Duration: 10/01/2004 to 09/30/2009

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

The need. Plant-parasitic nematodes cause major economic losses to horticultural and field crops in the U.S. Current control methods rely largely on nematicides (both fumigant and non-fumigant types) for most crops and pre-plant soil fumigation with the broad-purpose fumigant methyl bromide for high value fruit and vegetable crops. However, there are serious concerns about the use of nematicides in terms of food safety and environmental quality issues. In addition, methyl bromide is proposed to be withdrawn from use in the U.S. in 2005. Alternative environmentally compatible control strategies that use bio-based options (including resistant, non-host, or antagonistic crop cultivars and biological control agents) are urgently needed to replace high-risk chemical nematicides.

Importance of work and consequences if not done. Identification, characterization, and enhancement of resistant crop germplasm, antagonistic crops cover crops, and biocontrol agents are essential for the development of bio-based management options against plant-parasitic nematodes. Assessment of the effects of such management techniques on diversity of both plant-parasitic and free-living nematodes will increase our understanding of soil health and aid in development of sustainable crop management. If this work is not done, crop production will continue to move offshore where environmental standards for pesticide use are less stringent and our supply of safe, inexpensive grains, fruits, and vegetables will dwindle. Moreover, local farmers will lose market share and have reduced opportunities for economic development.

Technical feasibility of work. NE-171 is one of the most productive regional research groups nationwide. Members and collaborators of NE-171 (nematologists, molecular biologists, plant breeders, and extension specialists) have the demonstrated expertise to accomplish the proposed objectives and to continue serving stakeholders. During the first three years of the current project (2000 ? 2002), NE-171 members have published 95 refereed research papers, book chapters, technical and extension publications, and popular articles. Accomplishments of the current NE-171 project include: identification and characterization of fruit and vegetable germplasm and cultivars (pepper, strawberry, onion, and watermelon) that are resistant or tolerant to nematodes; validation of alternative nematode management tactics in vegetable crop production; demonstration that green manure crops suppressed plant-parasitic nematodes and weeds in orchard replant sites; and development of an immunoassay for quantitative measurement of P. penetrans endospores in soil and infected nematodes.

Advantages of a multistate effort. Interactions of scientists from several states who share the common goal of developing bio-based alternatives to manage plant-parasitic nematodes will be highly advantageous for several reasons: i) nematode-resistant and antagonistic crop germplasm and biocontrol agents must be tested in a wide range of environments against several nematode species and populations to determine their best utilization; ii) different strains of biocontrol organisms are likely to be discovered in different geographical areas and different cropping systems thus providing a potentially diverse array of new biocontrol agents; iii) the breadth of knowledge and experience of scientists participating in the northeastern region will lead to unique approaches and solutions to research problems that are beyond the scope of a single researcher or even a single institution.

Impacts of work. The long-term impacts of research efforts in the northeastern region will be a better understanding of bio-based cropping systems and lead to future alternatives for the reduction and dependency on nematicides and simultaneously increasing soil health. Specific examples of short-term impacts include management of potato early dying and black root rot of strawberry through development of green manure and antagonistic cover crops; development of cropping schemes using root-knot nematode (RKN) resistant vegetable and non-host cover crops that will allow growth of subsequent susceptible vegetable crops without nematicides; identification of low-risk nematicides and biological products on horticultural crops; development of orchard re-plant methods using antagonistic and/or non-host cover crops that will eliminate the need for nematicide applications; the molecular characterization of Pasteuria penetrans that will facilitate detection and quantification of P. penetrans populations, and further the development of methods to produce P. penetrans with nematode/host specificities for use in management of RKN species in a wide range of crops.

Related, Current and Previous Work

A review of CRIS projects was performed and results are listed in Appendix E. A number of projects from outside the region had similar objectives but focus on different nematodes or crop systems. There are three other multistate regional nematology projects. One focuses on persistence of soybean cyst nematodes and another involves the genetic variability in cyst and RKN. Our project has some overlap with the southern multistate effort, inasmuch as both include plant resistance for nematode management, but the crops and target nematodes have relatively little overlap.
The loss of multi-purpose soil fumigants such as methyl bromide as well as several traditional nematicides from the market due to environmental concerns and the costs of re-registration has focused attention on the development of crop rotation, and other alternative schemes to manage plant parasitic nematodes in both vegetable and field crops (Hirunslee et al., 1995; McSorley, R. and D.W. Dickson, 1995; McSorley and Gallaher, 1992; Rodriguez-Kabana and Kloepper, 1998; Weaver et al., 1995). Crop rotation is a beneficial production practice and has been extensively studied as a more sustainable nematode and pathogen control measure than chemical pesticides. Crop rotations often reduce nematode populations by reducing reproductive potential and survival, eliminating or reducing populations. Crop rotations and cover crops also can lead to disease reduction by increasing the population or activity of beneficial soil microflora. In such cases, the crop used in the rotation sequence is often selected for the development of a beneficial rhizosphere microbial community (Kloepper et al., 1991; Latour et al., 1996; Smith and Goodman, 1999; Weller et al., 2002). Among the organisms that are most likely favored by cover crops are fungal egg-parasites, nematode-trapping fungi, endoparasitic fungi, endomycorrhizal fungi, plant-health promoting rhizobacteria, and obligate bacterial parasites (Sikora, 1992). The use of legume cover or rotation crops provides organic nitrogen while also suppressing plant-parasitic nematodes. Unfortunately, many legumes are susceptible to RKN (Meloidogyne spp.), often the key nematode pathogens requiring management in many systems (McSorley, 1998, 1999). A number of legume crops have been identified that are highly resistant to various species and/or races of RKN. Some of these, cowpea (Vigna unguiculata), velvetbean (Mucuna deeringiana), and sunn hemp (Crotalaria juncea) have potential in cropping systems (McSorley, 1999; Rodriguez-Kabana, et al., 1988, 1989; Sipes and Arakaki, 1997; Weaver et al., 1995). In addition to nematode management and nitrogen provided by nematode-resistant legumes, crop residues may increase levels of soil organic matter, improving water-holding capacity and other soil properties (McSorley and Gallaher, 1995).

