Duration: 10/01/1999 to 09/30/2004

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Plant pathogens that are harbored in soil and crop residue continue to cause diseases that dramatically impact the yield and quality of crops and plants produced in the North Central region. There are limited effective management options for most of these pathogens, so biological control offers novel alternatives. This project addresses the interactions of applied and resident microorganisms with this group of pathogens and their host plants within the varied environments of the region. The ultimate objective is to develop practical disease management systems that maximize the benefits of applied and resident microbial agents.

JUSTIFICATION:

Crop and plant production and processing of agricultural products are important to the economy of the North Central Region of the United States. Some of the most important field and horticultural crops in the US, e.g., corn, soybean, wheat, barley, potato, tomato, common bean, sugar beet, turfgrass, and ornamental plants, are produced abundantly in this region. Almost all agriculturally-important plants are affected by soil- and residue-borne pathogens. Some pathogens cause substantial losses in the field, while others cause loss of crops in storage or affect the quality and safety of the agricultural product. In addition, managing these pathogens requires crop rotation and other practices that are costly to growers. The effect of these pathogens can be extremely damaging. For example, Fusarium head blight (scab) epidemics of the upper Great Plains from 1993 to 1997 caused several billion dollars in damage to the small grain industry, devastated the farm economy in many rural areas, and nearly eliminated mailing barley production (McMullen et al, 1997).

There are many other examples of important soil- and residue-borne pathogens (Agrios, 1988). With the shift from intensive cultivation to minimum tillage cropping systems, diseases caused by these pathogens have become increasingly prevalent. For example, Phytophthora sojae and the soybean cyst nematode, two important soybean pathogens in the USA, caused an estimated yield loss in 1994 of 2,500,000 metic tons (Wrather et al, 1997). Rhizoctonia solani, the cause of damping-off and root rot diseases found on almost all crops, has been a serious problem because of a lack of adequate controls. Pythium and Aphanomyces on pea, sugarbeet and other crops cause serious losses on seedlings. Sclerotinia is a major pathogen of bean, soybean, canola and other broadleaf crops. In the last ten years this pathogen has gone from being rare to become a major disease on soybean in the North Central Region. Other pathogens such as Pyrenophora tritici-repentis, the cause of tan spot of wheat, Gaeumannomyces graminis, the cause of take-all of grasses, and Cercospora zeae-maydis, the cause of leaf spot of corn, are all residue-borne pathogens of major importance. Diseases incited by pathogens such as Fusarium moniliforme, which causes corn ear and stalk rot and can contaminate grain with fumonisin mycotoxins, result in yield losses and in human and animal health problems (Munkvold and Desjardins, 1997).

Soil- and residue-borne pathogens are among the most difficult to control, especially those that infect plants through the roots. Genetic resistance is available to control some, but not most, of these pathogens. Cultural controls have been widely used for these pathogens, but vary greatly ineffectiveness, and are not always economically feasible for growers or agribusinesses to implement. Fungicides have not been economically feasible for most root-infecting pathogens because of the inaccessibility of the infection site and biological complexity of the soil environment. Current public sentiments towards pesticides suggest that there will be a decrease, rather than an expansion, in the use of synthetic pesticides. There is recognition in the agricultural community that alternative control strategies for soil- and residue-borne pathogens are needed that are compatible with agricultural production methods, environmentally safe, and fit into integrated pest management systems. Biological control is an alternative strategy that could play an important role in the future of US agriculture.

Biological control of plant pathogens has been intensely researched for the past 30 years. Progress has been made in identifying biocontrol agents, understanding their basic biology, testing efficacy of biocontrol against a wide range of pathogens, and evaluating them under commercial plant production practices. Indeed, in the past five years, new biological control products have been registered by the EPA and are sold commercially. Biological control of soil and residue-borne pathogens, however, has not been widely implemented in agriculture, especially in field crops. Much basic and applied research is needed to realize a consistent and economically sound use of biological control and an integration into current crop management practices. A greater understanding of the ecology, physiology and genetic variation of biocontrol agents, their methods of application, and their interactions with pathogens, hosts, environments and agricultural systems is necessary before there is widespread commercial-scale use of biocontrol.

The nature of biocontrol, using one or several organisms against another, is an intrinsically difficult biological problem that requires much understanding before it can be effectively used. In addition, cooperative efforts between scientists working on similar biocontrol agents and pathogens within similar agroecosystems are required to make progress toward implementation of biocontrol.2 Regional cooperation among scientists working on biocontrol can advance knowledge on how to use this important technology. Sharing of biocontrol agents and testing on various crops under a wide range of environments and soil types is one type of cooperation. Another is the development of techniques for producing, storing, and delivering biocontrol agents. The standardization of protocols for testing is an important result of cooperation. Also important, regional cooperation provides a forum for discussion where results and ideas are exchanged and debated and cooperative efforts are enhanced and broadened.

