . . . . . . . . . . . . . . SOil mOiStureo O O O O O O O I O O O O o I O O O O O O 0 Soil temperature . . . . . . . . . . . . . . . 3 . . . . Mycelial growth on agar media. . . . . . . . . . . . . . . . Mycelial growth on muck soil or DASS treated muck. . . . . . Production of sclerotia and monilioid cells on agar media. . Production of sclerotia in muck soil . . . . . . . . . . . . Lysis of mycelium on soil° . . . . . . . . . . . . . . . . Lysis of mycelium on agar medium with or without covering soil. . . . . . . . . . . . . . . . . . . . . . Survival of sclerotia on or in muck soil . . . . . . . . . . on muck SOil O O I. O O O O O O O O O O O O O O O O O O O In muCk soil 0 O O O O O O O C O O O O O O O O O O O C 0 Survival of Rhizoctonia mycelium in soil . . . . . . . . . . Soil microorganisms affecting survival of Rhizoctonia solani Testing antagonists of Rhizoctonia solani on agar. . . . . . Effect of p-dimethyl aminobenzenediazo sodium sulfonate (DASS) sensitive microorganisms on survival of Rhizoctonia solani . . . . . . . . . . . . . . . . . . Page 24 24 25 25 26 26 27 27 28 28 29 3O 3O 3O 31 32 32 33 TABLE OF CONTENTS (cont'd.) Page RESULTS. 0 O O O O O O O O O O O 0 0 O C O O O O I O I I O O O O 35 Comparative survival ability of 19 isolates of Rhizoctonia solani in muck soil stored in closed plastic bags. . . . 35 Comparative survival ability of isolates of Rhizoctonia solani in muck soil under open storage conditions. . . . 35 Comparative survival of Rhizoctonia solani isolates in mineral soil under open storage conditions . . . . . . . 37 Effect of volatile substances on growth of Rhizoctonia SOlani . O O O I O O O O O O O O O O O O O O O O O I O I 40 Effect of soil pH, moisture, and temperature on the survival of Rhizoctonia solani . . . . . . . . . . . . . 4O SOil mOistureo O O O O O O O O O C O O O O 0 O O O O O O 4]- Soil temperature . . . . . . . . . . . . . . . . . . . . 43 Growth capability of Rhizoctonia solani isolates . . . . . . 43 Ability of Rhizoctonia solani isolates to produce sclerotia or moniliOid cells I O 0 0 O O O 0 O O O O I 0 O O O O O 46 Survival of sclerotia of Rhizoctonia solani on or in muCk SOil. O O O O O O O O O O I O O O O O O O O O O O O 48 Lysis of Rhizoctonia solani mycelium on soil . . . . . . . . 54 Survival of Rhizoctonia solani mycelium in soil. . . . . . . 58 Effect of soil microorganisms on survival of isolates of Rhizoctonia solani . . . . . . . . . . . . . . . . . . . 62 Differential lysis of two Rhizoctonia solani isolates on agar plates treated with soils . . . . . . . . . . . . . 64 Influence of p-dimethyl aminobenzenediazo sodium sulfonate (DASS)-sensitive microorganisms on survival of Rhizoctonia solani . . . . . . . . . . . . . . . . . . . 66 vi TABLE OF CONTENTS (cont'd.) Page DISCUSSION. 0 O O o O 0 ° 0 C O O G O O 0 O 0 D O O O O O O I O 0 7O APPENDICES A. IDENTIFICATION OF THE SPECIES RHIZOCTONIA SOLANI. . . . . 80 B. COMPARISON OF SOIL SEGMENT AND BEET SEEDBALL ASSAYS FOR RHIZOCTONIA SOLANI. . . . . . . . . . . . . . . . 84 Assay soil infested with five Rhizoctonia solani isolates. . . . . . . . . . . . . . . . . . . . . 85 Assay soil infested with various numbers of propagules. . . . . . . . . . . . . . . . . . . . 87 Assay soil infested with different sizes of propagules. . . . . . . . . . . . . . . . . . . . 87 LITERATURE CITED. 0 O O 0 O O O O O O 0 O O O O O O O O O O O O O 91 vii Table LIST OF TABLES Selected isolates of Rhizoctonia solani used in the investigations. . . . . . . . . . . . . . . . . o . . . . Survival of 19 isolates of Rhizoctonia solani in natural muck soil after 2 and 30 days storage in closed plastic bags, as determined by colonization of beet seedballs . . Growth rate of four isolates of Rhizoctonia solani on natural muck soil and potato dextrose agar. . . . . . . . Production of sclerotia by four Rhizoctonia solani isolates on potato dextrose agar, potato dextrose yeast extract agar, and water agar after two weeks incubation at 24 C . Comparison of sclerotial numbers and size in two isolates of Rhizoctonia solani three weeks after infestation of soil with Rhizoctonia-vermiculite inoculum. . . . . . . . Bacterial and fungal contamination of sclerotia of two Rhizoctonia solani isolates after incubation on muck soil naturally infested with bacteria and fungi . . . . . Relative ability of four isolates of Rhizoctonia solani to colonize muck soil and survive at 2, 7, and 14 days incubation as determined by a soil-mycelium aggregate index, aggregate viability, and aggregate wet weight. . . Lysis of hyphae of two Rhizoctonia solani isolates on water agar with or without muck soil, as determined by the number of hyphae units with or without rose bengal staining. . . . . . . . . . . . . . . . . . . . . . . . . viii Page 18 36 47 49 SO 53 61 65 LIST OF TABLES (cont'd.) Table Page 9. Linear mycelial growth from sclerotia of Rhizoctonia solani isolate R57 on p—dimethyl aminobenzenediazo sodium sulfonate—treated muck soil in plastic slide chambers after 48 hr incubation at 24 C. . . . . . . . . 67 10. Survival of Rhizoctonia solani, isolate R57 in muck soil treated with p-dimethyl aminobenzenediazo sodium sulfonate (DASS) determined by beet seedball colonization and soil segment assays . . . . . . . . . . 68 11. Comparison of soil segment and beet seedball colonization assays of five Rhizoctonia solani isolates . . . . . . . 86 12. Comparison of soil segment and beet seedball colonization assays of two Rhizoctonia solani isolates with five inoculum levels. . . . . . . . . . . . . . . . . . . . . 88 13. Comparison of soil segment and beet seedball colonization assays with two different propagule sizes of Rhizoctonia solani at two inoculum levels. . . . . . . . 9O ix LIST OF FIGURES Figure l. 2. 3. 4. 5. 6. Survival of five Rhizoctonia solani isolates in natural muck soil stored in open cheese boxes for 2 to 150 days, as determined by colonization of beet seedballs. Survival of five Rhizoctonia solani isolates in natural mineral soil stored in open cheese boxes for 2 to 150 days, as determined by colonization of beet seedballs° Effect of four soil moisture levels on the survival of five isolates of Rhizoctonia solani in infested muck soil containing 0.2 g propagules (size 600 p - 800 u) per 100 g soil. Survival determined by recovery of Rhizoctonia from soil segments (left) and by beet seedball colonization (right)° . . . . . . Survival of two isolates of Rhizoctonia solani at various soil temperatures as determined by recovery of Rhizoc- tonia from soil segments and by beet seedball coloniza- tion. Soil infested with Rhizoctonia-vermiculite inoculum and incubated at 24 C for 2 days (designated as 0 day incubation) before incubating at various temperatures 0 O O O O O G 0 b O O o o O (a Growth rate of Rhizoctonia solani isolates on potato 6 O dextrose agar, potato dextrose yeast extract agar, and water agar at 24 C . . . . . . . . . . Growth of two Rhizoctonia solani isolates from sclerotia O 0 which had been incubated on muck soil for 0-42 days. Sclerotia washed before being placed on (A) PDA and (B) acidified PDA. (C) Washed sclerotia treated with sodium hypochlorite, and rewashed before being placed on PDA. Colony dia. in mm after 24 hr incubation . Page 38 39 42 44 45 52 LIST OF FIGURES (cont'd.) Figure Page 7. Mycelium of Rhizoctonia solani isolates RS4 (A) and R51 (B) on muck soil were stained with rose bengal and recovered with polystyrene membranes after 28 day incubation. . . . . . . . . . . . . . . . . . . . . . . . 56 8. Mycelium of four Rhizoctonia solani isolates lysed on muck soil, as determined by the percentage of hyphal units (Hyphal length 600 u unit) not showing stain). Hyphae were stained on the soil with rose bengal and recovered for examination with polystyrene membranes. Lysis index = 100(1 - number of hyphae units with stain at the indi- cated period of incubation/number of hyphal units w1th stain at l-day incubation). . . . . . . . . . . . . . . . 57 9. Aggregates of soil-mycelium—vermiculite formed in muck soil infested with Rhizoctonia-vermiculite inoculum of R54 (A) and R51 (8) and incubated for 1 week (16 X). Vermiculite (V) and Muck (black clumps, m) aggregated by mycelium of Rhizoctonia solani . . . . . . . . . . . . 60 10. Effect of naturally occurring muck soil organisms on surVival of three isolates of Rhizoctonia solani after 2, 7, and 21 days incubation of inoculum in test soil. Beet seedball (right) and $011 segment (left) assays used With two soils: autoclaved (o—-—-o) and mixture of autoclaved and natural soil (o----o) . . . . . . . . . 63 ll. Nuclei with HCl-Giemsa stain in cells of actively growing hyphae of Rhizoctonia solani (700 X). . . . . . . . . . . 82 12. Distribution of numbers of nuclei in the first and second hyphal tip cells of five isolates of Rhizoctonia solani HCL-Giemsa stain was used and counts were made of 260. cells of each isolate . . . . . . . . . . . . . . . . . . 83 xi INTRODUCTION Rhizoctonia solani is known to have resistant sclerotia and hyphae capable of surviving in soil and plant debris (10, 74), and the mycelium of this fungus is known to be more resistant than mycelia of other soil fungi to lysis in natural soil (36). Probably due to this resistance and wide host range, 5, solani is worldwide in its distribu- tion (57). Some soil types, however, have less Rhizoctonia than others (51). For example, little or no 5, solani exists in the muck soil of the Michigan State University muck experimental farm (18, 19, 44). Why .3. solani is absent in this muck soil of almost pure organic matter is unknown. Several factors could affect survival and persistence of 3. solani in soil, such as inherent survival ability of different 5, solani isolates (5, 41, 47), differing soil environments (47), and the influ— ence of other soil microorganisms (5, 47). Certain isolates of B. solani can survive well in muck soil during 10 days incubation, as was demonstrated by Pan (44), and it was considered possible that some iso- lates could survive in such soil for longer periods. This has been demon- strated in this investigation for one isolate and the capability must exist with some others. In order to explore causes for the absence of 5, solani popula- tions in muck soil,growth and survival of selected isolates of 5, solani were compared under various conditions in the laboratory. Lysis of hyphae and survival capability of mycelial and sclerotial propagules of several R. solani isolates was also tested under certain conditions. LITERATURE REVIEW Rhizoctonia solani is a well known and very important plant pathogen of many plant species causing such diseases as seed decay, seedling damping-off, wirestem and soreshin, root rot, hypocotyl and stem cankers, bottom or head rot, crown and bud rot, leaf blight, soil rot when aerial parts contact soil, and storage rots (3). The fungus has an indefinite number of strains, a wide host range, and is world- wide in its distribution (12, 31, 57). Because of the economic impor- tance of this fungus it has been studied extensively. Parmeter (54) has edited the most recent comprehensive review of 5. solani including its taxonomy, morphology, cytology, physiology, ecology, genetics, and pathology. Effective control of plant disease is the main goal of plant pathologists. For disease to develop there must be an association of a virulent pathogen and a susceptible host under favorable environmental conditions. Therefore, control of a disease can be performed 1) before the pathogen and host association occurs; 2) after it occurs (but be- fore infection); or 3) after infection and disease symptoms appear. Disease control is generally better and easier to accomplish in the other two stages (53). Therefore, understanding sources of inoculum 3 and characteristics of pathogen survival become an important subject of study for plant pathologists. B, solani is unlike spore-forming pathogens such as Fusarium spp. which are able to produce abundant secondary inoculum rapidly under suitable conditions for dissemination and infection. Pathogenic levels of B, solani inoculum must be maintained in the soil, by some mechanism, for extended periods and in the absence of a host in order for the fungi to survive. Soil fungi may survive by either active or inactive processes (53). Active survival may be through parasitism and saprophytism while inactive survival may be through resistant resting propagules. Both active and inactive survival of B. solani are well-known (4, 47). Parasitic survival has been considered by some workers (16, 59, 65) to be the most important way for 3, solani to persist. 