RKN resistant vegetable cultivars as rotational crops may limit damage in subsequently planted susceptible crops. Cucumber and muskmelons double-cropped after a RKN resistant tomato produced increased yields, had reduced root-galling, and lower densities of M. incognita second-stage juveniles (J2) in soil than the same cucumber and muskmelon cultivars grown after a susceptible tomato (Colyer et al., 1998; Hannah et al., 1994; Hannah, 2000). The RKN resistant pepper Carolina Cayenne was effective as a rotation crop for managing M. incognita in susceptible bell peppers (Thies et al., 1998). Yield of squash grown in the spring following a rotation crop of castor, cotton, velvetbean, or crotalaria, produced heavier yields than squash grown after RKN susceptible peanut (McSorley et al., 1994). When eggplant was grown following resistant ?Mississippi Silver? southernpea, root galling by M. incognita race 1 was less severe than when eggplant was grown following susceptible ?Clemson Spineless? okra (McSorley and Dickson, 1995). Crop rotations can reduce certain nematode densities, but may increase populations of other nematode pathogens, particularly in multi-species nematode communities (LaMondia and Halbrendt, 2003; Noe, 1998). Integration also must consider economic returns of rotation crops, additional capital and labor requirements, and grower and market acceptance of the rotation crop. In the northeastern region, rotations are already used as part of management programs for Globodera rostochiensis on potato (Brodie, et al., 1993; Mai and Lownsberry, 1952), Heterodera schachtii on sugar beet (Mai and Abawi, 1980), M. hapla on carrot (Kotcon, et al., 1985) and Xiphinema spp. on peach. Although crop rotations alone are unlikely to provide nematode control in all nematode-crop systems, their potential as one component of integrated pest management systems has not yet been fully realized. This potential, when integrated with other non-chemical management measures such as biological control, is likely to become increasingly important, as traditional nematicide options are lost. Several nematode-antagonistic crops including rotation and cover crops such as rapeseed, marigold, forage pearl millet, Avena strigosa, sudangrass and sorgho-sudangrass were identified in the current project (LaMondia and Halbrendt, 2003). Native prairie plants that are resistant to the northern RKN (LaMondia, 1996) may be useful as cover crops for managing this and other plant-parasitic nematode species. Various plant species may have differential effects on populations of each of these nematodes (LaMondia and Halbrendt, 2003). Furthermore, the incorporation of plant shoots as green manures appears to play a significant role in the nematode-antagonistic effects of these plants (Halbrendt, 1996; LaMondia and Halbrendt, 2003).

Biological control offers an alternative or supplemental management tactic to chemical or cultural control of plant-parasitic nematodes. Pasteuria penetrans is an obligate, mycelial, endospore forming bacterial parasite of endoparasitic nematodes, such RKN (Meloidogyne spp.), and ectoparasitic nematodes, such as sting (Belonolaimus) (Chen and Dickson, 1998; Giblin-Davis, 1990). This parasite is a promising biological control agent for management of the most important agricultural species of Meloidogyne (Dickson et al., 1994). Pasteuria penetrans is widespread in soils where RKN occur, however isolates of the bacterium vary markedly in specificity towards different RKN species and populations within species. These bacteria can reduce RKN populations below economic threshold levels in several different crops and environments (Chen and Dickson, 1998;). More than 35 publications reported that Pasteuria may reduce symptoms or disease severity caused by RKN in various crops (Chen and Dickson, 1998). In Florida, RKN suppression has been attributed to P. penetrans (Dickson et al., 1994; Chen et al., 1992; Weibelzahl-Fulton et al., 1996). A study of Pasteuria from nematodes in bermudagrass turf revealed an inverse relationship between the proportion of RKN J2 infected and the number of J2 present (Giblin-Davis, 1990). In 2001, soil samples from golf courses in the northeastern US that had been submitted for routine nematode assays contained Pasteuria-infected stunt nematodes (Wick and Dicklow, 2001).

