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SOJAE AND THEIR POTENTIAL FOR BIOLOGICAL CONTROL OF PHYTOPHTHORA ROOT ROT OF SOYBEAN presented by Susan Jane Humble has been accepted towards fulfillment of the requirements for Masters degree in Plant Pathology Major professor Dateof’VKM/r ? /779 f 0-7 639 OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to book drop to remove this checkout from your record. HYPERPARASITES OF OOSPORES OF PHYTOPHTHORA MEGASPERMA VAR. SOJAE AND THEIR POTENTIAL FOR BIOLOGICAL CONTROL OF PHYTOPHTHDRA ROOT ROT OF SOYBEAN By Susan Jane Humble 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 1979 ABSTRACT HYPERPARASITES OF OOSPORES OF PHYTOPHTHORA MEGASPERMA VAR. SOJAE AND THEIR POTENTIAL FOR BIOLOGICAL CONTROL OF PHYTOPHTHORA ROOT ROT OF SOYBEAN By Susan Jane Humble The potential of oospore hyperparasites for biological control of Phytophthora root rot of soybean (Phytophthora megasperma var. sojae) was investigated. Hyperparasites of oospores were present in all of the 15 field soils tested. An average of approximately 50% of oospores were parasitized in flooded soils and in soils at 50% of water holding capacity. The frequency of hyper- parasitism was not correlated with the disease potential of soils for Phytophthora root rot. The frequency of parasitism in natural soil increased as temperature increased from 16 to 28 C. Temperature differentially affected oospore invasion by fiyphochytrium catenoides and Pythium sp. Oospores of E. megasperma var. sgjag race 7 were generally more resistant to infection than oospores from races 1 and 3. Hyperparasites did not control disease in steamed soil artificially infested with E. megasperma var. sgjag. Control was inconsistent when hyperparasites were added to soils naturally infested with the pathogen. To my husband ii ACKNOWLEDGEMENTS The author wishes to express her appreciation to the following persons whose assistance and encouragement made this thesis possible. To Professor J. L. Lockwood for his understanding, guidance and assistance as the committee chairman, To Professor E. C. Cantino and Professor M. L. Lacy for their valuable suggestions while serving as committee members, To Dr. B. Sneh for his invaluable guidance and support, To the Department of Botany and Plant Pathology for its financial assistance during the period of this research, To my husband and my family, whose patience and assistance made this thesis possible. iii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION LITERATURE REVIEW Terminology Mycoparasitic Relationships Mycoparasites of Oospores Occurrence and Importance of Hyperparasitism in Nature Effect of Environment on Mycoparasitic Relationships Use of Hyperparasites for Biological Control of Plant Pathogens MATERIALS AND METHODS Production of Phytophthora megasperma var. so'ae inoculum Estimating the Water Holding Capacity of Soil Observation and Evaluation of Oospore Para- sitism Isolation and Culture of Oospore Hyperpara- sites Dual Cultures of Hyperparasites and Phytophthora megasperma var. sojae Host Range StudIES with Oospore Hyperparasites Pathogenicity Tests with Oospore Hyperpara- sites on Soybeans Soybean Seedling Bioassay for Phytophthora Root Rot of Soybean RESULTS Occurrence and Importance of Hyperparasitism in Nature . 7 Dual Cultures of Hyperparasites and Phytophthora megasperma var. sojae Host Range Studies with Oospore Hyperpara- sites iv ix OJ UIUMJNN H 13 17 17 19 20 21 22 23 27 28 3o 30 39 39 Pathogenicity Tests with Oospore Hyperpara- sites on Soybeans Comparisons of Hyperparasite Efficiencies of Oospore Parasites Effect of Temperature on Parasitism of Oospores Comparative Susceptibilities of 008pores of Three Races of Phytophthora megasperma var. sojae to Hyperparasitic Attack Biological Control of Phytophthora Root Rot of Soybean using Hyperparasites in Soil Artificially Infested with Oospores of Phytophthora megasperma var. sojae Biological Control of Phytophthora Root Rot in Field Soils Naturally Infested with Phytophthora megasperma var. sojae DISCUSSION LIST OF REFERENCES APPENDIX 52 57 68 7a 80 TABLE LIST OF TABLES Phytophthora root rot disease ratings of field soils as determined using a soybean seedling bioassay in the laboratory. Parasitism of cultured oospores of Phytophthora megasperma var. sojae race I in natural soils. Maximum amounts of parasitism of cultured oospores of Phytophthora megasperma var. sojae race 1 occurring in soils during three weeks incubation. Parasitism of oospores of Phytophthora megasperma var. sojae race 1 by different types of hyperparasites in flooded field soils. Comparisons of susceptibilities to hyperparasitic attack in natural soil of Phytoohthora megasperma var. sojae race 1 oospores from culture and from infected soybean seedlings. Effect of oospore hyperparasites on the number of nematodes hatched from cysts of Heterodera schachtii after 10 days. Germination, survival and growth of soybean seedlings (cv, Hark) grown in soil infested with oospore hyperpara- sites for 5 weeks in a greenhouse, 33 35 38 42 1+4 TABLE 10 ll 12 13 14 15 l6 Parasitism of oospores of Phytophthora megasperma var. sojae race I produced in culture and in soybean seedlings, by various hyperparasites in steamed soil. Comparative susceptibilities of 005pores of three races of Phytophthora megasperma var. sojae to hyperparasitic attack by Hyphochytrium catenoides and Pythium sp. after 10 days. Bioassay for Phytophthora root rot in steamed soil artificially infested with Phytophthora megasperma var. sojae race oospores from soybean seedlings. Biological control of Phytophthora root rot using hyperparasites in steamed soil infested with Phytophthora megasperma var. sojae race 1 oospores isolated from soybean seedlings. Pathogenicity of hyperparasites on soybean seedlings (cv. Hark). Biological control of Phytophthora root rot by hyperparasites applied to field soils naturally infested with Phytophthora megasperma var. sojae, Trial 1. Biological control of Phytophthora root rot by hyperparasites applied to field soils naturally infested with Phytophthora megasperma var. sojae, Trial 2. Biological control of Phytophthora root rot by hyperparasites applied to field soils naturally infested with Phytophthora megasperma var. sojae, Trial 3. Parasitism of oospores of Phytophthora megasperma var. soiae race l in natural soil infested with hyperparasites. 56 58 60 62 64 65 TABLE 17 l8 Parasitism of cultured oospores of Phytgphthora megasperma var. sojae race 1 in natural soils. Parasitism of oospores of Phytophthora megasperma var. sojae race I by different types of hyperparasites in flooded field soils. 