5. solani possesses characteristics of a wide host range, fast growth, and rela- tively simple food requirements, therefore it is commonly found asso- ciated with crops and weed plants. A high incidence of 3. solani in weed host including lanbsquarters (Chenopodium berlandieri), rough pig- weed (Amaranthus retroflexus), and puslane (Portulaca oleracea) was re- ported by Oshima and his co—workers (42). Boosalis and Scharen (11) re— ported that pigweed (Amaranthus retroflexus) plays an important role in the epidemiology and ecology of 5. solani inciting root and stem rot of irrigated crops in western Nebraska. Weed strains of 5, solani patho- genic to crops were also demonstrated by others (16, 28). During the growing season in eastern Kansas when the tempera- ture was too high for mycelial survival and sclerotial production, 5, solani depended on parasitic growth in the susceptible host for persis- tence (22). Survival of 5, solani on other crops was very important. Pitt (59) found that 5. solani formed sclerotia on potato tubers and stolons and that a high incidence of sharp eyespot disease occurred on wheat following potato crops. He also demonstrated that wheat isolates of_§. solani declined rapidly after artificial introduction into un- sterile soil, and the naturally infested cereal straws buried in soil supported only limited survival. Therefore he concluded that sapro- phytic survival of 3. solani isolates from wheat stems was not a major factor in the persistence and survival of the fungus of sharp eyespot disease. This conclusion was supported by Sanford (65) who reported ‘5. solani “disappeared" from heavily infested soil before 4 months in the absence of a susceptible crop, but survived up to 8 months in soil planted to susceptible crops. Although parasitic survival is very important for the survival of 3. solani in soil the significance of active saprophytic survival of '3. solani cannot be neglected. Papavizas and Davey (48) found that B. solani survived in naturally infested soil for at least 20 weeks. Olsen et a1. (41) and Baker et al. (5) infested natural soil with mycelial mats, and they found that some isolates of B. solani survived in the soil for at least 12 months (41) and 21 months (5), respectively. The conflicting results of these (5, 41, 48), and others (59, 65) should not be surprising as survival of 3. solani in natural soil differs from isolate to isolate (24, 41, 46). Under conditions unfavorable for parasitic or saprophytic activ- ity, 3. solani depends on resistant hyphae or sclerotia for survival. Sclerotia may be formed from moilioid cells, and monilioid cells are formed from new cells of vegetative hyphae. These cells may be hya- line of brown, barrel-shaped, pyriform, irregular, or lobate. The ratio of length and width of monilioid cells is approximately 1-3 : l. Monil- ioid cells have been called doliform cells, barel-shaped cells, short cells, sclerotial cells, thick wall cells, and chlamydospores. These cells may develop relatively thick walls but do not necessarily differ from other hyphal cells in this respect. Monilioid cells have the appearance of conidia, but there is no evidence that they serve this function. Chains of monilioid cells are formed in various patterns, mainly on or above the surface of a substrate, but also within host tissue. They may be few in number and scattered, or form loose to semicompact masses of varying sizes, or they may be aggregated into .- sclerotia (l4). Sclerotia are usually initiated on agar when the mycelium has grown to the edge of the medium (2). Perombelon and Hadley (58) indi- cated that sclerotial initiation first appeared on liquid media when the dry weight of the colony reached its maximum. Nutritional condi- tions such as high concentrations of glucose and sucrose were necessary for sclerotial initiation, but low concentration was required for their maturation. Longevity of R. solani sclerotia from several months to several years has been reported. Chowdhury (15) found that sclerotia retained their viability in dry sand over 2-1/2 years in the laboratory. Pitt (59) reported that sclerotia of 5. solani survived in soil for 6 months and remained infective to wheat seedlings. Palo (43) working with an isolate pathogenic to rice, found that the sclerotia survived in soil for 6 months. Potato isolates remaining viable in soil for 6 years were demonstrated by Herzog and Wartenberg (29). Boosalis and Scharen (10) showed that Rhizoctonia causing damping-off and seedling blight of sugar beet persisted in soil as hyphae and sclerotia within and on plant debris for up to 7 months after harvesting the crop. Townsend and Willetts (72) considered that the dense cellular contents, pigmentation and rather impermeable wall of the sclerotia cells were factors responsible for their resistance. However survival of R. solani sclerotia varied among isolates of the fungus. Naiki and Ui (37) compared the survival ability of the sclerotia of two 5, solani strains in soil. The strain which was a relatively good survivor pro- duced larger and fewer sclerotia than the strain which was a relatively poor survivor. Sclerotia of the good survivor could remain viable up to 360 days in soil, but the poor one only 40 days when tested on agar media. Baker et a1. (5) found that formation of sclerotia did not neces- sarily increase survival of this fungus in the presence of suitable an- tagonistic organisms. Some isolates which formed many sclerotia in the soil died out within 2—3 months. Therefore they concluded that the ability of 5. solani isolates to produce sclerotia was not related to survival. Although the mycelium of 5. solani was considered less resistant than the sclerotia under high temperatures (67) and adverse conditions (64), mycelium may be important for survival of E, solani. A strain of .5. solani which infects cereal roots in South Australia oversummered in the form of thin, dark brown hyphae (64). Similarly, Warcup et al. (76) found that a root-infecting strain, in contrast to other strains, per- sisted mainly as residual hyphae in soil during the dry summer period when sclerotia cannot be formed. Mycelium of B, solani was also reported to be more resistant to adverse environment than other soil fungi when in the presence of an- tagonistic microorganisms or toxic substances. Lockwood (36) found that, of several fungi tested, 5, solani was the only fungus resistant to lysis brought about by the action of natural soil placed on the top of agar cultures. Potgieter and Alexander (60) demonstrated that chiti- nase and 8-(1-3) glucanase, which incide lysis of Fusarium sp. cell walls, did not affect cell walls of R, solani. This resistance might be due to the presence of melanins in the hyphal cell wall of R. solani (60). The survival of R. solani (the fungus in general) in natural soil may be affected by several factors. Baker et a1. (5) concluded that the inherent survival ability of R. solani, the sum of the antagonistic abilities of other soil microorganisms against 3. solani, and influences of soil environments were the determining factors in survival of R, solani in soil. There are numerous reports that field isolates or basidiospore isolates of Thanatephorus cucumeris (R. solani) possess various survival abilities in soil (5. 24, 41, 46). Flentje, et al. (24) found that strains of R. solani differed markedly in survival ability in soil. Some survived indefinitely, others dying out in 2-3 months. Papavizas (46) tested the survival ability of twenty single basidiospore isolates each of B, praticola and R. solani within precolonized buckwheat stem segments placed in soil. He found that most single~spore isolates de— clined faster than the parent culture, but several were stronger 10 survivors than their parents. This phenomenon was confirmed by Olsen et a1. (41). They mixed hyphal fragments with aerated, steamed soil or non—treated soil, and survival of single-spore isolates was deter- mined by amount of living hyphae, organic matter aggreation of hyphae, colonization of organic matter, and percentage of radish seedling di- sease produced after a certain period of incubation. Some single basidiospore isolates survived in lower numbers than their parents, but some survived in higher populations over a 12 month period. Papavizas (46) concluded that karyogamy and segregation in the basidiospore stage were mechanisms which might be responsible for main- taining or even increasing the over-all survival ability of R, solani in soil. With a few exceptions, isolates of R, solani possessing high growth rates and saprophytic activity also possessed high survival ability in precolonized substrates (46). Therefore, Papavizas agreed with Garrett's concept that degree of saprophytism and longevity of R. solani are strongly interrelated (25). This conception can not be gen- eralized, however, within the entire species. Baker et al. (5) found that some strains of R. solani grew vigorously for a time and colonized organic particles in soil, but were dead in a few months. Therefore they concluded that there is no obvious relationship between growth or colonization and survival of R. solani in natural soil. 11 A number of papers report effects of soil microorganisms on growth, saprophytic activity and survival of R, solani in vitro. These effects may also occur in soil or rhizospheres. The associations and interactions of soil or rhizosphere microorganisms and R, solani may result in the lysis of R, solani, or a reduction in the ability Of.§- solani to initiate colonization of organic substrates, exploit the sub- strates, or to spread out from colonized substrates. Possible antagon- istic mechanisms between the soil microorganisms and R. solani include hyperparasitism, antibiosis, and competition. Naiki and Ui (38) studied the microorganisms associated with sclerotia of R. solani in soil and their effect on viability of the pathogens. They found that cells of the sclerotia in isolates F-lS (relatively poor survivor) and B-5 (relatively good survivor) were in- vaded by Trichoderma sp. to a different extent, being 13.1 and 7.2% respectively. Boosalis (8) introduced 5, solani into natural soil amended with green plant materials. He found that only 18% of the hyphae were invaded by Penicillium vermiculatum and Trichoderma sp. under conditions most favorable for parasitic activity. Another report of Boosalis (9) indicated that he was unable to detect any appreciable amount of hyperparasitism of 5, solani hyphae screened from several in- fested fields. Therefore he doubed the importance of hyperparasitism in the microecological behavior of R, solani in natural soil. 12 Competition for available nutrients and sensitivity to antagon- istic substances have been suggested as the most likely suppression mechanisms against 5, solani by antagonists in soil. Blair (7) reported that R. solani depression in soil amended with wheat straw and dry green grass amended soil was due to the nitro- gen starvation of the mycelium of R, solani. This was caused by rapid utilization of the available soil nitrogen by cellulose decomposing microorganisms multiplying upon the fresh organic matter. This was confirmed by Davey and Papavizas (17). Soil treated with ground oat straw hay enriched with various amounts of NH4NO3 apparently suppressed Rhizoctonia activity at all C/N ratios tested when the cellulolytic fungus Humicola sp. greatly increased in numbers in the presence of de- composing oat straw. In contrast, in soil treated with only NH4NO3 there was a high percentage of buckwheat colonization by B, solani and low numbers of Humicola sp. . Papavizas (47) in reviewing his own work reported that the saprophytic survival of B. solani