Pasteuria penetrans forms endospores as its final growth phase and these spores are the infective stage used for nematode control. The ability to form spores is a significant advantage if we can formulate these bacteria for agricultural use, as mature spores are highly resistant to drying and mechanical shearing. The relationship between soilborne Pasteuria endospores and suppression of infection of plants by Meloidogyne spp. has been well documented (Chen et al., 1997; Chen and Dickson, 1998; Weibelzahl-Fulton et al., 1996). The basis for biocontrol potential is due in part to the fact that propagation of the bacteria within the pseudocoel of the infected nematode host results in a loss of fecundity. The nematode host serves to amplify the infective endospores that are released in the soil to repeat a cycle of infection and amplification (Preston et al., 2003). As the density of soilborne endospores of a particular species and strain of Pasteuria increases, those soils may become more suppressive to nematodes susceptible to infection by that species and biotype of Pasteuria.

Other microbial pest control agents such as Arthrobotrys, Bacillus, Burkholderia, Paecilomyces, Paenobacillus, and Verticillium may be used to enhance systems for the management of plant-parasitic nematodes (Kerry, 1998; Kokalis-Burelle et al., 2000; Sikora and Hoffmann-Hergarten, 1993; Siddiqui and Mahmood, 1996; 1999; Stirling, 1991). In addition, natural products from nematode-antagonistic microbes can be toxic to plant-parasitic nematodes or disrupt the nematode life cycle (Anke and Sterner, 1997; Anke et al., 1995; Chen et al. 2000; Hallman and Sikora, 1996; Han and Ehlers, 1999; Hu et al., 1999; Köpcke et al., 2001; Meyer et al., 2000; Singh et al. 1991). Active compounds that affect egg hatch and second-stage juvenile mobility of M. incognita and Heterodera glycines were identified from nematode-associated fungi and from rhizosphere-inhabiting bacteria and fungi (Nitao et al., 1999; Meyer et al., 2000; Meyer and Roberts, unpubl.). Two of these fungi, Fusarium equisetei and Chaetomium globosum, were utilized in bioassay-guided isolation to identify compounds responsible for in vitro activity against M. incognita (Nitao et al., 2001, 2002). Continued research is needed to identify additional microbes with ability to produce compounds active against plant-parasitic nematodes. These compounds have potential for application as novel nematicides, and as live biocontrol agents.

Many biologically based approaches to pest management employ organisms that are directly antagonistic to pathogens (Cook & Baker, 1983; Nemec et al., 1996). In addition to directly affecting pathogens, a second mode of action for microbial biocontrol agents is the stimulation of induced systemic resistance in the host plant that minimizes or prevents infection (Smith et al., 1999). The advantage of using a microorganism to induce resistance is that once the crop plant is colonized, the stimulating effect is continuous. The addition of microbial agents to transplant mixes provides an ideal delivery mechanism for introducing biocontrol agents on transplanted vegetable crops. Combinations of microbial agents, PGPR and elicitors can be conveniently introduced in this manner. Research indicates that several new species of Bacillus increase transplant vigor and yield in many different transplanted crops including tomato, pepper, and strawberry (Kokalis-Burelle et al., 2002a; Kokalis-Burelle et al., 2002b; Kokalis-Burelle, 2003).

Cultural and biological approaches to nematode management may have numerous impacts on crop yield and sustainability beyond their impact on target plant-parasites. The success of cultural and biological control of plant-parasitic nematodes is usually evaluated based on the direct impact of management tactics on target nematode populations. However, the integration of crop rotations, cover crops, organic amendments, tillage practices, and other crop protection practices may have dramatic non-target effects on the physical, chemical and biological characteristics of soils. These non-target effects may substantially influence plant growth and yield. The concept of soil health uses certain characteristics such as low populations of plant disease and parasitic organisms, as well as high populations of organisms that promote plant growth, such as the majority of non-parasitic soil nematodes (Magdoff, 2001).

There are at least five trophic groups of nematodes in soils (Yeates et al, 1993). The change in nematode numbers, and nematode community analysis, has been successfully used as an indicator of soil health (Neher, 2001). The majority of soil nematodes are beneficial, having direct and indirect effects on soil nitrogen (Neher, 2001). The dynamics of nematode communities may be better predictors of soil health and crop sustainability than plant-parasitic nematodes. Cultural nematode management tactics such as rotation have been demonstrated to influence soil organic matter (Abawi and Widmer, 2000) and other physical, chemical and biological properties of soil. The number and diversity of free-living nematodes in soil reflect the availability of nutrients, water-holding capacity, soil structure, density, pH, buffering capacity and biological components (Widmer et al., 2002). Biological control tactics also may influence ecological characteristics of soils. Nematode community structure is likely influenced by a combination of previous soil use, soil factors, and initial species composition (Griffiths et al., 2002).

Objectives

1. Develop cultural controls for plant-parasitic nematodes based on resistant, non-host, or nematode-antagonistic rotation crops and green manures.
   