82 FIGURE LIST OF FIGURES Infection of Phytophthora megasperma var. sojae race 1 oospores from soybean seedlings by various hyperparasites in steamed soil. Effect of incubation temperature on parasitism of culture-produced oospores of Phytophthora megasperma var. sojae race 14in natural soil after 10 days. Effect of temperature on parasitism of oospores of Phytgphthora megasperma var. sojae from soybean seedlings by Hyphochytrium catenoides and Pythium gp. after 10 days. Effect of temperature on growth of hyperparasites. ix 50 51 53 INTRODUCTION Economically, the most important group of diseases affecting soybeans in Michigan is the root rot complex, of which the most widespread and damaging is that caused by PhytOphthora megasperma (Drechs.) var. sgjgg Hildb. As the soybean acreage has expanded, PhytOphthora root rot has increased in extent and severity. Resistant varieties have been available since the 1950's. Within the past few years, however, new races of the pathogen have developed for which resistance has not been bred. Given the pathogen's potential for genetic change, resistant varieties cannot be viewed as a permanent or complete method of control. Chemical control has not been prac- tical because of mammalian toxicity, lack of efficacy, or excessive costs. Alternative control methods therefore need to be investigated. Sneh et al. (59) discovered various fungal hyper- parasites of P. megasperma var. sojae oospores in natural soils. The purpose of this work was to evaluate their potential for biological control. LITERATURE REVIEW The following literature survey discusses fungal hyperparasitism in relation to biological control of plant pathogenic fungi. The review covers research which has contributed to the understanding of hyperparasitic inter— actions, and that which has directly involved biological control experiments using hyperparasites. Terminology Parasitism is a symbiotic relationship in which one member of the association derives nutrients from the other. If the host in such an association acts as a parasite in another relationship, the antagonist is termed a hyperparasite. When both parasite and host are fungi, the relationship is mycoparasitic. Two major classes of mycoparasites are recognized (9, IA). "Necrotrophs" kill host cells prior to or during their parasitic development and obtain their nu- trients from the dead cells. These parasites are also referred to as "destructive" parasites as they cause ob- vious damage to their hosts. "Biotrophic" parasites derive their nutrients from living host cells without inflicting noticeable injury to the host, at least until later stages 3 of their development. Parasites of this type which cause little or no damage to their hosts are also termed "balanced" (15). Mycoparasitic Relationships Reports of mycoparasitic relationships abound in the literature. Virtually all classes of fungi contain poten- tial hosts and all classes of fungi contain species which parasitize other fungi. Vegetative, sexual and asexual structures may be parasitized. Several excellent review articles summarize the various myCOparasitic associations which have been recognized (8, 9, 14, 15). Mycoparasites of Oospores Oospores are sexually derived spores considered relatively resistant and capable of remaining viable in soil for long periods. Their survival over time is partly attributable to their thick oospore wall, which presents a considerable barrier to attack by antagonistic micro- organisms. For example, several mycoparasites of Oomycetes are able to penetrate and destroy hyphae, sporangia and young oogonia, but are unable to parasitize oospores with fully thickened walls (26, 27). Dactylella spermatophaga overcame the physical barrier presented by oospore walls of Aphanomyces euteiches by penetrating antheridia and infecting the oospore via fertilization tubes (24). How- ever, many microorganisms exist which are able to directly penetrate and destroy mature oospores. 4 The actinomycete ActinOplanes missouriensis para- sitizes oospores of Pythium sp., Phytophthora megasperma var. sgjag, P. cactorum and Aphanomyoes euteiches (59). The class Chytridiomycetes contains several oospore mycoparasites. Rhizidiomycopsis japonicus and Canteriomyces stigeoclonii attack oospores of Pythium sp., Phytophthora megasperma var. sojae, P. cactorum and Aphanomyces euteiches (59). The host range of Hyphochytrium catenoides includes Pythium myriotylum, P. ultimum (6), Aphanomyces euteiches (6, 59), Phytophthora megasperma var. sojae, P. cactorum and Pythium sp. (59). Person et al. (#9) and Kenneth et al. (38) described Phlyctochytrium spp. parasitizing oospores of downy mildew fungi. Phytophthora megasperma var. sojae oospores also are internally parasitized by the Oomycetes‘Pythium sp. and Leptolegnia §p. (59). Hyphomycetes represent the largest known group of oospore parasites. The genus Dactylella is known for its parasitism of nematodes, and several species have been reported to infect oospores. Dactylella spermatOphaga has been cited most often for its parasitic activity. It is known to attack 18 Pythium spp., three Phytophthora spp., and Aphanomyces euteiches (2A, 48, 59). Dactylella stenocarpis, Q. anisomeres (29), Q. helminthodes (28) and Q. stenomeces (30) attack various Pythium spp. but their potential host ranges have not been fully investigated. 5 Other hyphomycetous fungi parasitic on oospores include Trichothecium arrhenopum (25), T. polyctonum (28), Trinacrum subtile (24), Diheterospora chlamydosporia, Humicola fuscoatra, Fusarium oxysporum, Cephalosporium s2. and Alternaria tenuis (59). Occurrence and Importance of Hyperparasitism in Nature Many researchers, in their reports of hyperparasitic relationships, recognize the biological control potential of these organisms. Unfortunately, most studies of hyper- parasites have been done in dual culture under laboratory conditions. We therefore know little of their behavior, importance, or even existence in nature. Direct observations of hyperparasitic associations occurring in nature are most likely to be made on fungi which develop above-ground. Rusts are highly visible and widely studied, so it is not surprising that most reports of hyperparasitism in nature involve destruction of pustules of various rusts. Darluca filum is the fungus most commonly reported to be parasitic on rusts (l, 21, 34, 36, 37, 40, 41, 45, 57). Other species associated with rusts include Fusarium bacterioides (68), Trichoderma gp., an unidentified Fungi Imperfecti species (42), Phyllosticta §E., Coniothyrium §2- (34), Monasporium uredinicolum (20) and Cladosporium §22. (37, 57). It should be noted that though the above species may destroy rust pustules, direct evidence of a parasitic relationship is often lacking. The importance of hyperparasites in controlling rust diseases in the field is difficult to assess. Cladosporium §p. was reported by Smith (57) to slow the spread of asparagus rust only under environmental conditions which favored hyperparasitism. Darluca filum was observed on over 90% of Cronartium strobilinum uredia in some oak stands (40, 41). These stands developed fewer telia than adjacent oak stands which contained little Darluca filum. Biological control therefore appeared localized (41). Ampelomyces quisqualis parasitizes perithecia of powdery mildews (35, 69). Griffiths (35) found this parasite effective mainly on lower leaves. On upper leaves the host was able to outgrow the antagonist and remain largely undamaged. Yarwood (69), however, reported it was responsible for almost complete control of clover powdery mildew in the field during 1931. Cephalothecium roseum reduced the number of viable perithecia in stroma of Dibotryon morbosum (39). Many other fungi, including Coniothyrium §E., were reported to infect Q. morbosum stroma, but evidence of true parasitic relationships was not given. Other above-ground hyperparasitic relationships observed in nature are Phgmg s2. on Taphrina mirabilis (34), and Fusarium §E., Cerebella §p. (56) and E. roseum (46) parasitizing the sphacelial stage of ergot. Natural control of above-ground plant pathogens through hyperparasitism appears to be intermittent, and 7 accounts for only a low level of pathogen population reduction. Parasitized fungal structures have been recovered from natural soil in several cases. Kenneth et al. (38) and Person et al. (49) found oospores of downy mildew fungi in infected plant material in contact with soil to be parasitized by Phlyctochytrium §pp. Sclerotia of Sclerotinia sclerotiorum and S. trifoliorum collected from natural soil were occasionally penetrated and destroyed by Coniothyrium minitans (61, 63). The frequency of para- sitism in these cases was generally low. Another approach to determining the presence of hyper- parasites in soil is to artificially infest natural soil with the potential host and observe recovered host struc- tures. Evidence of this sort may not be quantitatively reliable if the baiting inoculum has been produced in culture.