W3147: Managing Plant Microbe Interactions in Soil to Promote Sustainable Agriculture

(Multistate Research Project)

Status: Active

W3147: Managing Plant Microbe Interactions in Soil to Promote Sustainable Agriculture

Duration: 10/01/2013 to 09/30/2018

Administrative Advisor(s):


NIFA Reps:


Statement of Issues and Justification

The future of sustainable agriculture in the U.S. will increasingly rely on integrating various advanced technologies with traditional agricultural practices. Although genetic engineering of crop plants promises enhanced yields and new routes to disease resistance, it is also important to recognize that these plants associated with microorganisms, some of which cause plant disease while others protect against disease. Identifying, understanding and using microorganisms or microbial products to control plant disease and enhance crop production are becoming more central parts of sustainable agriculture. Biological control or biologically-based pest management (BBPM) 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 antagonistic or suppressive to plant pathogens through cultural, physical or biological means, including plant mechanisms. Biological control may affect the populations and/or activity of plant pathogens. Some of the benefits of utilizing microorganisms include:


- Reduced dependence on chemical pesticides, which is important because of expanding demand for organic produce and increasing costs of such petroleum-based inputs

- Reduction in the development of pathogen resistance to biological control organisms,

- More selective action against pathogens and not against beneficial organisms;

- Biodegradability of microbial pesticides and the by-products of their manufacture

- Reduced danger to humans or animals

- Improvement of soil quality and health

- Increased food safety

- Management of diseases in natural ecosystems

- Improve plant productivity via controlling abiotic stress

- Adaption to climate change, as pathogen distributions shift

- Increased N use efficiency and reduced N and P contamination of waterways and oceans



Demand for biopesticides has continued to expand dramatically in the last five to ten years. A 2009 report from research firm Frost & Sullivan put the value of biopesticides in the US and Western Europe combined markets at US $594 million in 2008 and forecasted that the market will nearly double by 2015 to a market value of $1.02 billion (Biopesticide Industry Alliance, 2012). The Biopesticide Industry Alliance, established in 2001, had 31 member companies in 2006 but now has 65 members in 2012. The International Biocontrol Manufacturer's Association currently has over 130 companies marketing microbial biocontrol agents. This growth has been driven by expanding organic markets as well as increased public sensitivity to the risks and hazards of chemical pesticides. Within the last four years, 15 microbial active ingredients have been registered by EPA. Presently, there are 23 bacterial and 17 fungal active ingredients registered in the US.



However, further research is required to overcome problems related to high-volume production, storage, delivery, compatibility with conventional products, and formulation of such products. With increased demand for biological options, new active ingredients or organisms will need to be identified and characterized. In addition, regulatory agencies are increasingly interested in understanding the mode of action of such novel microbials. Therefore, basic research into the physiology and genetics of biocontrol microbes will continue to be needed. More research is also needed on how to use these products with integrated pest management (IPM) programs that have been developed for many crops.



This proposed research fits a number of the Strategic Goals and Objectives for 2010-2015 established by NIFA:

Objective 4.4
Protect Agricultural Health by Minimizing Major Diseases and Pests to Ensure Access to Safe, Plentiful, and Nutritious Food;

Objective 3.3  Support Sustainable Agriculture Production in Food-Insecure Nations and

Objective 1.3  Support a Sustainable and Competitive Agricultural System.

These goals are tied together by biological control and biologically based pest management- using the idea that the environment is enhanced by reducing our reliance on fungicides and nematicides to manage disease. Whether in rural communities or in developing countries, this research is geared toward rational, low input and sustainable agriculture.



Why a Multi-State, Multi-Disciplinary Approach? Because biological control is the result of complex interactions between the agent, the environment, and the pathogen, research in this area must be multi-disciplinary and collaborative. No single research institution has sufficient resources and variety of expertise to solve the diverse disease problems that might be addressed through the use of biological controls. Many of the targeted pathogens occur in multiple states and a coordinated research effort could provide more cost-effective outcomes. Because the results of our efforts are only now beginning to affect U.S. agriculture and the biopesticide industry, continuation of the W-2147 project for another five years will lead to further improvements in the efficacy and adoption of biological controls in American agriculture. In addition, these biological and cultural control techniques need to be tested under a range of environmental conditions and cropping systems that reflect the diversity of U.S. agriculture. The more than 20 researchers in this multistate project also collaborate with researchers in the U.S. and around the world, providing further impact and cross-fertilization of knowledge, as well as conducting the needed outreach activities for implementation of biocontrol options. In addition, because of the Great Recession and strained state and now federal budgets, the number of faculty and researchers in plant protection has been significantly reduced. Because of this reduction in resources and human capital, it is more important than ever to gain synergy by leveraging resources with a multi-state group.



JUSTIFICATION:



Economic Costs Due to Soilborne Plant Pathogens

From 2001-2003, an average of 7% to 15% of the major world crops (wheat, rice potatoes, maize and soybean) were lost due to diseases caused by fungi, nematodes, and bacteria (51). Detailed studies on the wheat crops in the Pacific Northwest had documented loss of up to 36% due to Pythium, Fusarium, Rhizoctonia, and Pratylenchus (16,20, 65,66).


- Soilborne pathogens are an important constraint to vegetable production in the US, as they cause significant reduction in quantity and quality of yield and their control adds greatly to the cost of production

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

- About 90% of the 2000 major diseases of the principal crops in the US are caused by soilborne plant pathogens (35).

- Monetary losses due to soilborne diseases in the U.S. are estimated to exceed $4 billion per year (39), and losses due to parasitic nematodes exceed $100 billion per year world-wide (5). In soybeans in the U.S. alone, all diseases combined caused losses of $15 billion from 2000-2007 (76a).

- Since 2010, the stem and bulb nematode (Ditylenchus dipsaci) has re-emerged, with significant losses on garlic in NY, the NE region and Ontario, Canada.