2. Develop biological control agents, such as Pasteuria penetrans, for suppression of plant-parasitic nematodes.
   
3. Determine the effects of cultural and biological controls of plant-parasitic nematodes on nematode community ecology dynamics at the trophic group level.
   
4. 
   

Methods

Objective 1. Develop cultural controls for plant-parasitic nematodes based on resistant, non-host, or nematode-antagonistic rotation crops and green manures. Scientists will search for resistance to Meloidogyne hapla in vegetable crops (onion and carrot, NY; and pepper, ARS-USDA - SC) and also to root-lesion nematode in vegetable (onion and beans, NY), small fruit (strawberries, CT), and rotational grain crops (rye, wheat, soybean, vetch; CT, PA, and NY). We (ARS-USDA ? SC) have already evaluated more than 400 accessions of pepper (Capsicum spp.) from the U.S. Capsicum collection (USDA, ARS) and have identified one Plant Introduction (PI) with moderate resistance to M. hapla. Seed of this resistant PI will be increased for further testing against local isolates of M. hapla by collaborators in CT, MA, and NY. Resistant cultivars will be utilized to develop alternative nematode management strategies for managing root-knot nematodes in vegetable crops (ARS USDA ? SC). The potential of using a resistant bell pepper cv. as a rotation crop for managing M. incognita in subsequent, susceptible double-cropped cucumber and squash will be evaluated. New rotational combinations of vegetable crops will be developed and tested for effectiveness in managing Meloidogyne spp. including double-cropping systems (susceptible vegetable crops after resistant cvs.). Appropriate herbicides will be applied in order to reduce weeds problems in all plots. Although the principles and concepts for managing plant-parasitic nematodes using resistant cvs. in rotation with susceptible cvs. are well established and proven, the usefulness of M. incognita resistant bell peppers as a rotation crop for managing this nematode in double-cropped cucumber and squash has not been demonstrated. Furthermore, the effectiveness of resistant-susceptible vegetable cropping schemes for managing Meloidogyne spp. must be evaluated on a crop by crop basis because differences in levels of resistance and susceptibility of various crops (and cvs.) to various Meloidogyne species and races will affect their use. The use of summer cover crops of RKN-resistant cowpea and sunn hemp for managing RKN in vegetable crops and the effects of these practices on soil microbial and nematode communities will be investigated by scientists in FL and SC (FL, ARS-USDA- SC, and ARS-USDA- FL). Field experiments will be conducted on sandy soils infested with M. incognita, a common nematode pathogen of many vegetable crops (McSorley, 1995). The leguminous cover crops ?Iron Clay? cowpea and sunn hemp, both which are suppressive to M. incognita (McSorley, 1999), will be evaluated in a split-plot design with three summer cover crops (sunn hemp, ?Iron Clay? cowpea, or none) as main plots and three amendment treatments (sunn hemp hay, cowpea hay, or none) as sub-plots replicated five times. The cover crops will be grown during the early summer on plots receiving cover crop treatments. The cover crop will be harvested before pod development to make maximum use of its value as a green manure. Cut residues of the cover crops will be removed from some plots or added to others depending on the treatment involved. Nematode-susceptible eggplant will be planted in the plots mid-August. Data will be collected on nematode population density, nematode community structure, disease incidence and severity, root damage (nematode galling and disease rating), crop yield, weed density, nutrient levels in crops and soil, soil organic matter, and soil water-holding capacity. The same cultivar will be evaluated for the ability to manage the northern root-knot nematode, M. hapla, in CT. Scientists will assess the impact of nematode-antagonistic rotational or cover crops on populations of lesion and root-knot nematodes, and the impact of such antagonistic crops on nematode damage to vegetable crops in CT, PA, and NY. In NY, the mechanisms involved in such suppression will also be investigated. Results from the current project have identified several nematode-antagonistic rotation and cover crops including rapeseed, marigold, forage pearl millet, Avena strigosa, sudangrass and sorgho-sudangrass. These crops will be further evaluated for nematode suppressiveness against RKN, cyst, lesion or dagger nematodes in parallel or complementary studies. In addition, native prairie plants we have found resistant to M. hapla (LaMondia, 1996) will be evaluated for host status to lesion and dagger nematodes. We have determined that different plant species may have differential effects on populations of lesion and dagger nematodes. Aster and Rudbeckia are resistant to M. hapla (LaMondia, 1997), and Rudbeckia and marigold reduced lesion nematode densities and potato early dying. The incorporation of plant shoots as green manures also may impact the nematode-antagonistic effects of these plants (Halbrendt, 1996; LaMondia and Halbrendt, 2003). CT and PA will evaluate seven grass species, four Aster species, purple and yellow coneflower, and small black-eyed Susan for their host status and effects as green manures on lesion and dagger nematodes, and for their potential as antagonistic cover and rotation crops. These native plants are suitable for direct seeding, should compete well with weeds, and are available as reasonably priced seed. The antagonistic mechanisms of these plants, which reduce densities of plant-parasitic nematodes during plant growth or as a green manure, will be determined in the greenhouse, microplot, and field tests in CT, NY, and PA. The toxicity of root exudates or plant breakdown products such as glucosinolates released from Brassica residues (Sang et al 1984) or the nematicidal residues from oats, sudangrass or sorgho-sudangrass will be evaluated in vitro or directly on nematodes in soil to determine the most efficacious use against particular nematode species. Data and techniques developed in complementary systems will be shared to allow us to evaluate, compare, and increase efficacy of antagonistic plants on different nematodes. For example, the release of cyanogenic compounds and suppression of root-knot nematodes by sudangrass was increased when plants were incorporated prior to the first frost in NY. A low volume soil bioassay technique developed in PA will be used to further evaluate the toxicity of green or freeze-dried plant extracts on a variety of plant parasitic nematodes using soils and nematodes supplied by cooperators in CT, MA, NY, and WV. Objective 2. Develop biological control agents, such as Pasteuria penetrans, for suppression of plant-parasitic nematodes. Sequence relationships of host-specific P. penetrans spp. will be determined; bacterial genes that are involved in nematode host specificity or preference will be identified; nucleic acid