Duration: 10/01/2003 to 09/30/2008

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

The future of sustainable agriculture in the U.S. will increasingly rely on the integration of biotechnology with traditional agricultural practices. Although genetic engineering promises enhanced yields and disease resistance, it is also important to recognize that plants exist in intimate associations with microorganisms, some of which cause plant disease while others protect against disease. Identifying, understanding and utilizing microorganisms or microbial products to control plant disease and enhance crop production are integral parts of sustainable agriculture. Biological control has the potential to control crop diseases while causing no or minimal detrimental environmental impact. For this proposal, we define biological control as the manipulation of microbial populations through cultural, physical or biological means including plant mechanisms. Some of the benefits of utilizing microorganisms include:

7 reduced dependence on chemical pesticides, important because of the increasing restrictions on chemical usage due to environmental and public concerns;

7 lack of development of pathogen resistance to biological control organisms, important due to the observed increase in resistance to many chemical controls;

7 selective action against specific groups of pathogens and not against beneficial organisms;

7 biodegradability of microbial pesticides and the by-products of their manufacture;

7 lack of danger to humans or animals;

7 improvement of soil and enhancement of agricultural sustainability.

During the past project period, W-147 made major contributions towards our understanding of microbial biological control of plant disease. To date several commercial biocontrol products and processes are available and registered by the EPA. These include fifteen bacterial and seven fungal products, and three activators of plant defenses (http://www.apsnet.org/online/feature/biocontrol/). However, the complexity of this area requires further research in order to overcome continuing problems in production, storage, delivery, reliability, efficacy, establishment and the understanding of the mechanisms of action.

Why a Multi-State, Multi-Disciplinary Approach? Due to the broad nature of the complex problems, this research must be multi-disciplinary and collaborative. No single research institution has sufficient resources and diversity of expertise to solve the problems. Many of these pathogens occur in multiple states and need a coordinated effort for research and to prevent duplication of effort. Because the results of our efforts are only now beginning to affect U.S. agriculture, continuation of the W-147 project for another five years will lead to further improvements in the use of biological control in agriculture.

JUSTIFICATION:

Economic Costs Due to Soil Borne Plant Pathogens

Soil borne plant pathogens result in severe yield and economic losses for growers. Economic losses due to pathogens are estimated at 10-20 % of the attainable yield for many crops (Pimentel, 1991). Yield failures resulting from acute diseases such as vascular wilts, take-all of cereals, Phymatotrichum root rot, Verticillium and Phytophthora may be even more severe and have destroyed entire agriculture industries. The soybean cyst nematode and Phytophthora stem and root rot are the most severe diseases of soybeans, and reduced yields by 7 million tons in the northern states during 1997 alone (Wrather et al. 2001).

7 For root diseases of mature crops, there are few effective and economical post-plant strategies for control.

7 About 90% of the 2000 major diseases of the principal crops in the US are caused by soil borne plant pathogens (Lewis and Papavizas, 1991).

7 Monetary losses due to soil borne diseases in the U.S. are estimated to exceed $4 billion per year (Lumsden et al., 1995), and losses due to parasitic nematodes exceed $8 billion per year (Barker et al. 1994).

7 Several of the top 15 restricted, invasive quarantine pathogens listed by APHIS are soil borne, and could represent a biosecurity risk.

Environmental Costs of Soil Borne Plant Pathogens

The cost of soil borne plant pathogens to society and the environment far exceeds the direct costs to growers and consumers. The use of chemical pesticides to control soil borne pathogens has caused significant changes in air and water quality, altered natural ecosystems resulting in direct and indirect affects on wildlife, and caused human health problems. For example, methyl bromide, a fumigant used to control soil borne diseases, has become notorious in recent years for depleting the ozone layer and changing the climate of our planet. The production and importation of this product will be banned by 2005, and is the subject of an intensive search for alternative methods. Many other chemicals are being removed from the market due to regulatory and public concerns. Additionally, plants evolved in the presence of microorganisms and are dependent on them in order to carry out many activities necessary for growth and reproduction. Thus, long-term chemical applications may permanently alter the microbial community structure sufficiently such that sustainable agriculture may be impossible.

Societys Expectations

As is readily apparent from reading the popular press, consumers are demanding plentiful low cost but safe food while simultaneously requiring the use of fewer chemical controls. New specialty and organically-grown crops will also require non-chemical methods for management of diseases. This has resulted in numerous new pesticide regulations and the loss of more and more pesticides to control soil borne diseases. Several soil borne diseases, for example, those caused by Phytophthora, Verticillium, Gaeumannomyces and Fusarium, remain major problems after more than 100 years of study. Soil borne pathogens are well adapted to soil conditions, and once established are very difficult to eliminate by any known method of control. Chemical controls are often too expensive to be economically practical and chemicals effective against many pathogens have yet to be identified. Other approaches with great potential include the development of transgenic crops engineered with resistance genes to several pathogens. However, there is widespread public reluctance to accept these crops as evidenced by protests both here and in Europe. This public concern, combined with the natural ability of pathogens to overcome introduced resistance genes, has frustrated efforts to maximize this approach.