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

- New invasive species have been discovered in N. America in the last ten years, including Phytophthora ramorum, cause of sudden oak death, the potato cyst nematode, Globodera pallida in Idaho and most recently golden cyst nematode G. rostochiensis in Alberta and Quebec. The root knot nematode Meloidogyne enterolobii (syn. M. mayaguensis) was first detected in the U.S. in Florida a few years ago, and is aggressively spreading around the world. Once they become established in natural ecosystems, these pathogens cannot be easily managed.

- In the last three years, citrus greening (Huanglongbing disease) has decimated the citrus industry of Florida, and has been found in Texas and just recently (2012) in California.

- Laurel wilt, caused by Raffaelea lauricola and vectored by exotic ambrosia beetles, threatens the native laurels of the East Coast and the avocado industry in Florida and California.

- Changing climate will result in more plant stress, drought conditions, salinity or in some cases a wetter climate, which will predispose plants to more disease.



Environmental Costs of Soilborne Plant Pathogens

The cost of soilborne plant pathogens to society and the environment far exceeds the direct costs to growers and consumers. The use of chemical pesticides to control soilborne 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 soilborne diseases, has become notorious in recent years for contributing to the depletion of the ozone layer. The planned ban on production and importation of methyl bromide has been repeatedly delayed by a lack of cost-effective alternatives, and there remains an intensive search for replacement control methods. This fumigant was to be totally banned by 2005, but critical use exemptions for the U.S. resulted in 2011 usage that was still 10% of the 1991 levels. A potential alternative, methyl iodide, was recently (2012) withdrawn from the U.S. market for health concerns. Bayer, the maker of aldicarb (Temik), a widely used nematicide and insecticide on cotton and potatoes, has decided not to renew the EPA registration. A US distributor (Ag Logic) for Chinese pesticides picked up the aldicarb (Memik) registration, which raises serious questions concerning product stewardship of one of the most toxic compounds ever present in US agriculture. Telone (2,3-dichloropropene), a soil fumigant/nematicide widely used in potato production, has reduced supply and restrictions by township quotas in California. Larger buffers and restriction zones are needed for many pesticides. For example, in many counties in Florida, Telone is restricted to soils with a shallow hard pan in order to restrict water movement into the shallow water table. The registration for maneb, a protectant fungicide widely used for almost 70 years, was cancelled in 2010. Development of fungicide resistance continues to be a problem with the newer generation of low impact fungicides with specific modes of action, such as the strobularins.



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.



Society's Expectations

As is readily apparent from reading the popular press, consumers are demanding plentiful, low cost and safe food while simultaneously requiring the use of fewer chemical controls. In 2010, the organic industry grew almost 8% from the previous year, and the value of organic fruits and vegetables was $10.6 billion in the US, 12% of the total market (Organic Trade Association 2011 survey). Organic food sales have increased 337% from 2000 to 2010. In 2008, there were 2.6 million acres of certified organic cropland in the US, an increase of 117% since 2000 (NASS). Several other trends have accelerated since our last renewal. Organic food is now available from large retailers such as Wal Mart, Kroger Co and others. There is an increasing "locovore" movement where people want locally-grown food, usually grown organically, from farmer's markets, CSA (community supported agriculture) or community gardens. USDA has initiated a BioPreferred® Program for labeling certified biobased products and for encouraging their use by federal agencies. This labeling will tell the consumer the percent of biobased ingredients in a product.



Organically-grown crops require non-synthetic methods for management of diseases, and organic growers are seeking scientifically-based disease management methods. Recent national surveys (2004) by the Organic Farming Research Foundation have identified pest and disease problems as a major concern for organic growers. Many of our products are certified as organic with the Organic Materials Review Institute (OMRI). During the last few years, more and more pesticides that control soilborne diseases have been taken off the market or regulated, including methyl bromide. Soilborne pathogens are well adapted to soil conditions, and once established are very difficult to eliminate. Even if chemical products are available, they are often too expensive to be economically practical. However, for many pathogens, chemical remedies 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 has resulted in reluctance by growers to adopt such technology since consumer boycotts could be devastating, especially in small or specialty crop markets. These concerns, 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-3147 are to:

- Provide society with a safe, low cost food supply

- Reduce the environmental impact of soilborne disease control on ornamental, bioenergy, fiber and food crop production

- Protect natural ecosystems from invasive species

- Development of new industries and products for biologically based disease control



Biological Control and Soil IPM Systems As Attractive Alternatives

Biological control is an attractive approach for the control of soilborne diseases (18,19,28,35,72,73,53,74,75,48,49,40,13,32,19). 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 soilborne plant pathogens has made large strides over the past several years. Much of this success is due to activities of the members of W-3147. Today the EPA lists more than 40 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. Within the last few years, several new products containing Trichoderma and Bacillus have been released. However, most of these products are for seed and seedling diseases. W-3147 project is unique in emphasizing biological control of root diseases of perennial crops, including tree fruits and turfgrass, which are generally not treatable as annual crops with chemicals or other methods. Since our last renewal, members of the former NC-125 have joined our group, extending expertise to important field crops, including soybean, corn, and alfalfa.



Interest and enthusiasm about biocontrol continues within the science of plant pathology. Since 2000, over 2,200 peer-reviewed articles have been published on biological control of plant pathogens (Web of Science, April 2012). In fact, two new journals were launched in the 1990s-the journal Biological Control, which covers arthropod, nematode 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-3147 regional project is both very timely and successful. Commercial interest has also increased substantially. In the past five years, a number of new companies have been formed that develop and market biopesticides. The industry is forecasted to expand considerably in the next five years.



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

- Identification of more effective agents. Workers are isolating potential antagonists from soils where many pathogens originated and testing on a range of pathogens.