This five-year project will build on the past success of cooperative efforts on biocontrol in the North Central Region. We will compare efficacy of biocontrol systems on economic plants, and compare methods of production, delivery and enhancement of applied and indigenous biocontrol agents. Biocontrol systems have already been identified by cooperating scientists. We also will investigate the influences of host and environment on biocontrol efficacy. There also will be a major effort to determine the mechanism of action of biocontrol systems. We will study the nature of biocontrol agent-crop-pathogen interactions and elucidate the biochemical and genetic basis for interactions. We intend to foster the development of new ideas and approaches for biocontrol through the interaction of scientists during this project. The results of this project will have direct application to the use of biocontrol to protect important economic plants from soil- and residue-borne pathogens.

This project falls within several North Central Regional Association (NCRA) crosscutting research areas. Its primary involvement is in the Integrated Pest Management area and specifically addresses these priority objectives:

• Develop alternative controls based on biological control and cultural practices.


• Reduce pesticide use and the risk of human, animal and environmental exposure to pesticides through research and citizen/consumer education.

The project is also relevant to the Food Production, Processing and Distribution area in the objective.

• Develop improved animal, plant and microbial production, processing and marketing systems which are environmentally sound and profitable.

The project also addresses the Natural Resources and the Environment objective.

• Identify and apply ecosystem management principles and practices for the utilization and protection of resources, restoration of natural systems and management of rural landscapes.

In addition, some of the activities speak to the Genetic Resources Development and Manipulation area objectives:

• Collect, preserve, share, enhance and evaluate germplasm at the molecular, cellular and/or organismal levels; and


• Develop increased knowledge of the interactions and interrelationships of the various life forms.

Related, Current and Previous Work

Contributions of NC-125

The body of research on biological control of plant pathogens is extensive, and NC-125 scientists have contributed significantly to that knowledge. The microorganisms and host-pathogen systems investigated by NC-125 scientists are diverse, reflecting the diversity of cropping systems and environments found in the North Central region. Among the numerous species and strains of bacteria and fungi that have been evaluated for biological efficacy by NC-125 members during the current project, several are notable because of the wide range of locations and/or crop-pathogen systems in which they were found to be beneficial. Burkholderia cepacia strain AMMD, found previously to control Pythium damping-off of pea, was as effective as commercial fungicides in controlling damping-off in sweet corn. Stenotrophomonas maltophilia (=Pseudomonas maltophilia) strain C3 was effective against Rhizoctonia solani and Bipolaris sorokiniana in turfgrass (Giesler and Yuen, 1998; Zhang and Yuen, 1998a). Similarly, Pseudomonas fluorescens strain Pf-5 inhibited multiple turfgrass pathogens: Dreschslera poae and Sclerotinia homoeocarpa (Rodriguez and Pfender, 1997). As part of the extensive testing of Bacillus spp. across crops, B. megaterium was found to inhibit R. solani in soybean (Bertagnolli et al., 1994). Among fungal biocontrol agents, binucleate Rhizoctonia spp. (BNR), which were discovered previously by NC-125 members to control R. solani in soybean, turfgrass, and sugarbeet, were found to be effective on canola and white mustard. Sporidesmium sclerotivorum, a parasite of sclerotia developed previously by USDA-ARS, Beltsville to control Sclerotinia minor, reduced the viability of S. sclerotiorum sclerotia in IA soybean fields and reduced the incidence and severity of stem rot disease. Trichoderma virens (=Gliocladium virens) isolates discovered previously in Beltsville to provide commercially practical control of Pythium and Rhizoctonia diseases of potted plant crops, were effective also in controlling damping-off in sweet corn, and diseases caused by Sclerotium rolfsii in bean (Lewis and Fravel, 1994), carrot and tomato (Ristaino et al., 1994)

In addition to expanding the uses of known biocontrol agents, identification of new bacterial and fungal biocontrol organisms is an ongoing effort in this project. Novel rhizosphere-colonizing bacterial strains (Karakaya and Martinson, 1995) and fungi species such as Ulocladium atrum in KS and Cladorrhinum foecundissimum in Beltsville (Lewis et al., 1995) were identified. In MI commercially-available inoculum of the mycorrhizal fungus, Glomus intraradices, increased yield of potato pre-nuclear minitubers and reduced the severity of minituber dry rot caused by Fusarium sambucinum (Niemira et al., 1996).