in soil and numbers of antagonists in the rhizosphere were related to C/N ratio in amended soil. Buck- wheat stem segments precolonized by R, solani were incubated in soils amended to various C/N ratios with mature oat straw and free nitrogen. After certain intervals the percentage of viable Rhizoctonia in the precolonized segments was determined. The oat straw with C/N ratiOs 13 of 30 and 83 was more effective in reducing saprophytic survival of R, solani than oat straw with a C/N ratio of 10 or supplemental nitrogen only (52). Populations of antagonists were higher in the rhizospheres’ of bean plants as influenced by oat straw and supplemental nitrogen amendment at the higher C/N ratio of 30, 50 and 83. Soil amended to a C/N ratio of 10 or with free nitrogen only contained fewer antagon— ists (45). Volatile substances such as CO2 produced during decomposition of organic matter in soil may affect R, solani activity. Durbin (20, 21) indicated that subterranean isolates were less sensitive to CO2 than surface or aerial isolates. Papavizas (46) reported that with few exceptions, isolates of R. solani possessing high saprophytic ac- tivity also possess high tolerance to CO and high survival ability in 2 precolonized substrates. Smith and Ashworth (68) demonstrated that soil amended with rice hulls or oak sawdust reduced parasitic activity of R, solani on small white bean seedlings, and increased populations of soil micro- flora. Water extracts of rice hull, oak sawdust, and non-amended soil were nontoxic to R. solani, but similar extracts from soil amended with them strongly inhibited the fungus. It was demonstrated that chitin- amended soil reduced 5. solani activity and increased populations of antagonists (27). It was suggested (70) that substances toxic to R. l4 solani were produced. Toxic substances, extractable by n-butanol, have been demonstrated in chitin—amended soil. The active factors were pos- tulated to be non-specific cell constituents of microorganisms (69). Non-biotic factors may affect survival of R. solani directly or indirectly in soil. Growth of R. solani was strongly suppressed at pH 4.0 (49), but the growth was good at pH 4.5 to 8.1 (7). Usually the optimum soil reaction for growth of R, solani was neutral or slightly alkaline (49). Probably these same neutral or slightly alkaline condi- tions are also good for survival of this fungus in soil. Survival of .5° solani in soils of pH 6.3, 6.8, and 7.9 did not differ significantly (6). Usually longevity of R, solani is decreased by high temperature or high moisture in culture media or in soil. Cultures of B, solani survived on agar slants for 13 months at 0-25 C, 4-6 months at 30 C, and only 1 month at 35 C according to Nisikado and his coworkers (40). The optimum temperature for saprophytic activity of R, solani differed from one soil to another (49). Saprophytic activity was significantly greater at 20 C in Maryland soil than at other temperatures tested. In this soil, activity decreased markedly at 30 C. On the other hand, 3. solani was most active saprophytically at 26-30 C in Florida soil and significantly less active above and below these temperatures. 15 Ui et al. (73) found that two strains of R. solani existed in flax and fallow fields of Hokkaido in Japan. The strain attacking plants in May and June was called the "Spring" strain, in July and August the “summer" strain. Temperature favorable for growth of the Spring strain on potato dextrose agar and through sterile soil in a tube was lower than the optimum for the Summer strain. A mixture of these 2 strains was used to infest sterile soil and incubated at two temperature range: 5 to 18 C (average 13 C) and 12 to 28 C (average 20 C) for 0-21 days. Then, 3, solani was isolated by the trapping method using flax stem fragments. They found that R. solani recovered from the lower temperature storage was all Spring strain, but recover- ies from higher temperature storage included both strains. They sug- gested that the effect of temperature on the activity or survival of .5. solani differs with different strains. Chowdhury (15) demonstrated that under field conditions scler- otia of R. solani retained viability for 5 and 7 months on the surface of moist and dry soil, respectively. That sclerotia lose viability more rapidly in moist than in dry conditions, was also reported by others (39, 43). Saprophytic activity of R. solani in naturally infested soil was significantly higher at soil moistures from 20 to 50% moisture holding capacity (MHC) than at 70 to 80% MHC as measured by l6 colonization of buckwheat stem segments. At 90% MHC, activity was al- most eliminated (49). These conclusions were similar to those of Blair (7). Reduction of saprophytic activity and growth of R, solani in high moisture soil was attributed to declining aeration (7), increasing CO2 concentration (47), or increased microbial activity (33, 62). Blair (7) was able to increase growth of R. solani in 50% MHC soil by improving aeration. It was demonstrated by Papavizas and Davey (50) that CO2 reduced saprophytic activity of R. solani, and by Papavi- zas and Davey (49) that saprophytic activity is greater in soils at 20- 60% than at 70-90% MHC. Accumulation of CO2 in high moisture soil was suggested by Papavizas (47). Kovoor (33) demonstrated that there was no bacterial activity against 5. solani at low soil moisture levels (30% saturation); however, ‘5. solani on buried slides was attached by bacteria when the soil mois- ture was increased to 80%, complete lysis of mycelium occurred. In- creased soil microbial activity in high moisture soil was also reported to reduce survival of R. solani by Radha and Menon (62). They tested survival of R. solani in sterile and non-sterile soil at various soil moisture levels. They found that high soil moisture had no adverse effect on R. solani in sterilized soil, but in unsterilized soil growth and survival were considerably affected. Thus, at 25% and 50% MHC, R. 17 solani remained viable in 100% and 98% of precolonized coconut root segments after 24 weeks of incubation, respectively. But at 100% MHC the fungus survived in only 40% of the segments after 12 weeks of in- cubation. .R. solani depends for survival on parasitic or saprophytic activity and on its resistant sclerotia or hyphae. Success or failure to survive in natural soil is the result of interactions of a given isolate in a given soil under a given set of environmental conditions. These complex interactions when analyzed may explain why some soils have higher populations of B. solani than others. MATE RI ALS AND METHODS Sources of Rhizoctonia solani Isolates of Rhizoctonia used in these studies were identified as R. solani (see Appendix A). Isolates with the prefix "R" were ob- tained from Dr. Donald J. deZeeuw's laboratory at Michigan State Uni- versity, East Lansing, Michigan. Those designated "PR" were from Dr. George C. Papavizas, Crops Research Division, ARS, USDA, Beltsville, Maryland (Table 1). TABLE 1. Selected isolates of Rhizoctonia solani used in the investigations Isolates Isolated from Isolates Isolated from R41 - R55 vetch R42 - RS7 rutabaga R43 - R58 - R44 crown vetch PR58 - R45 pine PR108 - R47 pine PR56 - R49 red beet PR56-5 - R51 egg plant PR56-30 - R54 red beet PR56-32 - PR56-42 - 18 19 Culture and assay media Rhizoctonia solani was maintained and grown on potato dextrose agar (PDA), potato dextrose yeast extract agar (PDYA), and potato dex- trose yeast extract broth (PDYB). PDA was prepared similarly to the method of Altman (l). PDYA contained PDA and 0.5% yeast extract (DIFCO) and PDYB had the same ingredients as PDYA but without agar (26). The selective medium of Ko and Hora was used for assay of R. solani populations (32). In the beginning of this study acidified PDA at pH 4.0 (0.9 ml 50% lactic acid in 200 ml PDA) had been used. The selective medium was made in two parts. Part 1 was auto- claved and cooled to approximately 45 C before mixing with part 2. Part 1 contained 1 g KZHPO4, 0.5 g MgSO4°7 H20, 0.5 9 KCl, 10 mg FeSO4'7 H20, 0.2 g NaNOZ, 20 g agar and 1 liter distilled water; and part 2 con- tained 0.4 g gallic acid, 50 mg p-dimethyl aminobenzenediazo sodium sul- fonate (DASS), 50 mg chloramphenical and 50 mg streptomycin sulfate. Preparation of Rhizoctonia— vermiculite inoculum The procedure has been described previously (44). A fresh 4- day old PDA or PDYA plate culture of Rhizoctonia was chopped aseptically with 100 ml sterile distilled water in a sterilized monel metal Waring 20 Blender for 30 seconds. To prepare a large amount of inoculum, the suspension was mixed aseptically in one liter of sterilized vermiculite medium (1 volume dry vermiculite, 2-3 mm mesh, plus 1/3 volume potato dextrose broth) in a 2800 ml Fernbach flask. A smaller amount of inoc- ulum was made with 100 ml of vermiculite medium and 20 ml of suspension in a 500 ml flask. The Rhizoctonia-infested vermiculite medium was in- cubated at 24 C for 4 days and was then ready for use as soil inoculum at various designated proportions. Preparation of uniform_propagu1es from mycelial mat and sclerotia A modification of the method of Henis and Ben-Yephet (26) was used. .5‘ solani was grown on PDYA for 2 or more days and 7 mm discs were cut from the colony margin. The discs were transferred to 500 m1 flasks containing 50 ml PDYB, and grown at 24 C for 4-10 days depending on the experimental purposes. The resulting mycelial mat, which con- tained sclerotia in some isolates, was removed, washed 3 times with distilled water and then chopped in a monel metal Waring Blender with distilled water for 30 seconds. The chopped mixture was poured into a 500 m1 graduate cylinder and the large, rough particles allowed to settle. The upper mycelial suspension was decanted and discarded. 21 Distilled water was then added to resuspend the particles of mycelial mat and sclerotia. The procedure was repeated until the supernatant remained clear. The sediment containing fragments of mycelial mat or sclerotia was collected and further reduced in size by chopping with a stainless steel razor blade. The resulting chopped propagules in distilled water were passed through various mesh-size sieves to collect lots of uniform-sized propagules. Experimental soils Muck soil was obtained from Michigan State University's Experi— mental Farm on the Corey Marsh near Bath, Clinton County. The soil re— action was 6.9. Soil water holding capacity was 2.7-3.7 grams of water per gram oven—dry soil. All muck soil used in this study, unless otherwise indicated, was passed through a 3.15 mm mesh sieve and adjusted to 50% water hold- ing capacity. All the soil infested with Rhizoctonia-vermiculite inocu- lum was incubated at 24 C. Conover loam mineral soil was obtained from the Michigan State University farm at East Lansing. The soil was sifted to remove large debris. Soil reaction was 7.45.. Soil water holding capacity was 0.45 g HZO/g oven dry soil. 22 Assaying populations and survival capability_of Rhizoctonia solani Methods used for enumerating populations or evaluating survival of R. solani in soil included the red beet seedball colonization or trapping assay (32, 44) and the soil segment assay (32). Beet seedball colonization assay,--Autoclaved beet seedballs (Beta vulgaris Cv. Detroit Dark Red, Ferry Morse Seed Co.) were incu- bated in test soil at 24 C for 48 hr. The seedballs were then removed from soil, washed in running tap water for 10 seconds, blotted on a clean paper towel to remove excess water and transferred to acidified PDA or selective medium (10 seeds per plate). Assay seedballs were incubated on the plates at 24 C for 15 or 24 hr, respectively. Macro- and microscopic examination revealed Rhizoctonia colonization on the seedballs. Two different sizes of soil sample (one a 64 ml volumetric and the other a l g oven dry equivalent) were used in this type of assay depending on the experimental convenience. When 64 ml soil samples were chosen, half of the soil was layered in a petri dish, 50;beet seedballs arranged uniformly thereon and then covered with remaining soil. Usually 3 or 4 replications of similar samples were used. When a volume of soil equivalent to l g oven dry or 2 gram soil was used the soil was mixed with 10 beet seedballs in a vial and capped. These tests were replicated in 3 or 4 vials. 