- New bioinformatics information through the genomic analysis of the biocontrol agents and using microarrays to study gene expression in the plant. Within the last 3 years, seven Pseudomonas fluorescens isolates with biocontrol ability have been sequenced (38), while that of Lysobacter enzymogenes strain C3 is near completion. This initiative was spearheaded by members of W-3147, and included several biocontrol agent strains studied by members, including P. fluorescens Pf-5, Q8r1-96, 30-84 and Q2-87 and L. enzymogenes C3. Data mining of these genomes has resulted in the discovery of many new antimicrobial and insecticidal compounds.

- Advances in metabolomics and proteomics are also being used to study the biochemical pathways and in-situ detection of antifungal metabolites produced by biocontrol bacteria.

- -Omic advances have been used to identify and create new systems for the control of abiotic stresses, improve biological control, and increase abilities of plants to utilize nitrogen

- Understanding the genetic diversity of pathogens, biocontrol agents and beneficial microbes, using advances in DNA sequencing, such as pyrosequencing.

- Identification and characterization of natural disease suppressive soils. In the past 4 years, members have used next generation pyrosequencing to describe microbial communities associated with the natural decline of fungal and nematode diseases.

- Integration of biocontrol into current agronomic practices.

- Identification of parameters affecting efficacy and survival after application.

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

- Investigation of manipulation of cultural parameters that advance biological control (eg. uses of compost, green manures, and rotation crops) and improve soil health and productivity.

- Understanding the role of the plant in biological control (vis-a-vis induced resistance pathways).

- Use of microbial products or metabolites and other biorational approaches, such as compost teas, plant strengtheners, etc.

- Controlling replant diseases in apples and mitigating root-knot nematode crop damage by use of Brassica seed meals



The promise, public acceptance and environmental benefits of non-chemical management of root diseases continue to make research on this area both timely and of critical importance to the future of U.S. and world agriculture.



Clearly there is much to be done in order to improve biocontrol agents so that they will continue to become major factors in the control of soilborne diseases. Biocontrol agents isolated by participants of W-3147 at ARS-WA, ARS-CA, CA-R, OR, MT, AK, OH, NE 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 nematicides, fungicides and soil fumigants that agriculture has depended on for the last 50 years. 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-3147 regional project has excelled and will continue to depend on during the next five years.



This project fits the goals of numerous other NIFA and USDA initiatives. But the need for this project has become even greater in the last few years, given changes in funding priorities. The panel on Biologically-Based Pest Management was eliminated in 2004, leaving many biocontrol researchers with reduced or eliminated funding, and this research has not been funded by other programs. The recent changes in NIFA grants toward larger multidisciplinary cooperative (CAP) grants has also made it more difficult for individual researchers to work on projects that do not fit the grand themes of these programs. This project will support the need for multidisciplinary research, without the high administrative costs associated with large CAP grants.

Related, Current and Previous Work

There are a number of reviews on the use of biocontrol organisms to control soilborne plant pathogens (3, 17, 18, 19, 20, 28, 29, 30, 35, 61, 72, 73, 74, 75, 53 22, 48, 49). The EPA currently lists more than 40 biocontrol agents that are registered or pending registration with the EPA and available for commercial use in the U.S. (http://www.epa.gov/pesticides/biopesticides/product_lists/new_ai_2009.htmlt)



Identification of New Biocontrol Agents

A list of 44 biocontrol agents which have reduced disease are listed by Cook and Baker (19); however, many more are likely to exist and more recent references catalogue them (13, 23a, 71a). 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. Few are using methods that detect non-culturable organisms as an initial screen in searching for new biocontrol agents. 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 (26). Thus, we have barely scratched the surface in our hunt for biocontrol agents. W-3147 members have been instrumental in discovering many of these new biocontrol agents, including strains of Pseudomonas fluorescens with superior colonizing ability (Q8R1-96) (63), diacetylphoroglucinol-producing strains of P. fluorescens (62), Trichoderma atroviride 901C (45); Pseudomonas aureofaciens AB254 (40); and Dactylella oviparasitica (54) for control of cyst nematodes). From 2003-2008, this list has been expanded to include Muscodor (24, 69), Lysobacter enzymogenes (77), Bacillus and Paenobacillus spp. (23), Myxobacteria (Myxococcus spp.) (10), and Pochonia chlamydosporia (Borneman and Becker, unpublished). Other biorational products have evolved from biocontrol research. For example, rhamnolipids are biosurfactants produced by Pseudomonas aeruginosa, which lyse zoospores of Pythium and Phytophthora. This research has led to a commercial EPA-registered product (Zonix), Bli-nix. The bioactive cyclic lipopeptide orfamide A, was discovered using a genomisotopic approach from the sequenced genome of P. fluorescens Pf-5 (37). Since the last proposal, members have discovered new agents such as Chryseobacterium (ARS-WA), (12), new isolates of Bacillus amyloiquifaciens (MI) (50) and Pseudomonas (ARS-WA) (55). Members have sequenced a number of bacterial genomes, leading to the discovery of new compounds such as cyclic lipopeptides (ARS-WA) (43) anti-insecticidal compounds, (ARS-OR) (38) and macrocyclic lactams (NE) (36).



Tables 5, 6, and 7 in the Appendix outline some of the biocontrol agents and strategies that are being developed and researched by members of W-3147.



Disease Suppressive Soils and Plant Protecting Microorganisms

Over the last 20 years there have been surprising and exciting discoveries for natural methods to suppress or eliminate pathogens, and/or protect plants. Intensive studies of disease suppressive soils have led to the development of new methods of analysis (25, 9, 7, 4) and new insights into the nature of soilborne disease suppression (73, 27). Many of these advances have been made by members of W-3147 since the last proposal. This includes the first identification of new bacterial genera associated with Rhizoctonia decline in North America (112), the role of complex communities of phenazine producing Pseudomonas spp. (55), and the role of actinomycetes in suppression by glucosinalate biofumigation (14, 15, 44) Such advances indicate that active management of soil microbial communities can be an effective approach to develop natural suppression of soilborne diseases and improve crop productivity (42). Generally speaking, there are two approaches to actively managing crop-associated microbial communities.