- As will be discussed later, the nutritional status of a host often determines its susceptibility or resistance to a parasite. Culture-grown inoculum may therefore respond differently in soil than naturally produced inoculum. Boosalis (l3) infested natural soil with maize meal- sand cultures of Rhizoctonia solani. After eight weeks incubation, recovered hyphae were found to be parasitized by various soil fungi, but the maximum level of infection was only 18%. Sneh et al (59) baited natural soils with oospores of Phytophthora megasperma var. sojae, P. cactorum, Pythium pp. and Aphanomyces euteiches. Twelve different micro- organisms (Oomycetes, Chytridiomycetes, hyphomycetes and actinomycetes) were found to infect the fungal structures. Studies of two soils infested with different amounts of Phytophthora cactorum indicated that hyperparasites may be involved in determining the disease potential of the soils. Effect of Environment on Mycoparasitic Relationships If hyperparasites are to be used as biological control agents, it is essential that their responses to varying environmental conditions be understood. Unfortunately, studies of the effects of environmental variables on myco- parasitism have been sparse and rarely deal with the inter- actions as they occur under natural conditions. Effect of Temperature Mycoparasitic activities appear to be limited within a fairly narrow temperature range. Though the upper limits have seldom been established, most myCOparasites studied fail to infect their hosts at temperatures below 20 to 15 C. The significance of this trend, however, is not always clear. In many cases the decrease in parasitism observed at low temperatures may be cuased by a decrease in the growth rate of the fungi and not by a direct effect on the infection process. 9 Boosalis (l3) infested natural soil with Rhizoctonia solani and monitored recovered hyphae. At 28 C, up to 18% of the Rhizoctonia hyphae were parasitized by Trichoderma pp. and Penicillium vermiculatum, but no significant parasitism was observed at 18 C. Calcarisporium parasiticum is a balanced mycoparasite of several Phypalospora spp. The Optimum temperature range for its infection of P. obtusa in dual culture was 25 to 30 C. Parasitism was greatly reduced at 35 C and 10 C (10). The optimum temperature for parasitism of Sclerotinia sclerotiorum sclerotia by Coniothyrium minitans was 20 C, but in this case the relationship was not completely inhib— ited until 5 C (62). There are several instances in which temperature appears to directly affect the parasitic relationship. Ayers and Lumsden (6) studied the effect of temperatures from 35 to 10 C on invasion of oospores of Pythium myriotylum by Hyphochytrium catenoides zoospores over a period of six days. The rate of infection was greatest at 30 C, and parasitism did not occur above 30 C or below 20 C. The temperature range for production and motility of zoospores of the hyperparasite was wider than that for the infection of oospores. In dual culture, Rhizoctonia solani was an efficient parasite of Mucor recurvus at 30 or 25 C. At lower temperatures, however, the degree of parasitism dropped 10 and at 15 C M. recurvus was immune to attack (18). The infection process appeared to be affected, as growth of neither fungus was inhibited at the lower temperatures. Thamnidium elegans was highly susceptible to Piptocephalis virginiana at 25 C but was immune below 20 C. The parasite's reaction to another host, Helicostylum sp., over the same temperature range was very different. This host was resistant at the higher temperature and maximum parasitism occurred at 20 C or lower. Both hosts grew equally well over the temperature range (11). Gonatobotryum fuscum also parasitized different fungal hosts within different temperature ranges (53). Effect of Ph Trichoderma lignorum was studied as a hyperparasite of Rhizoctonia solani by Weidnling (65). He isolated a "lethal principle" from culture filtrates of T. ligporum which was toxic to host hyphae. This toxin appeared to play a role in the infection process. If the toxin was adsorbed by charcoal while the fungi were growing in dual culture, T. ligporum no longer killed its host, though patterns of physical contact remained unchanged. This toxin was rendered inactive at a high pH, and T. ligporum lost its parasitic activity under similar alkaline con- ditions. In this case, pH appears to have affected parasitism directly by inactivating a specific chemical factor necessary for death of host cells. ll Aytoun (7) found a similar effect of pH on the lethal activity of T. koningi and T. ligporum on Armillaria mellea and Polyporous schweinitzii. These two Trichoderma spp. effectively parasitized and destroyed colonies of both hosts on media at pH 3.4. The rate of destruction of hosts decreased with increasing pH and at pH 7.0, the parasites were unable to kill host hyphae. High pH apparently directly affected parasitism as host hyphae which encountered the parasite on the sides of glass petri plates above media at pH 7.0 were killed. The effect of pH cannot completely be explained by inactivation of a toxin since at high pH the parasites not only failed to kill their hosts but also failed to exhibit the normal patterns of contact with host hyphae. DeVay (23) summarized the work of Butler (17) on Rhizoctonia solani's parasitic responses to variable pH. Rhizopus spp. were most susceptible to R. solani from pH 6.7 to 7.1, while other hosts (Pythium spp.) were susceptible over a greater range of pH. Effect of Light Light intensity affects mycoparasitism in vitro but its importance as a limiting factor in nature has not been investigated. When Rhizoctonia solani was grown with various hosts on PDA under different light conditions, parasitism was found to be equal in the dark and under the diffuse daylight present in the laboratory (18). However, l3 (9, 12). Shigo (53) and Cerrato (19), on the other hand, found that high C/N ratios usually supported the greatest parasitic development by Gonatobotryon fuscum on Ceratocystis spp., and an unidentified Basidiomycete on various species. Parasitism of Physalospora spp. by Calcarisporium parasiticum was apparently unaffected by the C/N ratio of the medium (10). The previous studies were all done on artificial media in the laboratory. Boosalis (13) found that natural soil amended with green manure supported greater para- sitism of Rhizoctonia