Basic mechanistic and ecological research on these biocontrol systems have added to our understanding of how they function and what factors affect their activity. In addition, some of this information has led to strategies for enhancing delivery and efficacy in the field. Production of pyrrolnitrin was found to be the main mechanism of biocontrol by B. cepacia strain AMMD (Regner, 1996). Similarly, some of the biocontrol activity of P. fluorescens Pf-5 was attributed to pyrrolnitrin production (Rodriguez and Pfender, 1997). Extracelluar enzymes were implicated in the activity of B. megaterium and Trichoderma harzianum against R. solani (Bertagnolli et al., 1994; Dal Solglio et al., 1998). Chitinase production was associated with in vitro and in vivo inhibition of fungi by S. maltophilia strain C3 (Zhang and Yuen, 1998b), leading to the use of chitin amendments as a method to stimulate multiplication of the strain on plant surfaces and to enhance biocontrol efficacy (Zhang and Yuen, 1998a): Investigations into the dynamics of applied bacterial agents on plant surfaces have provided information as to the population levels required for efficacy and compatibility with host genotype (Giesler and Yuen, 1998; King and Parke, 1996). In NE, the influences of irrigation and shading on the turfgrass canopy environment and on establishment of applied bacterial agents were studied (Giesler, 1998). Information derived in Beltsville regarding the influence of nutrient substrates on G. virens led to the development of granular formulations incorporating fungal biomass with food bases in alginate prill orpre-gelatinized starch-flour forms (Lewis et al., 1995; Lewis et al., 1996). An alginate prill formulation also allowed effective delivery of Taleromyces flavus for control of Verticillium dahliae (Fravel et al., 1995). Control of R. solani by BNR fungi was found to require prior localized epidermal infection by the biocontrol agent; induced resistance was implicated as the biocontrol mechanism as the pathogen was found to be inhibited at some distance from the site of BNR infection but no evidence of antibiosis was found (Poromarto et al., 1998). Similarly, inhibition of Fusarium dry rot by G. intraradices on potato minitubers was attributed to induced systemic resistance (Niemira et al., 1996).

Basic and applied research into biocontrol systems involving resident microorganisms has been the other thrust ofNC-125. Swine manure has been investigated for its effects on soybean pathogens; some pathogens were unaffected or else stimulated by incorporation of manure into soils, whereas the hatching of soybean cyst nematode was inhibited by soluble components. Oat cover crops incorporated into soils suppressed root rot caused by Aphanomyces spp. in sugar beet (MN) (Windels, 1997)and in pea (WI) .The effects were attributed to decomposition products, such the saponins, that inhibit production of zoospores and sexual structures or cause lysis of zoospores. Isoflavones from legume roots were found to affect the germination and asexual reproduction of Phytophthora sojae in the absence of a host (Vedenyapina et al., 1996); this system may alter fungistasis and thereby subject the pathogen to attack by resident microbes. The role of manganese oxidation and reduction by the pathogen Gaeumannomyces graminis var. tritici and by other rhizosphere microorganisms in controlling severity of take-all disease of wheat was confirmed by micro-XANES spectroscopy (Schuize et al., 1995). Lignosulfates were evaluated as a means to prevent micronutrient immobilization and oxidation by the pathogens and other microbes. Composts made from various yard or agricultural wastes used in tomato production in OH varied in their capacity to decrease the severity of fruit rots caused by soilborne pathogens. Using a foliar bacterial pathogen as a bioassay, disease suppression was found to be due to induced systemic resistance.

Related Funded Regional Research Projects

Three other funded regional projects also address plant disease biological control. In the Western region, W-147 "Managing Plant-Microbe Interactions in Soil to Promote Sustainable Agriculture" is involved in identifying and characterizing biocontrol systems, investigating controlling mechanisms, and implementing these systems. The Southern region project S-269, Biological Control and Management of Soilborne Plant Pathogens for Sustainable Crop Production, pertains to the evaluation of applied microbial systems and cultural practices for control of soilborne pathogens. The North Eastern regional project NE-171, Biological and Cultural Management of Plant-Parasitic Nematodes, focuses on nematodes. Although certain objectives in these three projects are similar to those of NC-125, the pathogen, crop and environmental systems vary among the regions to such an extent that very little duplication in research is expected. The efforts of NC-125, W-147 and S-269 (formerly S-241) were found to be complementary in the past when research findings were shared in joint meetings of the three projects (Estes Park, CO, 1993; San Diego, CA, 1996). In addition to the fore mentioned projects, there are other funded regional research projects that have strong soil microbial and environmental components: S-257 relates to soil water and solute movement; S-262 focuses on beneficial rhizosphere microorganisms; and W-170 pertains to the chemistry of soils and organic wastes. None of these projects, however, pertain directly to plant disease or to microbial interactions.

Objectives

1. Compare efficacy of biocontrol systems on economic plants in the North Central region. a) Compare methods of production, delivery, and enhancement of applied and indigenous biocontrol agents; b) Determine the influences of host and environment on biocontrol efficacy.
   
2. Determine mechanisms of action of biocontrol systems used in the North Central region. a) Determine the nature of biocontrol agent-crop-pathogen association; b) Elucidate biochemical and genetic basis for the interactions.
   
3. 
   