The first approach is to develop disease suppressive soils through manipulation of carbon inputs. This involves adjusting the types and timing of organic inputs, such as cover crops, animal manures, composts, compost teas, and crop sequencing. Such approaches have been shown to provide site-specific reductions in disease and pest incidence (1, 11, 76, 64, 70, 21, 34). The advantage of this approach is that it relies on locally available resources to maximize soil health in a sustainable way. The disadvantage is that outcomes tend to be more variable depending on soil type, so knowledge of soils and available inputs is essential.



The second approach involves inoculation with disease suppressive microorganisms. All soils harbor detectable populations of disease suppressive organisms, however, the diversity, relative abundance and activities of these organisms can vary substantially from site to site. Historically, researchers recovered microbes and then screened them for disease suppressing activity. However, molecular tools now allow us to identify microbes that are suppressive in situ and recover them in a directed fashion (9). Such an approach has already proven useful at collecting indigenous fungi that acted as effective inoculants to suppress soilborne diseases caused by nematodes (54). In addition, analyses of the genetics and genomics of disease suppressive microbes (56, 2, 31) have led to new methods to isolate disease suppressive microbes in a directed fashion from any location. This approach too, has led to the development of effective and low cost inoculants (48).



Methods that transform resident microbial communities in a manner that induces natural soil disease suppression have potential as components of environmentally sustainable systems for management of soilborne plant pathogens to reduce the need for pesticides.



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 agent's ecology. Known mechanisms by which biocontrol organisms reduce disease include:

- Induction of plant resistance mechanisms and regulation of gene expression

- Antibiotic and toxin production.

- Cell-wall degrading and lytic enzymes.

- Siderophore production.

- Biosurfactant production

- Mycoparasitism

- Shifts in microbial communities

- Alleviation of ROS effects or toxicities

- Mycoviruses- in soybean on Fusarium
- Sex pheromone used to disrupt spore germination

- New mechanisms and chemistries are being discovered by genomic analysis, including lipopeptides, compounds with activity against insects and macrocylic lactams.



Several of these mechanisms were identified only recently by members of the W-3147 project. These include the role of biofilms (79, 67), the genetic and biochemical pathways for production of phloroglucinol and phenazine, (58, 46), the regulation of phenazine production (41), the role of phloroglucinol producers in suppressive soils (62) and the in situ detection and quantification of antifungal compounds produced by biocontrol agents in the soil and rhizosphere (71). 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. 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. A number of unknown "antibiotic islands" were detected in the genome of P. fluorescens Pf-5, which led to the discovery of the bioactive lipopeptide orfamide A (37). A screening of the bacterium Lysobacter enzymogenes strain C3 led to the discovery of HSAF (heat stable antifungal factor). The structure of this compound has been determined to be a dilrydromaltophilin, a unique macrocyclic lactam with a tetramic acid moiety (77). The production of HASF was proven to be a mechanisms in the biological control of foliar and soilborne pathogens (36) and was implicated in the biocontrol of plant parasitic nematodes by strain C3 (78) This group of compounds could be exploited for fungicides and antifungal drugs. A recent survey of regulatory genes in C3 reviewed genes involved in both antibiotic production and the production of type 4 pilus which is thought to be involved in motility and attachment to roots and fungal hyphae by the bacterium.



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.



In summary, research related to the objectives outlined above is in progress throughout the world. However, the lack of broad acceptance of biocontrol agents reflects two major obstacles. First is the complexity of the ecological systems in which the biocontrol agents must operate. Second is the current mindset of growers that biocontrol is expensive and inconsistent. To overcome both hurdles, we must better understand the factors that influence the efficacy of biocontrol agents once released in the field in order to begin to manipulate the system as a whole and maximize the benefits of biocontrol and its contribution to sustainable agriculture.



Differentiation from other regional workgroups

Currently only one other regional workgroup, SDC348 (formerly S-1028), is focusing on various aspects of microbial biological control of soilborne plant pathogens. This group is focusing on the genetic diversity of both the pathogen and the biocontrol agent, and is testing management techniques for controlling diseases. Biocontrol is only one of the management techniques they are testing, and their second objective is very applied. The W-3147 project has focused on characterizing new biocontrol agents, understanding the biological mechanisms of naturally suppressive soils, and applying these agents in agriculture. Each project utilizes a different approach to attain their objectives. Research conducted under W-3147 involves a greater emphasis on understanding the mechanisms involved in plant-microbe interactions. In addition, W-3147 research is unique in that biocontrol organisms and approaches must be customized for a more arid climate than that of the other regions. W-3147 project is unique in emphasizing biological control of root diseases of mature crops, which are generally not treatable with chemicals or other methods. The combination of approach, crops, pathogens, cropping systems and biocontrol agents distinctive to W-3147 provides a unifying theme that facilitates progress toward the objectives. Differences among the two regional projects will serve as a benefit by allowing these groups to compare results across the U.S. during the joint meetings held every 3 years.



Table 1 in the Appendix shows the differences in objectives between the two projects, and Table 2 provides a comparison of the disease systems and hosts to be investigated by members of the two regional projects. It is apparent that 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 3. The responsibilities of the states with respect to soilborne plant pathogens and objectives addressed are shown in Table 4.

Objectives

  1. To identify and characterize new biological agents, microbial community structure and function, naturally suppressive soils, cultural practices, and organic amendments that provide management of diseases caused by soilborne plant pathogens.
  2. To understand how microbial populations and microbial gene expression are regulated by the biological (plants and microbes) and physical environment and how they influence disease.
  3. Implement sustainable management strategies for soilborne pathogens that are biologically based and are compatible with soil health management practices.
  4. Provide outreach, education, extension and technology transfer to our clients and stakeholders- growers, biocontrol industry, graduate and undergraduate students, K-12 students and other scientists.