23 Soil segment assay.--Recovering Rhizoctonia from soil segments-- the equivalent of 1 g oven dry soil was moistened with distilled water to about 80% WHC and divided into approximately 10 equal parts. Each segment (each part) was transferred intact onto selective medium (5 seg- ments/plate) and incubated at 24 C for 48 hr. Three replications were used. Comparative survival ability of isolates of Rhizoctonia solani in muck soil under closed and open storage conditions Sieved soil was infested with 3% (v/v) Rhizoctonia-vermiculite inoculum. Under closed storage conditions, the infested soil was placed in plastic bags sealed to avoid loss of water. After 2 and 30 days incubation, soil samples of 64 ml were taken for the beet seedball assay. Under open storage cOnditions, the infested soil was placed in a plastic cottage cheese box (500 ml container) without covering. Soil moisture was adjusted to maintain approximately 50-60% WHC (1.5-1.8 g HZO/g soil) by adding water 2 or 3 times a day. Soil samples (64 ml) were taken at intervals of 2, 10, 20, 30, 60, 90, 120, and 150 days for the beet seedball assay. Acidified PDA was used to determine beet seedball colonization by Br solani under both storage conditions. 24 Comparative survival ability of isolates of Rhizoctonia solani in mineral soil under open storage condition The methods used here were similar to those described for test- ing the comparative survival ability of isolates of R. solani in muck soil under open conditions. At soil moistures of 50-60% WHC there was O.225-0.27 g water/g oven dry soil. Effect of volatile substances on growth of Rhizoctonia solani Storage dishes 100 mm in diameter and 80 mm high were used for test units. Each dish contained 100 ml of washed sand, natural muck soil, or natural muck soil infested with Rhizoctonia-vermiculite inocu— lum. Petri dish bottoms (90 mm die.) each containing a layer of Ko's selective medium (32) without p-dimethyl aminobenzenediazo sodium sul- fonate were seeded with, a 1000 11 dia. mycelial fragment of _R_. solani, then inverted to cover the storage dishes. Cover and dish were sealed with electrical tape to prevent the loss of gases. There were 4 repli- cations of each treatment. 25 Soil moisture and temperature effects on survival of Rhizoctonia solani Soil moisture.—-In these experiments washed fragments of mycelial mats and sclerotia were used as inoculum because poor survival of most isolates of B, solani in muck soil might have been due to an increase of certain antagonistic microorganisms invading the Rhizoctonia—vermiculite amendment. Mycelial and sclerotial fragments (600-800 u dia.) were prepared and excess water removed on filter paper in a Buchner funnel. Aliquots of muck soil, equivalent to 100 g oven dry, were infested with 0.2 g of the inoculum. After infestation, the soil moisture was adjusted to 20, 40, 60, or 80% WHC (0.6, 1.2, 1.8, and 2.4 g water per gram dry soil). All soil lots were stored in cottage cheese boxes, with lids on, inside closed plastic bags and held at 24 C. After 1, 4, and 10 weeks storage, aliquots (l g. oven dry equiv.) were taken from each treatment and assayed for the presence of Br solani by soil segment and beet seedball assays. For the beet seedball colonization assay, all soil samples were brought to 50% WHC just prior to assay. During 10 weeks of incubation under the above conditions, soil moisture.decreased.gradually: 26 Storage period after ' - Soil m ' t infestation, wks. 015 ure, % WHC 0 20 40 60 80 1 20 40 60 80 4 18 37 56 75 10 14 33 50 68 Soil temperature.--Isolates R54 and R51 were used in this ex- periment. Muck soil was infested with 10% Rhizoctonia-vermiculite inoculum, and Petri dishes containing the infested soil (70 ml/plate) were stored in a plastic bag for 2 days at 24 C. At this time 6 repli- cate assays (using 2 g soil samples) were made for each isolate by both soil segment and beet seedball colonization methods. The rest of the soils were transferred to storage temperatures of 4, 18, 24, 28, and 32 C for incubation periods of 7 and 43 days. Three replicate soil segment and 4 replicate seedball assays were made on these samples. Mycelial growth on agar media A disc (5 mm dia.) obtained from the margin of an R, solani colony growing on PDYA, was placed on culture media (15 ml/plate). There were 6 replications for each isolate on PDYA, PDA, and water agar (WA). Incubation was at 24 C. Mycelial growth was measured 24 hr after infestation. 27 Mycelial growth on muck soil or DASS treated muck Small polystyrene plastic microchambers were made by stacking and gluing together five Redi—Fix FTA antigen slides (Aerojet Medical and Biological Systems, EL Monte, CA 91734). The inside dimension of the chambers were 62 X 18 X 5 mm or 31 X 18 X 5 mm. A small sample of screened and moistened muck soil or p—dimethyl aminobenzenediazo sodium sulfonate (DASS) treated muck soil was placed in the chamber and the surface smoothed with a stainless steel spatula. A fragment of mycelium or a sclerotium (1000 u dia.) was placed on the soil. The slides with infested soil were kept in a plastic tray (25 X 17 X 6 cm), covered with moist chromatography paper and the entire assembly was placed in a plas- tic bag to prevent rapid evaporation of water from the soil. Mycelial growth was measured with a stereomicroscope 24 and 48 hours after inoculation. Production of sclerotia and monilioid cells on agar media Cultures were grown as described above for mycelial growth on agar media. Production of sclerotia and monilioid cells determined at 7 and 14 days. 28 Production of sclerotia in muck soil Sifted muck soil (300 u mesh) was infested with 10% Rhizoctonia- vermiculite inoculum. Infested soil were placed in four Petri dishes (70 ml soil/plate) and kept inside a plastic bag at 24 C. Three weeks after inoculation, the inoculated soil was sifted through a coarser screen (500 u mesh). Particles > 500 u remaining on the sieve were examined to determine the number and size of sclerotia. Lysis of mycelium on soil Mycelial colonies, as described above under "Mycelial growth on muck soil“ were used for this study. Lysis of mycelium was determined at weekly intervals by direct observation with a steriomicroscope and by mycelial staining. Mycelium was stained in situ with rose bengal before recovering from the soil by the method of Lingappa and Lockwood (35). The recovery medium was 1-2% polystyrene solution made by dissolving plastic Petri dishes in a 2 : 1 benzene-toluene solution. When the myce- lium was stained and recovered on the membrane, hyphal units (600 u in length/unit) within 5 mm of the original inoculum position were examined under the microscope (125 X) for lysis. Four replicate observations of 10 microscopic fields were made for each treatment. The percentage of 29 hyphal lysis was determined according to the formula: % lysis = 100 (l - number of hyphal units with stain as indicated period of incuba- tion/number of hyphal units with stain at 1 day incubation). Lysis of mycelium on agar medium with or without covering soil Testing lysis of R, solani on sterile soil in the plastic slide chambers was unsuitable, because of sterilization difficulties. There- fore, in order to prevent contamination of the sterile soil, Lockwood's technique (36) was modified and employed. Petri dishes each containing 20 ml water agar were seeded with isolates of R, solani (agar discs 5 mm die) and allowed to grow for 2 days. About 30 m1 of natural soil or autoclaved were placed on the surface of each agar plate. The plates were then incubated at 24 C for 1, 2, and 3 weeks. After incubation, the soil was removed and 1 cm square agar plugs 1.5-2.5 cm from the center of the original inoculum disc were taken for staining with rose bengal and examination for lysis. Four replications were made for each treatment. Each agar plug was examined by viewing 20 fields under the microscope at (125 X). Each hyphal strand 600 u in length was consid- ered as a unit. The numbers of hyphal units stained with rose bengal 30 were recorded. Water agar plates seeded with R, solani without added soil and with sterile soil were used as controls. Survival of sclerotia on or in muck soil On muck soi1.--Sc1erotia (100 u diam) of isolates R51 and R54 were placed on the smooth surface of muck soil in Petri dishes inside plastic bags and incubated at 24 C. After certain incubation periods, sclerotia were removed and washed with sterile distilled water 3 times. Twenty sclerotia were placed on PDA, and another 20 on acidified PDA. Some of the washed sclerotia were additionally treated with 0.525 % sodium hypochlorite for 5 min. rewashed 3 times with sterile distilled water and 20 were then plated on PDA. After 24-48 hr incubation at 24 G, percent sclerotial germination, growth diameter of the mycelium (after 24 hr incubation), and numbers of sclerotia contaminated with natural soil bacteria and fungi were recorded. In muck soi1.--Two grams air dry muck (300 u mesh) was mixed with 50 sclerotia (1000 u dia.) of R, solani and the soil moisture adjusted to 50 % WHC. Vials containing this sclerotia-soil mixture were capped and incubated at 24 C. After 1, 4, 9, 16 and 26 weeks of 31 incubation sclerotia were removed from the soil by washing the sclerotia- soil mixture on a 500 u mesh sieve with distilled water. Recovered sclerotia were next washed 3 times in sterile distilled water, blotted on a clean paper towel and placed on PDA to test viability. Forty sclerotia of each isolate, R54 and R51, from each incubation period were tested. Survival of Rhizoctonia mycelium in soil Muck soil was infested with 10% Rhizoctonia—vermiculite inoculum, placed in Petri dishes (70 ml/plate), and incubated in a plastic bag. After incubation the soil was passed through a 3.15 mm mesh sieve. Large particles remaining on the sieve were poured carefully into a clean dish and the mycelium-soil aggregates were picked up by forceps. Some mycelium-soil aggregates hung on the sieve after the rough particles were removed. The percent of sieve area covered with hanging mycelium- soil aggregates was determined. The presence of R, solani hyphae in the aggregates (on the sieve and removed) was determined under the micro— scope. Viability of R, solani hyphae on the sieve was determined by placing these aggregates on the selective medium. 