Methods

The following approaches are proposed to achieve the objectives of enhancing the availability and utility of biocontrol agents and systems and increasing the understanding of the mechanisms governing their effectiveness in suppressing diseases of economic crops in the North Central region. Results are expected to contribute to the sustainability of crop systems in this region. Cooperation between individuals and institutions in North Central and other states will enhance the widespread application of these systems in agricultural and horticultural production. The methodology is described according to the specific objectives.

I. Compare efficacy of biocontrol systems on economic plants in the North Central region.

A. Compare methods for production, delivery and enhancement of applied and indigenous biocontrol agents. The majority of the studies proposed will utilize biocontrol agents already discovered and characterized to some extent. Bacterial genera will include Azospirillum, Bacillus, Enterobacter, Pseudomonas, and Stenotrophomonas (IN, MN, NE, NJ, NY, OH). Species of binucleate Rhizoctonia, Fusarium, Gliocladium, Glomus, Sporidesmium and Trichoderma, will be among the fungal antagonists targeted for study (IA, IL, IN, MI, MN, ND, OH, USDA-ARS). However, in some instances additional biocontrol strains will be sought to fill specific needs, i.e. for crops and pathogens not previously emphasized. These will be isolated from North Central region crops or acquired from plant pathologists, microbiologists and commercial companies outside of the region. Sharing of strains and formulations among members of NC-125 will foster efficiency and allow determination of widespread efficacy of known biocontrol agents. In addition, some researchers will examine microbial communities in existing disease-suppressive soils and composts or as influenced by crop amendments and micronutrients (IL, IN, MN, NY, OH).

Because of the diversity of crops and environments represented in this project, it is difficult to identify host systems that are common to all of the participating states. Treatments will be evaluated on soybean and other legumes (IA, IL, IN, MI, ND, NE), cereals and turfgrass (IN, NE, NJ, NY), corn (USDA-ARS), and sugarbeet and vegetable crops (MI, MN, NY, OH). In general, studies will focus on several soil-borne pathogen species or groups, especially Rhizoctonia solani, (IN, MN, ND, NE, NY, OH), oomycetes (Pythium spp. and Aphanomyces cochlioides) (IN, MN, NY, OH), Sclerotinia sclerotiorum (IA, NE), and ectotrophic pathogens, e.g. Gaeumannomyces graminis var. tritici and Magnaporthe spp. (IN, NJ). In addition, the control of other major soil- or residue-borne pathogens, e.g. Bipolaris and Fusarium spp., also will be investigated (MI, NE, USDA-ARS). Laboratory, greenhouse and field evaluations will be carried out in soils infested with indigenous or introduced inoculum. Depending upon the crop and biocontrol agent, application of biocontrol agents will be accomplished by seed treatment (as seed coatings or in seed hopper), incorporation in soil or planting medium, or application to foliage. Individual biocontrol agents will be compared with combinations of organisms, commercial fungicides, biocontrol agent-fungicide combinations, and appropriate controls. Data to be collected will include emergence and stand counts, yield, infection frequency, disease severity, biocontrol agent population levels, and pathogen inoculum level.

In some states, research will be conducted on unique biocontrol systems or delivery methods. In IA, for example, investigations will focus on control of Sclerotinia sclerotiorum by the sclerotialparasite Sporidesmium sclerotivorum. The potential for commercialization of the agent will be evaluated. Sporidesmium inoculum will be provided to cooperators in other states for testing in growers' fields and tested for efficacy in controlling Sclerotinia diseases of soybean, commonbean, sunflower, alfalfa, and clover, and other crops grown in the North Central region. Research in NE will emphasize Stenotrophomonas maltophilia as an applied biocontrol agent species. Strains in this species typically produce chitinases. The importance of propagating strains in liquid media containing chitin will be investigated on the basis of rapidity of cell growth, induction of chitinolytic enzymes, and the ability to utilize exogenous chitin once applied to plant surfaces. The application of cells along with culture media constituents (containing antibiotics and enzymes produced by the bacterium and exclusive nutrients available for bacterial utilization) will be tested for enhanced establishment of the biocontrol agent population establishment and efficacy in biocontrol. Research conducted in NY will involve field studies to examine the relationships between application frequency and timing on the efficacy of selected microbial inoculants for controlling soilborne diseases on turfgrasses. The goal will be to optimize application scheduling for bacterial (Enterobacter, Pseudomonas, and Azospirillum species) and fungal (Trichoderma harzianum) inoculants, as well as other strains being studied by other NC-125 members, in an effort to maximize efficacy. Daily and weekly applications of bacterial inoculants, as well as daytime versus nighttime applications, will be compared. Efficacy, population dynamics, and possible non-target responses to introduced microbial strains will be examined. This research is designed to complement new technology development in the turfgrass industry for applying biological products. In MI, the effectiveness of selected species of arbuscular mycorrhizal fungi against root-pathogenic fungi such as Fusarium sp. will be tested. The biocontrol fungi will be applied to soil at planting to reduce root disease of several crops such as asparagus, potato and soybean. The arbuscular mycorrhizal fungi to be used will include laboratory and commercially-available strains. Research will also determine if the utility of indigenous mycorrhizal fungi can be increased by applying certain compounds found in root exudates, some of which are commercially available, to various natural soil systems. These natural systems will contain differing mixtures of arbuscular mycorrhizal fungi. Additionally, research will determine whether the effectiveness of arbuscular mycorrhizal fungi as biocontrol agents can be increased by the addition of other biocontrol agents such as Trichoderma sp. Research involving USDA-ARS will emphasize fumonisin-nonproducing strains of F. moniliforme as applied agents to control fumonis in mycotoxins in corn. A variety of strains and inoculation methods will be tested under field conditions, and efficacy will be monitored by analyzing fumonis in contamination of harvested grain.

