MSU LIBRARIES .——. ‘ RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. \ a mo }‘22!. :54 W II. STUDIES ON THE REACTION OF SOYBEAN TO CAUSAL AGENT OF STEM ROT OF SOYBEAN Further development of a laboratory assay for use in screening for resistance to stem rot, and field assessment of commercial cultivars Screening of soybean introductions for stem rot resistance by Laura Beth Kao A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1987 ./ fx \ /"/ k I d . J 2” ‘1‘? ABSTRACT STUDIES (Hi THE REACTION OF SOYBEAN TO SCLEROTINIA SCLERQTIORUM, CAUSAL AGENT OF STEM ROT OF SOYBEAN I. FURTHER DEVELOPMENT OF A LABORATORY ASSAY FOR USE NJSCREENING FOR RESISTANCE TO STEM ROT, AND FIELD ASSESSMENT OF COMMERCIAL CULTIVARS II. SCREENING OF SOYBEAN INTRODUCTIONS FOR STEMROT RESISTANCE By Laura Beth Kao Improvements were made upon a laboratory assay for detecting resistance in soybean to Sclerotinia gglgrggigrum. Stems of six-wk-old greenhouse-grown plants were excised, and the leavesand top portions of the plants were removed. Stems were inoculated with colonized disks of 2% millet agar. Resistance was assessed by measuring lesion lengths after seven days of incubation on moist vermiculite. A higher incidence of infection and greater uniformity in lesion lengths ‘were obtained Eur reducing the temperature of incubation from 25:3 C to 21:1 C, and by inoculating cut stem apices instead of the axils of the first trifoliate leaves. This improved assay was used to search for resistance in over 800 soybean introductions, among which several were superior to Corsoy, a known partially resistant cultivar. Field assessment of commercial soybean cultivars for resistance to stem Inn:‘was also done during three consecutive years. Wide differences in susceptibilityof cultivars were seen, with some reproducibility. To my partner in life, Cheng, and to my parents iii ACKNOWLEDGEMENT I would like to thank my graduate committee, Drs. Thomas Isleib, Melvyn Lacy, and especially my major professor, John Lockwood, for their guidance, the Soybean Promotion Committee of Michigan for its interest in this project and financial support, Rhonda Oyer for her technical assistance, various laboratory colleagues and relatives who have helped me in watering, recording,data, and planting and, finally, Cheng, who has cheerfully done more than his share of housework to allow me time to work on my thesis.IhL David Chun did the original work on this assay and trained me in its execution before leaving Dr. Lockwood's laboratory. Dr. Thomas Isleib and his staff planted and weeded the Sclerotinia-infested field plots, and Clifford Zehr and his staff irrigated the disease-infested plot and tilled both the infested and uninfested plots. My gratitude is extended for their work. iv TABLE OF CONTENTS Page List of Tables Vi List of Figures viii Literature Review IntrOduction O O O O O O O O O O O O O O O O O O O O O O O O l The organism . 1 Life cycle 2 Symptoms 2 Pathogenesis . 3 Host range and distribution 5 Economic significance . 6 Epidemiology 10 Disease control . . . 19 Assays for resistance to Sclerotinia sclerotiorum . . . . . . 22 Assays using hosts other than soybean . . . . . . 22 Assays utilizing soybean . . . . . . . . . . . . . 28 List of References . . . . . . . . . . . . . . . . . . .33 Part I. Further development of a laboratory assay for use in screening for resistance to stem rot, and field assessment of commercial cultivars Introduction . . . . . . . . . .. . . . .. . . 41 Materials and Methods . . . . . . . . . . . . . . .42 Results . . . . . . . . . . . . . . . . . . . . . .48 Discussion . . .. . . . . .. . . . .. . . .. . 69 Part II. Screening soybean introductions for stem rot resistance Introduction . . . . . . . . . . . . . . . . . 79 Materials and Methods . . . . . . . . . . . . . . .80 Results . . . . . . . . . . . . . . . . . . . . . 82 Discussion . . . . . . . . . . . . . . . . . . . . 92 List of References . . . . . . . . . . . . . . . . . . .95 Table LIST OF TABLES Page SECTION I Effect of two isolates of Sclerotinia sclerotiorum used as inoculum on lesion development in the excised-stem laboratory assay. . . . . . . . . . . . . . . . . . . . . 49 Comparison of mycelium-colonized agar and an aqueous suspension of ascospores as inocula in the excised stem assay.. . .. . .. 50 Effect of age of soybean cultivars Corsoy and Weber 84 on the development of Sclerotinia stem rot on excised stems in the laboratory.. .. . .. .. .. . .. .. 53 Effect of inoculation site on development of lesions in the laboratory assay.. .. .. . 54 Comparison of inoculation of A-cm-long petioles attached to excised stems vs. cut apices of stems. . .. . . . . .. . .H . 55 Comparison of inoculation on cut apices, chloroform-treated internodes or nail polish-treated internodes on development of lesions and percentage infection of stems in the excised stem assay.. . .. . .. . .. . 56 Effect of temperature on development of Sclerotinia stem rot in excised stems of soybean cultivars Corsoy and Evans.. . .. .. . 61 Effect of temperature of development of Sclerotinia stem rot in excised stem of soybean cultivars Corsoy and Evans. .. .. .. . 61 Harosoy isolines used to determine effect of flower color on resistance of sdybean to Eslesesisie sslssssiezse. -- . - -- ~ -- - 62 Vi Table Page 10 Mean lesion lengths resulting from cut apex inoculation of potted soybean plants Covered with a polyethylene bag and incubated on alaboratory bench for 8 days. .. . 63 ll Reactions of soybean cultivars to Sclerotinia stem rot in laboratory assays and in field tests in 1984 and 1985. . .. . . . .. . . .. . 65 12 Correlation coefficients between disease incidences of 16 commercial varieties tested in an infested field and lesion lengths obtained in laboratory assays of the same varieties grown in a non-infested field. .. . . . . .. . . . . .. . . . . 68 SECTION II 1 Plant introductions screened for resistance to stem rot by the excised stem assay.. .. .. 83 2 Plant introductions with average lesion lengths 70% or less of the average lesion length of Corsoy. . .. . . .. . . .. . . .. . 91 3 Reaction of representative plant intro- ductions to S. sclerotiorum in two laboratory tests. . . . . . . . . . . . . . . . . 92 vii Figure LIST OF FIGURES Page SECTION I Effect of age of soybean cultivars Corsoy and Weber 84 on the development of Sclerotinia stem rot on excised stems in the laboratory. .. . . .. . . .. . . .. . 52 Effect of temperature on lesion length development of Sclerotinia stem rot in excised stems of soybean cultivars Corsoy and Evans. . .. . . . . .. . . . .. . . .. . 59 Effect of temperature on percent infection of excised stems of soybean cultivars Corsoy and Evans in the laboratory assay for detecting resistance to Sclerotinia sclerotiorum. SECTION II Frequency distribution of results of screening soybean plant introductions (PI's) for resistance to stem rot caused by S. sclerotiorum using the excised stem laboratory assay. .. . . .. . .. 90 viii LITERATURE REVIEW Introduction- Stem rot or white mold of soybeans (Glycine max (L) MerrJ is causedlnrthe discomycetous fungus, Sglggggigig gglgggglgggm (Lib.) de Bary. This disease develops after bloom and causes spreading lesions on the above-ground parts of the plant accompanied by profuse growth of the fungus (72). The stem of the plant may become girdled, resulting in wilting and death of the foliage. Potential for yield loss ix: susceptible soybean cultivars is considerable (19,32). Due to the persistence of sclerotia, the fungal resting structures, and to the wide host range of the patho- gen, control is difficult and frequently unsuccessful if environmental conditions are favorable for disease develop- ment. Differences in resistance and susceptiblity among soybean cultivars have been noted (33,45). Recent interest in the development of new cultivars incorporating resis- tance to S. sclerotiorum has led to the development of assays to detect resistance (16,21,44,45L The organigm Sclerotinia gglerotlorum is an) inoperculate, discomy- cetous fungus that produces stromata (iJL, the sclerotia) free front host tissue (43). The sclerotia are hard, irregularly shaped bodies consisting of a white medulla covered by a dark, melanized rind. The sclerotia produce 1 2 stipitate apothecia which in turn produce ellipsoidal, hya- line ascospores. The name of S. sclerotiorum was changed to Whetzelinia sclerotiorum in 1972, but the previous name was restored by Linda Kohn in 1979 (43) Life cycle Summaries of the life cycle of Sclerotinia sclerotiorum have been provided by Grau (31), Purdy (66), and Sinclair (72). In brief, the fungus forms sclerotia on or in the infected plant. The sclerotia serve as the resistant, over- wintering structures and can germinate carpogenically to form apothecia when environmental conditions are favorable. Ascospores produced in the apothecia are forcibly ejected upward into the air stream, and are dispersed on air cur- rents. There is no known functional conidial or repeating stage. Hyphae derived from the ascospores colonize non- living or senescent plant parts or wounded plant tissue; from thence infection is established in healthy tissue. Sclerotia form on or in the diseased tissue, thus completing the life cycle. Alternatively, in the presence of a food source, sclerotia can germinate and infect plants directly. §ZEEEEE§ The predominant features of stem rot infection are the presence on infected plants of the causal fungus as fluffy, white mycelium and later the black, hard-rinded sclerotia. Symptoms are most often observed on the main stem 15tu>40 cm above the soil line (33). Lesions originate at leaf axils and advance up and down the stem from the node. Multiple stem infections can occur (72L Infection first appears as grayish, water-soaked lesions which become tan, then bleached white (31L The stem can be girdled, inhib- iting movement of water and nutrients to the upper foliage (72). The foliage of girdled plants wilts and assumes a grayish-green.castu This is often the first indication of stem rot infection in the field. Tissue between leaf veins is discolored and dries up while veinal tissue remains green. As the disease progresses, the leaves become tan, curl and shrivel, but remain attached to the stems. Fluffy fungal mycelium covers the diseased area during periods of high relative humidity (31). Large sclerotia that com- pletely occupy the normal pith region of stems are produced (64). Pod development and pod fill above the stem lesions are greatly reduced (33,72). Occasionally sclerotia are found in pods (72). Stem infections are rarely observed at the soil line (72), and roots do not develop symptoms (30,60). S. sclerotiorum is capable of attacking seedlings, but this occurs infrequently (66). Pathogenesis SS gglgggglgggm can initiate infection from either germinating ascospores or from mycelium derived from sclero- tia Or ascospores (46). In stem rot of soybeans, ascospores serve as primary inoculum. Ascospores infect mature or senescent flowers, and after colonizing these tissues, the mycelium invades the main stem. The process is similar to infection in beans. Much histological work on the infection process has been conducted on bean plants, and this information has been extrapolated to the infection process in soybeans. Ascospore germination and subsequent formation of an appressorial mass in 33559 were studied microscopically by Purdy (65). A germ tube emerges from the ascospore and upon contact with a surface, dichotomously branches to form a hand-like structure with the terminal cells becoming en- larged at the tips. This appressorial mass is darker in color than the hyaline hyphae from which it arises. Eventually, an infection peg appears in the circular area of each terminal cell in contact with the plant. Abawi 3; 3L (3) studied the time course of the infec- tion process in bean. Ascospores germinated within 6 hours of being atomized onto bean blossoms. Within 24 hours, extensive germ tube branching and appressorium formation occurred. The appressoria then formed infection pegs which penetrated the epidermal layer of the bean blossom and formed vesicles therein. The vesicles gave risetn>infec- tion hyphae which ramified through the petals. At 48-72 hours after inoculation, the flower parts were thoroughly colonized and had aerial hyphae growing from them. Hyphal strands extruding from the blossoms produced appressoria when in contact with other tissues, facilitating spread to other tissues. Lumsden (46) emphasized the intercellular nature of growth of the infection hyphae that arise from the vesicle. In infected stems, an organized, fan-shaped infec- tion front of hyphae growing beneath the cuticle develops and girdles the stem. Then ramifying hyphae, which are of a smaller diameter than the infection hyphae, invade the dead or dying tissue both inter- and intra-cellularly. These hyphae emerge through stomates or breaks in the cuticle, giving the cottony appearance to infected tissue. Various cell-wall degrading enzymes and the production of oxalic acid are associated with the process of pathogene- sis (46.78% The most important of the enzymes are endo- polygalacturonase, pectinase, and pectin methylesterase. These and other enzymes aid infection by digesting host tissues, thus providing nutrients for the pathogen and redu- cing mechanical resistance of the plant tissues to fungal growth. After tissue is thoroughly colonized, sclerotial ini- tials form. Microscopically, these appear as clumps of short, barre1~shaped cells. Mature sclerotia form 3-7 days after infection. Host range and distribution Purdy (66) published information on the host range of S. gglggggigrgm which included unpublished material from P.B. Adams. Adam's survey of the literature revealed that S, gglggggigrgm infects members of 64 plant families, 225 genera, and 361 species. At least one more species has since been added to this oft-cited list (9%. Given this wide host range which includes many economically important plants, it is not surprising that many crops grown in rota- tion with soybeans or in the same area as soybeans are susceptible hosts. Among these are cabbage (16,21,58), green bean (16),lettuce (l6,31),peanut (16,21L,sunflower (16,21,30,3l,37), rapeseed (28), safflower (37,55), mungbean (37), field peas (14,41), potatoes (31), celery (41), car- rots (41), dry edible beans (30,31,42), Brussels sprouts (58), cauliflower (58), kale (58), turnip (58), Jerusalem artichoke (37) and sugarbeet (30). Some weeds associated with these crops are also hosts (2,9,26). Distribution of the fungus is worldwide, although it does not occur in every locality of every country (66). Stem rot of