and antibody probes will be applied to determine the levels of Pasteuria spp. in soil and their potential for suppression of nematode infestation; and train scientists in the group on procedures for soil detection of Pasteuria spp. (UF - FL). Several genes, including sigE, atpF, and dnaE, have been sequenced for P. penetrans (Preston et al., 2003). Analogous and additional genes will be sequenced from Pasteuria spp. infecting sting, lance, and ring nematodes. Unique sequences will be used to develop DNA probes and PCR-primers that will be used to quantify different species of Pasteuria in nematode infected plants. Amino acid sequences of glycosylated adhesin proteins will be used to design primers to amplify encoding DNA sequences from P. penetrans P20. Definition of these sequences will allow design of degenerate primers to amplify analogous genes from other Pasteuria spp. Sequence analysis and identification of glycosylation sites may reveal patterns that distinguish the adhesins and support a structure/function relationship to host preference. Unique sequences encoding species and biotype-specific adhesins also will be used to detect these species and strains in environmental samples. Detection and quantification of endospores will be carried out using the monoclonal antibody that recognizes the glycan epitope shared only by endospores of Pasteuria spp. (Schmidt et al, 2003). This approach will quantify the level of Pasteuria spores in the soil and will be used to estimate the extent to which soils may be expected to be suppressive for root-knot nematodes. The application will extend to the quantification of Pasteuria spp. endospores derived from various nematode pathogens of turfgrasses in the northeastern US. The occurrence of Pasteuria species in soil will be determined based on a soil assay developed in Florida and demonstrated to the group during a workshop at the first multi-state meeting of the project. This procedure will be used during the course of the project for the detection and quantification of Pasteuria in soil. In addition to the use of the monoclonal antibody to detect the levels of mature virulent endospores of all Pasteuria spp. in the soil, specific nucleotide probes will be used to detect the levels of Pasteuria infection of differentiated nematodes in root samples (FL). The probes will be synthesized to be specific for regions in different genes that have unique sequences that allow the distinction of Pasteuria isolates unique to the different nematode hosts. These probes will be used as primers for PCR amplification and quantitative determination of Pasteuria spp. and biotypes that are able to infect and suppress different plant-parasitic nematodes. The use of these probes should allow the identification of target host, and thereby determine the potential for applying a particular Pasteuria spp. or strain to control a particular nematode. Correlations between the levels of a particular Pasteuria spp. or strain and the extent to which soil is suppressive for a particular nematode will be determined in field plots as previously described (Chen et al., 1996). To further test the suppressive potential of a Pasteuria spp. or strain, greenhouse experiments will be performed (FL) in which the levels of Pasteuria and nematode hosts can be controlled. Dose responses of nematode propagation to introduced Pasteuria spores will quantify the biocontrol potential for each. Suppressive soils containing a suppressive agent will be transferred to non-suppressive soil field sites. A suppressive soil containing P. penetrans will be moved to a RKN free field. The new site is to be inoculated with Meloidogyne arenaria and a peanut crop planted. The plot, a 3 by 3 factorial design, six replicates, will include three winter crops: wheat, vetch, and rye, a summer peanut crop with three soil treatments: chloropicrin, 1,3-D, and untreated for a total of nine treatments (UF - FL). A putative M. hapla-suppressive soil will be evaluated for Pasteuria biocontrol using a protocol developed in FL, transferred to a new site, inoculated with M. hapla and planted to tomato (CT). Methods for the amplification of P. penetrans will be improved (UF - FL). The aim here is to define the optimum conditions for the continuous amplification of P. penetrans. Microplots containing M. arenaria and P. penetrans will be used and planted with wheat in winter and cucumber, squash, and peanut in summer. Cucumber and squash are ca. 60-day crops, whereas peanut is a 135 to 150 day crop. As many crops cycles of squash and cucumber vs one crop of peanut will be compared. Cucumber and squash will be rotated within the same plots. The treatments will be arranged in a randomized complete block design replicated three times. The relationship between P. penetrans attachment and infection will be studied (UF - FL). The aim will be to determine the relationship between the number of endospores attached per J2 and the subsequent infection rates. P. penetrans attachment on M. arenaria will be achieved by centrifugation method with attachment levels of 1 to 5, 6 to 15, and over 15 endospores/J2. The three categories will be inoculated to tomato to determine the rate of infection and fecundity. The natural suppression and efficacy of commercially available biorationals will be evaluated (UF - FL, MA, & RI). The extent to which natural populations of the nematode parasites Pasteuria and Arthrobotrys build up to suppress plant-parasitic nematodes in golf greens will be determined; the length of time required for populations of Pasteuria and fungal antagonists such as Arthrobotrys to build up to suppressive levels in golf greens will be determined; and a model will be developed that will allow prediction of nematode activity based on the proportion of Pasteuria-infected individuals. In addition, golf course soil characteristics and cultural practices will be examined to determine what effect they may play on population levels of the assayed nematode antagonists. New commercially available formulations of Paecilomyces lilacinus will be tested to determine whether they will provide control of Meloidogyne spp. The material will be tested on tomato grown on raised beds covered with polyethylene mulch and drip irrigation. The fungus will be applied at least three times, preplant, at plant, and post plant. Natural products, antagonistic fungi, and Gram-positive bacteria including Bacillus spp. and Streptomyces spp. with nematode suppressive and plant growth-promoting capabilities will be developed for application on transplanted crops; and the efficacy in reducing nematode populations will be determined (NY, ARS-USDA, FL, and ARS-USDA MD). Initial experimentation for efficacy of biocontrol agents for RKN on tomato will be conducted under laboratory and greenhouse conditions. Once efficacious rates of organisms are determined, microplot and field experiments will be conducted in combination with methyl bromide alternative soil treatments or crop production practices. Treatments will be evaluated in