The ultimate goals of this collaborative work of W-147 are:

7 To provide society with a safe, low cost food supply.

7 To reduce the environmental impact of food production

Biological Control As an Attractive Alternative

Biological control is an attractive approach for the control of soil borne diseases (Cook, 1993; 1990; Cook and Baker, 1983; Jacobsen and Backman, 1993; Lewis and Papavizas, 1991; Weller, 1988, 2002, Paulitz and Belanger, 2001, Boland and Kuykendall, 1998, Whipps, 1992, 1997, McSpadden Gardener and Fravel, 2002, Mathre et al. 1999. Advantages of a biological approach to disease control include a lack of environmental damage, reduced human health risks, lack of resistance development in the pathogen, selectivity in mode of action, lack of activity against most beneficial microorganisms, and improved soil conditions and agricultural sustainability.

Biological control of soil borne plant pathogens has made large strides over the past several years. Much of this success is due to activities of the members of W-147. Today the EPA lists more than 24 commercial biocontrol agents that are registered and commercially available in North America. Nearly all of them have been registered during the past five to ten years. However, most of these products are for seed and seedling diseases. W-147 project is unique in emphasizing biological control of root diseases of mature crops, including avocado, citrus, wheat, and turfgrass, which are generally not treatable with chemicals or other methods (Tables 2 and 3).

Interest and enthusiasm about biocontrol have never been greater. A recent analysis of articles published in 1996 in Phytopathology, the premier plant disease journal in the U.S., shows that nearly 20% of the articles dealt with biocontrol. In fact, two new journals were launched in the 1990s-the journal Biological Control, which covers both arthropod and microorganism-mediated control methods, and Biocontrol Science and Technology. Combined with the increasing resistance in parts of the world to transgenic plants, it appears that the W-147 regional project is both very timely and successful.

In spite of the strides made in biological control research, there are many areas that require work before biocontrol will be used extensively. Current areas of research include:

7 Identification of more effective agents. Workers are isolating potential antagonists from soils where many pathogens originated.

7 Understanding the genetic diversity of biocontrol agents.

7 Identification of natural disease suppressive soils.

7 Development of lower cost production, storage, and distribution systems.

7 Improved quality control assays.

7 Improved stability of the agent during production, storage and application.

7 Integration of biocontrol into current agronomic practices.

7 Identification of parameters affecting efficacy and survival after application.

7 Understanding the mechanisms of action of control, especially at the molecular and biochemical level.

7 Investigation of manipulation of cultural parameters that advance biological control (compost, green manures, rotation crops).

7 Understanding the role of the plant in biological control (induced resistance, genetic resistance)

7 Microbial community and plant-microbe interactions with biocontrol agents.

The promise, public acceptance and environmental benefits of biocontrol continue to make research on this area both timely and of critical importance to the future of U.S. and world agriculture.

Despite over 30 years of research, biological control of plant pathogens is not widely used in commercial agriculture and sales of biofungicides represented less than $1 million compared to total fungicide sales of $5.5 billion (Powell and Jutsum, 1993). Powell (1991) summarizes the current status of plant biocontrol agents when he says, the real problem for biological control is to deliver an active agent to the site where it is required and keep it there while activity is required. We are yet unable to do that efficiently with most of our current biocontrol agents. Clearly there is much to be done in order to improve biocontrol agents so that they will become major factors in the control of soilborne diseases. Biocontrol agents isolated by participants of W-147 at ARS-WA, ARS-CA, CA-R, OR, MT, AK and NY have the ability to suppress a wide variety of plant pathogens that cause serious diseases of food, fiber and ornamental crops. The need for high quality biocontrol agents has never been more critical because of the pending loss of fungicides and fumigants upon which agriculture has been dependent for the last 50 years. Consider the billion-dollar-a-year commercial strawberry industry in California, which relies exclusively on soil fumigation with a combination of methyl bromide and chloropicrin at about 250 lb/acre for disease, nematode and weed control. The mandated 75% reduction in the use of methyl bromide by 2003 will leave this industry vulnerable to soilborne nematodes and pathogens. Biocontrol may provide a safe, environmentally sound alternative to methyl bromide and other valuable agricultural chemicals that may be lost in the future. Understanding the complex biological and environmental interactions that must occur for biocontrol to be effective requires the combined efforts of multiple investigators at multiple institutions focusing on different aspects of the problem, from applied to basic research. This logical approach is an area in which the W-147 regional project has excelled and will continue to depend on during the next five years.