solani by Penicillium vermiculatum and Trichoderma sp. than did non-amended soil. The effect, however, may not be totally attributable to a change in the nutritional content of the soil. Use of Hyperparasites for Biological Control of Plant Pathogens Hyperparasites which occur naturally in the field may contribute to reductions of plant pathogen populations. To be economically feasible control agents, their effi— ciency and reliability must be increased. Though control potentials of many hyperparasites have been studied, few are nearing the stage of commercial application. Biological control experiments carried out under sterile and controlled environmental conditions have generally been successful (3, 13, 55, 64). When research is carried one step further to non-sterile conditions, however, the results are often disappointing. For example, 13 (9, 12). Shigo (53) and Cerrato (19), on the other hand, found that high C/N ratios usually supported the greatest parasitic development by Gonatobotryon fuscum on Ceratocystis spp., and an unidentified Basidiomycete on various species. Parasitism of Physalospora spp. by Calcarisporium parasiticum was apparently unaffected by the C/N ratio of the medium (10). The previous studies were all done on artificial media in the laboratory. Boosalis (13) found that natural soil amended with green manure supported greater para— sitism of Rhizoctonia solani by Penicillium vermiculatum and Trichoderma pp. than did non-amended soil. The effect, however, may not be totally attributable to a change in the nutritional content of the soil. Use of Hyperparasites for Biological Control of Plant Pathogens Hyperparasites which occur naturally in the field may contribute to reductions of plant pathogen pOpulations. To be economically feasible control agents, their effi- ciency and reliability must be increased. Though control potentials of many hyperparasites have been studied, few are nearing the stage of commercial application. Biological control experiments carried out under sterile and controlled environmental conditions have generally been successful (3, 13, 55, 64). When research is carried one step further to non-sterile conditions, however, the results are often disappointing. For example, 14 Allen and Haenseler (3) found that Trichoderma lignorum controlled damping-off caused by Pythium deBaryanum and Rhizoctonia solani more effectively in sterile soil than in natural soil. Boosalis (13) reported that Penicillium vermiculatum gave almost complete control of Rhizoctonia damping-off and seedling blight of peas in sterile soil, but when natural soil was used control dropped consider- ably. Some trials carried out under non-sterile conditions in controlled environments have been successful. Fedorinchik (33) found that when wheat was artificially inoculated with Puccinia triticina and its hyperparasite Darluca filum, the vegetative stage of the rust was almost totally destroyed. Similarly, the bacterial hyperparasite Bdellovibrio bacteriovorus, when inoculated onto soybean leaves with Pseudomonas glycinia, inhibited both local and systemic symptoms of soybean blight (52). Ahmed and Tribe (2) infested natural soil with sclerotia of Sclerotium cepivorum and the hyperparasite Coniothyrium minitans. The resulting control of white rot of onions was as effec- tive as that from the fungicide calomel (mercurous chloride). The most crucial step in developing biological con- trol methods for hyperparasites is in carrying research from the laboratory or greenhouse into the field. The potential control agent must not only deal with the indig- enous microorganisms but also with variable and often 15 severe environmental conditions. Darluca filum (51, 60) and Fusarium pp. (50) have different temperature and/or moisture ranges for maximum growth than their rust hosts. This places obvious limitations on their use as control agents outside the greenhouse. Nevertheless, encouraging results have been reported from experiments conducted in the field. Wollenweber (68) sprayed natural infections of Cronartium spp. with a suspension of Fusarium bactridioides spores and found that all cankers had died within one year. Mower et al. (46) sprayed plots of rye with spores of Fusarium roseum, a hyperparasite of the sphacelial stage of Claviceps purpurea, and obtained over 90% control of ergot. In addition, the Fusarium hyperparasite was able to degrade potentially dangerous ergot alkaloids. In this case com— mercial control appears possible as both host and hyper- parasite develop best under equivalent environmental conditions. Control of soil—borne pathogens has also been achieved in the field with hyperparasites. Coating dry bean and sugar beet seeds with Corticum sensu lato resulted in a 100% increase in seedling stands compared to non-treated seed plots, reflecting control of damping-off caused by Rhizoctonia solani (47). Weindling (66) achieved control of damping-off of citrus seedlings, also caused by Rhizoctonia solani, by acidifying the soil with peat moss or aluminum sulfate, The control was apparently partially l6 attributable to stimulation of natural populations of hyperparasitic Trichoderma spp. T. harzianum inoculum used to infest field plots significantly reduced damage to tomato seedlings caused by Sclerotium rolfsii (67). In this case, the hyperparasite was added to soil with a substantial food base (primarily ground ryegrass seed) which may have increased its growth in soil and allowed more vigorous parasitic attack. De la Cruz and Hubbell (22) reported that an unidentified Basidiomycete, when inoculated into nursery soil (previously fumigated per common practice), provided complete control of charcoal root rot of slash pine seedlings caused by Macrpphomina phaseolina. In view of the literature presented, hyperparasites appear to have definite potential as biological control agents for plant pathogens. Further studies should emphasize development of methods which will stimulate or favor their parasitic activities. MATERIALS AND METHODS Production of Phytophthora megasperma var. sojae inoculum Aseptic zoospores were obtained from 4 to 6 day old cultures of P. megasperma (Drechs.) var. sojae Hildb. incubated at 23 i 2 C on 10 m1 Difco lima bean agar (LBA) per 10 cm diameter petri plate (32). Cultures were flooded 5 to 6 times with 15 ml sterile distilled water at 20 min intervals. After the final washing, culture plates were flooded once more and left overnight. Zoospores were harvested from the standing water in culture plates. The concentration of zoospores in aqueous suspension was determined with a hemacytometer. One drop of cotton blue in lactophenol was added to 1 m1 of the zoospore suspen- sion to stop zoospore motility and simplify the counting procedure. Oospores were produced in culture and in soybean seedlings. To prepare oospores in culture, blocks of 3. megasperma var. ppjpp mycelium (ca. 2 x 2 x 1 mm) were transferred from 4 to 6 day old cultures on LBA to 20 m1 V-8 juice broth supplemented with 30 mg cholesterol/l in 10 cm diameter petri plates and incubated in the dark at 23 1 2 C for 5 weeks (5). Mycelial mats were collected and ground in an Omni Mixer suspended in ice water for 17 18 30 min. The suspension was washed through a series of sieves (420, 177, 74 and 44,um, in order of use) to remove the bulk of mycelium. The