Methods

Objective 1: To identify and characterize new biological agents, naturally suppressive soils, cultural practices, and organic amendments that provide management of diseases caused by soilborne plant pathogens.

A. Assessing isolated microbes for biocontrol activity. In the past, extensive efforts were made to isolate microorganisms at random from soil and plant material and then identify, through in vitro, greenhouse and field tests, those with potential as biological control agents. This strategy tended to yield candidate species that occur in high populations or those that grow quickly in culture. While such "prospecting" is still important in the search for new biocontrol agents, isolation and assessment efforts in this project will be more directed. Members have developed a multivariate sampling and marker-based selection strategy that significantly increased the diversity of bacteria recovered from plants, and led to a 5-fold improvement in efficiency and recovery of several novel strains of bacteria with significant biocontrol potential. Studies on suppressive soils and agricultural practices described below will likely identify taxa that have not been investigated previously in the context of disease management. Experiments in controlled and field environments will focus on representative strains of these new taxa to determine their efficacy. Tables 5-7 show some of the biocontrol agents that are being investigated by W-3147.

B. Examining naturally suppressive soils. Suppressive soils hold considerable potential for managing soilborne pathogens. Such soils have been defined as "soils in which the pathogen does not establish or persist, establishes but causes little or no damage, or establishes and causes disease for a while but thereafter the disease is less important, although the pathogen may persist in the soil" (Cook and Baker, 1983, 19). When the suppressiveness has a biological origin, the crucial steps in realizing this potential are to identify the causal organisms and then to understand the agronomic and environmental factors that enable them to function. Armed with such knowledge, it may be possible to develop effective and sustainable pest management strategies through the application of the organisms and through agronomic practices that influence their populations.

To identify microorganisms involved in pathogen suppressiveness, we frequently employ a DNA-based population-based approach. Population-based investigations of suppressive soils can be performed by examining the microbial community composition in soils possessing various levels of suppressiveness. Members of W-3147 have examined microbial communities using a variety of culture or culture-independent methods, such as next-generation sequencing or 454 sequencing. Suppressiveness potentially can be used by growers to manage disease. For example, planting decision models for sugar beets could be enhanced by incorporating the population densities of the biocontrol fungus Dactylella oviparasitica (work by Borneman and Becker (CA-R). D. oviparasitica and similar fungi have a wide distribution in regions impacted by cyst nematodes. These fungi can also reduce cyst nematode populations in field conditions and in soils with different chemical and physical characteristics. Given that D. oviparasitica and related organisms comprise a clade of fungi containing effective biological control agents targeting several economically important nematodes, and that similar fungi have been identified on several continents, this general approach may prove to be useful for a wide range of crops in other geographical locations.

Members of our workgroup have successfully employed this strategy in several systems. For example, Paulitz (ARS-WA) identified bacterial taxa in the Sphingobacteria (Flavobacterium and Chryseobacterium) and Oxalobacteriaceae that may be involved in suppression of Rhizoctonia bare patch of wheat. Weller (ARS-WA) identified unique communities of phenazine-producing Pseudomonas in arid wheat producing areas, extending across hundreds of square kilometers of the Pacific Northwest. Anderson (OK) identified rhizobacterial taxa associated with higher and lower productivity in wheat. Hao (MI) identified sequences of Lysobacter and Acidobacteria associated with suppression of Streptomyces scab of potato. Borneman and Becker (CA) have used Illumina-based analysis of the small-subunit rRNA gene to examine root-associated bacteria from plants exhibiting different levels of peach replant disease symptoms and identified groups associated with increased peach growth.

One major advantage of investigating microorganisms associated with suppressive soils is that these organisms have demonstrated the ability to function in production agricultural systems. We will continue to identify and characterize such organisms towards the development of more sustainable and effective strategies to manage soilborne pathogens. In the future, we will also expand our search to fungal communities, using next-generation sequencing. There have been additional advances in this technology over the last 4 years that will allow us to move beyond just bacteria.

C. Examining cultural practices that influence soilborne pathogens. Understanding the effects of soil management practices on the incidence and damage of root diseases could lead to the ability to engineer soils that suppress diseases by a variety of mechanisms including the production of pathogen suppressive soils, stimulation of host resistance, and/or modifying soil environments by, for example, incorporation of cover crops and green manures.

Towards this goal, members of our W-3147 workgroup are participating in a collaborative and multidisciplinary project termed the Cornell Soil Health Program Work Team (PWT). The goal of the PWT is to address the progressive deterioration of agricultural soils and provide information that will aid growers in the assessment and management of their soils. The PWT is also interested in developing appropriate management strategies to maintain high quality productive soils without measurable deterioration. The project seeks to incorporate a broader range of soil tests to provide greater information to growers, identify soil health problems and means of addressing them, as well as to create a database by which to evaluate soil test results.

Members of the W-3147 workgroup have performed numerous trials examining how agricultural practices influence soilborne pathogens. For example, research in WA has demonstrated that applications of lime can suppress Fusarium wilt of spinach, a pathogen that can persist in the soil for over 10 years. Applications of brassica seed meals can suppress apple replant diseases, by shifting microbial populations to suppressive actinomycetes (ARS-WA). Proper timing of herbicide can control reservoirs of inoculum from crop volunteers and weeds, and reduce Rhizoctonia bare patch in onion and wheat (WA and ARS-WA). Disease suppression in long-term no-till is also being investigated. Soil solarization is being investigated as a method to disinfest nursery bed soils contaminated with Phytophthora spp., either alone or in combination with amendment with Trichoderma asperellum TA1 by Parke (OR) and Widmer (ARS-MD). Solarization for 2-4 weeks resulted in no recovery of P. ramorum from infested leaf disks buried in soil 5 cm and 15 cm below the surface in field trials in CA. In OR, solarization similarly reduced recovery of P. pini from 5 and 15 cm below the soil surface.