This project also fits the goals of other CSREES initiatives, including the National Integrated Food Safety Initiative of 1998, and other programs, such as Integrated Pest Management (IPM), Methyl Bromide Transition Program (MTB), Pest Management Alternatives Program (PMAP) and Sustainable Agriculture (SARE).

Related, Current and Previous Work

There are a number of reviews on the use of biocontrol organisms to control soilborne plant pathogens Becker and Schwinn, 1993; Cook, 1993; 1990; Cook and Baker, 1983; Jacobsen and Backman, 1993; Lewis and Papavizas, 1991; Powell, 1991; Weller, 1988, 2002; Whipps, 1992, 1997, Paulitz and Belanger, 2002). The EPA currently lists more than 24 biocontrol agents that are registered or pending registration with the EPA and available for commercial use in the U.S. (http://www.oardc.ohio-state.edu/apsbcc/productlist.htm).

Identification of New Biocontrol Agents

A list of 44 biocontrol agents which have reduced disease are listed by Cook and Baker (1983). However, few of these biocontrol agents are currently being used in mainstream agriculture. One promising approach is to search for new and better biocontrol agents. Most of these efforts involve isolating and selecting biocontrol agents on growth media and testing in the laboratory or the greenhouse. Few are looking for biocontrol agents in foreign lands where pathogens may have evolved. Few are isolating slow growing or non-cultivable biocontrol agents using the pathogen as bait (Lewis and Papavizas,1991). Estimates suggest that previous isolations have yielded only a tiny fraction of the microorganisms that exist in soils and on plant surfaces. For example, it is estimated that there are over 1.5 million species of fungi that have not been described (Hawksworth et al. 1991). Thus, we have barely scratched the surface in our hunt for biocontrol agents. W-147 members have been instrumental in discovering many of these new biocontrol agents, including strains of Pseudomonas fluorescens with superior colonizing ability (Q8R1-96) (Raaijmakers and Weller, 2001), diacetylphoroglucinol-producing strains of P. fluorescens (Raaijmakers and Weller, 1998), Trichoderma atroviride 901C (McBeath, 2001); Pseudomonas aeurofaciens AB254 (Mathre et al. 1999); Rozella sp., Lytobacter mycophilus, and Hyphodontia alutacea, (Menge, unpublished); Dactylella spp., and unclassified species of Rhizobium and a-proteobacteria for control of cyst nematodes (Becker and Borneman, unpublished).

Mechanisms of Disease Control

Biological control agents express a variety of mechanisms that are responsible for pathogen inhibition. Therefore, if we are to maximize the effectiveness of any biocontrol agent, we must understand the function of the mechanism in the biocontrol agents lifestyle. Known mechanisms by which biocontrol organisms reduce disease include:

7 Induction of plant resistance mechanisms.

7 Competition for nutrients or space.

7 Antibiotic and toxin production.

7 Cell-wall degrading and lytic enzymes.

7 Siderophore production.

7 Biosurfactant production

7 Mycoparasitism

Several of these mechanisms were identified only recently by members of the W-147 project. These include the role of biofilms (Zhang and Pierson, 2001, Stanghellini and Miller, 1997), the genetic and biochemical pathways for production of phloroglucinol and phenazine, (Pierson et al. 1995, McSpadden Gardener et al. 2000), the regulation of phenazine production (Mavrodi et al. 1998), the role of phloroglucinol producers in suppressive soils (Raaijmakers and Weller, 1998) and the in situ detection and quantification of antifungal compounds produced by biocontrol agents in the soil and rhizosphere (Thomashow et al. 2002). There are certainly many more mechanisms as yet undiscovered. Furthermore, although the mechanisms are known for some biocontrol agents, these agents do not control disease efficiently. This suggests that we do not yet understand the effects of nutrients, environment and growth stage on the control mechanisms. For instance Pierson and Pierson, (1996) have recently shown that environmental factors and other organisms regulate the amount of phenazine antibiotic produced by the biocontrol bacterium, Pseudomonas aureofaciens. This means that it is not enough to understand that phenazine production is the mechanism for biological control, but we must understand: when it is produced, where it is produced, why it is produced, how much of it is produced, which environmental factors affect its production and what influence the indigenous microflora has on its production.

These examples serve to illustrate the point that every biocontrol agent-plant pathogen-host crop system requires special insight on how best to utilize the biocontrol agent to maximize disease control. This maximization of biocontrol will be different for different regions of the United States. Biocontrol is a complex process, and therefore much research is needed to understand the mechanisms involved.