soybeans occurs in Brazil, Canada,Hungary, India, Nepal, South Africa, and the United States (72). Sggggmig significance S; sclerotiorum can cause severe but sporadic losses to crops (1,9,18,26,30,31,42,50,53,55,57,59,61,66,79JHD. Purdy (66) cites a table compiled by P.B. Adams from which Purdy estimates that crop production losses in the United States caused by S. sclerotiorum are in the millions of dollars. S. sclerotiorum is reported to cause significant economic losses in snapbean production in New York state, with 90% of plants being infected each year In! early August (57L disease in spring-sown oil-seed rape and turnip rape in Sweden where it may cause yield losses up to 50%. Head rot 7 one of four principal diseases of safflower in Montana (55). However, almost all researchers who study diseases caused by SS sclerotiorug acknowledge the sporadic nature of infection (26,31,33,42,51,53,59,61,62,72,78L Fbr example, Grau and Radke (32) conducted field tests of soybean cultivars for three consecutive yearsimia naturally infested field.Al- though a disease severity index of 34 on a scale of O to 100 occurred the first year, no disease was found in the same field during the second year. During the third year, the disease severity index was 39. Not only can disease vary from year to year, it can also vary from site to site within the same year. Coyne 35 El- (24) obtained varied and occasionally widely different infection ratings of the same dry bean cultivars planted in the same year in two different white mold nurseries. Phrases such as "potentially destruc- tive" (31), ”the scattered nature of the disease" (59), or "frequently causes serious, but unpredictable, yield losses" (78) are added as qualifiers to descriptions of Sclerotinia- incited diseases. Because of the sporadic nature of the disease, stem rot is not considered an extremely important disease of soybean. The American Phytopathological Society's ngpgggigm 9; S31; Sggg Slgggggg says, "The disease is considered of minor importance in the United States except for local outbreaks during prolonged wet periods, when plants may be killed before maturity" (72L To paraphrase LJL Purdy, who was asked to report on the impact of Sglgrggiglg diseases at a 1979 symposium on Sglggggggig; if it were more important, more work would.have been donecniit,(66L. However, there are reports that incidence of stem rot of soybeans is increasing (15,19, 30-33). Several factors contribute to this phenomenon. The practice of narrow row spacing creates a humid, comparatively stable environment conducive to disease development (32). Irrigation has :1 similar effect because carpogenic germination of sclerotia requires water, as does germination of ascospores (1,2,32,34,70). Finally, soybean production is expanding into land formerly cropped to susceptible hosts and/or soybeans are being grown in rotation with susceptible hosts such as dry edible beans, green beans, cabbage, peanut, sunflowers and sugarbeets (19,21,30-33). When conditions are favorable for Sglggggigig infec- tion, yield losses can be significant. In field tests done with various cultivars, irrigation schemes, and row widths, Grau and Radke (32) demonstrated that S. gglggggigggm reduces yield of susceptible soybean cultivars and that disease severity and yield are inversely correlated. By omitting some irrigations during the growing season, disease severity could be reduced, resulting in 10 and 22% yield increases in 1979 and 1981, respectively, over plots given more irrigation. Chun 3E El- (19) also found an inverse correlation between disease incidence and yield. In field experiments conducted using 16 commercial soybean cultivars of varying susceptibility to stem rot, they found that for every 10% increase in disease incidence, yield was reduced by 235 kg/ha or 7.8%. Although Grau and Heimann (31) sug- gest that yield is significantly reduced when 20% or more of the soybean plants are killed by Sglggggigig stem rot, the findings of Chun 33 El- (19) would indicate that even lower amounts of disease are capable of reducing yields. In dry beans also, an inverse correlation between white mold reactions and yields of various cultivars was shown in the field (24,42). Kerr 2E 51- (42) studied the relation of the disease to yield and yield components in dry edible bean. A survey of irrigated bean fields in western Nebraska over 4 years revealed that 30% of fields were infected with white mold resulting in an average loss of yield of 13%. Seed yield, weight per 100 seeds, number of seeds per five plants and number of pods per 5 plants were all decreased in diseased plants as compared to healthy plants. A higher proportion of smaller seeds was harvested from diseased plants than from healthy plants. Reduced seed number per plant was the major component of yield loss followed by reduced seed weight. Besides the direct loss of yield due to the activity of the pathogen, there are other sources of profit loss to the soybean grower. Presence of sclerotia in a shipment of soybeans intended for human consumption can result in the rejection of the whole shipment at a foreign port of entry (9,72). Cultural practices adopted to prevent stem rot can also reduce yields. For example, elimination of irrigations 10 can reduce yield while reducing stem rot severity (32% Also, planting lower-yielding but more resistant cultivars can compromise yield (42% Epidemiology The vast majority of time during-the life cycle of S. gglggggigggm is spent as sclerotia in or on the soil (8). Sclerotia can survive for 3 to 5 years or longer (7,8,63, 70). Some of the factors which reduce longevity in soil are flooding and desiccation, burial in soil, pesticides, soil fumigants, and the activity of soil inhabitants, both macro- and microscopic (7,8,10,41,50-54,62,82).' Laboratory studies on the effect of availability of water at various points in the life cycle of S. sclerotiorug revealed that water potentials fron:-1.to -64 bars did not hamper production of sclerotia, but sclerotia could not germinate to produce apothecia at water potentials of -6 bars or lower (34). There are contradictory reports as to whether sclerotia which have been desiccated can later pro- duce apothecia (34,63). Alternate wetting and drying of sclerotia is detrimental to survival (32). Flooding has been shown to kill sclerotia in 23 to 45 days in various soil types regardless of the depth at which the sclerotia were buried (53L Various workers have studied the effects of burial on survival of sclerotia in conjunction with cultivation methods (7,50-52). Buried sclerotia are degraded more quickly than those on the soil surface (50,51). Sclerotia 11 reach the soil surface from the plant on or in which they were formed by gravity, wind, or harvesting (70) and would be subject to burial by tillage. Only sclerotia in the top 2 or 3 cm of soil are functional; below this depth, the stipes of the apothecia have greatly reduced chances of reaching the soil surface (2,51). However, sclerotia recovered from soil depths greater than 3 cm were able to germinate carpo- genically when exposed to proper conditions in the labor- atory (70). This would indicate that buried sclerotia remain capable of producing apothecia should they be returned to the soil surface through further tillage. Since sclerotia can survive for several years in the soil, the possibility of this phenomenon occurring is not remote. Further, Phillips (63) showed that the longer sclerotia remain in soil, the shorter the length of time needed for carpogenic germination to occur. Thus, Merrimanfls advice (50) to practice deep plowing to reduce Sclerotinia-incited disease was modified later (51) to caution that if deep plowing is used as a control, further plowing should be avoided that might return viable sclerotia to the soil surface. The most important detriment to sclerotial survival in the soil is the activity of soil inhabitants. Various organisms have been shown to reduce sclerotial populations of S. sclerotiorum either by preventing their formation or ‘oy parasitizing and destroying sclerotia (8,10,41,50,53,54, 82). Twenty of 60 strains of bacteria isolated from the 12 soil degraded and/or prevented germination of sclerotia lg yi££2.<82). Several fungi. Izieheéesae sppu Seaieshysisa aiaiteas. and §22£iseeaies sslszetiyezea. ere noteworthy sclerotial mycoparasites (8). Larvae of the insect, Srggy; gig sp., form holes in the rind through which other organisms, such as Trichodermg, can invade (10). Weakening of the rind of the sclerotium, 'whether through the activity of other microbes or incomplete devel- opment, can result in decreased sclerotial survivability. Several fungal contaminants, including Rhiggpgg, Fusarium, 5222;. Iziehssesse and éltszeezie frequently grew from field-collected sclerotia (37,50). Scanning electron micro- scopy has revealed that the rind cells of sclerotia produced under natural conditions were collapsed and perforated com- pared to sclerotia produced in pure culture in the labora- tory (50). These breaks in the rind can serve as portals of entry for further colonization by parasitic microorganisms. Incomplete melanization of the rind was also found to be correlated with fungal contamination during formation of sclerotia (37). Light brown, incompletely melanized sclero- tia were prone to myceliogenic germination when placed on moist sand and exhibited no dormancy. Conversely, intact, fully-melanized sclerotia remained dormant until exogenous nutrients were provided or until the rind was damaged. Interestingly, while studying the efficacy of mycopara- discovered that the presence of mycoparasites frequently 13 increased apothecial production. Such a phenomenon would be an evolutionarily advantageous trait since expedient propa- gation when threatened with death would increase the chances of an organism's genes being carried to a new generation. Whether a sclerotium is formed on the surface of the plant or inside the pith can affect its survival and germi- nation in the soil, presumably because of the difference in the exposure to microbiota. Merriman (51) collected sclero- tia from the pith or from the outside of bean stems and buried them in the soil directly, or after first inserting them into lengths of healthy bean straw, the ends of which were plugged with cotton. The sclerotia which had been formed on exposed plant surfaces were degraded more rapidly than those formed in the pith. Sclerotia which were placed directly in the soil were degraded more rapidly than those inserted in bean straws; however apothecium production was increased idi‘buried sclerotia. These findings also relate to the observation of Phillips (63) that sclerotia formed on sunflower root surfaces "invariably" begin germinating carpogenically sooner than those formed in stem cavities when placed in moist conditions in the laboratory. Populations of sclerotia in natural soils ranged from zero to less than 10 sclerotia per kilogram of soil (8} Neither the number of sclerotia in the soil (62,70) nor the number of apothecia present (12,17,52,62,81) was correlated Witfli disease incidence. Despite this, a minimum population is evidently necessary to incite a moderately severe 9P1 demic (70). 14 Sclerotium population density can be increased in the absence of a susceptible economically important host by formation of secondary sclerotia (7,8,70), or by production of sclerotia on weed hosts (8,9,26). Adams (7) buried sclerotia in the soil and followed their population changes for three years. At some of his sampling times, he found more sclerotia present than he had originally buried, which he attributed to production of secondary sclerotia. Inocu- lum density was constant in winter and spring, but declined in the summer and autumn. The decline was attributed to the detrimental effects of drying and remoistening of soil. Sclerotia under fallow conditions survived as well as those cropped to hosts. Despite heavy disease development on susceptible hosts, inoculum density did not significantly increase. Schwartz and Steadman (70) followed the population of sclerotia in the soil of a field planted first to a susceptible crop and then to a succession of resistant hosts for three years. The population density decreased from the original 6.2 sclerotia/kg of soil to l sclerotium/kg of soil at the end of this four-year span. Sclerotia need a period of conditioning before they can germinate to form apothecia. In reviewing the literature pertaining to this subject, Phillips (63) found that the minimum resting period needed by sclerotia of S. gglgggg; igggm before carpogenic germination was variously reported to be from 7 to 141 days. His own research indicated that 90 days were required before unconditioned sclerotia began 15 to germinate. By these results, sclerotia should be able to germinate in the same season in which they are formed. However, another group found that only 10-20% of sclerotia collected from the field in the fall germinated in the laboratory, whereas 70% of those collected in the spring germinated (70). Protocols for producing ascospores in the laboratory call for incubating sclerotia at 4 C for several weeks, thus simulating the requisite conditioning period (56). Cool temperatures and ample moisture are conducive to carpogenic sclerotial germination and the development of epidemics caused fur S. gglggggigggm (2,34,56,61,63,72). Continuous moisture for approximately 10 days is required for apothecial development (2). In dry climates, irrigation provides this moisture (2,70). The most conducive environ- ment for apothecial production in a dry edible bean field in Nebraska was the irrigation furrow, especially where the plant canopy shaded the soil surface (70). In heavy- canopied crops or with narrow row-spacing, dew can provide moisture at the soil surface (28). In years with heavy rainfall, increased development of apothecia with a concom- itant increase in disease incidence has been noted (61). The temperature range suitable for apothecial production ‘was 10-25 C, with an optimum of about 10<3(]J. Growth of clie fungus $3 31532 and development of lesions occurs in a similar range with an optimum of 20 i 5 C; 30 C is too hot fc>r’ fungal growth or lesion development (1,18,60,64,69,7l, 16 80). Continuous exposure to 35 C for three weeks reduced the survival of sclerotia (7); however, it would be unlikely that such conditions would occur in the field. The natural range of temperatures encountered in temperate climates should not be prohibitive to carpogenic germination and disease development. Air temperatures 10 cm above the soil in a bean field were between 10 and 30 C during the period when white mold was first observed until it approached maximum severity (10 August to 31 August 1976) (80). Ascospores need free moisture at the plant/fungus interface and a source of nutrients for infection to occur on intact plants (1,3,34,48,65). However, once they have landed onziplant, ascospores can await theform new apothecia (27). The potential for production of ascospores by a single sclerotium was calcu- lated to be as high as 2.3 x 108 (70). Apothecial produc- tion lasted for 16 and 18 weeks at two sites in Victoria, Canada (51L.Apothecia can release ascospores for 2 to 17 days, with an average of 9 days (70). V Mycelium derived from sclerotia has low competitive saprophytic ability (1,59). Sclerotia placed 1 cm (1) or over 2 tun (59) away from a plant were unable to form mycelium that would traverse the distance to the plant and cause infection. Mycelial production apparently requires an exogenous source of nutrients (1,2,37,62,65). If a dead leaf bridges the distance between the sclerotium and the plant, infection can be accomplished (2,62,65). The primary function of sclerotia, therefore, lies in the production of apothecia. Spread of S.