resulting crude oospore suspension was centrifuged for l min at 320 xlg, the supernatant liquid was removed, and the pellet of oospores was resuspended in sterile distilled water. The centri- fugation process was repeated until the oospores were free of mycelial fragments (approximately 6 times). For production of oospores in soybean seedlings, soybean seeds (cv. Hark) were soaked in 5.25% sodium hypochlorite for 3.5 to 4 min and washed at least 6 times with sterile distilled water. Seeds were then placed on potato-dextrose agar (PDA) and incubated at 23 i 2 C for 5 days. Seedlings which subsequently showed contamination were discarded. Erlenmeyer flasks (250 ml) were filled. with 20 g Capac loam soil, flooded with 25 ml sterile distilled water, and autoclaved for 1.5 hr. Approximately 10 soybean seedlings and at least 1011L zoospores of P. megasperma var. sojae race 1 were placed in each flask. Flasks were incubated at 23 i 2 C for 5 weeks. During the incubation period oospores were produced in the diseased soybean tissues. To isolate the oospores, cotyledons were aseptically removed and the seedlings were washed in sterile distilled water, frozen for 48 hr at -15 C, and ground in an Omni Mixer suspended in ice water for 30 min. The suspension was then washed through a series of sterile sieves (420, 177, 74 and 443um, in order of use) to remove 19 plant material. The crude oospore suspension was then passed through a 28,um pore size nylon mesh sieve which retained the oospores but allowed the extraneous plant material to pass through. Oospores were rinsed off the sieve, suspended in sterile distilled water and centri- fuged at least 6 times, as for cultured oospores. Oospores from culture and from soybean seedlings were maintained in aseptic conditions throughout the isolation procedure. They were stored in sterile distilled water at 4 C. Estimating the Water Holding Capacity_of Soil Certain experiments required manipulation of the water content of soils. Measurements of soil moisture were based on the soil's water holding capacity (WHO). Soil was dried at 105 C for at least 18 hr, ground with mortar and pestle, and passed through a sieve with 2 mm openings. A cone of moistened Whatman #1 filter paper was fitted into a glass funnel and 20 g of dried soil was placed inside the cone. After weighing the cone, water was slowly dripped onto the soil until the first drcp of water broke from the paper and flowed down the funnel. At this point the soil was considered to be at saturation. The WHO was expressed as the ratio of the weight of water held by soil.at saturation to the original weight of dry soil. 20 Observation and Evaluation of Oospore Parasitism To study oospore parasitism in natural soil, 20 g of soil was placed in sterile 10 cm diameter glass petri plates and flooded with 25 m1 sterile distilled water or adjusted to 50% WHO. When individual oospore parasites were to be observed, sterile 10 cm diameter glass petri plates were filled with 20 g of steamed Capac loam soil. Soil was infested by distributing 6 ml of a suspension of hyperparasite inoculum over the soil using a 10 cc syringe, bringing the soil moisture to 75% WHO. Plates containing Hyphochytrium catenoides or Pythium pp., which invade oospores via zoospores, were flooded with 20 ml sterile distilled water. Nuclepore membranes were used to retain oospores during their incubation in soil. The methods for prepa- ration of membranes were adapted from those developed by Sneh (58). Drops of an aqueous suspension of oospores were applied to Nuclepore (polycarbonate) membranes (12.5 mmz, 0.4,um pore size) on a Millipore filter base. Excess water was removed by applying a vacuum to the apparatus. The membranes were then floated on flooded soil, or placed beneath a circle of nylon net under soil maintained at less than 100% WHC, for apprOpriate incu- bation periods. For microscopic observation, membranes were removed from the incubation system and inverted onto 3 drops of 1.5% molten water agar (WA) in the hollows of depression 21 slides. The membranes were removed after the agar had cooled and several drops of distilled water and cover slips were placed over the agar surface. The slides were incubated in a moist chamber for at least 3 hr before observation. Oospores were considered parasitized when fungal hyphae grew around or from oospores, and when the hyphae were associated with dead or apparently unhealthy oospores. Unhealthy oospores contained disorganized and granulated cytoplasm. Growth of reproductive structures inside or on the surface of oospores was also indicative of parasitism. Isolation and Culture of Oospore Hyperparasites Oospore hyperparasites were originally isolated from natural soils. Two Cephalosporium isolates, Dactylella ppermatqphaga Drechs., Diheterospora chlamydosporia (Komischko) Barron & Onions, Fusarium oxysporum (Schlecht.) Snyd. & Hans., Humicola fuscoatra Traaen., and Hyphochytrium catenoides Karling were isolated by Sneh et al. (59). Pythium pp. was isolated by the author. Nuclepore membrane filters carrying cultured oospores of PhytOphthora megasperma var. ppjpp race 1 were floated on flooded Capac loam soil. The membranes were removed after 6 days incubation in the dark at 23 i 2 C and the oospores were transferred to 1.5% WA for microscopic observation. Hyphae associated with parasitized oospores were cut from 22 the agar surface and transferred to acidified PDA (pH 5.5). After several transfers to acidified PDA the fungus was in pure culture and identified as Pythium pp. All of the hyperparasitic fungi were stored on PDA slants at 4 C. To prepare hyperparasite inoculum for infestation of soil, four 3- to 4-week-old cultures were scraped from the surface of PDA. The cultures were ground in sterile distilled water with a glass tissue homogenizer and the suspension was then centrifuged at 575 x g for 3 to 5 min. After the liquid supernatant was removed the mycelial pellet was resuspended in 40 m1 sterile distilled water for storage at 4 C. For use, 10 ml of the suspension was diluted in 40 m1 sterile distilled water. Dual Cultures of Hyperparasites and PhytOphthora megasperma var. sojae Petri dishes (10 cm diameter) containing corn—meal agar (CMA) or PDA were inoculated on one side with blocks of mycelium (ca. 2 x 2 x 1 mm) from 2 week old PDA cultures of oospore hyperparasites. Two PDA and two CMA plates were prepared for each hyperparasite. The Opposite sides of plates were inoculated with mycelial blocks (ca. 2 x 2 x 1 mm) from 5 day old LBA cultures of 3. megasperma var. ppjpp_race l. Plates were incubated in the dark at 23 i 2 C for 3 weeks. At approximately 1 week intervals, plates were observed for the presence of inhibition zones. Two blocks of agar (ca. 12.5 sq mm) 23 were removed from each plate from areas of hyphal inter- action and observed microscopically for evidence of para- sitism of Phytophthora hyphae. Host Range Studies with Oosppre Hyperparasites Glomus fasciculatus Sand containing chlamydospores of p. fasciculatus, a mycorrhizal fungus of soybeans, was provided by Dr. Gene Safir (Michigan State University). To isolate spores, the sand/spore mixture was washed through a sieve with 2 mm openings into a 3 gal metal bucket. After swirling the bucket to bring chlamydospores into aqueous suspension, the water was poured into centrifuge tubes and