Production, Formulation, Storage and Application

In order for biocontrol organisms to be commercially accepted, strategies must be developed for their production, formulation and application in the field. Only when these additional conditions are met will biocontrol be accepted as a major tool for reducing disease. For most biocontrol organisms, this has not been accomplished. Indeed, commercial development of biocontrol agents for disease control has lagged far behind that which has occurred in entomology for the control of insects. Commercial formulations and delivery systems for biocontrol agents have been reviewed by Lewis and Papavizas (1991). However, Cook, (1990) cautioned against this rigid approach and recommended that field utilization of biocontrol agents be placed into three broad categories:

7 Maximization of the use of naturally occurring biocontrol through manipulation of crop rotation, mulching, composting and other cultural practices. Crop systems management for biocontrol of soilborne disease may be the most practical and successful method of biocontrol and could lead to the discovery of more biocontrol methods. Cook (1990) included the utilization of naturally occurring disease suppressive soils in this category.

7 Introduction of well-adapted rhizosphere competent biocontrol agents via the methods described by Lewis and Papavizas (1991) with the hope that they will survive and reproduce. With the introductive approach, amendments, additives, habitat modifications, or partial sterilization of the soil may enhance the establishment of the biocontrol agent. This method has been the one most commonly attempted, but mostly with seed pathogens, seedlings or crops grown in potting media. It has yet to be practiced widely under field conditions with a wide variety of crops.

7 Redundant application of biocontrol agents multiple times per growing season. This approach utilizes microorganisms as microbial pesticides. This technique is virtually untested, but it has many advantages since it does not require that the organisms establish or survive and they can be applied in large numbers in a condition when they are most effective. The technology to attempt this type of biocontrol has only become available in the last few years. Regardless of the methods used to produce, formulate and apply biocontrol agents, few have proved successful in a large scale. Improved methods and more research are required under field conditions if biocontrol is to be widely used on a commercial scale.

Members of W-147 have developed and are testing novel strategies in the formulation and application of biocontrol agents. For example, researchers at UCR have demonstrated that biocontrol agents can be continually produced and applied to crops using a fermenator-injector system, allowing multiple applications over a season, and eliminating the problem of storage and survival of biocontrol agent inoculum (Steddom et al. 2002) .

In summary, research related to the objectives outlined above is in progress throughout the world. This indicates that the objectives are valid and timely, and that the potential for success is great. In spite of this effort, biological control agents have not resulted in great changes in agricultural methods or strategies. This lack of broad acceptance reflects two major obstacles. First is the complexity of the ecological systems biocontrol agents must operate in. Second is the current mindset of growers that biocontrol is expensive and inconsistent. In order to overcome both hurdles, we must better understand the factors that influence the efficacy of biocontrol agents once released in the field. Then we can begin to manipulate the system as a whole in order to maximize the potential benefits of biocontrol to long-term sustainable agriculture.

Differentiation from other regional workgroups

Currently three regional workgroups are focusing on various aspects of microbial biological control. Each group is focusing on complementary issues and concepts using different microorganisms in different geographic regions. W-147 project is unique in emphasizing biological control of root diseases of mature crops, which are generally not treatable with chemicals or other methods. A comparison of the objectives of the regional projects NC-125 and S-302 that are most closely related to the objectives proposed by W-147 is presented in Table 1.

Although a quick glance at the above table might suggest that there is considerable overlap among the objectives of the three projects, each project utilizes a different approach to attain their objectives. Research conducted under W-147 involves a greater emphasis on understanding the mechanisms involved in plant-microbe interactions. In addition, W-147 research is unique in that biocontrol organisms and approaches must be customized for a more arid climate than that of the other regions. The combination of approach, crops, pathogens, cropping systems and biocontrol agents distinctive to W-147 provides a unifying theme that facilitates progress toward the objectives. Differences among the three regional projects will serve as a benefit by allowing these groups to compare results across the U.S. during the joint meeting held every 3 years.

Table 2 provides a comparison of the disease systems and hosts to be investigated by members of the three regional projects. Table 3 gives more detail on common areas of research. It is apparent even where there is overlap of host crop and pathogen, different research approaches and objectives differentiate the projects. Again, regional, soil, climate and farming practices also separate what appear to be similar projects. Research leaders, area of specialization and resources are listed in Table 4. The responsibilities of the states with respect to soil borne plant pathogens and objectives addressed are shown in Table 5.

Objectives

1. 1. To identify and characterize new biological agents, naturally suppressive soils, cultural practices, and organic amendments that provide control of diseases caused by soil borne plant pathogens
   
2. 2. To understand how microbial populations and their gene expression are regulated by the biological (plants and microbes) and physical environment and how they influence disease.
   
3. To develop and implement biological control in agriculture.
   
4. 
   