B. Determine the influences of host and environment on biocontrol efficacy. In NE, S. maltophilia strains will be isolated from local crops and obtained from NC125collaborators to create a collection from rhizospheres and phyllospheres of different host plants. Strains will be compared for their ability to colonize rhizospheres and phyllospheres of turfgrass, cereals, and legumes (dry bean and soybean). Colonization ability of the strains will be related to efficacy in controlling fungal pathogens. In field experiments, canopy and soil environmental conditions (e.g. moisture, temperature, and carbohydrate nutrient availability) will be electronically monitored. The ability of applied bacterial strains to colonize and multiply on roots and foliage will be related to specific environmental parameters. To confirm that environmental parameters have a direct influence, the activity of applied agents will be tested under controlled environmental conditions.

Participating researchers will examine the potential of various soil amendments for disease control. In experiments planned for IN, plant tissue analyses for Cu, Mn, N, P, and Zn will be correlated with disease incidence and severity in wheat and corn. Micronutrient formulations will be developed and evaluated in the field to improve uptake efficiency and enhance biologically mediated disease suppressing ability. In MN, micronutrients will be evaluated alone and in combination with extracts from oat plants or soil in which oat plants are decomposing for direct effects on structures of A. cochlioides and for changes in microbe populations. The fungus will be observed for effects on formation of zoosporangia, zoospores, oogonia, antheridia, and oospores. In greenhouse and field experiments, soils infested with A. cochlioides will be left fallow or planted to oat; then the oat crop will be incorporated into soil and allowed to decompose. After sugar beet seeds are sown, various fertilizer and micronutrient combinations will be added. Bulk and rhizosphere soils will be assayed on selective media for microorganisms, including pseudomonads, manganese-oxidizing and -reducing bacteria, and Trichoderma spp., associated with pathogen suppression in other crops. Data will be collected on emergence, control of Aphanomyces damping-off and of mid- and late-season root rot, population of A. cochlioides, rate of breakdown of soil-incorporated cereal residues, amounts of nutrients available, uptake of nutrients by sugar beet, and yield and quality of sugar beet.

Research in IA will address the factors that affect survival of Sporidesmium, a mycoparasite of Sclerotinia sclerotia. To determine the potential for long-term survival of Sporidesmium in field soils and spread from point sources, areas infested since 1995 and neighboring fields will be monitored through soil sampling (baiting with sclerotia) and disease incidence. This will indicate possible long-term residual effects of the mycoparasite and whether its spores are moved by air, water, or soil. Experiments will be designed to determine if sclerotial soil fungi other than Sclerotinia are providing a food base for Sporidesmium survival, since this biotrophic organism has been found in soils with no history of Sclerotinia. Soils yielding Sporidesmium will be sampled for sclerotia and sclerotial-forming fungi, and sclerotia produced in pure culture will be challenged with Sporidesmium. Whether Sporidesmium is native to Iowa prairies or has been introduced will be determined by searching for it in native undisturbed soils, and/or looking for native fungi that may be its host. This is important in order to know how added Sporidesmium may alter the soil ecosystem or possibly affect beneficial fungi. Furthermore, the potential for organisms in the soil that may parasitize Sporidesmium or limit its activity will be explored.

II Determine mechanisms of action of North Central region biocontrol systems.