Members from OR have developed a systems approach for managing pests and diseases in nurseries based on knowledge of critical control points and implementation of best management practices. This strategy was initially developed to manage Phytophthora diseases but is now being considered for broader application to pests and pathogens of nursery and greenhouse crops by USDA-APHIS.

Table 8 in the Appendix shows W-3147 efforts in the area of suppressive soils and cultural 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 microbes influence disease.

Previous cooperation among W-3147 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, and must be understood in order to improve the consistency of biocontrol.

The following is a short list of the mechanisms of primary of interest and the microbial systems in which they will be investigated. Other mechanisms and organisms will be included as they are elucidated.

- Induction of plant resistance mechanisms. This includes both systemic acquired resistance (SAR) and induced systemic resistance (ISR). Researchers in NY and MT are looking at commercial plant inducers, testing them in the field, and looking at plant defense response gene expression. Researchers in NE are looking at ISR induction by Lysobacter. Researchers at ARS-WA are looking at how antifungal compounds such as phloroglucinol induce resistance in Arabidopsis. Microarray technology is being used to determine what genes are stimulated by colonization with Pseudomonas (ARS-WA)
- Secondary metabolite production. Researchers in ARS-WA and TX will focus their investigations on antimicrobial compounds produced by Pseudomonas such as phenazine, phloroglucinol and other anti-microbials in suppressing soilborne pathogens. Previously, researchers in TX elucidated the 3-dimensional structure of enzymes in the phenazine biosynthesis pathway. They also demonstrated the importance of these secondary metabolites for biofilm formation and plant host colonization. Future work involves using 'terminal modifying enzymes' to alter the structure of the phenazines produced and discern the roles of the altered structures in biofilm formation and host interactions. The WA-ARS group also found a unique group of phenazine producers in eastern Washington and will be examining its role in suppression of Rhizoctonia. Researchers at OR will investigate a class of lipopeptides (orfamide A) newly discovered from the genomic sequence of P. fluorescens PF-5. Researchers at CA-R will study rhamnolipids produced by Pseudomonas in hydroponic systems which lyse zoospores of Pythium and Phytophthora. Studies in NE will focus on production of HSAF, a new class of antibiotics with a unique macrocyclic lactam with a tetramic acid moiety, by Lysobacter. The biosynthetic gene cluster was elucidated in the previous project and this information will allow studies on altering the antibiotic structure through gene modification, which may lead to new natural products. Bacillus from suppressive soils produce the lipopeptide LCI that shows antimicrobial activity against a range of soilborne pathogens. Work in MI will identify the role of LCI production in disease suppression. For all of these systems, the environmental factors that stimulate antibiotic production and the genes involved in regulating production will be explored in the new project. For example, researchers at TX and NE will be working on understanding bacterial communication via QS and two-component signal transduction systems that enable bacterial biocontrol agents to sense their biological and physical environments. New research will focus on understanding how these regulatory networks are interconnected to control the expression of secondary metabolite production. W-3147 researchers will also investigate the role of antibiotics in ecology of biocontrol bacteria. Workers at ARS-WA have discovered a widespread distribution of phenazine producers across low rainfall areas of eastern Washington. New research will investigate whether phenazines may help bacteria adapt to dry conditions by the production of biofilms and as function in electron exchange under O2 limiting conditions. They also will examine the selection for tolerance of pathogens to phenzines. Mutant strains of Lysobacter enzymogenes with enhanced or inhibited antibiotic production will be used in NE to investigate the role of antibiotics such as HSAF in microbial competition and colonization of soil and roots.

- Genomic sequencing and analysis. The first genome sequence of a biocontrol agent was accomplished by members of W-3147 (P. fluorescens Pf-5). This has led to a new paradigm of discovery of mechanisms, based on 'mining' the genome and looking for undiscovered chemistries and enzymes. Since the last renewal, we have sequenced over 10 strains of Pseudomonas, and genomes of Bacillus and Lysobacter. New genomes will be sequenced, as the price drops significantly, using Illumina and Ion Torrent and other next-generation systems.

OBJECTIVE 3: Implement sustainable management strategies for soilborne pathogens that are compatible and integrated with good soil health practices.

Intensive production of crops has contributed to gradual deterioration of soil health, resulting in reduced yield and profitability. Characteristics of deteriorated soils include crusting, compaction, low content of organic matter, the increased incidence and damage of root diseases caused by soilborne pathogens, and the prevalence of other pests. In addition, it is well documented that damage due to diseases caused by soilborne pathogens is greater in poor quality and unhealthy soils. Thus, there has been an increasing interest in addressing soil health constraints in a holistic, sustainable, and environmentally compatible manner. Among the long-term management practices promoted to maintain and/or improve soil health status are various reduced tillage systems, more diverse crop rotations, numerous cover crops, green manures and the application of composts. All these soil and crop management practices have profound direct and indirect effects on the populations and disease potential of soilborne pathogens as well as on the diversity and dynamics of the soil microbial communities. The increased implementation of soil health management practices; specifically reduced tillage systems are impacting many of our current recommendations and strategies for controlling plant pathogens. Thus, there is a great opportunity and a challenge for scientists associated with W-3147 to become involved in the multi-disciplinary investigations dealing with soil health management to assure the development and implementation of soil health management programs that are also suppressive to soilborne pathogens and plant diseases in general.