centrifuged 4 min at 575 x g. After the super- natant liquid was removed, the pellet containing spores was resuspended in distilled water saturated with sucrose and centrifuged for 1.5 min at 575 x g, The supernatant which now contained spores was immediately poured through a 44,nm sieve which retained the spores. After several washings to remove the sucrose, spores were transferred to test tubes and suspended in distilled water for storage at 4 C. Chlamydospores were impacted on Nuclepore membranes and placed over 20 g steamed Capac loam soil in sterile 10 cm diameter petri plates. Soil was inoculated with hyperparasites and flooded with 20 m1 sterile distilled water. Control plates contained steamed soil flooded with 24 26 ml sterile distilled water. After 10 days incubation in the dark at 23 I 2 C, chlamydospores were transferred to water agar for micrOSCOpic observation. To test spore viability, a portion of the original chlamydospore suspension was distributed over the surface of 10% WA in 10 cm diameter petri plates. The number of germinated spores after 1 week incubation at 23 1 2 C was counted under a dissecting microsc0pe. Thielaviopsis basicola Endoconidia and chlamydospores of T. basicola were tested for susceptibility to oospore hyperparasites. The methods used to isolate endoconidia were adapted from those of Maduewesi and Lockwood (43). Six day old cultures of T. basicola race 157, grown on Czapek's agar with 5 g yeast extract per liter, were flooded with sterile distilled water and rubbed with a cotton swab. The resulting spore suspension was centrifuged at 320 x.g for l min and the liquid supernatant was removed. Cen- trifugation was repeated 5 to 6 times as needed to clean the spore preparation. Chlamydospores were isolated following the methods of Maduewesi and Lockwood (43). Four week old cultures of T. basicola race 157, grown on Czapek's agar with 5 g yeast extract per liter, were flooded with sterile distilled water and rubbed with a cotton swab. The standing water was discarded to remove the bulk of 25 endospores. Plates were then scraped and the scrapings ground in a sterile glass tissue homogenizer for approx- imately 2 min. The suspension was passed through a 37 pm mesh sieve which retained the chlamydospores. After several washings with sterile distilled water the chlamydospores were washed off the sieve into centrifuge tubes. Centrifugation followed at 320 x,g for 20 sec. The spores were then dried with suction filtration by passing the suspension over a 0.4/xm pore size Nuclepore membrane on a Millipore filter base. Dried spores were refrigerated in test tubes at 4 C and resuspended in sterile distilled water for use. Drops of chlamydospore or endoconidial suspensions were impacted onto Nuclepore membranes at the rate of 5 x 103 spores per membrane. The membranes were floated on sterile Capac loam soil extract in 6 cm diameter petri plates. One drop of a mycelial suspension of an oospore hyperparasite was applied to membranes. Membranes carrying spores without hyperparasites served as controls. After 1 week incubation in the dark at 23 1 2 C, spores were transferred to water agar for microscopic observation. The viability of each original spore suspension was tested (44). Nuclepore membranes carrying spores were floated on carrot root extract in 6 cm diameter petri plates. The endoconidia or chlamydospores were transferred from membranes to water agar after 4.5 to 7 hr or 17 to 24 hr, respectively. The germination of at least 100 spores 26 on each of 2 replicate membranes in 1 plate was observed microscopically. Heterodera schachtii A non-sterile aqueous suspension of cysts of the sugar beet cyst nematode H. schachtii was provided by Dr. G. W. Bird (Michigan State University). Ten cysts were individually placed in a circle on Nuclepore membranes. Agar disks (ca. 2 x 2 x 1 mm) from PDA cultures of oospore hyperparasites were placed in the centers of the rings of cysts on duplicate membranes. Control membranes contained cysts only. Membranes were floated on sterile distilled water and incubated at 23 i 2 C in the dark for 2 weeks. After incubation, cysts were transferred to water agar and observed microscopically for evidence of parasitism. Throughout the 2 week incubation period, nematodes hatched from the cysts and migrated to the bottom of the petri plates. The effect of test fungi on the number of nematodes hatched was determined by counting the number of nematodes found on the bottom of plates at the end of the incubation period. Because the number of cysts avail— able was limited, the experiment could not be repeated. To determine the variability in hatch rate from cysts, therefore, the hatch rates from two separate control plates were compared. 27 Pathogenicity Tests with Oospore Hyperparasites on Soybeans Mycelia of two Cephalospprium isolates, Dactylella ppermatophagp and Hyphochytrium catenoides were scraped from 2 week old PDA cultures grown at 23 i 2 C. Mycelia of Fusarium oxysporum, Diheterospora chlamydOpporia and Humicola fuscoatra were each grown for 2 weeks at 23 i 2 C in 250 m1 Erlenmeyer flasks containing 50 m1 PDB. The mycelia were washed with sterile distilled water, ground with a tissue homogenizer and suspended in 100 m1 sterile distilled water. Twenty-five m1 of hyperparasite inoculum were mixed with steamed soil (3:2, field soil/ sand or greenhouse mix, v/v) in plastic cups approximately 12 cm in diameter. Four cups were infested with each hyperparasite. Controls contained non-infested soil. Soybean seeds (cv. Hark) were soaked in a 30% H202 solution for 30 sec and washed 6 to 7 times with sterile distilled water. Four seeds were planted in each con- tainer. Soybean plants were harvested after 5 weeks in a greenhouse. The effects of the test fungi on soybean plants were determined quantitatively from seedling emergence, survival, and oven dry weights of plants. The overall conditions of roots and above-ground parts of each plant were qualitatively compared to control plants. 28 Soybean Seedling Bioassay for Phytophthora Root Rot of Soybean Bioassay methods were adapted from those developed by Eye et a1. (32). Ten cm diameter sterile glass petri plates were filled with 20 g of soil, naturally or arti- ficially infested with Phytqphthora megapperma var. sojae, and flooded with 25 ml sterile distilled water. Four to five plates were used per soil or soil treatment. Plates were covered and incubated at 23 i 2 C for at least 5 days. Two day old soybean seedlings (cv. Hark), grown in vermiculite at 23 i 2 C, were placed on the flooded soil (4 to 5 per plate). Incubation was continued at 23 i 2 C for an additional 3 to 5 days, or until symptoms of Phytophthora root rot were evident on seedlings. Seed- lings were transferred to distilled water approximately 5 hours before they were rated for disease. The rating of PhytOppthora root rot was based on both disease incidence and symptom severity. The seedlings, immersed in distilled water, were observed under a dissecting microscope for characteristic sporangia of P. megasperma var. sojae. The presence of sporangia con- firmed that seedlings were infected with Phytophthora root rot. Seedlings were then assigned to categories based on symptom severity; 0 = no disease, 1 = mild, 2 = medium, and 3 = severe disease. Healthy seedlings which did not have sporangia were