A. Determine the nature of biocontrol agent-crop-pathogen associations/interactions. Interactions of biocontrol agents with the host and environment will be explored in relation to several fungal and bacterial systems. In IA, the interaction between living Sclerotinia sclerotia and the mycoparasite Sporidesmium will be investigated for factors that may explain the compatible parasitic relationship and provide clues for better in vitro culture of Sporidesmium. The three-way interaction of binucleate Rhizoctonia (the antagonist), R. solani (the pathogen), and the soybean host plant will be investigated in ND. The association of S. maltophilia strains with host roots and foliage will be studied in NE, to determine the influence of the biocontrol strain, host species, environmental conditions, and time after application on colonization of various plant parts. The spatial distribution of colonies of the bacterium on root and leaf surfaces will be examined to determine the relationship of colony numbers, size and proximity to the inhibition of fungal spore germination and infection. Research in NY will be designed to determine the structure of microbial communities associated with both suppressive and non-suppressive soil environments, and these community structures will be correlated with Pythium suppressiveness. The types of culturable antagonistic bacteria that may impart disease-suppressive properties to compost-amended soils will be identified, and the relatedness of isolated Pythium-suppressive bacteria to other members of the community will be determined. Results of the research will provide a strong ecological context for the study of biological control systems and will provide amore rational basis for selecting organic amendments and microbial antagonists for use in sustainable disease management systems. Research in NY also will be directed towards the ecological mechanisms of tolerance in different plant genotypes. Emphasis will be on the role of seed- and root exudation, that influence microbial communities, in regulating levels of Pythium infection. The strategy will be to determine whether tolerance plant to Pythium infection can be explained on the basis of quantitative or qualitative changes in fatty acid exudation or by tolerant plants have greater populations and activities of fatty acid metabolizing organisms. The basis for these studies comes from other work that established a role for fatty acid metabolism in Enterobacter cloacae in the biological control of Pythium damping-off.

Information exchange within NC-125 will facilitate the development and execution of methods that are common to many of the biocontrol systems. All of the research proposed in Objective IIA, to some degree, will employ selective media for isolating microorganisms from soil and plant parts. Use of drug resistance (e.g. carboxin resistance in binucleate Rhizoctonia [ND] and rifampicin resistance in S. maltophilia [NE]) will facilitate detection and identification of the applied agents. These microorganisms also will be genetically transformed with GUS or GFP reporter genes to provide a means of visualizing the interactions of the biocontrol agents with the host and pathogen. Testing of recombinant organisms will be conducted with the approval of the participating institution. On the other hand, unique methodology will be required for some of research areas. For example, a molecular approach will be used for characterizing microbial taxa potentially involved in the suppression of Pythium damping-off of turfgrasses. This will involve comparisons of 16S rRNA gene sequences in DNA extracted from Pythium-suppressive soils with those from non-amended, non-suppressive soil. By defining suppressive soil properties on the basis of a specific microbial community structure, it will be possible to investigate microbes that have not been cultured previously, some of which may have major impacts on Pythium suppression.

B. Elucidate the biochemical and genetic basis for the interactions. Several biocontrol systems will be investigated using a biochemical approach. The research on some systems also will be supported by analysis of the genes controlling the mechanisms of action. In ND, the biochemical basis of biocontrol of R. solani by BNR will be studied by examining the phenolic compounds released from soybean when challenged by BNR, and then determining if any compounds inhibit the growth of R. solani. Research in IN will address the effects of biological and abiotic suppression of Mn-oxidation by G. graminis var. Gaeumannomyces graminis var. tritici, which is related to virulence, on pathogenesis and disease expression. Experiments will be conducted in culture using high energy X-ray fluorescence (micro-XANES) techniques to measure Mn-oxidation states and in field plots by evaluating nutrient composition of plant tissue. The relationship of soil biological components involved in mineral cycling to virulence and pathogenesis will be determined by adding specific Mn-oxidation inhibitors and various mineral amendments to soils. Peptidase profiles of pathogens and potential biological control agents will be compared for indications of compatibility/ incompatibility relationships affecting virulence and pathogenesis.

In previous work in NY, two (-oxidation genes (fadB and fadL) in E. cloacae strain EcCT-501R3 that affect the ability of the strain to suppress cotton seed infection by P. ultimum were identified. Genetic and biochemical evidence indicated a role of linoleic acid metabolism in the interaction between E. cloacae, P. ultimum, and germinating cotton seeds. The results also supported a competitive exclusion model for the biological control of soilborne plant pathogens. Research in NY will continue to elucidate the biochemical basis for this interaction in the spermosphere and to further understand the regulatory aspects of (-oxidation in E. cloacae. Focus will be placed on FadR (encoded by fadR), a DNA-binding protein that controls both fatty acid biosynthesis and fatty acid degradation. Fatty acid exudation into the spermosphere of plants treated with wild-type and fad mutants of E. cloacae will be examined to verify the linoleic acid dynamics observed in vitro. Furthermore, fadR mutants that constituatively express FadR will be generated to alter the temporal dynamics of linoleic acid exudation and seed infection by Pythium spp.