field trials conducted over 24 months. Various types of microbes and natural product formulations will be tested for production of compounds toxic to nematodes (Nitao et al., 1999). Testing will include an examination of microbe-nematode interactions, ultrastructural studies at various stages of the microbe life cycle (Meyer and Wergin, 1998), and greenhouse studies on efficacy of potential management agents in the soil (methods in Meyer, 1994, 1998; Meyer et al., 2000, 2001; Nitao et al., 1999; Sardanelli, 1997). Objective 3. Determine the effects of cultural and biological controls of plant parasitic nematodes on nematode community ecology dynamics at the trophic group level. Agricultural systems in the northeastern states are diverse and often specific to certain locations or markets. As a result, nematode pathogens may be as diverse as the specialized crop systems they attack. Our research involves both direct collaboration and complementary studies by individual researchers designed to extend results from model systems for wider applicability. Current concepts of ?soil health? relate to the growing conditions under which crops are produced. This includes all components that are needed in crop production such as physical, biological, and chemical aspects of the soil. An optimization of all these components should reduce pest and pathogen damage and therefore result in higher yields and less input cost. In Alabama, the nematode community structure under cotton in conventional tillage systems has little diversity (R. Huettel, personal observation). This may relate to poor ?soil health? as the lack of diversity is especially evident in cotton fields where plant parasitic nematodes are present. The use of nematicides as well as herbicides with Bt cotton and fungicides to control diseases most probably contributes to the lack of diversity in these fields. On several research units within the Alabama Agricultural Experiment Station, there are ongoing studies that include conservation tillage regimes with cotton as the major crop. One such program has been underway for several years at the E.V. Smith Research Center located in central Alabama. This program includes no-tillage, minimal tillage, and conventional tillage systems that measure all agronomic effects as well as pest and disease monitoring. However, there is no monitoring for nematode communities in the program. This research will compliment this conservation tillage program by providing information on nematode biodiversity for each of the tillage regimes being studied. In preliminary soil samples, root-knot nematodes were observed as the primary plant parasitic nematodes and there were varying numbers of free-living nematodes found under the tillage systems. Plots will be monitored using several ecological measures such as maturity index, community structure indices, etc. These will be correlated with other soil characteristics being measured within the systems being used. Plots will be sampled for nematodes monthly during the growing season. Pre- and post-sampling will also be conducted. The nematode genera will be assigned to trophic groups and the ecological indices will be calculated. This research will be conducted with the ARS Soil Conservation Laboratory, Auburn, AL. Collaborations will also be established with NE-171 members, especially on ecological indices and sampling methodology. Similarly, extensive collaborative efforts on soil health are underway in New York. Multidisciplinary faculty, extension educators and growers are involved in the soil health initiative for vegetable cropping systems. Populations and diversity of plant-parasitic and free-living nematodes will be determined at all the demonstration sites on commercial fields (organic and conventional) and long-term soil health sites have been established at research sites. Nematode data will be related to soil management practices, crop yield, and also to specific physical, chemical and biological parameters obtained from the same sites. Scientists in six states will conduct complementary or collaborative studies to assess the impact of rotation and cover crops on plant-parasitic nematode populations and nematode community structure. Results from our current project have identified several nematode-antagonistic crops such as rapeseed, marigold, forage pearl millet, Avena strigosa, sudangrass and sorgho-sudangrass. These crops and other potential nematode-antagonistic plants will be used to evaluate the effects of plant growth and/or green manure shoot incorporation on target nematode populations and nematode community structure. Long-term population dynamics of plant parasitic and other nematode trophic groups will be monitored at the Kellogg Biological Station LTER site (MI) and at the West Virginia University Organic Research Farm (WV), where ongoing farming systems studies are underway. Multi-year rotations are being compared under several conventional and organic farming systems to evaluate interrelationships among farming practices on nematode community structure. In addition, effects of farming systems on other nematode biocontrol agents (trapping fungi and nematode predators) will be evaluated (WV) to identify farming practices that best enhance indigenous nematode biocontrol activity. Scientists will either determine nematode community structure based on trophic group (AL, CT, MA, NY) or on more detailed identification to genus (MI, WV). Maturity and diversity indices will be calculated and correlated to crop yield. We propose to determine the effect of plant species on nematode communities. We will grow and incorporate the same amount of green manure material of four Aster species, A. laevis, A. nova-angliae, A. pilosus, and A. ptarmicoides in microplots infested with lesion or root-knot nematodes. Butyric and propionic acid are extremely effective as preplant nematicides (RI). The biocidal activity of these chemicals is nonselective, however, and at extremely high rates, these chemicals can even have a deleterious effect on soil structure. Multiple rates of acetic, propionic and butyric acid will be applied to soil microplots over the course of 3 years and the treated soil will be assayed monthly to determine the effect of each of these treatments on populations of free-living and fungal-feeding nematodes. Acetic acid is currently used as a low impact herbicide in organic systems and this study will help clarify its effect on nontarget fauna. Researchers have documented that organic acids are typically selective towards plant parasitic nematodes, having marginal effect against free-living forms at comparable rates (Browing et al., 2004; Dijan et al. 1994; Sayre et al. 1965). These studies have all been conducted in sand pots in the greenhouse or glass vials but show that nematodes such as Heterorhabditis and Steinernema are anywhere from 10-1000X less sensitive to butyric acid. We will examine the effect of butyric acid against plant parasitic nematodes and free-living trophic groups at the same time, in field soil.