32 Soil microorganisms affecting survival of Rhizoctonia solani Natural muck soil was sterilized in the autoclave for 2 hr the first day and again sterilized for 2 hr the second day. The autoclaved soil was tested on PDA to make certain it was free of microorganisms. The sterilized soil lots were inoculated with 3 isolates of R. solani inoculum (Rhizoctonia-vermiculite inoculum, 10%). Part of the inocu- lated soil was further amended with 5% non-sterilized muck soil. Samples of each experimental soil were then placed in Petri dishes (70 ml/plate), enclosed in a plastic bag and incubated at 24 C. After 2, 7, and 21 days 2 g soil samples were taken for soil segment and beet seedball colonization assay. Three replications were used for soil segment and 4 replications for beet seedball assay. Testing antagonists of Rhizoctonia solani on agar Soil microorganisms from natural muck soil, were also isolated and tested for interactions on agar media with R. solani. The follow- a U I ing media were employed: NPX-acidified£—'PDA (23) was used for isolation AENPX detergent (nonyl phenyl polyethlene glycol ether, Union Carbide Corp.) added to PDA at 1000 ppm prior to autoclaving then acidi- fied with 0.9 m1 of 20% lactic acid per 200 ml. 33 of some fungi while delaying other fungi and inhibiting actinomycetes . . / b . and bacteria. SOll extract-—'agar (61) was used for bacterial and the . . /c . . . . chitinr—-medium (34) for actinomycete isolations. PDA was used as a general growth and testing medium for fungi and bacteria. Peptone /d . . . glucose-—-agar (75) was used as a growth and testing medium for actino- mycetes. To test antagonisms, the microorganism and R. solani were grown on opposite sides of the plate. If the test microorganism was known to be a slow grower, it was allowed to grow for 2 to 3 days be- fore the R. solani was added. Effect of p-dimethyl aminobenzenediazo sodium sulfonate (DASS) sensitive microorganisms on survival of Rhizoctonia solani High populations of Pythium spp. naturally occurring in muck soil (19) may affect survival of R. solani. Para-dimethyl aminobenzene- diazo sodium sulfonate (DASS) at 20 lb/acre may be used to control b £—'Soil extract agar contained glucose 1 9, K HPO 0.5 g, KNO 0.1 9, soil extract 100 ml, and agar 15 g in 900 m1 disti led water. lE-Chitin medium contained colloidal chitin 2 g, agar 20 g, and distilled water 1000 ml. d ZL-Peptone glucose agar contained peptone 5 9, glucose 20 g, agar 20 g, and distilled water 1000 ml. 34 Pythium in muck soil without damage to R, solani (18, 44). Based on this information and preliminary trials, it was decided to use DASS to control naturally occurring Pythium spp. and other microorganisms such as Aphanomyces spp. or Phytophthora spp. in muck soil that could be sensitive to it. This inhibition made it possible to study effects of these microorganisms on the survival of isolate R57 in muck soil. Muck soil infested with 10% Rhizoctonia-vermiculite inoculum was treated with DASS, 5% granules, at rates of O, 22, 44, and 66 mg active ingredient/liter soil (approximately 0, 20, 40, and 60 1b/acre, 4 inch deep). Lots of soil so treated were kept in 500 m1 covered cottage cheese boxes inside a plastic bag. Soil samples (2 g were taken for beet seedball and soil segment assay after 2, 7, and 14 days incubation at 24 C. RESULTS Comparative survival ability of 19 isolates of Rhizoctonia solani in muck soil stored in closed plastic bags All of the isolates tested showed poor survival in muck soil except isolate R54 as determined by beet seedball colonization assay (Table 2). Among the 18 relatively poor survivors, 5 isolates were poorly established in muck soil from the beginning as indicated by the 2-day soil samples. The other 13 isolates had become well established at the same time. Comparative survival ability of isolates of Rhizoctonia solani in muck soil under open storage conditions The poor survival ability of R, solani isolates in muck soil under closed storage may have been due to an accumulation of toxic in- hibitory substances or poor aeration. Five isolates, R47, R51, R54, PR56, and R57 which possessed high ability to colonize beet seedballs 35 36 TABLE 2. Survival of 19 isolates of Rhizoctonia solani in natural muck soil after 2 and 30 days storage in closed plastic bags, as determined by colonization of beet seedballs. ColonizationlE-of beet seedball, % Isolate 2 days 30 days Control 0 0 R44 2 1 R41 6 0 R45 10 0 R43 25 2 R49 35 3 R42 90 0 R55 90 3 PR108 90 5 PR56-42 100 0 PR56—30 100 2 PR56—32 100 5 PR56-5 100 5 R57 100 5 R58 100 5 PR58 100 10 R51 65 17 R47 100 20 PR56 100 22 R54 100 75 12 Average of 4 replications. Each replicate consisted of 50 beet seedballs incubated in 64 ml of test soil. 37 after 2 days in muck soil were chosen to study survival in muck soil stored at 24 C in open cottage cheese boxes. Isolate R54 had a very good survival in this experiment and apparently retained a high inoculum density during 150 days of open storage (Figure l). Isolates R47 and PR56 survived relatively better than did isolates R51 and R57, but relatively poorer than R54 (Figure 1). Comparative survival of Rhizoctonia solani isolates in mineral soil under open storage conditions Survival ability of R. solani isolates in muck soil and in min- eral soil may differ. Therefore, the survival of 5 R. solani isolates in mineral soil was tested under open storage conditions (Figure 2). Survival of isolates R47, R51, PR56, and R57 in mineral soil were similar to those in muck soil (Figures 1, 2). In this experiment, isolate PR56 was a relatively good survivor, isolate R47 was a good survivor for the first 30 days of incubation, but thereafter inoculum density dropped rapidly. Isolates R51, R54, and R57 were relatively poor survivors in mineral soil. Isolate R54 survival in mineral soil apparently was different from that in muck soil (Figures 1, 2). 38 100 \ 00 O l 0 O I A O T BEET' SEEDBALL COLONIZATION,% to O I 210 30 60 90 120 150 DAY AFTER INOCULATION FIG. 1.--Survival of five Rhizoctonia solani isolates in natural muck soil stored in Open cheese boxes for 2 to 150 days, as determined by colonization of beet seedballs. 39 BEET SEEDBALL COLONIZATION, °/o 210 30 6O 90 120 150 DAY AFTER INOCULATION FIG. 2.--Surviva1 of five Rhizoctonia solani isolates in natural mineral soil stored in open cheese boxes for 2 to 150 days, as determined by colonization of beet seedballs. 40 Effect of volatile substances on growth of Rhizoctonia solani There were no indications for the presence of inhibitory vola- tile substances inhibiting growth of R. solani isolates R51, R54, PR56, and R57 during 48 hr exposure in sealed storage dishes. Growth of these 4 isolates on selective medium about 50 mm above muck soil or muck soil infested with Rhizoctonia-vermiculite inoculum did not differ significantly from cultures above sand. Effect of soil pH, moisture, and temperature on the survival of Rhizoctonia solani All muck soil infested or non-infested with Rhizoctonia isolates at various incubation period was taken for measuring pH. The pH of in- fested or of non-infested with any isolate of R, solani remained con- stant in the range 6.8-7.1 throughout the experiment. Therefore, the effect of soil reaction on the survival of R. solani in this muck soil was probably not important. The soil moisture (SO-60% WHC) and tempera- ture (24 C) used in the above experiment may not have been suitable for survival of a given 5? solani isolate in muck soil. Therefore, various soil moisture and temperature conditions were tested to determine whether a given condition was favorable for survival of a given isolate. 41 Soil moisture.--Isolates of R, solani differed in their rela- tive survival in response to moisture level using beet seedball and soil segment assays(Figure 3). Isolates R47 and R51 survival were not affected differentially by soil moisture either during the first week or over the longer term (P=0.05). R54 survived quite well at all moisture levels for the first week. When using the soil segment assay at 4 weeks, soil moisture seemed to have had little effect on the sur- vival of R54. But when using the beet seedball assay, high soil mois- ture was significant more favorable for survival than low soil mois- ture. At 10 weeks the low soil moisture (20% WHC) was comparatively unfavorable to survival as shown by both assay methods. There was little effect of soil moisture in the 40-80 WHC range on survival of R54 indicated in soil segment assay, but when using the best seedball assay in high soil moisture level tests (80% WHC) R54 survival was sig- nificantly superior to survival at 40-60% WHC. The different results obtained with soil segment assay and beet seedball assay may be due to the different measuring standards employed in the two methods (see Appendix B and Discussion). However, R54 survived relatively well at soil moisture levels 40 to 80% WHC as indicated by both assay methods. PR56 survived well only during one week at higher moisture, but at 4 to 10 weeks these differences in moisture had little effect. Isolate R57 responded unfavorably to both extremens of moisture, particularly 42 []20 a so ) .40 I so NOMINAL wuc (°/. ‘00 ‘ R47 . R47“°° soT a so Li: . -— :. —— 100* R51 R51-100 4! C3 IHIHIHI 100 a! G” JL 3. ‘n J} .L C, C) IHIHIHIHIHIHIHI RECOVERY FROM SOIL SEGMENIS 7. am SEEDBALL commzmon .\° 100 E 50 g 50 O :— o ‘I 4 1O 1 4 10 WEEK INCUBATION AT VARIOUS MOISTURES FIG. 3.--Effect of four soil moisture levels on the survival of five isolates of Rhizoctonia-solani in infested muck soil contain- ing 0.2 g propagules (size 600 u - 800 u) per 100 9 soil. Survival determined by recovery of Rhizoctonia from soil segments (left) and by beet seedball colonization (right). *Horizontal lines drawn above groups of four bars include the bar(s) within the groups that do not differ from each other (L.S.D. 5%). 43 high moisture at the first week. At 4 weeks a reverse effect was ob- tained, both low and high moisture gave a little better survival than the intermediate moisture levels. At 10 weeks the survival of R57 was low in all moisture levels. In general, this experiment confirmed the previous test using Rhizoctonia—vermiculite inoculum amended muck soil (Table 2, Figure 3)- Isolate R54 survived well but the other 4 isolates survived poorly in muck soil. The relatively poor survivors, R47, R51, PR56, and R57, were not benefited in a given soil moisture at 10 weeks. Soil temperature.--Figure 4 illustrates survival at 5 storage temperatures. The lowest temperature gave the best survival in muck soil for R51 and R54 but the temperature differences were less important for R54 which survived well at all temperatures. Inoculum density of R51 decreased rapidly at 43 days incubation under all temperature con- ditions. Growth capability of Rhizoctonia solani isolates When the growth rate of 4 Br solani isolates (R51, R54, R57, and PR56) on PDA, PDYA and WA, isolate R51 grew much faster than the other 3 isolates(Figure 5)«Tbis relatively poor survivor grew faster than the “l. RECOVERY FROM SOIL SEGMENTS \ U4C $1“: 24C \28C '32c \ R51 R51 100- ._ ~1OO F— 50- Illllllllllllllllllll, R54 R54 100* 100 I ca (3 so? llIll|||IlII|Illl|lllllllllllllllllllllllU ’////////// '///////// fi'fi'lio’o’.’-’o.’ooouu 00000000000000.0000... .— —— —- —— —- .— — —! .— ——4 —4 -—< _. Inn—na— -— -—¢ -——¢ —-. u—a r—n — h—— b—1 b— \ \ \ \ \ \ \ \ '//////. lllllllllllllllllllllllllll 0 O 7 43 O 7 43 DAYS INCUBATION AT VARIOUS TEMPERATURES FIG. 4.--Survival of two isolates of Rhizoctonia solani at various soil temperatures as determined by recovery at various soil temperatures as determined by recovery of Rhizoctonia from soil segments and by beet seedball colonization. Soil infested with Rhizoctonia-vermiculite inoculum and incubated at 24 C for 2 days (designated as 0 day incubation) before incubating at various temperatures. *Horizontal lines drawn above groups of five bars includes the bar(s) within the group that do not differ from each other (L.S.D. 5%) “I. BEET SEEDBALL COLONIZATION 45 1.0 __. F... .E : E . E E 0.5- —, In __:___ O: :2 "" E F: F 3 .- O 1 L9 q, 10 E~ 1- at 10 E~ v- Sf 19 " “5 “5 “T “1 “5 U5 “5 “5 "5 “fl “5 “5 __ a: a a: a a: a: a: a: a: a a: n. n. a. O PDA PDYA WA FIG. S.--Growth rate of Rhizoctoni solani isolates on potato dextrose agar (PDA), potato dextrose yeast extract agar (PDYA), and water agar (WA at 24 C. *Horizontal lines drawn above groups of four bars include the bar(s) within the groups that do not differ from each other (L.S.D. 5%). 46 the other 3 isolates on all 3 media. PR56 was an isolate of intermedi- ate survival capability but grew faster on PDA and PDYA plates than either the poor survivor R57 or the good survivor R54. PR56 and R54 did not differ significantly in ngWth rate on water agar but PR56 was faster than R57. The relatively good survivor, R54, did not grow faster than any of the other 3 isolates on the 3 media. Thus the sur- vival of R, solani isolates in muck soil was not positively correlated with growth rate on culture media. Comparative growth of four R. solani isolates on natural muck soil was also determined. A fragment of mycelial mat (1000 u in die.) was placed on natural muck soil at one side of a microchamber or placed on one side of PDA plate as control. Four replications were made in this experiment. The growth rate of test isolates on muck soil appear to be a little faster than on PDA (Table 3). Here also the relatively poor survivor, R51, and the intermediate isolate, PR56, grew faster on soil than the good survivor, R54. Ability of Rhizoctonia solani isolates to produce sclerotia or monilioid cells Isolates R51 and R57 which are relatively poor survivors in muck, as well as the good survivor R54, produced abundant sclerotia 47 TABLE 3. Growth rate of four isolates of Rhizoctonia solani on natural muck soil and potato dextrose agar (PDA). Relative /a . Growth rate, mm/hr——' surVival Isolate . . . ability in , , Muck 5011 PDA muck 3011 R54 Good .34 c412 .31 c112 PR56 Intermediate .53 b .50 b R57 Poor .41 c .39 c R51 Poor 1.12 a .90 a -—3 Average for 48 hr growth period. ——'Different letters following the values indicate statistically significant differences between isolates (L.S.D. 5%). 