Methods

One of the difficulties of improving biological control of soil borne plant pathogens is the complexity and diversity of hosts, pathogens, competing microflora, soils and cultivation practices. The greatest strength of the regional approach to research by W-147 is the broad and diverse expertise each member brings to the group. In studying such a diverse group of plant-pathogen systems, common procedures are not practical and even interfere with novel discoveries. Thus, each collaboration described below will entail unique aspects of methodology and analysis. Objective 1: To identify and characterize new biological agents, naturally suppressive soils, cultural practices, and organic amendments that provide control of diseases caused by soil borne plant pathogens. To accomplish this objective, all states will continue to isolate and test individual and mixtures of microorganisms against diseases of local concern. This is necessary because often a single biocontrol agent will not provide control of a disease in all agroecosystems in which that pathogen occurs. Furthermore, the best agents against a pathogen often come from a local soil because the performance of biocontrol agents is greatly affected by biotic and abiotic factors. Participants will continue to screen biocontrol agents against the most important pathogens causing serious disease on crops in the western region. These include Verticillium dahliae, Sclerotinia, Myriosclerotina, Typhula, Monosporascus cannonballus, Thielaviopsis, Macrophomina, and Fusarium. In addition, intensive bi- and tri-state cooperative screening efforts will continue for antagonists of Gaeumannomyces graminis var. tritici (ARS-WA and MT), Phytophthora (CA-R, AK), Pythium (ARS-WA, ARS-CA., CA-R, AK, MT), Rhizoctonia (AK, ARS-WA, MT, NY) and plant parasitic nematodes (CA-R, WA and NY). A central database of biocontrol agents will be established on a website. Members will exchange and share promising collections, which include fungi, bacteria and other organisms which suppress plant pathogens through a variety of different mechanisms. We will also have extension researchers and county agents include these biocontrol agents in demonstration plots. Strains will be added to the collections as new organisms become available. It is hoped this approach will facilitate identification of those premier biocontrol agents that perform on a broad range of hosts, in different soil types or environments, at low doses and on a variety of diseases. The search for new biocontrol agents will continue. Putative biocontrol agents will be selected from bulk soil, the rhizosphere, the spermosphere, inside plant tissue and pathogen propagules. Special attention will be given to slow-growing or fastidious microorganisms using culture-independent techniques, such as oligonucleotide fingerprinting of ribosomal RNA genes (OFRG), a method that has been adapted and perfected by W-147 members at CA-R (Borneman and Hartin, 2000). This method has identified organisms (Dactylella spp. and Fusarium spp.) that may be involved in beet cyst nematode suppression. These organisms may have been missed in the past because of the use of nutrient agar as the substrate and the difficulty of culturing them. Natural soils suppressive against Phytophthora cinnamomi (CA-R), Pythium (ARS-WA, CA-R), Heterodera schachtii (CA-R), and Gaeumannomyces graminis var. tritici (ARS-WA), will be assayed for potential biocontrol agents. Induced suppressive soils such as those that develop after mulching, plowing in crop residues or rotating crops will also provide a source of biocontrol agents (ARS-CA, CA-R, CA-D, MT, NY). The development of suppressiveness under direct-seed or no-till cereal systems in the Pacific Northwest (PNW) will be a focus of research by ARS-WA. One of the limitations of direct-seed systems is the initial buildup of certain residue-borne pathogens such as Rhizoctonia solani AG-8 and R. oryzae. However, after long-term no-till, suppressiveness may develop, as demonstrated in Australia with R. solani. Researchers at ARS-WA will perform cycling experiments by planting barley monthly into intact soil cores in the greenhouse, in an attempt to accelerate the development of monoculture decline. If suppressiveness develops in these experiments, the responsible microorganisms can be isolated. Researchers at ARS-WA (Prosser) will study the succession of pathogens and biocontrol agents in the transition from natural virgin soil (never cropped) to irrigated annual crops. During this transition, certain species of Pythium become predominant, and the changes in microbial communities that accompany this pathogen shift may provide insights on the pathogen equilibrium maintained in native sites. New biocontrol agents will also be pursued through foreign exploration. For example J. Menge (CA-R) has traveled to New Guinea and surrounding areas to search for biocontrol agents of Phytophthora cinnamomi in areas where this fungus supposedly originated, and will continue to receive samples from collaborators in Southeast Asia. This is a strategy currently employed successfully by entomologists to control introduced insect pests. D. Weller (ARS-WA) has collected take-all suppressive soils from the U.S. and will collect in Europe in summer, 2003, as part of a sabbatical. The process of selecting candidate biocontrol agents will utilize methodologies which includes: 1) testing for antagonism against a target pathogen or a variety of soilborne plant pathogens; 2) testing putative agents in a seedling assay in the laboratory, greenhouse or growth chamber; 3) testing strains that show promise in small field plots; and, 4) testing the most effective agents in large-scale field plots using commercial practices. Selection methods (i.e., type of media, environmental conditions, soil type, etc.) cannot be standardized because the antagonists and pathogens to be studied in this project vary. Molecular genetic techniques will increasingly be used to facilitate the selection process. For example, potential biological control agents can be identified by correlating population levels with suppressiveness, using culture-independent approaches. In addition, biosynthetic loci for many metabolites known to be involved in biocontrol have been cloned and sequenced. Examples include phenazine-1-carboxylic acid, 2,4-diacetylphloroglucinol, pyrrolnitrin, pyoluteorin, rhamnolipids, siderophores and hydrogen cyanide. These genes can be used as markers to select for superior strains. For example, certain genotypes of the PhlD gene for biosynthesis of phloroglucinol are