Biocontrol mechanisms and traits of bacterial strains will be evaluated jointly in NE and NJ by biochemical and genetic means, using S. maltophilia strain C3 as the initial model system. The production of lytic enzymes (e.g. chitinases, proteases, glucanases, and lipases) and antibiotics in vitro will be investigated by electrophoretic separation and column purification. Purified materials will be tested for potential biological effects on fungal spore germination and hyphal growth. The role of these metabolites alone and in combination in biological control will be determined using genetic approaches. Random transposon mutagenesis will be conducted on strain C3, and mutants blocked in specific phenotypes associated with biocontrol, including the production of lytic enzymes will be selected by in vitro assays. Since strain C3 produces multiple isoforms for each of the various lytic enzymes, mutants isolated via this method are expected to be blocked primarily in regulatory genes that control expression of multiple phenotypes. Therefore, to effectively evaluate the role of a specific enzymatic activity in biocontrol, mutagenesis of structural genes for that enzyme activity will also be targeted. Selected structural genes will be isolated by direct cloning through heterologous expression in Escherichia coli or Burkholderiacepacia. Once isolated, genes will be further characterized by determining the nucleotide sequence. Mutations of each gene, disrupted at appropriate sites by sequence deletion or insertion of an antibiotic marker, will be incorporated into the genome of strain C3 by marker-exchange mutagenesis. For multiple mutations within a single strain, gene deletions will be performed using a marker rescue method. Once selected, mutant strains will be evaluated in comparison to the wild type for plant colonization and biocontrol efficacy in selected pathosystems, such as P. ultimum, R. solani and B. sorokiniana on grasses and cereals. The importance of induced systemic resistance (ISR) as a mechanism for biocontrol will be determined by applying the biocontrol agent to one part of a host plant and then challenging the same and distant parts with pathogens. To confirm the occurrence of ISR, host metabolites associated with ISR, e.g. chitinases and phytoalexins, will be assayed. These procedures will later be expanded to other microorganisms provided by NC-125 members.

Measurement of Progress and Results

Outputs

Outcomes or Projected Impacts

• Within the five-year period of this project, basic research conducted by NC-125 members will further knowledge regarding interactions among microorganisms and interactions of microorganisms with the plant host and the environment. The project will establish the fundamental scientific basis on which practical biocontrol systems will be developed and improved. This information, disseminated in scientific meetings and through publications, will assist researchers outside of the project in selecting, developing, and implementing biological control systems. Microorganism strains with commercial potential will be identified and methods for their production and delivery will be developed. The immediate beneficiaries of these results will be the microbial products industry. The project also will evaluate commercial biological control products in field setting and will identify cultural methodologies that will be easily implemented by crop producers, creating a sound scientific basis on which disease management recommendations can be made. This component of the project will be of direct benefit to growers and the crop production industry. Results from this project will be disseminated to the beneficiaries through the Cooperative Extension system and by agricultural and industry publications. Most of the products and cultural systems derived from this project will likely reach commercialization and implementation after the time frame of this project, but their successful use could lead to increased crop yields, lowered production costs, and reduced chemical use in some production systems.

Milestones

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Projected Participation

View Appendix E: Participation

Outreach Plan

Organization/Governance

The project will be administered by a technical comm. consisting of the project leader from each participating state or agency. An exec. comm. will consist of the chairperson, secretary, immediate past chairperson, and the Administrative Advisor (AA). The officers will serve 1 year, after which the secretary automatically becomes chairperson. The exec. committee will conduct business between meetings. Annual meetings to review research accomplishments and coordinate research will be authorized by the AA. Research will be reported by objective. The secretary will prepare minutes of the annual meeting and disseminate information among the committee members between annual meetings. The chair will conduct the annual meeting and prepare the subsequent annual report. Procedures outlined in the Manual for Cooperative Regional Research (Revised 1992) will be followed.



To increase the number of participating institutions and diversity of expertise, new members have been brought into the committee, including A.E. Desjardins, representing the USDA Nation Center for Agricultural Utilization Research, and L.L. Kinkel, a second representative Univ. of Minnesota. In addition, there will be new members from outside of the NC region-E.B. Nelson, Cornell University, and D.Y. Kobayashi, Rutgers University.



Cooperative Aspects: Time at each annual meeting will be reserved for coordinating the cooperative, regional aspects of this project. Specific strains of biocontrol agents, formulations, precautions, and procedures/techniques to be used in planting, inoculum delivery, monitoring and evaluations will be addressed. Standardized techniques and assay procedures will be established on a regional basis to ensure comparability of data. Specific methodologies available in one location can be made available to other researchers, with specialized training provided if appropriate. Identification, verification and evaluation of isolates can capitalize on the various project leaders expertise. Cooperation between members of this committee is best illustrated by our cooperative efforts on biocontrol of Rhizoctonia. Researchers from eight states (IL, IN, LA, OH, MN, NE, ND, and NY) will be comparing efficacy of biocontrol agents against Rhizoctonia on economically-important plants. The individuals involved will be sharing isolates of biocontrol agents for testing against various anastomosis groups of Rhizoctonia. In addition, three states are investigating mechanisms of action of biocontrol agents against Rhizoctonia. Comm. members will share their ideas, methodologies, and results of experiments to enhance the understanding of how to control the pathogen. Collaboration among members also has been, and will continue to be, in the forms of joint publication of research articles. Members also have collaborated in co-authorship of competitive grant proposals. As an example, G. Yuen (NE) and J.Parke (WI) obtained a NC IPM grant in 1994, "Biological control of Sclerotinia sclerotiorum on legumes in the North Central Region". Collaboration among committee members in seeking competitive grant funds will continue in the new project.