48 on agar (Table 4). However, PR56 (an intermediate survivor) pro- duced no sclerotia. All isolates except R51 produced monilioid cells on water agar. Production of sclerotia in muck soil is shown on Table 5. Much soil infested with isolate R54 had an average of only 2 small sclerotia per 70 m1 soil, but soil infested with isolate R51 had 17. R51 sclerotia varied from 1 mm to 4-5 mm in die. Survival of sclerotia of Rhizoctonia solani on or in muck soil An experiment was conducted to compare the ability of sclerotia of isolates R51 and R54 to maintain viability during extended incuba- tion on or in muck soil. The relation of this capability to the pres- ence of other soil microorganisms was also investigated coincidentally. Sclerotia of both isolates incubated for 0-42 days on soil germinated 100 percent and grew on PDA. Percent germination of R51 sclerotia was reduced slightly on acidified PDA, being 85 and 90% on 29 and 42 days samples, respectively. The precentage of viable sclerotia remained high after incuba- tion in soil for 26 weeks. Viability of R54 sclerotia tested on PDA was 100, 95, 97, 90, and 80% after 1, 4, 8, 16, and 26 weeks incubation, 49 TABLE 4. Production of sclerotia by four Rhizoctonia solani isolates on potato dextrose agar (PDA), potato dextrose yeast extract agar (PDYA), and water agar (WA) after two weeks incubation at 24 C. Relative Numbers and size (dia.) of sclerotiaéi I l t survival 5° a e ability in PDA PDYA WA muck soil No. mm. 1V0. mm. N0. mm. /b R54'—- Good 32 1-1.5 37 1-2 2 1 ll 10 PR56 Intermediate 0 0 0 0 0 0 [.12. R51 Poor 160 1-3 250 1-3 2 1 26 5 R57 Poor 125 1-2 110 1-3 2 l / -—'Two different size groups sclerotia observed on plates. a -. -- Average of 6 replicated plates 50 TABLE 5. Comparison of sclerotial numbers and size in two isolates of Rhizoctonia solani three weeks after infestation of soil with Rhizoctonia-vermiculite inoculum. . . a Number and Size of sclerotia £1. R51 R54 (Poor survival) (Good survival) No. mm. N0. mm. 2 1 X l 1 l X l 11 1-2 X 1-2 1 1-2 X 1-2 2 2 X 2-3 0 0 l 2 X 3—4 0 0 l 2 X 4-5 0 0 Total 17 2 lE'Average of 4 replications of soil each 70 ml. 51 respectively, and that of sclerotia of R51 was 100, 90, 85, 80, and 85%, respectively. Growth diameter of R54 sclerotia transferred to assay media showed little or no decline during 42 days incubation on the surface of muck soil, but the growth diameter of hyphae from sclerotia of isolate R51 declined with time (Figure 6). Bacteria and other fungi growing from sclerotia onto the assay media were noted and the number of sclerotia contaminated with micro- organisms were counted at 48 hr. Sclerotia of both R51 and R54 were highly contaminated with bacteria for all incubation periods on muck soil, but very few contaminating fungi grew out on PDA (Table 6). Contamination by fungi was not significantly different for the two iso- lates. No contaminating bacteria or fungi were observed on acidified PDA. Residual contamination of the sclerotia with bacteria after treatment with sodium hypochlorite was greater for isolate R51 than for R54. This difference was significant at the 5% level for all in- cubation periods. ‘Four bacterial isolates obtained from R51 and R54 sclerotia inhibited the growth of both R51 and R54 vegetative hyphae. However only R51 had hyphal tip lysis. 52 .coflumnsocw up cm Hmumm BE CH .MHp >coHou .«0m :0 Umomam mcwwn wuowmn Uwcmmz Ion pom .mUHuoanoom>£ Edflpom SUHS poummuu mfiuoumaom cosmos AUV «no Amy :0 pmomHm moflmn oHOan pmsmm3 mauoumaom .«om commapwom Amv pom .m>mp Nvuo How HMOm x09& :0 Umumnsocfl coma pm: £0H£3 mwuouwaom Eouw mmmeOmfl womaom casewoowflm o3u mo SD3OHOII.0 .OHm NV am 352: .:0m 20 <_._.Omm.._Om m— w No as d 1 <1 me - u q q om 2 w «o (“'W') 'VICI HlMOHE) pmm 53 TABLE 6. Bacterial and fungal contamination of sclerotia of two Rhizoctonia solani isolates after incubation on muck soil naturally infested with bacteria and fungi. . a . a Bacteria 1-- Fungi 1-- PDA after sodium PDA hypochlorite PDA Days on soil before assay R51 R54 R51 R54 R51 R54 2 20 13 11* l 0 0 8 20 20 15* 2 1 2 15 20 19 12* 2 1 1 29 20 20 11* 2 ' 4 2 42 20 20 16* 0 5 4 /a --Numbers of sclerotia contaminated with bacteria or fungi determined by placing them on potato dextrose agar (PDA) or on PDA after treat- ing with sodium hypochlorite. Sample size 20 sclerotia. * Differences between the values for R51 and R54 in the same treatment are significant (p = 0.05). 54 Lysis of Rhizoctonia solani I # _ mycelium on soil In a preliminary experiment, little or no mycleium of isolate R51 remained on the muck soil after 28 days incubation. On the other hand, mycelium of isolate R54 was abundant (Figure 7). The later experiment (Figure 8) showed that more than 50% of the hyphal ‘units (600 u in length/unit) of isolate R51, PR56, and R57 on muck soil lysed between 14 and 21 days incubation. Ninty-five percent of R51 hyphae lysed by the let day. Only a low percentage of hyphal lysis was ob- served for R54 between 7 and 35 days incubation. The percentage of hyphal lysis for R54, PR56, R57, and R51 at 35 days incubation were 32, 70, 87, and 100, respectively. Similar results were observed by direct examination of the soil with a stereomicroscope. After 7 days incubation, hyphae of R54 were much darker stained than were hyphae of the other isolates. No contaminating microorganisms were recovered on the polystyrene membrane at 1 day incubation, but .abundant bacteria associated with hyphae were recovered at 7 days. 55 FIG. 7.-—Mycelium of Rhizoctonia solani isolates R54 (A) and R51 (B) on muck soil were stained with rose bengal and recovered with polystyrene membranes after 28—day incubation. FIGURE 7 57 50- d I HYPHAL UNIT LYSIS, INDEX 1, 1 28 35 1 7 14 21 INCUBATION PERIOD,DAYS FIG. 8.--Mycelium of four Rhizoctonia solani isolates lysed on muck soil, as determined by the percentage of hyphal units (hyphal length 600 u/unit) not showing stain. Hyphae were stained on the soil with rose bengal and recovered for examination with polystyrene mem— branes. Lysis index = 100 (l - number of hyphae units with stain at the indicated period of incubation/number of hyphal units with stain at 1-day incubation). 58 Survival of Rhizoctonia solani mycelium in soil In a preliminary experiment, where soil had been infested with isolate R54 and incubated for 1 week, there was an abundance of soil- mycelium-vermiculite aggregates. On the other hand, there were few such aggregates when R51 was the initial inoculum (Figure 9). The mycelium associating with the aggregate was identified as R, solani with a micro- sc0pe. When a similar test was later made with isolates R54, R51, PR 56, and R57 (Table 7), there were many more soil-mycelium aggregates in R54 and PR 56 infested soil than in R51 and R57 infested soil at 2-days incubation. These soil mycelium aggregates were reduced rapidly in PR56- infested soil at 2-7 days incubation, but only slightly in R54 infested soil through 14 days incubation. The aggregate index of R57-infested soil increased at 14 days incubation. This was due to other fungal growth and association of other fungi in these aggregates. When the total wet weight of soil-mycelium aggregates was measured at 14 days in- cubation, isolate R54 was 2.1 g which was about 7.7, 12.3, and more than 200 times heavier than those of R57, PR56, and R51, respectively. That viable mycelium of Rhizoctonia existed in mycelium-soil aggregates was determined on Ko's selective medium (32). There was more viable mycelium in R54 infested soil than for other isolates. The percentage of viable mycelium of R54 in the aggregates declined 59 FIG. 9.--Aggregates of soil-mycelium-vermiculite formed in muck soil infested with Rhizoctonia-vermiculite inoculum of R54 (A) and R51 (B) and incubated for 1 week (16 X). Vermiculite (V) and Muck (black clumps, m) aggregated by mycelium of R, solani. 60 FIGURE 9 III-l" 1 l I]: .l l 61 TABLE 7. Relative ability of four isolates of Rhizoctonia solani to colonize muck soil and survive at 2, 7, and 14 days incu- bation as determined by a soil-mycelium aggregate index 12, aggre- gate viability 124 and aggregate wet weight 12, Soil-mycelium aggregate Relative . [3. /br . /c surVival , Index Viability,%-- Wet weight, g -' Isolate . , ability + 2 7 14 2 7 '14 14 days days days days days days days R54 Good 90 90 80 45 24 30 2.10 I _ ‘. PR56 nter 90 4O 3O 25 0 0 , 0.17 mediate . R57 Poor 20 10 40 15‘ 5 0 0.27 R51 Poor 10 1 l 20 0 0 0.00 Control -— 0 0 0 0 0 0 0.00 lE'The soil—mycelium aggregate index was determined by estimating the percent area of a sieve upon which the aggregates were retained after sieving infested soil and dumping out soil particles or clumps too large to pass through the sieve. ZE-Viability of individual soil-mycelium aggregates still hanging on the sieve was determined by placing them on selective medium for growth of Rhizoctonia mycelium. /c . . . .. -‘Wet weight of SOil-mycelium aggregates was taken after picking them from the screen with forceps. Soil particles and clumps without mycelial aggregation were discarded. 62 slowly between 2 and 14 days incubation, but declined rapidly in the aggregates of PR56, R57, and R51 between 2 and 7 days incubation. Effect of soil microorganism on survival of isolates of Rhizoctonia solani In order to obtain information concerning the effect of soil microorganisms on survival of R. solani, an experiment was designed using combinations of sterilized and non-sterilized muck soil (in Petri dishes enclosed in plastic bags) to which the R, solani inoculum was added (Figure 10). Isolate R51 did not survive well in sterilized soil or in mix- tures with non-sterilized soil. Isolate R57 behaved differently. It survived poorly in sterilized soil amended with natural non-sterilized soil, but survived very well in autoclaved sterilized soil. In con- trast to the above two isolates, isolate R54 survived very well in all soils tested. Another test of antagonism by soil microorganisms was made using the selective and differential media NPX-acidified PDA, soil extract agar, chitin agar and peptone sucrose agar described above (Materials and Methods). Microorganisms isolated from natural muck soil were tested against isolates of B, solani on the appropriate agar media. 63 ...I \ \ \ \ \ 50- \ :3 R51 \ g 0 V— ————————— ‘iio z O 3 100.. ¢ a:\\ a e a.\\\ #100 E W \ \\ \\ ‘\\ :4- : \\\ \\ z o ‘\ ‘ O m ‘ -5o 8 I 50" U 2 .1 _l "' 2‘. E R54 R54 0 “I () MI > G 3'; 8 100- Q— o a k‘\ 0:100 '5 3 \\ ‘K m x a: .\° \ .\. \ 50F. \\\\ \\\ ‘50 \ \ \ \ \\\ \\\ R57 \\\ R57 \\ o 1 I 1 1 40 2 7 21 2 7 21 INCUBATION PERIOD, DAYS FIG. 10.--Effect Of naturally occurring muck soil organisms on survival of three isolates of Rhizoctonia solani after 2, 7, and 21 days incubation of inoculum in test soil. soils: autoclaved (o Beet seedball (right) and soil segment (left) assays used with two O) and mixture of autoclaved and natural soil (o----O). 64 Although some antagonisms were observed there was no differential effect of any of the isolated fungi, bacteria, or actinomycetes against particular isolates of E. solani. Thus soil microorganisms that in- hibited the growth of relatively poor survivor Rhizoctonia isolates on agar also inhibited growth of the good survivor. Differential lysis of two Rhizoctonia solani isolates on agar plates treated with soils Because isolate R51 survived poorly in both autoclaved and natural soil, and isolate R57 survived poorly only in natural soil, the resistance to lysis of various 3. solani isolates was considered to be an important factor in determining their survival capability in muck soil. Therefore it was speculated that isolate R51 lysed exten- sively in both autoclaved and natural soil, and isolate R57 lysed significantly only in natural soil. To test this assumption, a com— parison of the lysis of both R51 and R57 was made on agar plates treated with soil. Within 7 days incubation, hyphae of isolate R57 lysed on water agar with natural soil on the top. No unlysed hyphae left on agar for this isolate between 7 and 21 days incubation. Similar results was obtained with the isolate R51 (Table 8). 