§£lg£gglg£gm can occur by various means. Sclerotia-containing soil adhering to seedling transplants, to equipment or to animals can be a means by which the fungus is transported (8). Irrigation water can spread plant debris which contains sclerotia through a field or to another field. Debris containing 8 sclerotia per 10 g dry weight of plant material was collected from the water sur- 18 face of irrigation furrows in.bean fields (70). Sclerotia can survive passage through the digestive tract of some animals, and consequently can be spread by movement of animals which have eaten food contaminated by S. gglgggglgggm (8). Infection by ascospores adhering to pollen carried by honeybees has been shown to occur experi- mentally (76), but its prevalence as a means of dispersal under natural conditions has not been studied. There are reports of the seeds of bean and other hosts being internal- ly and externally infected with mycelium of S. sclerotiorum (58,74). Sclerotia mixed in with seed lots can spread the fungus as well (72). Air-borne ascospores are the primary means of dispersal (”3 §- gglggggigggm (1,2,15,26,31,59,62,66,80,81). Asco- spores are important in local infections (2,12,59) and in spreading disease from field to field (1,2,62,81). Williams (8) trapped ascospores 30-150 cm above the soil surface and 150 m from a source. Patterson and Grogan (62) found no apothecia in their disease study plots, but did find them in adjacent fields and orchards. Thus, they attributed disease in their plots to ascospores produced elsewhere. There is even the possibility that ascospores are carried by wind from sites of production extremely remote from infected fields. Spores identified as Sglgggglgig sp. were among those trapped during airplane flights at altitudes of 500- 18,000 feet (49). After initial infection by ascospores, secondary spread 19 may occur through plant-to-plant contact (79). This process is considered to have only a minor role in epidemics, how- ever (2). There is no functional conidial stage in Sglerg; Sigig (43), thus no secondary spread occurs by this means. Disease Control Control measures utilized against S. Sglergglgggm include tillage practices (32,36,51), weed control (26), decreased irrigation (32,52), flooding (53), and fungicides (31,69). Other potential means of control include biological control (10,41,54,7TL fumigation (12), disease forecasting (28,61), and planting resistant cultivars (31) or cultivars with.eu1 open plant architecture (9,13,23,24,29,33,7l). Deep plowing was advocated since it was more effective in burying sclerotia than harrowing (50,51). Planting in wide (76 cm) rows was shown to be less conducive to disease development than narrower rows (32). Weed control eliminates weed hosts which can harbor the pathogen and also provide nutrients for ascosporic infection of crop plants (9,26%. Steadman (75) pointed out that Sclerotinia-incited diseases are not con- trolled consistently or economically. After reviewing control options, he concluded that "resistance and micro- climate modification.appear'to be the most useful control measures for field cropsfl Although differences in resistance among cultivars of soybean and other plants have been noted, (6,11,14,16,17,20- 22,25,33,35,38-40,44,45,47,55,67,68,78,79), the resistance 20 mechanisms are not precisely known. Plant architecture has been investigated as a possible factor in disease escape (11,13,23,24,29,32,33,57,7lfih as has row spacing (32,36L The microenvironment.in dense-foliaged cultivars or close row spacing of beans or soybeans is more conducive to disease development than open-canopied cultivars or wide row spacings, respectively. Two groups (13,57) found that the microenvironment between rows was less subject to wide variations in temperature and humidity than the environment above the canopy once the plants had formed a dense leaf canopy. Blad 35 El- further found that the canopy air temperature was 7-8 G lower in heavily-irrigated plots than in plots irrigated less than half as much (13). Grau and Radke (32) hypothesized that the more stable environment beneath the canopy might keep sclerotia from wetting and drying fluctuations, which can be lethal. Their studies indicated that disease severity increased as row width decreased. In peanut, cultivars with an upright growth habit and with small leaves were less susceptible to Sglggg; Elglg giggg infection than cultivars with dense, spreading- type canopies (22). In beans, whether the growth habit was determinate or indeterminate was not the sole determinant of disease reaction; rather, the distribution of the leaf area, especially near the ground was more important (71). Physiological resistance also exists (11,22,24,39,78). Growth of the fungus through a tolerant white bean cultivar was slower than through more susceptible cultivars (78L 21 Early maturation has been associated with disease avoidance (24,29), although other reports (11,33) claim that there is no relationship between resistance and maturity group. A reddish brown, restricted lesion frequently has been noted in association with plant resistance to S. sclerotio- E m (16,21,30,64,71,73). The fungus is localized within a 10-40-mm-diameter area around the site of inoculation; color of the plant tissue in this area ranged from light brown to reddish-brown (71,73). The relation of resistance to S. gglegggigggm and flower color has been investigated. Grau 33 El (33) ob- served in field trials that soybean cultivars with white flowers were relatively susceptible, while some cultivars with purple flowers were less so. However, they doubted that resistance was expressed in the flowers themselves. Although Grau 25 El- do not appear to have been aware of it, two groups had previously shown that blossoms of beans became colonized when inoculated in the laboratory, irres- pective of flower color or genotype (71L,or’resistance of the plant to white mold (40). Additional evidence which contradicts Grau's proposal was the finding of Cline and Jacobsen (21) that two white-flowering soybean cultivars were moderately resistant in inoculations made in the green- house. Boland and Hall (16) also obtained results which failed to correlate resistance with flower color. 22 Assays for resistance 22 fislsretiaie sseretiezsa A range of reactions to S. sclerotiorum has been noted in soybean (l6,17,20,2l,33,44,45), bean (6,11,25,35,39,40, 78,79), safflower (55), sunflower (38,67), peanut (22), field peas (14), alfalfa (68), and lettuce (47). Exploit- ation of these differences could lead to development of more resistant cultivars of these crops. In temperate climates, detecting resistance in the field is usually limited to one trial per year. Furthermore, field trials are subject to the seeming capriciousness of the fungus and environment (24,32). Therefore, the development of assays utilizing greenhouse- or growth chamber-grown plants has been explored” The advantages of such assays are: l) ability to produce test material year-round and to thus test more varieties and plant lines, and 2) ability to standardize factors such as the amount of and method of application of inoculum and environmental conditions. A.disadvantage is that conditions inherent to field experiments such as row- spacing and development of a closed plant canopy that might affect disease development cannot be reproduced in the greenhouse, growth chamber, or laboratory. Assays using hosts other Ehan soybean Three-hundred and eighty-eight plant introductions (PI's) of field pea were screened for resistance to white mold using a mist-chamber assay (14). An oat kernel infested with S. sclerotiorum was placed against the base of 23 a lO-day-old pea plant which had been conditioned in a mist chamber for 24 hours previously. Inoculated plants were returned to the mist chamber for 3 days, after which they were rated on a visual scale based on lesion length. Thirty-eight PI's that had the shortest lesions were assayed a second time. The range of disease severities was higher in the second test, but the relative ranking of the lines was the same. In two other experiments, disease ratings of 10 varieties which were included inlnnfliexperiments were significantly correlated (r-0.84). Thus differences in disease reaction were identified, and results of the assay were reproducible. Various methods of inoculation were examined by Madjid g; 51. (47) during the development of an assay to detect the reaction of lettuce to S. sclerotiorum. Lettuce seedlings, 14-31 days old, were inoculated l)tnrp1acing agar blocks colonized by mycelium 1 cm from the base of the plants, 2) with a mycelium-agar suspension sprayed on the plants or 3) with sclerotia which were incorporated into the potting mix. The percentage of survivors was determined 15 days later. Of these inoculation methods, the agar block technique was most satisfactory because the mycelial spray method required undefined "optimal environmental conditions", which were not always available and the sclerotial inoculum failed to infect. Significant differences in survival percentage were detected, but all cultivars tested showed a low level of resistance. 24 Oxalic acid was applied to alfalfa plants in an attempt to develop an assay for detecting resistance to S. trifolio- £22 (68)- Like é. esleresiersa. §- trifoliersa produces oxalic acid during pathogenesis. When the oxalic acid method was compared with the applicationlof kernels colo- nized with S. trifoliorum to the surface of a tray of seed- lings, no significant relationship between results of the two assays was shown. The authors concluded that the oxalic acid procedure was unsatisfactory. Tu (78) investigated differences of reaction of different cultivars of white bean to oxalic acid. He placed the petioles of excised bean leaves in vials con- taining different concentrations of oxalic acid. After different periods of time, the percentage of leaf area with symptoms was determined. Symptoms increased with concentra- tion of oxalic acid and with duration of time the leaves were exposed to the oxalic acid solution. Although the apparent rate of diffusion of oxalic acid in leaf tissue paralleled the growth of S. sclerotiorum in bean, he did not advocate an oxalic acid assay for detection of resistance since the intent of his paper was rather to prove that oxalic acid had a role in pathogenesis. Huang and Dorrell (38) devised an assay for screening sunflower seedlings which has similarities tofhfls assay. The roots of 3-week-old seedlings were washed and the intact seedlings were placed in a vial containing autoclaved cul- ture filtrates of S. sclerotiorum. Ratings based on degree 25 of wilting were made after 24 hours. Autoclaved culture filtrates which had been lyophylized and reconstituted worked as well as those which were freshly prepared. They detected a rather narrow range of reactions, from moderately resistant to highly susceptible, in the 16 cultivars tested. They recommended that this test be used as an adjunct to field testing. Ascospores often have been used as inoculum in resis- tance assays. Tu (78) inoculated lO-day-old white beans by having apothecia discharge ascospores directly onto misted seedlings inaiplastic container. After inoculation,pots were covered with a clear plastic bag and kept in a growth room. Plants were monitored for enlargement of lesions on leaves at 2-day intervals. After 8 days, two known suscep- tible cultivars were totally collapsed while a tolerant variety showed brown flecks that rarely reached 2 mm in diameter. If the tolerant variety did begin to rot, the rate of disease progress was approximately one-third that of the susceptible cultivars. Tu tested only three cultivars and did not repeat his results. His results seem contradic- tory to those of others (3,21,48,69), who found that pre- bloom plants could not be infected unless wounds were created, and that ascospores would not infect unless a food source was present. Although not quantified, the inoculum concentration used was very high; perhaps the ascospores which did not infect were used as a nutrient source by those that did. 26 Two laboratories employed assays utilizing excised leaves in moist chambers (1,6,69L.Inoculum was placed on the center of leaves and the lesion diameters were later recorded. Saito (69) utilized ascospores cu: mycelium growing on agar as inoculum, and found that agar inoculum was preferable since ascospores could not invade directly into host tissue unless a wound was made or nutrients were added to the spore suspension. Abawi (6) used agar inoculum and found that all cultivars and breeding lines tested by this method were highly susceptible. An assay utilizing plant tissue culture methods to test bean germplasm is being developed at North Dakota State University (35L. Relative growth of bean calli on tissue culture medium containing culture filtrates of different isolates of S. sclerotiorum is taken as an index of suscep- tibility or resistance. In an investigation of inheritance of resistance in bean to white mold, Abawi 35 31. (4) developed an assay designed to simulate the natural infection process. Plants were grown in a greenhouse to the blossom stage, then were inoculated by spraying 2 m1 of a suspension containing 2000 ascospores/ml onto their blossoms. Immediately afterwards, the plants were put in a mist chamber where they were incu- bated for 1 week before being rated on a scale based on number and severity of lesions. Although they claimed that "the ascospore inoculation