Directly or indirectly, all members of W-3147 will be involved in this objective (ARS-WA, CA-R, MD, MI, MN, MS, MT, NE, NJ, NM, NY, OR, and WA). Results obtained from the first two objectives of this proposal, results accumulated from previous W-2147 investigations and the wealth of experience of the participating scientists will be utilized to evaluate, fine-tune and implement ecologically-based control options against soilborne pathogens as integral components of long-term and sustainable soil health management programs. The latter will be possible by assessing the impact of soil health management practices and the mechanism(s) involved in soilborne pathogen control. It will also be crucial to educate growers on how to augment the promoted soil management practices with previously identified microbial biocontrol agents or products applied to seeds or planting mix, how to enhance native soil suppressive communities by modifying cropping sequences, and the best approach to applying appropriate composts and/or the use of selective and low risks chemical products. It is expected that the management programs which have been developed for, and are effective in one location might be different than those needed or effective at other sites due to the differences in the occurrence of major soilborne pathogens, main crops grown/production systems, soil types, and/or environmental conditions. Thus, close collaborations among the involved scientists in this project remain critical and are much needed for the success of the regional project. For example, the development and validation of accurate and cost-effective diagnostic protocols such as DNA oligonucleotide arrays, which have been developed by several W-3147 members, are essential. These arrays enable the detection and identification of the major root pathogens present on a farm, and provide the information necessary to select the appropriate management options. Research on the value of various cropping systems, such as cover crops, appropriate use of plant resistance, reduced tillage systems, and their appropriateness under different geographical and environmental conditions is critical for a wider acceptance and subsequent implementation by growers. The disease suppressive effect of applied or endemic biocontrol organisms is strongly influenced by cropping system alterations. Members of the W-2147 group have demonstrated benefits of using such strategies in their specific regions, and this group fosters effective interaction and sharing of expert knowledge.

The above multi-faceted research and demonstration activities proposed under this objective will be conducted both on Research Stations and on commercial fields of collaborating growers. Clearly demonstrating the benefits of effective management strategies under commercial growing practices will promote their adoption and also enhance multi-disciplinary collaborations. The successful completion of this objective will contribute to greater root health (reducing damage caused by soilborne pathogens), improving soil health and productivity, and reducing environmental risks; thus to more sustainable and resilient agroecosystems.

Objective 4. Provide outreach, education, extension and technology transfer to our clients and stakeholders- growers, biocontrol industry, graduate and undergraduate students, K-12 students and other scientists.

This objective is outlined in detail in the section titled OUTREACH PLAN

Measurement of Progress and Results

Outputs

  • Publications- peer reviewed, extension and technical bulletins, meeting proceedings, abstracts, book chapters.
  • Outreach and education materials- grower and extension talks, teaching materials, web-based training modules and certification programs
  • Highly qualified personnel- Trained students, educators and consultants that will serve the agricultural and biocontrol industry.
  • Management recommendations for soilborne diseases- ways of managing soilborne diseases with cultural methods, crop rotation, cover crops, organic amendments, and suppressive soils.
  • Biocontrol products- novel organisms with new modes of action, improved formulations and production systems, quality control assays, and application guidelines.

Outcomes or Projected Impacts

  • Reduced use of chemical pesticides and increased use of biologically based products.
  • Reduced damage by soilborne pathogens and increased crop productivity and profitability.
  • Safe, low-cost agricultural products
  • Benefits to 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.
  • Knowledge of the genomic and biochemical diversity of microbial communities and biocontrol agents, and how they function in agroecosystems.
  • Understanding of how the biocontrol agents interact with the plant and the environment, to predict their limitations and inconsistency in the field.
  • Expanded tool kit of disease management options for both organic and conventional growers, leading to improved agricultural productivity and sustainability.

Milestones

(0): Objective 1. Survey soils for suppressive activity against soilborne pathogens.
Objective 2. Sequence bacterial genomes.
Objective 3. Establish and maintain research and demonstration sites
Objective 4. Develop industry liaison to determine common goals

(0): Objective 1. Survey soils for suppressive activity against soilborne pathogens, - identify causal antagonists or suppressive microbial communities (including fungi) with culture-dependant and/or independent (DNA based) techniques.
Objective 2. Sequence bacterial genomes, annotate sequences.
Objective 3. Develop application and management practices.
Objective 4. Develop and implement disease management guidelines for organic and conventional producers.

(0): Objective 1. Identify causal antagonists or suppressive microbial communities (including fungi) with culture-dependant and/or independent (DNA based) techniques.
Objective 2. Annotate sequences, mine for novel sequences.
Objective 3. Develop application and management practices.
Objective 4. Develop and implement disease management guidelines for organic and conventional producers.

(0): Objective 1. Identify causal antagonists or suppressive microbial communities (including fungi) with culture-dependant and/or independent (DNA based) techniques and provide experimental evidence for cause of suppression (analogous to Koch's postulates).
Objective 2. Annotate sequences, mine for novel sequences.
Objective 3. Develop application and management practices, determine cost-benefits, and foster stakeholder involvement.
Objective 4. Develop and implement disease management guidelines for organic and conventional producers.

(0): Objective 1. Provide experimental evidence for cause of suppression (analogous to Koch's postulates).
Objective 2. Mine for novel sequences.
Objective 3. Develop application and management practices, determine cost-benefits, and foster stakeholder involvement.
Objective 4. Develop and implement disease management guidelines for organic and conventional producers.

Projected Participation

View Appendix E: Participation

Outreach Plan

This outreach plan is Objective 4 of the proposal, given in detail below.