65 TABLE 8. Lysis of hyphae of two Rhizoctonia solani isolates on water agar with or without muck soil, as determined by the number of hyphae units with or without rose bengal staining. a No. hyphal units stained 1-' . Days, R51 R57 incubation Natural Sterile No Natural Sterile No soil soil soil soil soil soil 7 1.1 5.3 7.5 2 8 7.8 8.3 14 0 1.8 0.8 0 5.9 6.0 21 O 0 0 0 7.1 5.7 /a -—'Hyphae 600 u in length was considered as units for examination. Each agar plug (1 X 1 cm) was examined for 20 microscopic fields (125 X). Results are averages of 4 replications. 66 The hyphae of isolate R51 also lysed on water agar either with autoclaved soil or without a soil covering. Isolate R57 was different. Apparently no significant amount of hyphae lysed in the absence of natural soil within a 21-day incubation period (Table 8). Influence of p-dimethyl aminobenzenediazo sodium sulfonate (DASS)—sensitive micro— organisms on survival of Rhizoctonia solani Other studies indicated that Pythium spp. may be effectively eliminated from muck soil without apparent damage to R. solani by application of DASS. The effect of 4 rates of DASS on growth of iso- late R57 in muck soil is shown in Table 9. DASS at 22 mg active ingre- dient per liter soil did not significantly (p = 0.05) inhibit linear growth of isolate R57. However at 44-66 mg per liter it significantly inhibited growth of this isolate. Since competition from Pythium may affect survival of certain other organisms it was decided to test that effect on R57 by treating soil with different levels of DASS (Table 10). There were no differences in survival in either soil segment or beet seedball assay at 2, 7, or 14 days attributable to the amount ' of Pythium suppression. Elimination of Pythium spp by DASS did not improve the survival of isolate R57 in muck soil (Table 10). Thus it 67 TABLE 9. Linear mycelial growth from sclerotia of Rhizoctonia solani isolate R57 on p-dimethyl aminobenzenediazo sodium sulfonate (DASS)—treated muck soil in plastic slide chambers 13 after 48 hr incubation at 24 C. DASS mg. active Growth 12' ingredient per mm. liter soil 0 20.3 A; 22 18.8 44 15.3 66 14.2 I} Chambers constructed from 5 Redi-Fix FTA antigen slides. Ia Average of 4 replications. I} LSD at 5% level was 2.55 mm. 68 TABLE 10. Survival of Rhizoctonia solani, isolate R57, in muck soil treated with p—dimethyl aminobenzendiazo sodium sulfonate (DASS) as determined by beet seedball colonization and soil segment assays. DASS, mg. zé-active Recovery from Beet seedball ingredient in 1 soil segments, % colonization, % liter of soil 2 days 7 days 14 days 2 days 7 days 14 days O 100 50 3 100 75 5 22 97 50 0 100 55 2.5 44 93 43 3‘ 100 43 o 66 100 57 0 100 46 O lE-l.1 mg/l is approximately 1 lb/acre 4 in. Effective control of Pythium in M.S.U. muck soil may be Obtained in the range 20-40 lb/A. 69 may be that some of the other DASS sensitive microorganisms such as Aphanomyces spp. and Phytophthora spp. also are not responsible for poor survival of R57 in muck soil. DISCUSSION Muck soil of the Michigan State University Muck Experimental Farm is known to contain very little or no Rhizoctonia solani (18, 19, 44). Frequent attempts to isolate Rhizoctonia directly from this soil were made during present research, always without success. Why this soil with high organic matter is lacking in R. solani has not been previously determined. Results of the present experiments suggest some factors affecting or not affecting the presence of R. solani in muck soil. Aeration is known to affect survival of certain isolates of R. solani (7) and this was also demonstrated here for isolates R47, R54, and PR56. Poor aeration may cause accumulation of CO2 in soil. According to reports in the literature, growth, saprophytic activity and survival of R. solani may be suppressed by some volatile substances such as CO2 (20, 50). In my experiments there was no evidence for the existence of volatile inhibitory substances in natural muck soil or in the muck soil infested with Rhizoctonia-vermiculite inoculum because there was no suppression of the growth of four 5, solani isolates 70 71 tested on assay medium. Similarly there was no indication of muck soil inhibition of 3. solani growth when the fungus was allowed to grow on the natural muck soil for 48 hr. Thus, the results all suggested that failure of R. solani isolates to survive in muck soil probably was not due to growth inhibitory substances, if any, existing in muck soil. That one 5, solani isolate "X" may survive better in "A" soil than isolate "Y," does not mean that isolate "X" will do better than "Y" in "B9 soil. In these experiments, isolate R54 was superior to PR56 for surviving in muck soil but the opposite was true in mineral soil. Baker and his associates (5) found that isolates of R, solani varied in their ability to survive in a given soil because the isolates varied in their resistance to soil-borne antagonists. Why R54 sur- vived well in muck soil but poorly in mineral soil is not known, but several explanations may be ventured: l) Nutrients needed by R54 may be unavailable in mineral soil. 2) R54 could be sensitive to certain antagonists in mineral soil that are not present in muck soil, or 3) The physical structure of mineral soil is in some way unfavorable for maintenance of viability of this isolate of R, solani. Suppression of growth and survival of R. solani has been re- ported as due to poor aeration (7), accumulation of CO (47, 50), and 2 increased activity of bacteria (33, 62) under high soil moisture. 72 However, using the beet seedball assay, the present work showed that R54 survived better in high moisture soil than in low moisture soil during 4 and 10 weeks incubation. Isolate PR56, gave similar results during the first week of incubation. The reason for the beneficial effect of high soil moisture, 80% WHC, is not known but probably the high soil moisture was favorable for production of resistant R, solani propagules in muck soil--because high humidity is essential for sclero- tium formation (66). In some cases the results of soil segment assay did not correlate with those in a parallel beet seedball assay. Where R54 infested soil was assayed by the two methods at 4 and 10 weeks the beet seedball assay showed that 80% WHC was better for R54 survival than 40-60% WHC, but the soil segment assay did not show this differ- ence. The beet seedball assay value is measured by the number of beet seedballs being colonized with R, solani. Factors influencing percen— tage of beet seedball colonization (Appendix B) are l) capability of the isolates of B? solani to grow and colonize beet seedballs, 2) num- bers of inoculum propagules in the soil, 3) size of the inoculum prop- agules in the soil. Soil segment assays measure the number of soil segments containing viable propagule (or propagules_of R. solani. This value is determined (Appendix B) by 1) distribution of B, solani in soil, and 2) number of inoculum propagules in soil. KO and Hora (32) claimed that one propagule Of.§° solani can colonize more than 2 beet 73 seedballs, so they indicated that soil clump (segment) assay was better for the quantitative assay of R. solani in soil. However, soil segment assay does not differentiate for size of propagules in soil. The beet seedball assay does differentiate between two amounts of inoculum in soil more certainly than the soil segment assay (Appendix B). It is also suggested that the beet seedball assay is more reliable for deter- mining saprophytic activity and populations of R, solani in soil. In none of the experiments with various soil moisture levels were there any differential effects on the survival of R47, R51, PR 56, and R57 after 10 weeks incubation. Since Rhizoctonia—vermiculite inoculum (RVI) used for infesting soil acted to increase populations of bacteria (18), it was thought that increased populations of bacteria might influence survival of R. solani in muck soil. Therefore experiments using washed propagules (mixtures of mycelial mat and sclerotial fragments) instead of RVI were made to eliminate the extra nutrients encouraged bacterial growth. Nevertheless the results using RVI and using washed propagules were similar. It can be concluded that the isolates of R. solani which survive poorly in muck soil are not differentially inhibited by use of RVI and accompanying bacterial growth. Both high and low soil temperature are unfavorable for survival of R. solani. Sherwood (67) in reviewing the literature notes that temperature down to 5 C are favorable for survival. Temperatures of 74 45 C or above are injurious. Ui and his associates (73) found that the "spring" strain of R, solani was active in May and June in the field (or at 20—25 C on agar medium). The "summer" strain was active in July and August (or 25-28 C on agar). In the present work, a temperature of 4 C was the most favorable for survival of both isolate R54 and R51 in muck soil. Temperature of 4 C may be unfavorable for activity of an- tagonists and also slow down the metabolic activity of R, solani. Therefore, 5, solani was little influenced by antagonists and there was little autolysis due to starvation at this temperature. Isolate R54, however, survived well in muck soil at all temperatures (4-32 C) tested compared to R51 which was relatively poor at all temperatures in a 43-day test. Therefore, no temperature between 4 and 32 was par- ticularly beneficial to survival of R51 in muck soil. In other words, the poor survival of R51 had little relationship with soil temperatures in the range of 4 to 32 C. Soil reaction (pH) of muck soil appeared to be unimportant as a factor affecting the survival of R, solani in these studies. The soil reaction varied only between pH 6.8 and 7.1 in all test samples and there were no significant pH differences between Rhizoctonia in- fested and non-infested soils. Growth rates of isolates R51 and PR56 were faster than those of isolate R54 on PDA and PDYA or on muck soil, indicating that 75 survival capability of an R, solani isolate was not correlated with growth rate. This confirmed the reports of Baker and his associates (5) but not the findings of Papavizas (46, 47). Papavizas found that growth, saprophytic activity, and survival of R, solani isolates were positively correlated. The differences in these conclusions with those of Baker and myself may be due to different isolates of R. solani used or by types of soil used for study. Sclerotia of R51 were able to survive in muck soil for 26 weeks. This may explain why R51 had not completely disappeared and could be detected by beet seedball assay after 150 days incubation (Figure 1). This finding and others (10) suggested the importance of sclerotia for survival of R. solani in soil. Nevertheless, there was no correlation between ability to produce sclerotia and mainten- ance of inoculum level in muck soil in the present work. For example, isolate R51 produced more sclerotia than isolate R54 on either agar media or in muck soil yet R51 populations dropped rapidly during a short incubation period. Baker and his associates (5) reported that a certain isolate of R. solani, producing abundant sclerotia, died out after 2-3 months but another isolate producing less sclerotia survived more than 1 year. When sclerotia of R51 and R54 were placed on muck soil for 0 to 6 weeks R51 was highly infested with bacteria. On the other hand 76 sclerotia of R54 incubated on soil for the same time had considerably less bacterial infestation. Bacteria which were isolated from such infested sclerotia were tested for their antagonistic action in vitro. Growth of both isolates R54 and R51 on PDA was inhibited by the bac- teria, but hyphal tip lysis was noted only on R51. After sclerotia of R51 had been incubated on soil for a period