associated with a strong colonization ability on peas (Landa et al. 2002). Knowledge of these genes and their sequences can be useful to select superior biocontrol agents. Colony hybridization and PCR in combination with probes and primers specific for sequences within the loci encoding these metabolites can be used to rapidly select strains that contain these known biocontrol traits. Biological control of plant pathogens may be divided into three categories: 1) biological control of the plant pathogenic inoculum; 2) biological protection of plant surfaces; and 3) biological control of post infection, i. e. induced resistance or cross protection. This objective will continue the long history of cooperation that has occurred in this regional project since its inception. Tables 6, 7, and 8 in the appendix show a small sample of the extensive amount of on-going cooperation within the project based on the three categories of biological control. Objective 2: To understand how microbial populations and their gene expression are regulated by the biological (plants and microbes) and physical environment and how they influence disease. Previous cooperation among W-147 members has led to the identification of several new mechanisms of disease control. Environmental and biological factors in the field affect both the populations of biocontrol agents and the expression of genes within the biocontrol agents, which are responsible for disease control. In order to improve the consistency of biocontrol, the effect of biological and environmental factors on populations of biocontrol agents, on populations of pathogens, and on the genetic expression of the mechanisms responsible for disease must be understood. Members of W-147 are utilizing both genetic and environmental approaches to understand the causes of inconsistency and methods to overcome it. W-147 members have now established an impressive collection of potential biocontrol agents that utilize different antagonistic mechanisms against soilborne plant pathogens. These biocontrol agents include bacteria, fungi and actinomycetes currently being studied in the laboratories of each member (AK, AZ, CA-R, MT, NY, OR, WA). (Table 6 and 7). These strains will be utilized for a number of purposes in more than one of W-147s objectives. Several members of W-147 are focusing on the genetic approach to solving problems with the consistency of biocontrol organisms. There is a major focus by OR, AZ and ARS-WA to understand the regulatory mechanisms utilized by biocontrol agents to control the expression of genes encoding products involved in disease suppression. For example, because antibiosis is involved in the efficacy of many bacterial biocontrol agents, researchers will be focusing on the regulation, biosynthesis and mutations of the pathways leading to antibiotic production. This work has already characterized the biosynthetic pathways for phenazine and phloroglucinol production. Using the primary model of phenazine production in Pseudomonas aureofaciens strain 30-84, we have shown that antibiotic production is regulated in this bacterium by quorum sensing and multiple interconnected regulatory pathways; and that the indigenous rhizosphere community influences gene expression in strain 30-84. Further research plans include characterization of negative signaling strains and their effect on antibiotic gene expression in the rhizosphere, identification of genomic loci regulated by the second quorum sensing system, and analysis of the effects of mutations in each quorum sensing system on cell structure, colonization and biofilm development. For the first time ever, the genome of a biocontrol agent will be completely sequenced and annotated- Pseudomonas fluorescens Pf-5. This project will be carried out by members of W-147 from AZ, ARS-WA, and ARS-OR. This will provide valuable information for all Pseudomonas research, and enable the identification and function of genes crucial for biological control. For example, workers at ARS-WA are attempting to understand why the DAPG-producing strain Q8r1-96 has such a superior colonization ability, compared to the closely related DAPG-producing strain Q2-87. Using subtractive hybridization techniques, they identified unique genomic DNA fragments present in strain Q8r1-96 but not in Q2-87. Full-length copies of these loci will be retrieved from a genomic library and sequenced, in order to evaluate the role of these genes in the unique root colonizing ability of Q8r1-96. These sequences may also be present in the genome of Pf5, and research by other Pseudomonas workers may help to identify the function of these colonization associated genes. By defining the genetic basis for the aggressive colonization activity of strains such as Q8r1-96, other strains can be selected with improved biocontrol effectiveness and reliability. Workers at ARS-OR are establishing DNA hybridization arrays for better assessing gene expression by Pf-5 on seed surfaces and identifying factors that influence the in situ expression of key biocontrol genes by this bacterium. Understanding the genetic diversity and population structure of biocontrol agents in suppressive soils will be a goal of research at ARS-WA. Using DNA fingerprinting techniques such as BOX-PCR, ERIC PCR, and restriction analysis of amplified 16S rRNA gene sequences, they identified seventeen genetic groups or subspecies within populations of DAPG producers. Certain groups were shown to have superior abilities to colonize the roots of wheat or pea or both crops, and control root diseases. Studies will continue to examine populations of DAPG producers under different crop rotation and tillage practices. Using this information, the performance of DAPG-producing biocontrol agents will be greatly improved, because agents can be targeted for use on crops or against diseases for which they are most adapted. Researchers at OR will explore the natural occurrence and diversity of Burkholderia cepacia complex bacteria in soil. The Burkholderia cepacia complex (Bcc) consists of at least nine bacterial genomovars that differ in their ecology, biocontrol efficacy, and potential to cause human disease. Using culture-independent methods, they have shown that natural populations of Bcc occur with high frequency (>80%) in soils with which people commonly have contact. This may be important in evaluating the risk assessment of potential biocontrol strains of Bcc. They will determine the role of cultural practices (rotation crop, soil type) on the abundance and diversity of Bcc. In order to understand biocontrol interactions, we need to better