Measurement of Progress and Results

Outputs

• Research on cultural controls for plant-parasitic nematodes using resistant, non-host, or nematode-antagonistic rotation crops and green manures will increase our knowledge on nematode dynamics and damage, and will result in the publication of numerous peer-reviewed journal articles and technical articles.
• Information will be developed about the efficacy of candidate biocontrol products.
• The documented increases or decreases in nematode diversity will be correlated with management practices and soil properties such as moisture, fertilizers, chemical treatments, rotation crops, etc. Increased nematode diversity should relate to increased crop yields and/or reduced input costs for disease or plant-parasitic nematode control. This should provide tillage systems that can be used by growers to better manage soil systems and reduce cost for disease and plant parasitic nematode control.
• Technology will be developed that improves the application efficiency of biological products for control of plant pathogenic nematodes. These products and practices will include commercially available transplants, transplant mixes, and microbial products that enhance plant growth, reduce nematode infestation, and increase yield.
• Research at golf course sites throughout Southern New England will document nematode species present, fungal and bacterial antagonists, a wide variety of accompanying soil characteristics and the chemical and cultural practices implemented on the sample sites. The research will result in basic knowledge about the biology of potential management agents and the modes of actions of such agents, which will be reported through scientific publications and presentations. The work will lead to development of nematode management products containing microbes or microbially based compounds.
• Publication and distribution of extension publications on the host status of different cover and rotational crops to major nematodes and effects of soil management practices on nematodes and their damage.