procedure.nappears reliable and effective in detecting high levels of resistance to white 27 mold", subsequent work has cast doubt on this claim. Hunter, one of the authors of the above-mentioned paper, and other researchers (40) attempted to repeat this work. Lines that had been reported to be resistant pre- viously were susceptible in the repeated tests. The fact that contact between blossoms and healthy green tissue was left to chance in this method was considered to'beziflaw. To remedy this, they sprayed detached blossoms with asco- spores, and then placed them in the axils of the leaves. This method produced more uniform disease incidence and severity. A subsequent experiment showed that pieces of celery petiole colonized with S. gglegggigggm were as effective as ascospore-sprayed blossoms in inciting disease. Either 95% or higher relative humidity in a growth chamber or a mist chamber was required for disease development. An experiment was conducted to determine if earlier removal of colonized celery petiole inoculum would reduce disease severity and thereby help differentiate levels of resistance (40). Inoculum was removed from 2-3 week old plants 12, 24, 36, 48, and 72 hours after inoculation. Optimal length of inoculum exposure was reported to be 24-48 hours, even though another experiment had indicated that if inoculum was left on for more than 24 hours, separation between susceptible and partially resistant cultivars was difficult. The same laboratory employed a modification of the 28 limited term inoculation method to screen Phaseolus PI's for resistance to white mold (39), then to evaluate progenies of crosses made to increase resistance (25). The change in protocol was as follows: after plants were inoculated with colonized celery pieces and incubated in a mist chamber for 48 hours, they were rated as having collapsed or upright stems. Plants with upright stems were grown on a greenhouse bench for 5 more days after which time the number of sur- viving plants was recorded. Of the 449 PI's screened, 29 had 50% or more survivors. Cultivars and breeding lines which had a low incidence of white mold in the field were highly susceptible when inoculated by this method. All lines could be killed if the inoculum was left on the stem in a humid chamber for longer than 48 hours. Breeding to build up minor genes for resistance was carried out, resulting inail7% increase in survivorship to the limited term inoculation procedure by the F4 generation (25). Assays utilizing soybean Soybean cultivars have been tested for reaction to S. 531332539333 in the field by groups located in Wisconsin (31.33), Ontario (17), and Michigan (45). Results from these tests often determined the selection of cultivars used in development of laboratory assays (l6,19,21,45,73). Cline and Jacobsen (21) in essence repeated the work of Hunter 95 91. (40) using soybean instead of bean. Asco- spores were sprayed onto blooming plants which were 29 incubated in a mist chamber, but no differences in stem rot reaction could be detected, since all cultivars except two had nearly 100% plant death. Detached flowers sprayed with ascospores and placed in the leaf axils resulted in higher disease incidence than spraying whole flowering plants with ascospores. When ascospores were sprayed on the whole plant, more disease was obtained when plants were incubated in a mist chamber than in polyethylene bags placed in a growth chamber; however, variability was greater in the mist chamber; Plants inoculated with colonized carrot pieces, then placed in polyethylene bags had no significant differ- ences in disease severity among 16 cultivars; disease severity was high in all. Finally, pieces of colonized celery were applied to the second or third node of a 4-week- old soybean by wrapping moistened cotton around the stem and inoculum, the plant was bagged and placed in a growth chamber. After 24 hours, the inoculum was removed, the plants were wetted, rebagged, and placed back in the growth chamber for a week; Statistically significant differences in the resulting disease severities were detected, but the experiment was not repeated. In order to identify stem rot-resistant cultivars adapted to Ontario, Boland and Hall (16) employed the limited term inoculation procedure as described by Hunter 35 El (40) with two modifications. Colonized green bean was used instead of celery as inoculum. Inoculated plants were incubated in the mist chamber for 50 to 144 hours (mean of 30 101 hours), before the inoculum was removed.and the plants were placed in a growthroom. This is considerably longer than the 24 hours advocated by Hunter eg 51, (40). The cultivar Evans was included in all experiments as a susceptible control. To compare the different experiments, disease ratings (0-5) were expressed as percentages of the rating for Evans in each test. Ten of the 43 cultivars tested had significantly lower ratings than Evans in one trial; the average of three trials resulted in only four cultivars having significantly lower ratings than Evans. They encountered considerable variability in their assay. For example, Corsoy 79, the least susceptible cultivar tested had a percentage disease rating of 6, 62, and 50 in three trials. The assay of Chun and Lockwood (20) utilized plants grown in a greenhouse for 3-4 weeks without fertilizer. The plants were excised at the soil level, the lateral leaves were removed, and stems were placed on 500 cc of silica sand moistened with 200 ml of distilled water in a tray lined with plastic film. The trays were tilted at 30 degrees from the horizontal.to keep the sand moist but not saturated. The stems were inoculated with 5-mm-diameter disks of S. sclerotiorum grown on 2% millet seed agar which had been coated withtL3% water agar to aid in.adhesion of the inoculum to the stem. Inoculum was placed on the axil formed by the stem and petiole of the first trifoliate leaf. Trays were covered with plastic wrap and incubated on a 31 laboratory bench for 6 days. Lesion lengths were measured from the point of inoculation to the end of the water-soaked lesion. Further tests showed that tilting the trays was not required and that moist vermiculite could be used instead of moist sand as the substratum (David Chun, personal commun- ication). Results of this assay showed significant differ- ences among cultivars, but the incidence of infection was very variable. Attempts to improve the assay of Chun and Lockwood were made by Sneller (73), who tried two modifications, both involving inoculations made on an internode of the excised soybean stem. In one, the inoculation site was rubbed with a chloroform-soaked cotton swab and allowed to dry before inoculation. In the other, nail polish was applied to the inoculation site, allowed to dry, then peeled off. Both inoculation techniques resulted inaihigh number of stems which escaped infection with resulting increase in varia- bility. High variability was also noted when the inoculum was placed on intact tissue. Sneller tried removing inoc- ulum after various periods of time, as in the limited term inoculation technique of Hunter 35 gl,(40), but found no significant relationship between cultivar reaction and dura- tion of inoculation. The concentration of millet seed in 0.3% from the usual 2.0% in an attempt to better discri- minate between reactions of susceptible and resistant varie- ties. However, inoculation with the less concentrated millet 32 seed agar resulted in many more stems escaping infection. The lesions that developed were no different in length than lesions produced by inoculation with 2.0% millet seed agar. Multiple inoculations were made along the full lengths of stems of 5- to lO-week old soybean plants, at approximately 6 cm intervals. The older the tissue,(i.e” the lower on the stem), the shorter the lesions that developed from inoculations made to sites treated with nail polish which had been stripped off after drying. This thesis describes studies done to improve the laboratory assay designed by Chun and Lockwood (20). In addition, soybean plant introductions were screened for resistance to stem rot using an improved assay in an attempt to identify resistant germplasm that could be used in breeding. Field trials of commercial soybean varieties grown in a S. sclerotiorum-infested plot were also conducted and disease development in the field was observed. LIST OF REFERENCES 10. LIST OF REFERENCES Abawi, G.S. and R.G. Grogan. 1975. Source of primary inoculum and effects of temperature and moisture on infection of beans by Eastaslinie asleresisrsa- Phytopathology 65:300-309. Abawi, G.S. and RJL Grogan. 1979. Epidemiology of diseases caused by Sclerotinia species. Phytopathology 69:899-904. Abawi, G.S., F.J. 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Flooding as a means of destroying the sclerotia of Sclerotinia sclerotiorum. Phytopathology 39:920-927. Mueller, J.D., M.N. Cline, J.B. Sinclair, and B.J. Jacobsen. 1985. An in vitro test for evaluating efficacy of mycoparasites on sclerotia of Sclerotinia sclerotiorum. .Plant Dis. 69:584-587. Mundel, H.-HH ILC. Huang, and G.C. Kozub. 1985. Sclerotinia head rot in safflower: assessment of resistance and effects on yield and oil content. Can. J. Plant Sci. 65:259-265. Mylchreest, SAL and BJLJ. Wheeler. 1987. A method for inducing apothecia from sclerotia of Sglegggigig sclerotiorum. Plant Pathology 36:16-20. Natti, JHL 1971. Epidemiology and control of bean white mold. Phytopathology 61:669-674. Neergaard, P. 1958. Mycelial seed infection of certain crucifers by Sclerotinia sclerotiorum (Lib.) d By. Plant Disease Reptr. 42:1105-1106. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 38 Newton, H.C. and L. Sequeira. 1972. Ascospores as the primary infective propagule of Sclerotinia sclerotiorum in Wisconsin. Plant Disease Reptr. 56:798-802. Nicholson, J.F” (LD. Dhingra, and J.B. Sinclair. 1973. Soil temperatures and inoculation techniques affect emergence and reisolation of Sglgggglgig sclerotiorum from soybean. Mycopathologia et Mycologia applicata 50:257-260. Nordin, K. 1986. Investigations of Sclerotinia stem rot (Sclerotinia sclerotiorum on spring sown oil crops in Sweden. (Abstr.) Can..L Plant Pathol. 8:352. Patterson,C.L.and.RJL Grogan. 1985. Differences in epidemiology and control of lettuce drop caused by Sclerotinia 3332; and S. sclerotiorug. Plant Dis. 69: 766-770. Phillips, AJLL. 1986. Carpogenic germination of sclerotia of Sclerotinia sclerotiorum after periods of conditioning in soil. J. Phytopathology 116:247-258. Phipps, P.M. and D.M. Porter. 1982. Sclerotinia blight of soybean caused by Sglggggigig minor and Sclerotinia sclerotiorum. Plant Dis. 66:163-165. Purdy, IHH. 1958. Some factors affecting penetration and infection by Sclerotinia sclerotiorum. Phytopathology 48:605-609. Purdy, IHH. 1979. Sclerotinia sclerotiorum: History, diseases and symptomology, host range, geographic distribution, and impact. Phytopathology 69:875-880. Putt, E.D. 1958. Note (“1 differences in susceptibility to Sclerotinia wilt in sunflowers. Can. J. Plant Sci. 38:380-381. Rowe, D.E. and R.E. Welty. 1984. Indirect selection for resistance to sclerotinia crown and stem rot on alfalfa. Can..L.Plant Sci. 64:145-150. Saito, I., M. Takakuwa, and T. Baba. 1968. A laboratory method for evaluating the effectiveness of fungicide on bean stem rot. Bulletin of Hokkaido Prefectural Agricultural Experiment Station 18:98-106. Schwartz, H.F. and J.R. Steadman. 1978. Factors affecting sclerotium populations of, and apothecium production by. Esleresinie sclerotiorum- Phytopathology 68:383-388. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 39 Schwartz, H.FH JZR. Steadman, and DJL Coyne. 1978. Influence «of Shgggglgfi yglgggig blossoming characteristics and canopy structure upon reaction to Sclerotinia sclerotiorug. Phytopathology 68:465-470. Sinclair, J.Bu ed. 1982. Compendium of Soybean Diseases. American Phytopathological Society, St. Paul, Minnesota, 104 pp. Sneller, C.H. 1987. Development of an assay for resistance to 2212£22ini2 221222212222 infection of soybean and investigations into the cause of variability within a genotype for tolerance to the fungus. Master of Science thesis, Michigan State University. Steadman, J.R. 1975. Nature and epidemilogical significance of infection of bean seed by Hhetzelinia sclerotiorum. Phytopathology 65:1323-1324. Steadman, J.R. 1979. Control of plant diseases caused by Sclerotinia species. Phytopathology 69:904-907. Stelfox, D” JflR. Williams, U. Soehngen, and R.G. Topping. 1978. Transport of Sclerotinia sclerotiorum ascospores by rapeseed pollen in Alberta. Plant Dis. Reptr. 62:576-579. Trutmann, P., P.J. Keane, and P.R. Merriman. 1980. Reduction of sclerotial inoculum of Sglegggigig 22l222212£22 with 222122by£i22 21222222- Soil Biol- Biochem. 12:461-465. Tu, J.C. 1985. Tolerance of white bean (Shggeglgg zulaeris) to white mold (22122221212 2212222i2222> associated with tolerance to oxilic acid. Physiological Plant Pathology 26:111-117. Tu, J.C. and W.D. Beversdorf. 1982. Tolerance to white mold (Sclerotinia gclerotiorum (Lib.) De Bary) in Ex Rico 23, a cultivar of white bean (Shggeglgg yglgggig LJ. Can.Jfl Plant Sci.62:65-69. Weiss, A., E.D. Kerr, and J.R. Steadman. 1980. Temperature and moisture influences on development of white mold disease (Sclerotinig sclerotiorum) on Great Northern beans. Plant Dis. 64:757-759. Williams, J.R. and D. Stelfox. 1979. Dispersal of ascospores of Sclerotinia sclerotiorum in relation to Sclerotinia stem rot of rapeseed. Plant Dis. Reptr. 63:395-399. 4O 82. Wu, H. and B. Nelson. 1987. Screening for mycoparasites of sclerotia of Sclerotinia sclerotiorum. (Abstr.) Phytopathology 77: In press. PART I. FURTHER DEVELOPMENT OF A LABORATORY ASSAY FOR USE IN SCREENING FOR RESISTANCE, AND FIELD ASSESSMENT OF COMMERCIAL CULTIVARS Introduction Sclerotinia stem ret (m: white mold caused by 221222212i2 2212222i2r22 (Lib ) deBery is a sporadically occurring but potentially destructive disease of soybean. In Michigan, soybeans are a major crop, grown frequently in rotation with corn and other graminaceous crops (13). Currently advocated cultivational practices such as planting in narrowly-spaced rows help to create beneath the plant canopy a cool, moist environment which is conducive to the development of the disease (11L. The pathogen has a wide host range, many members of which are economically important (21). Stem rot can be prevalent when soybeans are planted on land formerly cropped to susceptible hosts such as dry beans. Differences in susceptibility of soybeans to stem rot have been demonstrated in the field (5,6,12) and in the greenhouse (4,8,16,17). Evaluation of varietal resistance in the field is extremely laborious and permits only one cycle of evaluations during a growing season. Moreover, disease may be variable (5,11), such that selection of single resistant plants in a breeding program would be impossible. Consequently, interest in developing assays to detect resis- tance to stem rot using greenhouse- or growth chamber-grown plants has arisen (4,7JL16,17,22L This thesis describes studies done to improve the laboratory assay designed by Chun and Lockwood (7). In addition, field trials of commercial soybean varieties were conducted in a field infested with S. sclerotiorum. 