designated to have "no disease." Those in the "mild" category showed root 29 length and secondary root development approximately equal to that of healthy control seedlings, with only localized browning of the roots. Seedlings with stunted roots, some secondary root development and obvious brown lesions were designated "medium" in disease severity. The ”severe" category contained seedlings which were entirely brown, showed stunted roots and had no secondary root development. For an individual petri plate, the number of seedlings in each category was multiplied by the value assigned to that category. The sum of these scores represented the disease rating for that particular plate. Thus, the maximum rating for a plate with 5 seedlings was 15. The final disease rating for a particular soil or soil treatment was the average of the disease ratings from each of 3 to 5 replicate petri plates. RESULTS Occurrence and Importance of Hyperparasitism in Nature An experiment was designed to determine whether oospore hyperparasites are common in natural soil, and to determine if their presence may be significant in control- ling Phytophthora root rot in the field. Soil samples were collected from 33 soybean fields in the Michigan Counties of Shiawassee, Clinton, Monroe and Lenawee. The water holding capacity of each soil was determined. A bioassay using soybean seedlings to bait Phytophthora megasperma var. sojae was performed for each soil. Four seedlings were used per petri plate, and disease ratings varied between the maximum and minimum values of 12 and 0, respectively. Soils were then grouped into three categories (Table l). Soils showing disease ratings from 10 to 7 were designated to have high Phytophthora root rot potentials. Those with ratings from 5 to 3.5 were categorized as having medium disease poten— tials. There were no soils with ratings between 5 and 7. The remaining group contained soils which did not have detectable populations of P. megasperma var. ppjpp. To reduce the number of soils to a manageable number, five 30 31 TABLE 1. Phytophthora root rot disease ratings of field soils as determined using a soybean seedling bioassay in the laboratory. Disease potential Disease rating of soil of soila Soil Number 10.0 10* . 8.0 1*, 2*, 7*, 11*, 14 High 7.5 4, 8, 12, 15 7.0 5, l8 5.0 3. 27. 30 Medium 4.5 20*, 28* 4.0 6*, 9, 16* 3.5 19*. 33 Low 0 17, 21*, 22, 23, 24, 25*. 26*. 29; 31*. 32? 3‘Based on the average ratings of two replicate plates. 0f the soils chosen for further study, standard deviations were 0 for high and low disease potential soils and varied from 0 to 4.95 for soils of medium potential. fioils chosen for further study of parasitism in natural 801 . 32 soils from each grouping were chosen as representatives for further study (Table 1). To determine if hyperparasites of oospores were present in the soils, air-dried, cultured oospores of T. megasperma var. sojae race 1 on membranes were incubated in the dark at 23 i 2 C on each of the 15 test soils. Each soil (20 g per 10 cm diameter petri plate) was flooded with 25 ml sterile distilled water or adjusted to 50% WHC. Control oospores were incubated on autoclaved Capac loam soil. Oospores were transferred to water agar and observed microscopically at weekly intervals over a three week period for evidence of oospore para- sitism. Parasitism of oospores occurred in all soils, indi— cating that oospore parasites are widely distributed throughout natural soil (Table 2, Table 17). Under flooded conditions, the hyperparasites Hyphochytrium catenoides and various unidentified actinomycetes were common to all soils. Rhizidiomycopsis jappnicus and Canteriomyces stigeoclonii were also frequently observed. Parasites which infected oospores via hyphae (predom- inantly in soils at 50% WHO) could not be identified. After the first week of incubation, levels of co— spore parasitism varied between 6.5% in flooded soil 6 and 87.5% in flooded soil 32. Rates of increases in parasitism varied with the individual soil and with soil moisture. Maximum parasitism averaged 51.5% over all 33 TABLE 2. Parasitism of cultured oospores of Phytgphthora megasperma var. sojae race 1 in natural soils. {Disease po- tential and OosporesparasitizedL %a Flooded soils Soils at 50% WHC number of l 2 3 l 2 3 soil week week, wee§_pg week__4myfl;___1ugflg_ High 1 14.0 50.5 31.5 62.0 0 0 2 42.5 2.5 50.0 25.5 25.0 14.6 7 8.0 2.5 30.0 73.0 5.0 -- 10 29.5 45.5 53.5 49.0 43.0 9.0 11 31.5 33.5 44.5 69.5 35.0 35.6 Mean 25.1 40.9 41.9 55.8 21.6 14.8 Medium 6 6.5 37.0 47.0 26.0 51.0 8.6 16 19.5 57.5 42.0 40.0 41.0 25.9 19 31.5 50.0 46.5 47.0 70.0 59 5 20 52.5 20.5 58.0 43 5 30.0 7.5 28 7.0 44.0 28.5 -- -- -- Mean 23.4 41.8 44.4 39.1 48.0 25.4 Low 21 28.0 48.0 44.0 46.5 54.5 34.0 25 21.5 45.0 46.5 27.0 17.0 3.5 26 24.5 35.0 43.0 34.5 20.0 10.0 31 20.5 64.5 41 5 51 5 54.0 12.7 32 8705 7500 "" -_ -- -- Mean 36.4 53.5 42.8 39.9 36.4 15.1 Controlb 0 0 0 0 .0 0 3Based on the average number of oospores parasitized on each of 2 replicate membranes in 1 plate; 50-100 oospores were counted per membrane. bSteamed Capac loam soil. 34 soils and was generally reached during the second or third week. It should be noted that with many soils at 50% WHO the number of infected oospores apparently decreased with time (Table 2). Many of the oospores observed after the first week were devoid of internal contents and were mis- shapen, but not associated with parasites. Presumably in these cases, oospores had originally been infected and destroyed, with parasites lysing after oospores were emptied. These destroyed oospores were not counted as infected because direct evidence of parasitism was lacking, and were ignored. These dead oospores resulted in a decrease in the oospore population available for infection. The decrease in parasitism with time was probably a reflection of a decrease in the rate of oospore infection. The average maximum levels of parasitism for each soil disease potential grouping were compared using Student's t-test (Table 3). There were no significant differences between soil groupings, whether soils had been flooded or held at 50% WHC. Levels of parasitic activity therefore did not correlate with the disease potentials of soils. Furthermore, within a particular soil grouping, maximum levels of parasitism were not sig- nificantly different between the two moisture levels. Soils of each disease potential category apparently supported equivalent levels of parasitism, regardless of 35 TABLE 3. Maximum amounts of parasitism of cultured oo- spores of Phytpphthora megasperma var, sojae race 1 occurring in soils during three weeks incubation. Disease potential and number Maximum pprcent of oosporesparasitizeda of soil Flooded Soil Soil at 50% WHC High 48.2 i 5.1 55.1 i 20.6 1 2 7 10 ll Medigm 51.3 + 0.9 51.4 13-3 l6 19 20 28 I + Low 57.8 + 18.9 42.5 1 15.4 21 25 26 31 32 aThe average of the maximum values obtained for each soil within the three week incubation period. Comparison of maximum values, using Student's t-test, indicated values did not differ significantly (3:0.05). 