understand the make-up and diversity of soilborne pathogens that a plant will encounter and that the biocontrol agent must antagonize. Researchers at ARS-WA and ARS-CA are using molecular techniques such sequencing the ITS-1 region of rDNA and the mtDNA of the cytochrome C oxidase II to study diversity and phylogeny of Pythium at the species and subspecies level. For example, the composition of Pythium species in the wheat producing areas of inland PNW is relatively unknown. Aided by ITS-1 sequences, Paulitz et al. (2003) discovered a new species of Pythium (P. abappressorium) on wheat in eastern Washington. Although this species in widespread, it has never been included in biocontrol screening. This information will be useful for biocontrol testing, since some strains of the pathogen may be less sensitive to antifungal metabolites, and this may explain some of the inconsistency in biocontrol efficacy. Amplified fragment length polymorphisms will be used by researchers at ARS-WA to study the population structure of Rhizoctonia spp. in the Pacific Northwest. Another area of research pursued by members of W-147 is how the plant influences the biocontrol agent and pathogen. For example, researchers at UC-R have demonstrated that ascospores of Monosporascus cannonballus only germinate in the rhizosphere of melon or cantaloupe, but not other hosts. If these natural chemicals that stimulate germination can be identified, they be could be applied as a pheromone to stimulate germination before planting in the absence of the host plant, therefore causing the propagules to commit suicide through germination-lysis. While some researchers are looking for the genetic basis of biocontrol in the biocontrol agents, others are looking at genes in the plant that may stimulate biocontrol colonization in the rhizosphere. Workers at ARS-WA have observed that some wheat cultivars support higher populations of an aggressive P. fluorescens colonizer as compared to a weaker colonizer, whereas some cultivars do not support high populations of either the strong or weak colonizer. Using wheat root ESTs developed through the NSF and ITEC wheat genome projects, they will attempt to monitor specific host responses and identify genes that are up regulated in the presence of the biocontrol agent and pathogen. Many members of W-147 will pursue the mechanisms of biocontrol utilizing the environmental approach. Many participants have identified soils that are naturally suppressive to specific soilborne plant pathogens. Soils suppressive to Fusarium (NY), Meloidogyne (NY), Pratylenchus (NY), Pythium (ARS-WA, ARS-CA, CA-R, MT, NY), Phytophthora (CA-R), Rhizoctonia (ARS-WA, NY), Thielaviopsis (NY), Gaeumannomyces (ARS-WA), Heterodera (CA-R), and Verticillium (ARS-CA) have been identified. Those soils are influenced strongly by cropping sequences, organic amendments and crop management. Those soils suppressive against Heterodera and Phytophthora (CA-R) appear to be naturally suppressive. The suppressive factors in all of these soils will be sought by treating the soil with pesticides, fumigants, steam or heat to identify fractions with the biocontrol factor. Isolations, baiting, RAPD analysis, and microarray-based ribosomal RNA analyses, will be utilized to try to identify the organisms responsible for biocontrol. Researchers at OR will examine potential suppressiveness Phytophthora ramorum, causal agent of Sudden Oak Death, a newly described, apparently non-indigenous pathogen with a broad host range including native and horticultural vegetation. The effect of introduced biocontrol agents on non-target microbes in the rhizosphere is being investigated by researchers at ARS-WA. Using techniques such as T-RFLP (terminal restriction fragment length polymorphisms) analysis of the 16S ribosomal DNA, a culture independent technique, they will study the shifts in populations of non-target microbes in the rhizosphere of wheat grown in the field from seeds treated with Pseudomonas strains. This information will be useful for determining the long-term impacts, if any, of introduced strains. Finally, biological control agents will be labeled with genetic markers and introduced into the soil (CA-R) to follow the population dynamics. Methodologies include quantitative real-time PCR, DNA fingerprinting and labeling organisms with green fluorescent protein (GFP). These are new and novel ways to determine which soil organisms are predators or parasites of soilborne plant pathogens even though these biocontrol agents may not be cultivable. Using this method, the organisms responsible for soil suppressiveness may be discovered. Objective 3: To develop and implement biological control in agriculture. All participants of W-147 are either directly or indirectly involved in this objective (ARS-WA, ARS-CA, AK, AZ, CA-R, MT, NY, OR and WA). To date, Objectives 1 and 2 have done much to lay the ground work for the implementation of the biological control of plant diseases and results are providing reasons for optimism. With the continuation of W-147, we can now utilize results developed previously to obtain practical and beneficial economic results for the grower. During the next five years the W-147 project proposes to use all three of Cooks (1990) strategies for biocontrol to accomplish Objective 3 (Tables 9, 10, and 11): 1) the treatment of plant material and soil with biocontrol agents to reduce plant disease and maintain soil quality; 2) to encourage natural biological control with mulches, soil composts, and/or cropping practices to increase and support biocontrol agents; and, 3) the continuous application of biocontrol agents into irrigation water. Although target pathogens differ between states, crops, and soil conditions, members of this project are working cohesively toward the same goal of sustainable agriculture. Without exception, all states are cooperating with at least one other state associated with W-147. For specific examples of projects and methodology see Tables 9, 10, and 11. During the last 10 years a dramatic shift has occurred from the use of biocontrol agents primarily as seed treatments, to the use of specific cropping practices to sustain biological control. In addition to effects with biocontrol agents, more general effects are also being achieved by the use of mulches that favor the build up of organisms and green manures and rotation crops that are shown to significantly affect the microflora in the soil to bring about biological control. We propose to maximize these cultural controls and maximize the biocontrol abilities of these crop management practices.