41 42 MATERIALS AND METHODS The pathogen 22l2222i2i2 2212222i2222 isolate G from soybean was obtained from W. L. Casale of our department. In 1986, an isolate was obtained from soybeans growing in a field plot at the Michigan State University Plant Pathology farm and was designated isolate L. The two isolates were maintained on millet seed agar (per L: 20 g millet seed ground in a Wiley mill to pass through a sieve with 0.85 mm openings [20-mesh], 20 g agar) in 9-cm-diameter petri dishes, each containing 20 m1 agar. The agar plates were inoculated in the center and cultures were grown at 22 i 2 C for 4-6 (usually 5) days. The inoculum consisted of 5-mm-diameter disks cut withaicork borer from two concentric circles 1- 1.5 cm from the edge of the culture. Two experiments were conducted to compare the two isolates. In some experiments, ascospores were used as inoculum. To produce ascospores, sclerotia were either rinsed in 17 C running tap water for 4 wk or were placed in moist vermiculite at 4 C for 6 wk before being placed on moist sand or vermiculite and incubated at 22:2 C by a north- facing window. Ascospores were collected onto Millipore membrane filters (2 um pore size) with a vacuum apparatus as described previously'(23) and were stored at 22:2(3either in petri dishes for short periods of time (1-6 wk) or in vials over silica gel crystals for longer periods. 43 Soybean plants Seeds were germinated in moist vermiculite for two days. Uniform seedlings were transplanted into ll-cm- diameter X l4-cm-high plastic pots of 946 cc (32 on capacity, containing a potting mix consisting of a steamed mixture of sandy loam soil, sphagnum peat, and sand (523:2, vzvzv) with 3-7 (usually 3) plants per pot. Later, seeds were planted directly into the potting mix. Plants were grown for 3-7 wk in a greenhouse at various times of the year, ‘without fertilizer unless stated otherwise. The temperatures in the greenhouse ranged from 20-35 C, and day lengths were extended to 12 hr in the winter time by the use. of fluorescent lamps which were later replaced with high- intensity sodium lamps. Plants were cut off at soil level, put into polyethylene bags, and brought into the laboratory where leaves and growing tips were excised prior to inoculation. The cultivars Corsoy and Evans were used routinely in the development of the method. Corsoy was known to be partially resistant to stem rot in the field (11,12) whereas Evans was regarded as susceptible (12L Later‘, Weber 84, which was highly susceptible in the field experiments, was substituted for Evans. Inoculation procedure Excised stems were laid on 1000 cc vermiculite (2 cm depth) in plastic trays of 261(18 X 61cm dimensions. The trays were first lined with.a single layer of plastic film 44 (Borden Sealwrap, Borden Chemical Co” North Andover, MA), three layers of which also were used to cover the trays to retain moisture. The vermiculite was moistened with 500 ml distilled water, and 7 to 16 stems were arranged parallel to each other in the tray. Stems were inoculated by applying a disk of inoculum at various sites on the stems. In most experiments, inoculum was applied either to the axil of the first trifoliolate leaf or on the cut apex of the stem. In some experiments, inoculation was performed on sites on an internode which had been treated with either chloroform or clear nail polish which had dried then been stripped off. To aid in adhesion, inoculum disks were dipped iniLS% water agar before being applied to stems. Trays with inoculated stems were incubated on a laboratory bench for 5-10 days at ca. 2513 C, or in later experiments at 2111 C. In an experiment to determine whether inoculations made on cut stem apices of otherwise intact plants would yield results similar to the excised stem assay, intact plants in pots (l/pot) were brought to the laboratory where the top of the plant was excised 30 cm from the soil level leaving 3 or 4 expanded trifoliate leavescnithe plant. Colonized agar inoculum was placed on the cut stem surface. A stake was inserted in the soil and a polyethylene bag was placed over the plant and stake and bound around the lip of the pot with tape. In another experiment, the same design was used except that inoculation consisted of placing soybean flowers of unknown genotype which had been sprayed with an.ascospore 45 suspension in the axil of the second trifoliate leaf. Lesion lengths were measured as the distance from the site of inoculation to the farthest extent of the lesion as determined by scraping the edge of a ruler down the base of the lesion until it was obstructed by healthy tissue. Where more than one stem was used per treatment in a replication, mean lesion lengths in each replication were calculated as the sum of individual lesion lengths divided by the total number of inoculated plants, whether infected or not. Experimental design Various designs were employed. In general there were 5- 16 stems per treatment, arranged in 3-14 replications, with 1 to 3 (usually 1) trays serving as a replication. Signif- icant differences between means were determined by Tukey's Honestly Significant Difference test following analysis of variance. Experiments were repeated one or more times unless otherwise stated. Field experiments During three consecutive years, 1985-1987fi l6 soybean cultivars from maturity groups O-III were evaluated for reaction to Sclerotinia stem rot in a field on the Michigan State University farm. Planting and maintenance of the field were performed, respectively, by Dr. Thomas Isleib and Clifford Zehr. The field had been artificially infested in 1982 by broadcasting screenings containing sclerotia of S. gglgmgmmgmmm, which were obtained from elevators used to 46 store dry edible beans. The field was planted to soybeans in 1983, and in 1984, a test similar to the field tests of 1985-1987 was performed with 20 soybean cultivars. In each of the three years, 1985-1987, four 4-m (14 ft.) rows of each of 16 cultivars were planted 50 cm (20 in.) apart in each of five replications in a balanced 4 X 4 lattice design, using a different randomization each year. Beginning at flowering, ca. 4 cm water was applied weekly by overhead irrigation, unless otherwise stated. In September, disease was determined in the center two rows of each plot as follows: 0-no disease, l-infected plant, remaining alive, and 2-infected plant, dead. Since analyses of data showed no differences in rankings of cultivars by disease indices or incidence, the data are expressed as disease indices. In 1985, planting was done on 20 May, and disease ratings were made on 9 and 10 September. In 1986, due to late spring rains, planting was delayed until 18 June. The summer was a wet one, with rainfall occurring approximately weekly. In addition to the 16 commercial varieties, 10 soybean plant introductions (PI's) were planted in the infested field in a randomized complete block design with two replications. Disease ratings were made on 23 and 24 September. .Another plot removed from the infested field was planted on 18 June 1986 to the 16 commercial varieties and 10 PI's using 20-fpot rows of each. This latter field received no irrigation. It was weeded manually in mid-July. 47 Plants grown in the uninfested field were brought into the laboratory periodically and assayed for stem rot resistance by the excised stem method. Visual inspections for presence of apothecia and disease development were made throughout the summer of 1986 at approximately weekly intervals. Plates of exposed Sglempgmmim-selective medium (3) were placed for 3 and 4 hours during the afternoons of 8 and 18 August at six locations in the infested field, 50 cm above the ground, facing west. Both days were clear with moderate westerly winds. The plates were brought into the laboratory, wrapped with Parafilm (American Can Company, Greenwich, CT) and inspected for development of S. sclerotiorum. In 1987, planting was done on 15 May. Irrigation was initiated soon after emergence of the seedlings. Since no apothecia were found by visual inspection the previous year, sclerotia were placed at 18 sites in the field at marked locations so that apothecial development could be monitored at these known locations as well as in the rest of the field during the biweekly field monitoring. On 6 June, sclerotia from three sources were placed in styrofoam cups which had the bottoms removed and were buried at different depths in the infested field. The three sources of sclerotia were as follows: 1) sclerotia which had overwintered in soybean debris in the infested field were collected on 20 April 1987 and stored in the laboratory at 2212 C, 2) sclerotia were grown aseptically'on green beans in the laboratory and did 48 not receive a cold treatment, 3) sclerotia were collected from millet seed agar cultures of various ages which had not receivedaicold treatment. Disease ratings were made on13 and 4 September. The styrofoam cups containing the soil and sclerotia were gathered and brought back to the laboratory where the sclerotia were recovered and observed for physical damage. Upon observation in the field that some stems were considerably thicker than others, an unrepeated experiment was conducted comparing 3-field-grown plants with thin (2-4-' mm-diameter) stems and thick stems (6-10-mm-diameter). Data were analyzed by analysis of variance and differences between means were distinguished by Tukey's Honestly Significant Difference or Fisher's Protected Least Significant Difference tests. RESULTS 1221222 22 222222 Isolates G and L induced lesion lengths which were not significantly different (Table 1). Thus, after November 1986, isolate I; was used routinely. Since there were ongoing attempts to correlate the laboratory results with field results, it was considered advantageous that the isolate used in the laboratory tests be one obtained from the infested field. 49 Table 1. Effect of two isolates of Sclerotinia sclerotiormm used as inoculum on lesion development in the excised-stem laboratory assaya. Mean lesion length (cm)2 Isolate Isolate 2212;222 2 2 Corsoy 7.5 a 6.4 a Weber 84 7.8 a 6.8 a Hodgson 78 10.4 b 10.3 b aPlants were grown in the greenhouse for 6 wk. The stems were arranged in a randomized complete block design with three replications and two stems of each treatment per replication. Plants were fertilized with 0.6 g 20-20-20 NPK 2 and 4 wk after planting. bLesion lengths followed by tflu: same number were not significantly different by Tukey's Honestly Significant Difference test (£20.05). There was no interaction between cultivar and isolate of fungus by analysis of variance. 2222222222 22 i2222l22 An unrepeated experiment was designed to compare two types of inocula: mycelium-colonized agar and ascospores. Either a 5-mm-diameter plug of colonized millet seed agar or a 10 ul drop of water containing a suspension of 5500-6000 ascospores was applied to the cut end of 7-wk-old stems of four varieties of soybean (Table 2%. The resulting lesion lengthsfrom the two inoculation techniques werestrongly correlated (r-0.97), and lesions originating from the two kinds of inocula were comparable. However, in a later experiment 100 ul drops containing 300 ascospores each in a 0.1 M sucrose solution applied to cut ends or internodes largely failed to infect. The lack of infection in the 50 latter experiment was probably because the larger drops tended to roll off the inoculation sites and because the inoculum density used was lower. The first experiment indicated that the lesions originating from ascospores are comparable to those incited.by colonized agar. Because of the ease of producing and handling mycelium-colonized agar, and the precedent set by Chun and Lockwood (7), agar inoculum was used routinely. Table 2. Comparison of mycelium-colonized agar and ascospores in a water suspension as inocula in the excised stem assaya Average lesiom length (cm)§ 22l22222 2222 Ascospores Corsoy 6.3 ab 4.2 a Weber 84 11.2 abcd 7.2 abc Hodgson 78 15.2 cd 12.7 bcd Gnome 16.3 d 15.0 cd 8Plants were arranged in a completely randomized design with 6 observations per treatment. Plants were fertilized three times with 0.6 g 20-20-20 NPK 2, 4, and 6 wk after planting. bLesion lengths followed by the same letter do not differ by Tukey's Honestly Significant Difference test (2fi0.05L There is no interaction between form of inoculum and variety of soybean by analysis of variance. 22221222 22 222222 Intervals of 1 or 4 hr between excision and inoculation of stems, or keeping excised stems on ice or at ambient temperature, did not influence subsequent disease develop- ment. Therefore, cut stems were kept at ambient temperature 51 and were inoculated within 2 hr of cutting. In an unrepeated experiment, stems of six soybean varieties were excised at the base, placed in polyethylene bags and put in the coldroom at 4 C for 5 days prior to being assayed. Resul- ting lesion lengths were similar to those encountered when there was no time delay before inoculation. Despite this evidence that refrigerating or keeping plants on ice before assaying caused no difference in the resulting assay, plants were routinely assayed within 2 hr after excision in the greenhouse. Stems usually were incubated under the ambient light of the laboratory. In some instances, trays were covered with aluminum foil for 3 days to prevent upturning of the stem tips, which sometimes dislodged inoculum disks, then were incubated in the ambient light of the laboratory. 222 22 222222 Lesion development was compared in stems of Corsoy and Weber 84 plants 3, 4, 5, 6 and 7 wk old. These ages corres- ponded, respectively, to growth stages V2, V3, V4, V5-V6 and V7-Rl (9). Plants were fertilized by applying 0.6 g 20-20- 20 (NPK) to the soil in each pot once weekly after the plants reached 2 wk old. Stems were inoculated.at the cut apices and were incubated at 21:1 C. In Corsoy, lesion lengths tended to decrease in older plants, whereas in Weber 84, only the lesions in 7-wk-old plants were shorter than those in yeunger plants (Figure 1L.Differences between the 52 .AHOumuoan m5u cw mEmum powwoxm :0 Don Emum mficwuoumaum mo ucwEdon>me wcu :0 cm um>m3 cam zomuoo mum>wuaso :mmnkom we owe mo uommwm .H wuswwm 8V meatéuazme on on on n— . c . on . on V» IN. . on n.