36 the parasite species present or the Phytophthora root rot disease potential of the soil. In flooded soils, in which parasites could be dif- ferentiated, distribution varied (Table 4, Table 18). For example, in soil 31, actinomycetes were the predominant parasites while in soil 25, parasitism was mainly attri- buted to chytrids. The Phytpphthora root rot potential of soils could not be correlated with particular parasite distribution patterns. A second experiment was designed to determine whether oospores isolated from diseased soybean seedlings differed from cultured oospores in their susceptibility to hyper- parasitism in natural soil. Six field soils were used. Soil plates were prepared as in the previous experiment, and oospores were observed after 1 and 2 weeks incubation. Oospores from soybean seedlings and from culture were equally susceptible to hyperparasitic attack in flooded soil (Table 5). There was significantly more parasitism of oospores from culture than of those from seedlings in soils ll, 16, 20 and 31 at 50% WHO, but only at l incubation time. The two types of oospores therefore do not appear to have inherently different susceptibilities. In general, parasitism of cultured oospores was much lower than that seen in the previous experiment (Table 2), particularly in soils at 50% WHC. The experiments were separated in time by several months, during which the soils had been stored in a cold room at approximately 4 C. 37 .mpmam a CH mmcmhnEmE mpdoaamms 039 00 £600 :6 woNHeflmmsmm mohommoo 00 009852 owmsm>m one So panama .. 0 0.0 -- 0.00 0.00 -- 0.0 0.0 mm 0 0 0.0 0.00 0.00 0.00 0.00 0.00 0.00 00 0 0 0 0.00 0.00 0.00 0.00 :.00 0.00 00 0 0 0.0 0.00 0.00 0.: 0.00 0.00 0.00 00 0 0 0.00 0.00 0.00 0.00 0.00 0.0: 0.00 00 304 0 0.0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 00 0.0 0 0.: 0.00 0.0: 0.00 0.00 0.00 0.00 00 0.0 0.0 0.00 0.00 0.0: 0.00 0.00 0.00 0.00 0H 0 0.0 0 0.00 0.00 0.00 0.00 0.00 0.: 00 0.0 0.0 0 0.00 0.00 0.00 0.00 0.00 0.00 0 E0000: 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0: 00 0 0 0 0.00 0.00 0.00 0.00 0.00 0.00 00 0.0 0 0 0.00 0.00 0 0.00 0.00 0.000 0 0 0 0.00 0.0: 0.0: 0.00 0.00 0.00 0.00 0 0 0 0 0.00 0.00 0.00 0.00 0.00 0.00 H :00: Mom: #003 Mom; goo; #003 sec; Emma x803 Mom; 000m 0 0 0 0 0 H 0 0 a 00 00050: mwumpomm bum mmvoo%Eonmhm mopeohfioflwwofl mpfiswzso 0:0 HmHPCmP «R .posflpHmMQmm mmsommoo tom mmmomfim .maflom 00009 pmvooam CH mopammsmmpmmms Mo 00909 #:0060000 an H 0600 00060 .sm> mathmNmme macsprmopmnm 0o mmsommoo Mo Swapflmmsmm .: mgm 00 0W000>0 0:0 80 0000m0 HN.0 H 20.0 0 H 000 o H 000 0000080000 8800p0moocm0: 00.0 H mm.0 0N H 00 0N H 00 mwmnmop0800mm 0000000000 00.0 H 00.0 mm H mm mm H 00 N 0000000 88000MW0000000 00.0 H 00.0 0 H 000 0 H 000 . 0 0000000 20000000008000 00.0 H 00.0 0 H 000 0 H 000 0000800 00.0 H 00.0 :0 H mm 00 H mm 000000080 0000088: 0m.0 H 00.0 an H 00 mm H m0 8800m00wo 88000080 00.0 H no.0 NH H :0 ma H :0 00000000080030 0000000000800 00.0 H 00.0 00 H 00 00 H 00 0000000 0w .P8mam,00m 0H0>0>08m 00080m008m 0 080800000 H00m 000003 000 80>0 00000000 0 .008058000w 0 80 00003 m 00% 0000000000000: 0000000 8003 00000080 0000 80 8300w A0000 .>0V 008000000 8009000 00 cpzo0w 080 H0>0>080 .80000808000 .0 mgm CD I s \» [I I ‘S x’ H. CATENOIDES 4:. C) \ MAXIMUM COLONY DIAMETER, % 3 I (, l L_ l l 16 20 24 28 1' EM PERATURE, °C FIGURE 4. Effect of temperature on growth of hyperparasites. Measurements are the mean of 3 replicate PDA plates. Standard deviations were less than or equal to 6.0. 54 TABLE 9. Comparative susceptibilities of oospores of three races of Phytophthora megasperma var. sojae to hyperparasitic attack by Hyphochytrium catenoides and Pythium gp. after 10 days. Oospore Oospores Hyperparasite Race parasitized, %a Hyphochytrium catenoides 1 80.5 1 4.6 3 77.6 1 9.3 7 47.3 1 15.6 Pythium sp. 1 75.8 i 15.9 3 76.2 : 8.2 7 49.3 + 14.0 aBased on the mean of 3 replicate membranes in 1 plate; 200 oospores were counted per membrane. Values for races 1 and 3 were significantly greater than those for race 7 with a. catenoides. Values for race 3 were significantly greater than those for race 7 with Pythium sp. (3:0.05; Student's t-test). 55 10 cm diameter petri plate) at various rates (see Table 10). Soil was air-dried to kill residual Phytophthora hyphae. The results indicated that infestation rates between 165 and 825 oospores per g of soil would provide sufficient amounts of Phytophthora root rot without overloading the system. In further experiments, 500 oospores per g of soil were used for testing the ability of hyperparasites to decrease Phytophthora root rot of soybean. For biological control experiments, Humicola fuscoatra, Hyphochytrium catenoides or Pythium sp. were used. Control plates contained only steamed soil. Petri plates containing 3. catenoides and Pythium sp. were flooded and incubated at 23 1 2 C for 10 or 20 days before adding five, 2 day old soybean seedlings to each plate. Previous experiments (Figure 1) had shown that E. fuscoatra required a longer period of time to show parasitic activity. Therefore, soil containing fl. fuscoatra was incubated at 75% WHC for 20 or 30 days, then flooded for 5 days before soybean seedlings were added. After 3 days, seedlings were rated for disease severity. Seedlings from plates with Pythium gp. were washed in a mild Ivory detergent solution (2 drops per 250 ml distilled water) and rinsed in distilled water. Three root sections from each seedling were placed on PDA and 1.5% WA, incubated in the dark at 23 1 2 C, and observed 1 week later for hyphae of Pythium. 56 TABLE 10. Bioassay for Phytophthora root rot in steamed soil artificially infested with Phytophthora megasperma var. sojae race 1 oospores from soybean seedlings. Oospores per Seedlings Disease ggof soil infected, %a ratingb 0 O 0 16.5 0 0 165.0 60 1 54 7.2 1 6.7 825.0 100 1 O 12.0 1 2.0 1,650.0 100 1 0 15.0 1 0 3Mean number of infected seedlings from each of 5 replicate plates with 5 seedlings per plate. bMean disease ratings from 5 replicate plates per treatment. The maximum possible disease rating was 15.0. 57 Replicate plates were also prepared which contained Nuclepore membranes impacted with oospores of 2. megasperma var. sgiag race 1 from soybean seedlings. Prior to the addition of seedlings for bioassay, the membranes were removed for microscopic observation of oospore parasitism. None of the hyperparasites tested significantly reduced the amount of Phytophthora root rot as compared with controls (Table 11). Additional eXperiments with hyperparasites in artificially infested soil also failed to result in control of Phytophthora root rot. Seedlings from soil which was incubated for 20 days with Pythium sp. had significantly higher disease ratings than the control. The increased amount of disease was probably due to damage caused directly by Pythium sp. All three hyper- parasites were active during the period of incubation. At least 75% of all oospores on the membranes were infected. Results from the control plates with hyperparasites in the absence of oospores of P. megasperma var. sojae indicated that Pythium sp. caused a mild root rot of seedlings (Table 12). Though the symptoms were mild, the hyperparasite colonized the roots of at least 72% of seedlings. Biological Control of Phytophthora Root Rot in Field Soils Naturally Infested with Phytophthora megasperma var. sojae Two soils naturally infested with Phytophthora megasperma var. sojae were used to test the effectiveness 58 mmwom .pm> mesmmwmwme .m m.:H o.am m.mH m.mm *m.oH 6.:H s.mH 0.: + .mm esflnpwm mmwom .hm> I I I I mgommmwoe . 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