Measurement of Progress and Results

Outputs

• Success of the project will be measured via publications, a web site, and presentations to growers and at scientific meetings. Additional outputs will be new biological control approaches: new organisms, cultivation systems, or formulations.

Outcomes or Projected Impacts

• The impacts of the proposed work will be substantial. The results of the work will benefit growers, consumers, and the environment by making significant progress in producing low cost safe agricultural products. A greater understanding of the basic molecular and biochemical mechanisms will allow a better selection and improvement of existing new strains, and a more rational implementation of these organisms. By understanding how the biocontrol agents interact with the plant and the environment, we can better understand their limitations and inconsistency in the field.

Milestones

(2004): Establish web site, database for suppressive soils, list of biocontrol agents, identify novel suppressive soils.

(2005): Identify novel biocontrol agents from suppressive soils

(2006): Test potential organisms in lab, greenhouse, and field plots.

(2007): Elucidate mechanisms.

(2008): Implementation and application at commercial scale.

(0):0

Projected Participation

View Appendix E: Participation

Outreach Plan

The W-147 project will be made publicly available to growers and other researchers using several formats, including:

7 Refereed publications in scientific journals.

7 Peer-reviewed publications and brochures.

7 Talks by several members of W-147 to grower groups.

7 Communication of work to stakeholders by committee members with extension appointments

7 Posting of relevant research projects, approaches, results and discussion on a newly-designed web site.

7 Triannual joint meetings with the other two biocontrol research project groups. These were begun several years ago and have afforded great communication among the three groups.

Organization/Governance

The W-147 regional research program will be administrated by a technical committee consisting of a project leader from each of the participating states. Officers of the committee will be the Chairman and Secretary. The Secretary will be elected each year and will advance to Chairman the following year. For 2003-04 the committee officers will be Chairman- L. S. Pierson, Secretary: N. Callan. Meetings will be called each year by the administrative advisor, and a local arrangements coordinator will be determined for each annual meeting. At those meetings research accomplishments will be reviewed and recommendations made for coordination and publication of results. Several new members have been recruited into the project- Niklaus Gr|nwald, ARS-Prosser, Washington; Frank Martin and Carolee Bull, ARS-Salinas, CA; Krishna Subbarao, Univ. Calif. Davis; Jennifer Parke, Oregon State, Corvallis; Fred Crowe, Oregon State University, Pendleton; Joyce Loper, ARS-Oregon State University, Corvallis; Ekaterina Riga, Washington State University, Prosser; Natalie Goldberg, New Mexico State University, and Patricia Okubara, ARS-Pullman, WA, and Wesley Chun, University of Idaho.

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Attachments

Land Grant Participating States/Institutions

AZ, CA, HI, IL, ME, MN, MT, NM, NY, OH, OR, WA

Non Land Grant Participating States/Institutions

ARS-WA