— O U 0 N a on mz<>m Iml >Ommoo .ITI , o: 53 two cultivars were greatest in plants 5, 6 or 7 wk old (S - 0.05) (Table 3). Table 3. Effect of age of soybean cultivars Corsoy and Weber 84 on the development of Sclerotinia stem rot on excised stems in the laboratory8 Plantage 222222222222 122).h (wk) Corsoy _ Weber 84 3 9.3 ab 10.3 a 4 6.7 bcd 8.3 abc 5 5.8 cd 10.3 a 6 4.7 de ‘ 9.9 a 7 2 4 e 6.2 bcd a Stems (2 per treatment per cultivar in each of 7 replications) were inoculated at excised apices with mycelial inoculum disks. b Means followed by the same letter did not differ by Tukey's Honestly’ Significant Difference test (g-0.05L There was no significant interaction between agechplant and cultivar of soybean. Smpplementary fertilizer During some of the assays, plants grown for longer than 4 wk showed symptoms of nitrogen deficiency. Several experiments indicated that the use of fertilizer to correct the deficiency resulted in reduced mean lesion lengths. For example, 0.6 g fertilizer (20-20-20, NPR) was applied to the soil surface of pots in which four cultivars were growing weekly, after the second wk. Stems from 5-wk-old plants were inoculated at cut tips and incubated at 2111C. Mean lesion length for stems of fertilized plants was 3.8 cm and 54 that for unfertilized plants was 7.6 cm. These differences were statistically significant (S - 0.05). Site of inoculation The axil of the first trifoliolate leaf and the cut stem apex were compared as inoculation sites, using stems from 4- and 5-wk-old plants of Corsoy and Evans, and an inoculation temperature of 21:1 (L Inoculation of cut terminals gave longer lesions and more infected stems than did inoculation at the leaf axils of plants at either age (Table 4). Table 4. Effect of inoculation site on development of lesions in the laboratory assaya EEEEQX EXEBE Inoculation Lesion % Lesion % site length, cm infection length, cm infection Leaf axil 3.8 58 3.7 67 Cut apex 9.6 100 12.6 100 aStems were arranged in a completely randomized design with 12 stems per treatment. Based on the field observation that leaves and petioles of some cultivars were colonized by S. pglgmpgmpgmm, but that the fungus often failed to invade stem of the plant, and that a reddish-brown resistant reaction was often seen where the colonized petiole met the main stem, experiments were conducted in which 4-5 cm of petiole of the first 55 trifoliate leaf was left on the excised stem andagar inoculum was place at the end of the petiole. Inoculation on cut apices was also included as a treatment. The lesion length resulting from inoculation of a petiole tip was a reflection of petiole length rather than of resistance of the main stem to invasion (Table 5). Thus there was no advantage to detecting differences between cultivars by this technique over inoculation of the cut stem apices. Table 5 . Comparison of inoculation of 4-cm-lonm petioles attached to excised stems vs. cut apices of stems Lesion length (mm: Inoculated on Inoculated on cut 22222222 222 2222 222 22 2222222 Corsoy 9.0 a 4.2 b Weber 84 10.9 a 4.9 b Beeson 80 10.0 a 4.3 b aStems were arranged in a randomized complete block design with five replications, with three plants of each treatment per replication. There is no interaction between variety of soybean and inoculation site. Application 2: :mggmlmm £2 treated internodes In an attempt to reproduce the findings of Sneller (22), the internodes between the first and second trifoliate leaf of soybean stems were inoculated after having been either swabbed with chloroform or treated with nail polish which was allowed to dry, then removed. Table (5 gives the results of an experiment representative of five experiments done to test the 56 different inoculation techniques. Six cultivars were employed. Corsoy and Hodgson 78 are considered to be resistant varieties; Ozzie is susceptible in laboratory assays, but escapes disease in the field; Elgin is moderately susceptible; Gnome anui Weber 84 are highly susceptible. The average lesion lengths were generally longest and incidence of infection was 100% when inoculations were made on cut stem apices. With the nail polish treatment, incidence of infection also approached 100%; lesions were usually slightly shorter than with cut end inoculation. Inoculationcuichloroform-treated sites resulted in highly variable infection incidence which was reflected in the average lesion lengths, as well. Table 6. Comparison of inoculation on cut apices, chloro- form-treated internodes or nail polish-treated internodes on development of lesions and percentage infection of stems in the excised stem assaya Lesion length (cm) Percentage infection 22222222 22213222222E 2222 222292229 22222292222 2222g Corsoy 9.8 3.5 8.1 100 66.7 100 0 Ozzie 9.9 2.2 3.8 100 50.0 66.7 Elgin 9.5 4.1 6.7 100 83.3 100.0 Hodgson 78 13.0 4.5 7.4 100 100 0 100 o Gnome 11.3 5.3 8.5 100 83.3 100.0 Weber 84 10.3 4.5 6.4 100 40.0 100 0 aThere was no interaction between cultivar and inoculation method by analysis of variance. bInoculation on cut stem apices. cInoculation on chloroform-treated internodes. dInoculation on internode treated with nail polish which was allowed to dry, then was peeled off. 57 In one test,a treatment in which the internodes were rubbed with a dry cotton swab was included as a control lest breakage of trichomes due to rubbing was complicating the chloroform-treatment inoculation technique (data not shown). Incidence of infection with this treatment was extremely low (25%) and lesions tended to be short:(4 out of 6 were less than 1 cm long). The results obtained with this treatment were similar to those obtained when inoculations were made on an untreated internode. Since the results indicated that any mechanical damage resulting from applying chloroform with a cotton swab did not result in increased disease, the dry cotton swab treatment was left out of smbsequent chloroform-treatment experiments. In another test, inoculum was placed on a shallow cut made with a razor blade on an internode. Resulting average lesion lengths for Corsoy, Ozzie, Elgin and Hodgson 78 were 2.1, 6.9, 14.5, and 5.7 cm. The lengths of the lesions were similar to those initiated at sites of nail polish application and removal. 22222222222 22 incubation To assess the effect of incubation temperature on disease development, stems of cultivars Corsoy and Evans were incubated at 15, 20, 25 and 30 C in the dark and on the laboratory bench at 25:3 C in ambient laboratory light. Plants were 4 wk old and were inoculated at cut ends. Stems incubated at 15 and 20 C had longer mean lesion lengths and a greater proportion of infected stems than those incubated 58 at 25 and 30 C (Figures 2 and 3, Table'FL Stems in trays incubated on the laboratory bench.had the least disease of all. In an experiment in which the lesion length was measured every 24 h, it was noted that once infection was establishedm can zomuoo mum>fiuasu somehow mo mEoum emmwuxm aw Do» Eoum chHuouoHum mo acoanHm>me cuwcma cowmma co musumumanu Lo uuoumm .N muswwm 8V mantémazme on nu cu m. n n N 1 m In B O N ql 3 .. N O 1 H ._ m In W ( mz<>u Iml Emmoo + .. 2 60 .Esuowuoumfium mHCHuouoHum Cu mucmumwmmu museumuoe no“ xmmmm mucumuonma mnu ca mcm>m ecu momuoo mum>wuasu :monxom we madam emmwuxo mo :oHuumMCM ucmuuma :o manumumaEmu mo uuomwm .m magma; 8V “2322523 on on cu m— L _ on row Z w. .05 mu 2 nu MN :0m 12: ngzu IAWI >Om¢oo 8. cp— 61 Table 7. Effect of temperature on development of Sclero- tinia stem rot in excised stems of soybean cultivars Corsoy and Evansa Lesion lgngth Infected glants Temperature :gml- :3)— igl QQEEQI EXEEE QQEEQX EXEBE 15 6.3 ab 7 6 ab 100 a 100 a 20 9.0 a 10.8 a 100 a 100 a 25 4.2 bc 6.7 bc 57.3 b 81.0 a 30 4.8 bc 3 0 cd 57.0 b 38.0 b Room C 1.1 c 1 8 d 14.3 c 23.7 b aStems were arranged with 7 stems per treatment in each of 3 replications. bMeans in a column followed by the same letter did not differ by Tukey's Honestly Significant Difference test (2-0.05). There was no significant interaction between variety and temperature of incubation. c25:3 0. Table 8. Effect of temperature (”1 development of Sclerotinia stem rot in excised stem of soybean cultivars Corsoy and Evans8 Lesion length (cm) Infected plants SS: Temperature (C) Corsoy Evans Corsoy Evans 20 2.7 bcd 6.5 a 42 bc 72 ab 22 4.4 abc 6.0 ab 89 a 89 a 24 0.9 d 3.0 cd 11 cd 31 cd 27 0.3 d 0.0 d 6 cd 0 d 8There were 3 stems per treatment in each of 6 replications. There was no significant interaction between variety and temperature of incubation. 62 lesion lengths were not detected in either experiment (Table 9). Variability was considerably higher using the chloro- form-treatment inoculation technique. Flower color was not related to stem reaction. Table 9. Harosoy isolines used to determine effect of flower color on resistance of soybean to EElEEBEiBifl sclerotiorum M.S.U. Illinoi Mutant 522; £5 622; E- 5222 Phenotxpe IEEE 19 Eggs 29 183096 Harosoy-L2 none Purple 8.4 6.7 183184 L62-0906 wl White 8.2 5.0 183186 L72-ll38 ”4 Near white 7.7 7.9 183185 L72-1078 W3w4 Purple 9.8 6.3 throat 183187 T235 wm Magenta 9.9 4.0 183188 L63-l612 mepsl Magenta 9.2 5.1 183097 L6l-5047 Rpslrxp Purple 8 8 4.3 8Michigan State University Accession Number bIllinois Accession number cStems of S-wk-old plants were arranged in a randomized complete block design with 5 replications, with 3 stems of each isoline per replication. Plants were fertilized with 1.5 g 12-12-12 NPK 2 and 4 wk after planting. dStems of 6-wk-old plants were arranged in a randomized complete block design with 14 replications, one plant of each isoline per replication. éggay with potted plants An experiment was done to determine whether inoculations made on cut stem apices of otherwise intact plants would yield results similar to the excised stem assay and perhaps be more closely related to results of field 63 testing. Potted plants which had their tops excised 30 cm above soil level were inoculated on the cut apex, then were covered with a polyethylene bag. Lesion lengths were significantly correlated (r-0.59, 2-0.05) with the 1985 field disease indices but not with the 1984 field data (6) (r-u27) (Table 10). A variant of this experiment in which soybean flowers of unknown genotype were sprayed with ascospores and placed in the axils of the second trifoliate leaves gave no lesions. Table 10.Mean lesion lengths resulting from cutapex inoculation of potted soybean plants covered with a polygthylene bag and incubatedinfect. There wastu>significant dif- ference between the use of agar blocks or mycelial suspen- sions as inoculum; both were effective. However, the mycelial-agar suspension required undefined optimal environ- mental conditions which were not always available, thus making colonized agar blocks the preferable inoculum. Some other assays require laborious procedures for the production of inoculum (1,2,4). For example, Boland and Hall (4) have 70 a multi-step procedure in which cultures are transferred to three different media over a l6-day period with the end result being inoculum consisting of colonized green.beans which are wrapped around soybean stems. Use of ascospores requires production of apothecia, a process that requires a conditioning period in a cold environment that has been variously reported to be from 1 to 20 wk long (20). A recently published protocol for inducing apothecia from sclerotia requires 15 wk and includes a cold treatment and exposure to near-ultraviolet light before sclerotia form fertile apothecia (l9). Ascospore use also requires making a microscopic count of ascospores and adjusting the inoculum density. Despite the innate advantages of a laboratory assay which uses agar inoculum and has no special equipment requirement, the Chun and Lockwood (7) assay was not com- pletely satisfactory because of the number of stems escaping infection and because of variability due to asynchronous initiation of lesion development. These problems limited the ability to distinguish differences among treatments or cultivars. In my work, these disadvantages were overcome to some extent by standardizing such factors as age and stem- diameter of plants, but variability was reduced most by placing the inoculum on cut stem apices rather than at the leaf axils, and by reducing the incubation temperature after inoculation from 25:3 C to 2111 C (Table 7). With these changes, infection was usually close to 100% and variability 71 in lesion lengths was also reduced. Results using plants of different ages suggested that differences in cultivar res- ponse may be greatest in plants at least 5 wk old. A remaining problem is that of reproducibility of results. Work with single plant families indicated that variability of lesion lengths resulting from inoculation was not due to genetic heterogeneity within a cultivar (22L Lack of reproducibility may be due to varying environmental conditions in the greenhouse. Cline and Jacobsen (8) made the observation that disease severity resulting from their limited-term inoculation method was affectedlnrthe light intensity under which plants were grown, with etiolated tissue being more susceptible than non-etiolated tissue. Although no experiments were done to quantify the phenomenon in the current research, it was observed that when the fluorescent lamps were replaced by high-intensity sodium lamps, the plants were shorter, developed shorter lesions, and gave less variable results than previously. However, in the course of the year, conditions within the greenhouse were still quite variable: in the summer, the lamps are turned off and white-wash is applied to the exterior of the greenhouse to reduce the temperature and light intensity, while in the winter, the days are lengthened with artificial lighting and the average temperature is lower than during the summer. It is not yet known what growing conditions will produce plants whose reaction to stem rot most closely mimics that in the field. 