The research in NC-125 will be coordinated with related investigations in other regions through the exchange of publications, annual reports, personal contacts, etc. Jt meetings with other project committees will be held periodically. In addition, biocontrol agents will be exchanged with members of W-147 and S-269 to increase the range of crops and environments in which they are evaluated. NC-125 will sponsor a symposium on biological control in the NC region in conjunction with a regional group, such as the NC Division of the American Phytopathological Society. The committee will be involved in assembling information on biocontrol technologies used in the region. This information will be incorporated into an assessment of the technology to be compiled by the ESCOP-Biological Control Working Group.

Literature Cited

Agrios, G. 1988. Plant Pathology. Academic Press, New York. 803 p.

Bertagnolli, B.L., F.K. Dal Soglio, J.B. Sinclair, and D.M. Eastbum. 1994. Extracellular enzymes involved in the potential biocontrol of Rhizoctonia solani by Bacillus megaterium.and Trichoderma harzianum. Phytopathology 84:1136.

Dal Soglio, F., K., B. L. Bertagnolli, J. B. Sinclair, and D. M. Eastbum. 1998. Production of chitinolytic enzymes and endoglucanase in the soybean rhizosphere in the presence of Trichodermaharzianum and Rhizoctonia solani. Biological Control 12:111-117.

Giesler, L.J. 1998. Environmental effects on bacterial strains applied to the turfgrass phylloplane. Ph.D. Dissertation. University of Nebraska-Lincoln.

Giesler, L. J., and G. Y. Yuen. 1998. Evaluation of Stenotrophomonas maltophilia strain C3 for biocontrol of brown patch disease. Crop Protection 17:509-513.

Lewis, J.A, and Fravel, D.R. 1994. Biocontrol of damping-off and blight of bean by Sclerotium rolfsii. Biol. Cult. Tests 9:44.

Karakaya, A. and Martinson, C.A. 1995. The effect of some rhizosphere bacteria on development of maize. Taria Bitkileri Merkez Arastirma Enstitusu Dergisi 4:7-10.

King, E. B., and J. L. Parke. 1996. Population density of the biocontrol agent Burkholderia cepacia AMMDR1 on four pea cultivars. Soil Biol. Biochem. 28:307-312.

Lewis, J.A., Fravel, D.R. and Papavizas, G.C. 1995. Cladorrhinum foecundissimum: A biological control agent for the reduction of Rhizoctonia solani. Soil Biol. Biochem. 27:863-869.

McMullen, M., Jones, R., and Gallenberg, D. 1997. Scab of wheat and barley: a re-emerging disease of devastating impact. Plant Disease 81:1340-1348.

Munkvold, G.P.. and Desjardins, A.E. 1997. Fumonisins in maize- can we reduce their occurrence? Plant Dis. 81:556-565.

Poromarto, S. H., Nelson, B. D., Freeman, T. P. 1998. Association of binucleate Rhizoctonia with soybean and mechanism of biocontrol of Rhizoctonia solani. Phytopathology 88: 1056-1067.

Regner, K.M. 1996. The role of pyrrolnitrin in the suppression of damping-off of pea by Burkholderia cepacia strain AMMD. Ph. D Dissertation, Univ. Wise., Madison. 145 pp.

Ristaino, J.B., Perry, K.B., and Lumsden, R.D. 1996. Soil solarization and Gliocladium virens15 reduce the incidence of southern blight (Sclerotium rolfsii) in bell pepper in the field. Biocontrol Sci. Technol. 6:583-593.

Rodriguez, F., and W.F. Pfender. 1997. Antibiosis and antagonism of Sclerotina homoeocarpa and Drechslera poae by Pseudomonas fluorescens Pf-5 in vitro and in planta. Phytopathology87:614-621.

Schuize, D. G., T. McCay-Buis, S. R. Sutton, and D. M. Huber. 1995. Manganese oxidation states in Gaeumannomyces-infested wheat rhizospheres probed by micro-XANES spectroscopy. Phytopathology 85:990-994.

Wrather, J., Anderson, T, Arsyad, D., Gai, J., Ploper, L., Porta-Puglia, A., Ram, H., and Yorinori, J. 1997. Soybean disease loss estimates for the top 10 soybean producing countries in 1994. Plant Disease 81:107-110.

Zhang, Z., and G. Y. Yuen. 1998(a). Biological control of Bipolaris sorokiniana on tall fescue by Stenotrophomonas maltophilia strain C3. Phytopathology (in press).

Zhang, Z., and G. Y. Yuen. 1998(b). Chitinase production by Stenotrophomonas maltophilia strainC3 and its involvement in biological control. Phytopathology (in press).

Attachments

Land Grant Participating States/Institutions

CA, IA, IL, IN, MI, MN, ND, NE, NJ, NY, OH, WI

Non Land Grant Participating States/Institutions