of time, and then placed on agar medium there was a rapid decline in hyphal growth from the sclerotia. In contrast, the activity of R54 under the same circum- stances did not change significantly. This reduction in activity of the R51 hyphae from sclerotia probably was due to the death of some sclerotial cells and lysis of the growing hyphae. The hyphae of relatively poor survivors (R51, R57) and inter— mediate survivor (PR56) lysed readily on the surface of muck soil. On the other hand the hyphae of a good survivor (R54) were relatively resistant to lysis. The hyphae of R54, after incubation on soil for 2 days, stained dark brown with rose bengal but hyphae of other iso- lates did not. This dark brown stain may demonstrate a specific cell wall constituent responsible for resistance to lysis. Potgieter and Alexander (60) reported that chitinase and B-(l-3) glucanose, which incite lysis of Fusarium sp. cell walls, did not affect the cell wall of 3. solani. They related this resistance to the presence of melanin in the hyphal cell wall of R, solani. I have concluded as a result of 77 these tests, that resistance to lysis in the hyphae of various isolates of Br solani probably can be predicted by relation to the amount of melanin in the cell wall. The ability of 59 solani isolates to colonize organic matter and to retain viability in the soil, seems very important in determining success or failure of the isolate to survive in the soil. Isolate R54 was more able to colonize organic matter in muck soil and to maintain viable mycelium in the aggregated organic particles than isolates R51, R57 and PR56. Isolate PR 56 colonized organic particles very well in the beginning, but the mycelium in the colonized particles died out rapidly after 1 week incubation. The cause of lysis in R, solani was different from one isolate to another. In the case of R57 a significant part of the lysis was due to soil microorganisms naturally occurring in muck soil. The lysis of R51 hyphae, on the other hand, was not caused by the soil microorganisms alone. The hyphae of this isolate also lysed in the absence of soil microorganisms (autolysis). An experiment testing the relative survival of B, solani in sterile soil and in sterile soil amended with a small amount of natural soil agree that R51 was much more susceptible to autolysis than R57. The lysis of R51 may result from an accumulation of toxic substances produced in metabolism, toxic substances produced by the fungus degradation of organic matter, or as a result of nutrient depletion. 78 The experimental results indicate that isolate R57 was unable to survive in muck soil essentially due to the influence of soil micro- organisms. No single microorganism has been determined to affect the survival. Pythium spp. are known to occur naturally in muck soil (18, 19, 44). However, there was no evidence that Pythium spp. affected the survival of isolate R57 in muck soil when the Rhizoctonia infested soil treated with p—dimethyl aminobenzenediazo sodium sulfonate (DASS) to eliminate the Pythium spp. was compared with soil not so treated. Therefore the antagonists must not include Pythium. This experiment also suggested that the antagonists were not in the group of other DASS sensitive organisms such as Aphanomycetes spp. and Phytophthora spp. Butler (13) reported that R, solani acted as a parasite of Pythium. This could hardly be the case in our experience as Rhizoc- Eggi§_population are low in the very muck soils where Pythium is abun- dant (l8, 19, 44). The lack of R. solani in muck soil apparently is due to absence of a resistant isolate such as R54 which may have arisen as a mutant elsewhere. Antagonists naturally occurring in muck soil would appear to be the essential cause of lysis of R, solani hyphae, reducing activ- ity of sclerotia and consequently reducing survival of this fungus in muck soil. 79 '3. solani is recognized as a soil inhabitant (25). The find- ings of Baker and his associates (5), however, suggest that the term 'soil inhabitant' can not be applied to R. solani as a species because the survival capability of Br solani varies from isolate to isolate. My findings agree with this concept. APPENDICES APPENDIX A IDENTIFICATION OF THE SPECIES RHIZOCTONIA SOLANI Because of similarities in mycelial morphology there can be some confusion between R? solani and other Rhizoctonia species (55, 56). Therefore, before studying the survival of R, solani isolates in muck soil, it is necessary to be certain of identity. Character- istics Of R, solani were reviewed by Parmeter and Whitney (55) and it is not necessary to repeat them here. The fundamental difference be- tween mycelial morphology of Thanatephorus cucumeris (R, solani) and Ceratobasidum sp. is in the number of nuclei contained in young vege- tative hyphal cells. 3, solani possesses more than 2 nuclei in a hyphal cell, but Ceratobasidium sp. has only 2 nuclei. A modification of Saksena's (63) and Hrushovetz's (30) HCl- Giemsa stain was used. Squares Of cellophane (2.5 X 2.5 cm) were auto- claved in distilled water for 15 min. to sterilize and remove wax or grease. Treated squares were placed on a water agar plate near the center of which an inoculum disc (5 mm dia.) of the test Rhizoctonia was placed. After mycelium had grown over the cellophane, squares with mycelium attached were removed and stained. HCl-Giemsa staining 80 81 procedures: 1) cellophane squares bearing Rhizoctonia mycelium were fixed in Carnoy's solution (anhydrous ethyl alcohol 60 ml, acetic acid 10 ml, and chloroform 30 ml) for 10 min.; 2) stored in 70% ethyl al- cohol for 15 min. or longer; 3) treated in 60 C HCl (1 N) for 8-10 min.; 4) rinsed in distilled water; 5) stained in Giemsa staining solution for 30 min.; 6) rinsed in distilled water for 3-5 min. and 7) placed on a clean slide with a drop of phosphate buffer solution (0.2 M) pH 6.9 for microscopic examination. Numbers of nuclei in running hyphal cells were counted. For comparing the distribution of nuclear numbers in hyphal cells of 5.5. solani isolates, 260 of the first and second hyphal tip cells were examined. All isolates of Rhizoctonia used in this study tested typically showed more than 2 nuclei per active young hyphal cell (Figure 11). Distribution of the nuclear numbers is illustrated for five isolates, R47, R51, R54, PR56, and R57 (Figure 12). The average number of nuclei of these five isolates was 6.3, 5.3, 5.5, 5.5, and 7.9 respectively. 82 FIGURE 11 FIG. ll.--Nuclei with HCL—Giemsa stain in cells of actively growing hyphae of Rhizoctonia solani (700 X). 83 I 5 .. 10 15 20 NUC LEI PER CELL FIG. 12.--Distribution of numbers Of nuclei in the first and second hyphal tip cells of five isolates of Rhizoctonia solani. HCL- Giemsa stain was used and counts were made Of 260 cells Of each isolate. APPENDIX B COMPARISON OF SOIL SEGMENT AND BEET SEEDBALL ASSAYS FOR RHIZOCTONIA SOLANI To study survival Of 3, solani in soil, it is necessary to have a reliable method for enumeration populations of R, solani. Unfortu- nately, at the present time no single method can precisely detect amounts Of R, solani in soil. Baiting materials such as plant stem segments (49, 74) or seeds (32, 44), and soil clump (32) assays have been used to enumerate populations Of Br solani in soil rencently. In this thesis, a beet seedball colonization assay and Ko's soil clump (segment) assay were used throughout all experiments. In some cases the results of these two methods did not agree very well and it is not known which method would be more reliable under all conditions. KO and Hora (32) claimed that the soil clump (segment) assay was better for quantitative assay of Rhizoctonia solani in soil than beet seedball colonization assay, because one propagule of R, solani may colonize more than one beet seedball in soil after 2 days incubation. In my Own experiments, beet seedball colonization assay proved to be more practical, and less time consuming than the soil segment (clump) assay especially when working with large numbers of soil samples. 84 85 In order to understand some advantages and disadvantages of the beet seedball colonization and soil segment assays, they were compared experimentally. The two methods were used to assay populations of five 3, solani isolates in soil: to assay different inoculum densities of R, solani; and to assay different sizes of Rhizoctonia propagules in soil. The general materials and methods were previously described in Materials and Methods. In this experiment, an equivalent of l g oven dry muck soil was infested with a given number and/or size of R. solani mycelial fragments. Infested soil was assayed immediately. Assay soil infested with five Rhizoctonia solani isolates.-- Ten 500 u dia. mycelial fragments of isolates R47, R51, R54, PR56, or R57 were added to each muck soil sample. Three replicate samples were made for each isolate and assay. The soil segment assay, when used to measure numbers of soil segments containing 5. solani, gave less variation between isolates of R, solani than did the beet seedball colonization assay (Table 11). Large variations between isolates of R, solani assayed by beet seedball colonization, due to differences in saprophytic colonization capability of the isolates. In other words, the beet seedball assay may be able to differentiate degrees of saprophytic activity for R, solani isolates in soil, but the soil segment assay does not. 86 TABLE 11. Comparison of soil segment and beet seedball colonization assays of five Rhizoctonia solani isolates. /a %, Recovery from 12' %, beet seedball 12' Isolates “’ . . . SOil segments colonization R47 47 43 R51 60 73 R54 47 63 PR56 57 100 R57 40 6O ——'One 9 soil infested with ten 500 u in dia. propagules. -—— Average of 3 replications. ‘til‘llIll'jl 87 Assay soil infested with various numbers of propagules.--Soil samples were infested with l, 2, 4, 8, 16, or 32 mycelial fragments 500 u in die. Four replications of each were made. If the soil sample contained 1, 2, 4, 8, or 16 uniformly dis- tributed propagules, the theoretical seedball colonization or soil seg— ment recovery of R, solani should be 10, 20, 40, 80, and 100%. Enumer- ation of R, solani populations in soil by the two methods (Table 12), showed that the beet seedball assay over-estimated if the number of propagules in the test soil was low. However, if the numbers of propa- gules in soil was high (8 to 16 for R54, 16 for PR56), agreement between the beet seedball assay and theoretical assay value was very good. On the other hand, the soil segment assay usually tended to underestimate populations Of R, solani in soil. The exception in this experiment was for low (l~4) numbers of propagules of PR56. There the theoretical and experimental values were in good agreement. Underestimation of R. solani populations by soil segment assay may be due to some soil seg- ments contained more than 1 propagule per segment, while other segments had none. Assay soil infested with different sizes of propagules.——Lots of soil were infested with one or ten propagules of isolate R54 250 u dia. There were four replications of each treatment. . s y. .5 I). I 88 OOH OOH he mm 00 OH om mm mm mm cm w ov mm mm ow mm v ON 0m On No NH N OH 0v 5 ON m H mucmeomm muamfiomm COHHONHGOHOO HHOm Scum cOHuMNHGOHOO HHom Scum w .dem> HHmnooom omen w wnw>oowu w HHmnoOmm noon w wnm>oomu w HHOm 0 “mm HOOHOOHOOQB medmmmOHm .Oz Ommm vmm .me>mH EDHDOOGH m>Hw suH3 moumHOmH HcmHOm chouOONHnm OBu mo m>Mmmm COHDMNHGOHOO HHmnommm noon cum mucmfiowm HHOm mo comHummEOO .NH mqmda . II! II 89 Results of this experiment showed that the beet seedball colonization assay was differential for size of R, solani propagules in the soil, but the soil segment assay was not (Table 13). 90 TABLE 13. Comparison of soil segment and beet seedball colonization assays with two different propagule sizes Of Rhizoctonia solani at two inoculum levels. Propagules 12' ' %, recovery from %, beet seedball of R54 - soil segments colonization Size, n No. Ave.£§- Ave.£2- 1 5 5 250 10 48 40 l 5 25 500 10 43 75 /a -'Propagules per g muck soil. -—-Average of 4 replications. 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