72 In comparing the response of a number of cultivars in the field with stem lesions of plants grown in the green- house, correlation coefficients ranged from r--0.53 to r-0.86. Correlations are not enhanced by such cultivars as Ozzie which had no disease in the field in 1985, yet is fully susceptible by the laboratory assay. Boland and Hall (5) encountered results similar to those reported here in comparing field trials and greenhouse evaluations. Correla- tion coefficients for cultivars common to both kinds of test ranged from 0.03 to 0.41 and were not significant. Dis- couragingly, my work shows that plants grown in the field also do not respond to the laboratory assay in a manner consistently reflective of their disease reaction in the field. It would intuitively seem that assays using stems from the field would be more highly correlated with field results than would stems from greenhouse-grown plants. The results of the original laboratory assay using field-grown material showed a significant correlation coefficient with field results (r-0.68, 2-0.05L However, subsequent assays never reproduced this phenomenon. In fact, the tests did not always correlate well with each other. There was a tendency for high correlations between assays done in suc- cessive weeks (Table 12) such as in the last four tests done in 1986, which gave correlation coefficients of r-0.90, 0.52, 0.72, and 0.71. However, correlation coefficients between tests which were performed after a greater time interval than one week were usually not significantly correlated. 73 Besides the work done at Michigan State University, two other groups have done field-testing of soybean cultivars in Sglgrppipia-infested fields 1J1 recent years (5,11,12L Results in one year were never identical to those of another year. Sometimes a researcher has identified the tendency of a cultivar's reaction to the pathogen, such as ”moderately susceptible" or "resistant", but later work has not con- firmed such placement. For example, Grau gg 51410,12) found the cultivar Evans to be moderately susceptible in one field study. Based on this report, the original work done to improve the assay of Chun and Lockwood (7) included Evans as the susceptible control. Then, two years of results from the field at Michigan State indicated that Evans was among the more resistant cultivars, so it was replaced by Weber 84 in experiments done to study factors influencing experi- mental variability. Boland and Hall (4) also worked exten- sively with Evans, using it as the standard against which all other cultivars were judged in their growthroom studies. However, in their field trials in 1981, 1982, and 1984, Evans had 62.4, 23.1, and 7.5% diseased plants, respec- tively. Moreover, Evans was not consistent in disease ranking relative to other cultivars that they tested. Other cultivars that they tested in two or three field trials also gave contradictory results: some were among the most sus- ceptible one year and among the most resistant another year. This phenomenon was also noted in the cultivar Hobbit in field tests done at Michigan State University (6, this thesis). 74 The problem of inconsistent disease reactions of culti- vars in field tests is complicated by the sporadic nature of disease development from year to year. In tests done three years .in a row in an infested field in Wisconsin, no disease developed during the second year (11). That study was concerned with the effects of irrigation on disease development, and its general finding was that the greater the amount of water, the greater the disease development. During the second year, the optimally irrigated plots received more natural precipitation and irrigation water than in the other two years, so "the absence of Sclerotinia stem rot”.was not expected” (11%. Similarly, the lack of disease development in the Michigan State plot during the wet summer of 1986 was surprising. Boland and Hall (5) had a general decrease in disease incidence in the three years for which they provide data. Maximum percentages of diseased plants were 82.0, 56.8, and 22.4% in 1981, 1982, and 1984, respectively. In studies at Michigan State (6,this thesis), the maximum disease indices for 1984-1987 were, respectively, 39.5, 52.4, 0.5, and 11.7%. These results and those of Boland and Hall (5) illustrate the dilemma of whether field data should even be considered valid if disease is below a certain level. The tendency has been to regard the data as insignificantzin years in‘which disease is low. Thus, field testing is rife with unresolved problems, as has been shown with variable disease develop- ment in Wisconsin (11,12), Ontario (5), and Michigan State 75 University (6,this thesis). There is the possibility that a microbiological or physical factor unfavorable to S. sclerotiorum develops in the test plots repeatedly cropped to soybean, but this would not explain a high incidence of disease development following a year in which there was none (11». The field- and laboratory-produced sclerotia that were placed in the infested field as part of the studies reported in this thesis did not appear visibly damaged when they were recovered in the fall. ‘ThiS'would.beievidence against the build-up of a microbiological component that was causing the demise of sclerotia. Another problem with the field studies done to date is that different researchers test only cultivars grown in their region and they frequently do not use the same set of cultivars in their tests each year. This results in little overlap 111 the cultivars tested by different research groups. Even when common cultivars were tested, results were sometimes discrepant, as has already been illustrated in the case of Evans. 'Howevery in some instances, results with common cultivars have been similar. As an example, field tests from Michigan State (6,this thesis) agree with those of Grau SE 51. (12) in that Corsoy and Hodgson 78 were among the more resistant cultivars, whereas Gnome was highly susceptible. Ideally, the laboratory assay should be compared with the disease reactions ofziset of cultivars with a wide range of reactions to the pathogen, based on 76 many years of field testing done at many locations. Cur- rentLy, due to the sporadic nature of the disease and varying choice of cultivars tested by the different researchers, this body of knowledge is unavailable. Another possible reason for the frequent lack of corre- lation between field and laboratory results is that an assay based on the rate of lesion expansion in the excised stems may by-pass other components of resistance that may be operating in the field. The inoculum in field conditions is presumed to be ascospores which infect the flowers; from this initial infection, the pathogen invades the soybean stem (8,10,12). Therefore, components of resistance may also reside in the flowers, or in the cuticle or wax layers on the stem. The treatments of Sneller (22) using chloroform or nail polish treatment of stems were repeated in my work to exa- mine additional components of resistance. Sneller mistaken- ly believed that treatment with chloroform removed the cuticle. Treatment with chloroform dissolves epicuticular waxes, but not the cuticle (M. Bukovac, R. Hammerschmidt, personal communications). Sneller also claimed that the nail polish treatment removed cuticular wax only. However, this treatment strips off the numerous trichomes on the stem, resulting in holes that could serve as portals of entry directly into the plant epidermal cells. (M. Bukovac, personal communication). In any case, both Sneller's and my work showed that treatment of stem internodes with chlo- roform (n: nail polish resulted in increased infection over 77 that on untreated internodes, which largely failed to develop any lesions upon inoculation. The nail polish technique yielded results similar to those obtained when the cut end was inoculated or when the epidermis and possibly some underlying cortex cells of the stem were removed with a shallow cut of a razor blade. However, variability was higher in both of the alternative inoculation techniques, and they were more labor-intensive as well. Thus, inocula- tion on the cut apices was found to be preferable to either of the alternative inoculation techniques. Flower color was proposed as another possible factor in resistance of soybean plants to infection by S. Sglggpgip; 53m, based on the observation by Grau (12) that soybean cultivars with white flowers were relatively susceptible while some cultivars with purple flowers were less so. Two groups have reported exceptions to this proposal (4,8L Work reported here with Harosoy isolines indicates that the stem tissue of plants differing only by genes that regulate flower color does not vary significantly in its ability to support fungal colonization. It is perhaps noteworthy that Corsoy frequently has been the most resistant of the cultivars tested in the laboratory as well as demonstrating relatively little disease in field tests reported in this thesis and in studies of others (11,12). Corsoy (8) and the closely related cultivar, Corsoy 79 (4), also were relatively resis- tant in growth chamber tests. 78 Whether a consistently high correlation between field and laboratory reactions of plants to stem rot can be achieved is not known. Boland and Hall (5) identified cultivar height, lodging severity, and maturity as factors which affected disease development in one or more of their three years of field trials. They rightly say, "the identi- fication of cultivar characteristics that affect the responses of soybean cultivars to disease under field condi- tions increases the difficulty of identifying sources of field resistance to this pathogen in the growth room". This, of course, applies to laboratory assays as well. Despite this, a cultivar or introductionvin which.a lesion is severely limited after stem inoculation, would seem like- ly to be highly resistant to the disease. PART II. SCREENING OF SOYBEAN INTRODUCTIONS FOR STEM ROT RESISTANCE Introduction Sclerotinia sclerotiorum (Lib.) de Bary causes stem rot or white mold, a minor but potentially destructive disease of soybean (Slygipg Egg) (Ln) Merrill (10). Control of this pathogen through cultural practices such as planting in widely spaced rows (11) can help reduce disease in wet years when it occurs, but if environmental conditions are unsuitable for disease development, then the loss in yield due to wide row spacing would be for naught. Chemical control of diseases has not been.found to be economically feasible for Michigan soybean growers (13). Control of diseases of low-value field crops such.as corn and soybean is best done by planting resistant cultivars. Cultivars resistant to the major soybean disease in Michigan, Phytophthora root rot, are available and utilized (13L Breeding for resistance to stem rot of soybean has not historically been employed. However, in the early 1980's, interest in this pathogen increased because of increasing incidence of stem rot, due to planting in narrowly spaced rows and to soybeans being planted in fields formerly cropped to other susceptible crops (6). Researchers working with dry bean developed a growth chamber assay which they used to screen plant introductions of Shgggplpg spp. in order to identify lines that could be used for breeding resistance to white mold of bean caused by S. gglggpgipgpm (14,15). This "limited-term inoculation procedure" was adapted for use in soybean by Cline and Jacobsen (8), and 79 80 was reported to be a."feasible" method for evaluating soybean cultivars for resistance to stem rot. However, this group has not pursued screening. Lockwood and Isleib (16,17) became interested in developing an assay for evaluating soybean reaction to stem rot using greenhouse- grown plants. A method using excised stems incubated on moist sand in trays in the laboratory was developed (7) and used to screen 50 plant introductions (17). Working independently, :1 group at Guelph, Ontario has further adapted the limited-term inoculation assaytu>evaluate 43 soybean cultivars for resistance to S. sclerotiorum (4). The preceding portion of the thesis described further development of the laboratory assay described by Chun and Lockwood (6). In this section, screening of plant introductions (PI's) obtained from the United States Department :of Agriculture (U.SJLA.) Soybean Germplasm Collection located at Urbana, Illinois for resistance to stem rot is described. This work was funded by the Soybean Promotion Committee of Michigan and is still in progress. Saterials and mgphpgg S. sclerotiogpm isolate L was obtained from soybean on the Michigan State University Plant Pathology farm. The fungus was maintained on 2% millet seed agar as described previously (this thesis). Agar disks were used as inoculum. Disks (5-mm diameter) were cut with a cork borer from two or three concentric circles 1-2 cm from the edge of 4-6-day-old cultures. 81 Soybean seeds were planted in ll-cm-diameter X l4-cm- high plastic pots of 946the short axis of plastic trays of 54 X 26 X 6 cm dimensions on 2000 cc vermiculite moistened with l L distilled water (1.5 cm depth). Stems were inoculated by applying a disk of agar 82 inoculum to the cut surface at the stem apices. Disks were coated with 0.3% water agar to aid their adhesion. The trays were covered with three layers of plastic film (Borden Sealwrap, Borden Chemical Co., North Andover, MA), one layer of which was also used to line the tray before addition of vermiculite. The trays were incubated on a laboratory bench at 2111 C for 6-9 (usually 7) days before rating. Resistance was determined by measuring the length of the lesion from the site of inoculation to its farthest extent as determined by scraping the edge of a ruler down the the lesion toward the stem base until it was obstructed by healthy tissue. The experimental design consisted of a randomized complete block design. There were usually eight replications, with one plant of each PI per replication, but the 60 most recently assayed PI's had only four replications each. Data were analyzed by Tukey“s Honestly Significant Difference test following analysis of variance. The cultivar Corsoy, which has been shown to be a resistant cultivar in both growthroom (8) and field (6,12) studies, was included in all experiments. Beaglse Fifty-six experiments have been done to date. Seven hundred and ninety-eight PI's have been screened (Table 1). Average lesion lengths of the PI's were expressed as percen- tages of the average lesion length of Corsoy for ease of comparing among experiments (Figure 1). Six categories were 83 Table 1. Plant introductions (PI) screened for resistance to stem rot by the excised stem assay. Maturity Maturity Maturity £1 E Erase £1 E 95222 El 2 Erase 19.986 IV 54.859 II 68.427 11 30.594 II 862 II 430 II 599 II 865 I 436 II 600 II 873 II 439 II 36.653 I 55.089-1 III 443 II 47.131 II 887 IV 446 11 54.583 III 56.563 IV 448 II 591 111 57.334 III 449 IV 592 III 58.955 IV 454 II 600 IV 59.849 IV 455 II 54.604 II 60.269-2 IV 68.457 II 606-1 IV 272 111 457-1 11 606-2 IV 279 II 461 II 607 II 296-1 11 461-1 II 608 II 296-2 III 465 11 608-1 11 970 IV 465-1 11 608-2 111 61.940 III 466 11 608-3 III 944 IV 470 III 608-4 IV 947 IV 474 II 608-5 III 62.199 IV 474-2 I 54.609 III 62.202 III 68.475 II 610-1 III 202-2 IV 475-1 II 610-4 IV 248 IV 479 III 613 III 483 III 479-1 III 614 IV 63.271 1 480 II 615 III 468 IV 481 II 615-1 III 945 IV 483 III 615-2 IV 64.698 IV 484-1 11 617 IV 747 IV 484-4 II 618 III 65.338 II 488 11 54.619 II 65.341 II 68.494 III 620 III 346 II 500 11 620-2 III 354 II 503 II 809 I 379 III 508 II 818 II 388 II 516 II 834 I 68.011 IV 521 II 853 I 398 III 521-1 III 854 I 410 II 522 II 855 00 421 II 523 III 857 I 423 III 526 II Table 1 (cont'd.). 