1. Outreach and education to growers: presentations and workshops.

Over 50% of our members have extension appointments and regularly meet, consult, and teach growers, pest control advisors, industry representatives, as well as with members of the general public. Proposed outreach will be in the form of field days, workshops, grower meetings, and websites. In the last 4 years, our members have given 118 extension talks and published 67 extension bulletins. We publish extension publications, pamphlets and grower manuals, including IPM guides such as the Organic Strawberry Production manual published by UC Press. Our members publish chapters in the disease compendium series of the American Phytopathological Society (APS) Press and results of biocontrol trials are published in Plant Disease Management Reports. Some specific web-based examples include contributions to the Pacific Northwest Plant Disease Control Handbook, University of California IPM series, UC IPM Crop Management Guidelines (http://www.ipm.ucdavis.edu/PMG/crops-agriculture.html), High Plains IPM Guide, and the Annual Integrated Crop and Pest Management Recommendations from Cornell. We provide diagnostic services through the local and National Plant Disease Diagnostic Networks. Members of this project in NY are collaborating with other colleagues in presenting participatory training workshops throughout the NE-Region dealing with the diagnosis, biology and management of soilborne pathogens of vegetable crops and their sustainable management options with funding support from the NE-SARE program. Members also provide training for Certified Crop Advisors and pesticide credits for pesticide applicators. This training increases the level of safety and reduces overuse of fungicides. Members of our project reach large numbers of stakeholders at regional meetings such as the Great Lakes Expo (200 participants), the University of California Nursery and Floriculture Alliance Ornamentals Disease Symposium (100 participants) and the NW Ag Show. Presentations at these meetings increase adoption of sustainable agriculture practices.



2. Online Outreach:

A very innovative online outreach tool has been constructed by our members at Cornell University- The Cornell Soil Health Website (http://soilhealth.cals.cornell.edu/). Growers can send in samples to have soil health status evaluated and to learn of major soil quality constraints that need to be addressed. They are also able to access the Soil Health Manual that contains detailed information on what is measured, what soil processes are impacted and potential remedial practices. Another example is the Online Phytophthora Course: Training for Nursery Growers, developed by members from OR for growers and nursery workers to promote best management practices to eliminate Phytophthora. This free course is available in English and Spanish language versions. Course participants who pass an optional fee-based exam may obtain a certificate of mastery and earn continuing education credits. A new website, Forest Phytophthoras of the World (www.ForestPhytophthoras.org), was recently established to provide science-based information to aid in the understanding and management of the world's forest Phytophthora species, all of which have a soilborne phase. A companion online journal, Forest Phytophthoras, provides immediate open access to its content on the principle that making research freely available to the public supports a greater global exchange of knowledge. We envision an even greater web presence in the future, making use of webinars and distance education. A member in NY has given a series of three webinars on the soilborne pathogen Phytophthora capsici and including strategies that can be utilized to control the spread of the pathogen. One popular topic is the use of cover crops as green manures to increase the beneficial microbe population in the soil, including cover crop research and the use of pathogen detection arrays (as described in previous objectives). A member in OR has given webinars on systems approaches for managing Phytophthora in nurseries and water disinfestation techniques through the Water Education Alliance for Horticulture website.



3. Outreach to the biocontrol industry and agro-industry.


Many of the products developed by our group are being marketed by the biocontrol industry. This includes products such as Root Shield and Plant Shield, Trichoderma products developed by members in NY and ARS-MD. Other biocontrol products such as Bacillus amyloliquafaciens BACO3 from MI (Marrone Organic Innovation Inc.) are in the pipeline. Our members facilitate this technology transfer through patents and other IP protection vehicles with their respective agencies. We will continue our liaisons and cooperation with the agroindustry. For example, our members have organized symposia with speakers from industry to show scientists the state of the market. We plan to invite the director of the Biopesticide Industry Alliance, Bill Stoneman, to join W-3147.



4. Training the next generation of biocontrol scientists and practitioners.

Almost all of our members have teaching responsibilities, both at the graduate and undergraduate levels. They teach courses in biological control and plant pathology, train students in the lab and supervise graduate students. Many undergraduate students do special or honors projects in our labs. For example, over the last four years, one member (NE) has reached 240 undergrad students in intro plant pathology and over 80 grad students in his distance education grad level course on biocontrol of pests. We train and mentor postdoctoral research associates in our labs and research stations. Finally, we have extensive international collaborations, and travel to other countries to give seminars, and host international scientists in our labs. Some of our members (NE) also teach in long-distance web-based courses. Over the last 4 years, project members supervised 39 undergraduate projects, 11 MS students, 15 Ph.D. students and 15 postdoctoral associates.



5. Public education to general community and grades K-12.

Our members participate in science fair judging, Master Gardener Training, 4-H and FAA. Members from ARS WA have taught science modules at grade and middle schools and science summer camps for underrepresented groups on the Colville Indian Reservation. A member from NY has developed an elementary science education program in which she interacts with over 200 third and fourth-grade students each year. Additionally, she runs a five-week summer science camp (four days per week) for elementary school students that focuses on plant science, agriculture and sustainability. More information can be seen at http://web.pppmb.cals.cornell.edu/smart/esop/index.html

6. Providing information to policy makers at the local, state and national level.

In the past four years, members of W-2147 were involved in the development of microbial sequencing priorities list assembled by APS and have contributed to materials prepared by the APS Public Policy Board. One of our members is President-Elect of our professional society (APS), and in this capacity will serve on the Public Policy Board which advocates for agricultural sciences funding, training of future scientists, consortia of affiliated professional societies and private industries. Another member is editor-in-chief of APS Press, which publishes books for a wide audience. A member from OR has served on state, regional, and national advisory committee on Phytophthora ramorum and sudden oak death, and led a technical working group on systems approaches for APHIS. In this latter capacity she helped develop a national set of minimum best management practices for producing healthy nursery and greenhouse plants.



7. Publishing results in peer-reviewed scientific journals.

Finally, we must transmit our findings and results to or peers. W-3147 has an excellent publication record, which will continue. In the last 4 years, we have listed over 150 peer reviewed publications and 68 conference proceedings/abstracts by our members.

Organization/Governance

The W-3147 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 2013-14 the committee officers will be Chairman- Gary Yuen; Secretary: Ole Becker. 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 since the last renewal- Michael Anderson (OK), Betsy Pierson (TX), Kathryne Everts (MD) and James White (NJ).

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