68. 68. 68. .528 530 530-2 533—1 533-2 535 543 551-2 551-3 554 555 560 562 564 572 576 585 586 587 598 599 600 604-1 604-2 609A 609B 610 621 622 627 629 639 642 644 648 655 658 661 663 666 Maturity 95222 III II III III III III II II I I II III II II I I II I II II III II III III II III II II II II II IV III II II II II II 68. 84 .670-1 670-2 671 676 679 679-2 680 680-2 683 685 687 692 692-2 694 696 701 704 706 708 709 68.710 68 712 713 715 718 722 725 728 729 731 .732 732-1 736 741 746 748 748-1 756 759 761 Maturity Erase II II II II III IV II II II II II IV III II II III II II II II III II II II II 0 II II II III II III II II I II III III III II 68. 68. 69. 69. 70. 70. 70. .027 70 761 762 763 765 768 770 778 788 795 806 815 500 501 503 507 507 512 515 532 533 991 992 993 995 996 001 009 013 014 016 017 019 021 023 036 076 077 078 080 -3 Maturity Erase III II II II IV I II II II III II II II II I IV II III II I II II III III II III II IV III I I III II III I II III II II III Table l (cont'd.). 70. 70. 70 70 70 .084 087 089 .091 188 189 192 197 199 201 202 208 212 213 224 228 229 241 242 242-2 .242-4 243 247 .251 253 453 456 457 458 459 .460 461 462 463 466-3 466-4 467 469 469-1 470 Maturity Erase II I II II III III III II III III III IV III III II II IV I 11 IV 0 IV III II III II II II II II II II III II IV III IV III III III 70. 70. 71. 72. 72. 73. 78. 85 Maturity Erase III III I II II I IV II III III II II III II III I III III III II III IV IV IV III II III IV III II II II II III II II II II II 79. 79. 79. .243 .583 586 587 593 596 602 609 610 613 616 617 620 627 628 645 648 691 691 692 693 694 695 696 699 703 710 712 726 727 732 732 737 739 743 745 746 747 756 760 I b -3 -4 Maturity QEQEB I III II III II II II II I II III I III III III III I III III III III I II IV I II III II III I IV IV 11 0 IV II II II II III 86 Table l (cont'd.). Maturity Maturity Maturity £1 a Erase £1 a Erase £1 a Erase 79.761 II 80.828-2 IV 81.764 IV 773 II 831 III 765 I 797 111 834-1 IV 766 III 825-1 IV 834-2 Iv 767 II 835 III 837 IV 768 II 846 II 841 III 770 II 848 II 844-2 III 771 II 848-1 111 844-3 111 772 I 862-1 II 845-1 III 773 II 863 II 845-2 III 775 I 79.870-1 I 80.847-1 111 81.777 IV 870-2 III 847-2 IV 780 III 870-4 IV 81.023 IV 785 III 870-6 IV 027 III 971 II 872 III 029-1 IV 82.183 I 874 III 029N II 184 II 874-1 III 030 IV 210 IV 885 II 030-1 III 218 IV 80.459 III 031-1 III 232 III 461 111 031-2 111 235 III 80.466-1 III 81.033 I 82.246 IV 466-2 Iv 034-1 Iv 246-1 III 469 11 034-2 IV 259 IV 470 , III 035 II 263-1 IV 471 II 037-1 IV 263-2 II 471-1 III 037-2 III 263-3 11 473 Iv 037-3 III 264 IV 479 Iv 037-4 I 278 III 480 III 037-5 IV 291 IV 481 III ' 038 III 295 IV 80.485 II 81.040 I 82.296 IV 488 IV 041 III 302 111 488-1 II 041-1 III 307 IV 494 II 042-1 Iv 308 111 498-1 IV 042-2 IV 312N Iv 536 II 044-1 III 315 IV 671 II 044-2 111 325 IV 822 III 667 III 326 IV 825 III 761 III 509 IV 828-1 IV 763 II 527 IV Table 1 (cont'd.). 83. 83. 84 84 .532 534 544 554 555 558 581 853 858 868 881 881A 889 891 892 893 915 923 925 945-3 .580 609 637 665 666-1 668 668-1 673 673-1 674 .681 683 683A 686 750 810 896 921 928 954 Maturity Erase 11 IV IV IV IV IV IV IV IV IV IV IV IV IV IV IV IV IV IV I II II II II II I II II II I II II II II II II II II 85 86. 86 87 .964 965 992 .012 014 021 340 492 508 625 .671 86. 002 021 022 023 031 038 045 046 050 069 089 102 112 113 115 122 133 137-1 410 .411 416 443 454 463 737 741 878 878-2 972-1 Maturity Erase I II II II II II II II II II II II I II II II II II II II II II II II II II II I II I I I II II II II II II II 88. 88 88. 88 I43: .065 524 531 619-1 628 631 288 293 293A 294 .294-1 295 295-1 296 298 301 303 304 307 308 309 311 313 351 352 355 356 357 358 442 .443 455 468 479 484 495 497 508 777 787 Maturity E3222 II II I II II II I II II II II I II II II II II II II II II II II II II II II II II II I II II II I II I II II II Table 1 (cont'd.). 89. 89. 89 89 .pa .797 798 803 804 805-2 805-4 810 825 997 000 001 003-1 004 005-5 006-2 008 012 013 014 053 .055 055-1 056-3 057 058 059 060 061-1 063 064 .065 065-2 070 072 073 075 138 153 154 154-1 Maturity QEQEP I II II I I II II II II II 0 II II II II II II II II II I II I I I II I I II II II II II II II II II II II II 90. 91. 91. 91. 91. 88 .156 167 170 171 180 560 567 570 091 102 104 107 109 110 110-1 114 115 116 117 119 120 120-2 123 124 126 129 132-2 138 141 144 150 156 161 164 167 171 180 557 559 725-3 Maturity QEQBB II II II II II II I II II II II II II I I II II II II II II II I II II II II II II II II II II II II II II II II II 92. 92. 92 .732-1 732-2 733 .109 460 464 465 468 469 470 561 563 565 569 570 571 572 573 576 580 582 583 589 592 595 596 598 603 607 611 .625 627 629 630 633 639 649 660 661 671 Maturity Erase I I I II II II II I I I II II I II II II II II II II II II II II II II II II II II I II II II II II I II II II 89 Table l (cont'd.). Maturity Maturity Maturity £1 a Erase £1 a Erase £1 a Erase 92.677 11 132.205 0 153.233 0 681 II 206 I 234 00 683 II 207 0 235 0 684 II 214 00 236 I 687 II 215 I ' 237 0 694 II 217 00 240 0 696 11 135.589 II 247 I 698 II 590 II 249 0 705 11 142.491 I 250 I 706 I 151.249 00 252 00 92.717 11 152.361 0 153.255 I 719 II 573 00 261 0 733 II 153.203 00 263 I 734 II 208 00 264 II 748 II 209 0 265 0 93.217 II 210 00 270 0 560 II 211 00 273 0 565 II 212 00 274 I 96.152 I 213 0 171 II 214 I 96.188 11 153.215 I 193 I 217 00 195 II 218 00 201 II 219 00 97.605 II 221 00 103.414 11 222 00 131.531 I 225 00 132.201 I 226 II 203 00 229 I 204 0 230 00 I!) Suld :10 HEBWHN // 2 AVERAGE LESION LENGTH, 7 OF THAT OF CORSOY 91 arbitrarily chosen: <70%, 70-90%, 90-110%, 110-130%, 130- 160%, and >160% of the average lesion length of Corsoy. Seventeen cultivars had average lesion lengths less than 70% of those of Corsoy (Table 2). Of these, six were significan- tly different from Corsoy. Table 2. Plant introductions with average lesion lengths 70% or less of the average lesion length of Corsoy 54.600 68.708 * 80.218 54.604 70.077 84.666 * 54.606-2 70.243 * 84.750 * 54.608-2 70.485 84.625 54.608-3 79.737 86.021 54.855 * 80.459 * * Denotes significantly different than Corsoy by Tukey's Honestly Significant Difference test (£-0.05). The vast majority of PI's had longer average lesion lengths than Corsoy. Corsoy consistently had the lowest or one of the lowest average lesion lengths, although its average lesion length varied from 1.7 to 14.7 cm (average - 8.5) in different experiments. One hundred and fifty cultivars have been identified for further assaying either because they had short lesion lengths or because the experiment in which they were performed had a high coefficient of variability, making retesting desirable. About half of these have been retested with varied reproducibility of results. Table 3 shows representative results of retesting, with data expressed as average lesion lengths and percentages of the lesion length of Corsoy. In 92 many instances, resistance was confirmed in subsquent testing. Table 3. Reaction of representative plant introductions to S. sclerotiorum in two laboratory tests Iaar l Iaar 2 Ave. lesion % of Ave. lesion % of £1 assaar lasers Iasl Earaaz lasers Iasl Earaax 68.768 11.9 81 8.6 223 79.699 8.5 107 11.5 107 79.732-4 5.7 72 10.4 96 79.870-6 8.2 99 8.4 77 80.459 6.2 76 5.5 51 -80.841 7.5 90 9.8 92 81.035 3.0 60 7.9 107 81.761 3.0 62 10.5 143 82.555 5.8 298 9.9 126 Discussion Corsoy was chosen as a resistant control due to its resistance in tests done in the field (12) and laboratory tests (6,8,this thesis). There was variability in the average lesion length of Corsoy from experiment to experiment as can be seen in Table 3. For example, PI 68.768 had a lesion length of 11.9 cm in the first test, which corresponded to 81% of Corsoy's lesion length. In a second test, PI 68.768 had a shorter average lesion length, 8.6 cm, but was 223% of that of Corsoy which had an extremely short lesion. Conversely, PI 82.555 had a lesion length 298% of that of Corsoy in the first test, despite having a short average lesion length U18 cm). A second test resulted in longer lesion lengths for both Corsoy and 93 PI 82.555, so that an average lesion length of 9.9 cm for PI 82.555 was only 126% of that of Corsoy. These two examples are extreme; usually Corsoy was not so widely varying in its reaction in the assay. In fact, the author became more convinced of the validity of the laboratory assay due to Corsoy's repeatedly having one (H? the shortest lesion lengths in all but a very few tests. In the exceptions, Corsoy was intermediate among the PI's tested. EH75 were liberally selected for repeated testing either because they gave reactions comparable tn) or better than Corsoy or because the validity'of the experiment was in doubt due to Corsoy's giving an intermediate reaction. There are over 7,000 soybean PI's in the U.SJLA. Soybean Germplasm Collection. These accessions were obtained from all over the world, particularly from eastern Asia, where the soybean was first domesticated. The PI's represent a wide range of genotypes. Seeds are of widely varying sizes, shapes, and colors, and produce plants varying widely in morphology. Of the over 800 PI's subjected to the laboratory assay so far, several have been more prohibitive to the growth of S. gglgrppippgm through the stem tissue than Corsoy, the resistant control. These PI's require further testing in the laboratory, and more practically, in the field. If the resistant reaction is reproducible, these PI's can be used as sources of resistance in a breeding program. Originally, the first 500 PI's were requested and tested in numerical order, so that PI's from maturity groups 00-IV were assayed. At the 94 suggestion of the president of the Soybean Promotion Committee of Michigan, the next 400 PI's included only maturity groups 00, 0, I, and II, which represent only those adapted to Michigan. If genes for resistance are identified by this assay, chances of transferring these genes to cultivars that can be grown in Michigan are enhanced. During the screening to date, several PI's have been identified which limit the ability of S. gglggppipgpm to grow through the stem tissue. Although further testing is needed, PI's identified as being resistant in the excised stem assay can be used in breeding for resistance to stem rot. Given the range of reactions shown thus far (Figure 1), it is likely that resistance is polygenic. To increase resistance, it should be possible to combine genes through crossing lines which show resistance. LIST OF REFERENCES 10. 95 LIST OF REFERENCES Abawi, G.S. and R.G. Grogan. 1975. Source of primary inoculum and effects of temperature and moisture on Phytopathology 65:300-309. Abawi, 0.8” R” Provvidenti, and JJL Hunter. 1975. Evaluating germplasm for resistance to Whgggglipig sclerotiorum. (Abstr.) Proc..Am. Phytopathol. Soc. Ben-Yephet, Y. and S. Bitton. 1985. Use of a selective medium to study the dispersal of ascospores of Sclerotinia sclerotiorum. Phytoparasitica 13:33- 40. Boland, G. J., and R. Hall. 1986. Growthroom evaluation of soybean cultivars for resistance to Sclerotinia sclerotiorum. Canu.L Plant Sci.66z559- Boland, GgL and.Rw Hall. 1987. Evaluating soybean cultivars for resistance to Sclerotinia sclerotiorum under field conditions. Plant Dis. 71:934-936. Chung D., L.B. Kao, J.L. Lockwood, and T.G. Isleib. 1987. Laboratory and field assessment of resistance in soybean to stem rot caused by Sglggppipig sclerotiorum. Plant Dis. 71:811-815. Chun, D. and J.L. Lockwood. 1984. A laboratory method of possible use in assessing resistance of soybeans to white mold. (Abstr.) Phytopathology 74:869. Cline, MNN” and B.;L Jacobsen” 1983. Methods for evaluation of soybean cultivars for resistance to Sclerotinia sclerotiorum. Plant Dis. 67: 784-786. Fehr, W.R. and C.E. Caviness. 1977. Stages of soybean development. Iowa State Univ. Coop. Ext. Service Special Report 80. 11 pp. Grau, CJL, and H.I“ Bissonnette.l974w Whetzelinia stem rot of soybean in Minnesota. Plant Dis. Rep. 58:693-695. ll. 12. l3. 14. 15. l6. 17. 18. 19. 20. 21. 22. 96 Grau, C.R., and V. L. Radke. 1984. Effects of cultivars and cultural practices on Sclerotinia stem rot of soybean. Plant Dis. 68:56-58. Grau” CLR., V. L. Radke, and F. L. Gillespie. 1982. Resistance of soybean cultivars to §El££2£lfllé sclerotiorum. Plant Dis. 66: 506-508. Helsel, Z.R., TUJ. Johnston, and IAP. Hart. 1981. Soybean production 1J1 Michigan. Michigan State University Extension Bulletin E-1549. Hunter, J.E., M.H. Dickson, M.A. Boettger, and J.A. Cigna. 1982. Evaluation of plant introductions of Shgggplpg spp. for resistance to white mold. Plant Dis. 66:320-322. Hunter, J.E., M.H. Dickson, and J.A. Cigna. 1981. Limited-term inoculation: A method to screen bean plants for partial resistance to white mold. Plant Dis. 65:414-417. Lockwood, JJ; and TJL Isleib. 1983. Resistance of soybeans to white mold. Annual Report to the Soybean Promotion Committee of Michigan 1983-84. Lockwood, J.L. and T.G. Isleib. 1984. Resistance of soybeans to white mold. Annual Report to the Soybean Promotion Committee of Michigan. 1984. Madjid, A., S. Honma, and M.L. Lacy. 1983. A greenhouse method for screening lettuce for resistance to Ealaraaiara aalararrarss- Scientia Hortic- 18:201-206. Mylchreest, S.J. and B.E.J. Wheeler. 1987. A method for inducing apothecia from sclerotia of Sclerotinia sclerotiorum. Plant Pathology 36:16-20. .Phillips, AHJJ“ 1986. Carpogenic germination of sclerotia of Sclerotinia Sglerotiorum after periods of conditioning in soil. J. Phytopathology 116:247-258. Purdy. LJL 1979. Ealararrsia aalararrarss= History, diseases enui symptomology, host range, geographic distribution, euui impact. Phytopathology 69:875-880. Sneller, C.H. 1987. Development of an assay for soybean and d-n—v-e-s—f-i—g-a-t-i—o_n-s--i:1_t-o—-the cause of variability within a genotype for tolerance to the fungus. Master of Science thesis, Michigan State University. 97 23. Steadman, J.R. and G.E. Cook. 1974. A simple method for collecting ascospores of Hhetzelinia Sglgpgpiprgm. Plant Dis. Reporter 58:190. I'IICH IGRN STATE UNIV. LIBRARIES “WI I“ W “I W NI W '1 W IW IWI "H II "WI 9301 06881 03 312