Outcomes or Projected Impacts

• Knowledge generated during this project will lead to new components for integration into effective nematode management systems and will reduce losses in crop quality and yields. Growers will use new and improved methods for managing plant pathogenic nematodes developed under this project that will reduce the need for methyl bromide and other nematicides resulting in safer working conditions and a safer food supply.
• Biorational products or rotation and green manure crops that reduce plant-parasitic nematode populations will replace the use of organophosphate and fumigant nematicides. Effective nonchemical controls will help to maintain production in the absence of methyl bromide, and enhance economic opportunity in rural communities. For example, the current application of Vydate (up to 4 gal/acre) on carrot for nematode control costs growers about $250.00 per acre annually. Thus, there is a great opportunity to develop a safer alternative to Vydate at a lower cost.
• Plant-parasitic nematodes of turf are difficult to control using traditional chemical methods and only a rudimentary understanding of their ecological relationships exists. This project will help to develop an understanding of the impact of environment and biological antagonists upon the life history of these nematodes. This information will ultimately contribute to the formulation of effective cultural and biological management strategies that seek to mitigate nematode damage.
• Formalization of advisory programs for nematode management.
• Use of nematode diversity as indicators of soil health and quality.
• Development of specific nematode research methodologies including ELISA, trophic groups identification protocols, and suppressive soil assessment methods.
• Nematologists will be in a better position to advise agricultural stakeholders regarding the development and importance of plant parasitic nematodes. This information can reduce the application of organophosphate and fumigant nematicides and lead to long-term health, environmental and food safety benefits due to reduced pesticide exposure.
• Products that prove to reduce plant-parasitic nematode populations will replace the use of organophosphate nematicides.
• Training of onion, carrot, and other vegetable growers to conduct their own bioassays for root-knot nematodes will allow targeted nematode management only in fields with damaging nematode populations, thus reducing human health risk, environmental exposure, pesticide residues in food, and reduced production costs.
• Our efforts to sequence and annotate the genomes of different Pasteuria spp. may ultimately lead to defining conditions for the in vitro propagation and application of Pasteuria spp. and biotypes for the effective biocontrol of different species of plant-parasitic nematodes. The ability to determine the level of virulent endospores or biotypes in the soil will allow the judicious selection of an appropriate approach for suppressing a target nematode. Virulent Pasteuria at suppressive levels would preclude treatment. Sub-suppressive endospore levels may allow effective treatments with lesser amounts of nematicides delivered less frequently. The absence of Pasteuria strains effective on the target nematode will require the use of chemical nematicides at effective doses. It may also be possible, especially for root-knot nematodes, to amplify the endospores and deliver them as an amendment that will allow biocontrol for high value crops.
• Research accomplished in this project will lead to a better understanding of the relationships among soil properties, cultural practices, nematode populations and antagonist populations that are key factors in the development of healthy and productive soils.

Milestones

(2005): Develop preliminary data on efficacy of various organisms for RKN control on horticultural and agronomic crops in greenhouse and microplot trials.<p> Development of suppressive soils in golf courses: i) 40 golf courses will be sampled four times annually and analyzed; ii) soil composition will be analyzed; iii) fungal antagonist DNA will be extracted from soil samples; iv) fungal ITS sequences will be amplified; v) golf course cultural and management practices will be compiled.<p>Cover and rotational crops and green manures appropriate for each states research efforts will be identified.<p>Screening of vegetable germplasm (carrot, onion, pepper) for resistance to M. hapla.<p>Adequate sites for trials, and commercially available products will be identified to carry out biocontrol product efficacy trials.<p>The occurrence of Pasteuria species in soil will be determined based on a soil ELISA assay developed in Florida and demonstrated to the group during a workshop at the first multi-state meeting of the project. This procedure will be used during the course of the project for the detection and quantification of Pasteuria in soil.<p>A protocol for the identification of nematode-suppressive soils developed in FL will be presented as a workshop and used to evaluate potential suppressive sites in other states.<p>Identification of biological products and low-risk chemicals for nematode control as alternatives to high-risk nematicides.

(2006): Development of suppressive soils in golf courses: Sampling and DNA extraction will be repeated and the incidence of P. penetrans and fungal antagonists will be documented.<p>Complete regression analysis of the effects of soil characteristics, turf species, cultural practices on plant-parasitic nematodes and nematode antagonists.<p>Assessment of the impact of soil management practices to promote soil health on nematode diversity and damage in organic and conventional vegetable production systems.<p>Confirmation of the efficacy of biocontrol organisms against root-knot nematodes in tomato and other crops.<p>Determine carbon utilization preferences using the Biolog and optimize colonization and efficacy of RKN control in microplots.<p>The development of appropriate and statistically-based sampling methods and the location of field sites with adequate natural infestations of Pasteuria.<p>A workshop on transferring ecological concepts to production agriculture will be presented at the annual meeting of this committee. Protocols for evaluating community structure will include when to sample, and standardization of trophic types among free-living nematodes.

(2007): Evaluation of identified resistant vegetable germplasm sources against root-knot and lesion nematodes under microplot and production filed conditions.<p>Development of suppressive soils in golf courses: Previously undertaken surveys will be repeated and the entire dataset will be analyzed. Predictive factors conducive for nematode antagonist reproduction on golf courses will be determined from this data.<p>Effective laboratory assays for biocontrol agents will be evaluated in greenhouse and field microplot tests.<p>Determine field activity in spring microplot trials and establish treatments for fall field trials for RKN control on tomato.<p>Establishment of demonstration trials on the efficacy of sustainable soil management practices against plant-parasitic nematodes and root diseases in collaboration with vegetable growers.

(2008): Develop and validate integrated nematode management options based on results obtained on effective cover and rotational crops, biocontrol agents and resistant crop germplasm. Establish demonstration trials in experimental and production fields.<p>Statistical model of the effects of organic acids on free-living nematode populations in agricultural soils.<p> Field evaluations of identified biocontrol agents in commercial fields and the assessment of their impact on yield and profitability.<

Projected Participation

View Appendix E: Participation

Outreach Plan

The Multi-State Committee on biologically based alternative management systems for plant-parasitic nematodes will present programming in the participating states to its stakeholders including growers, commodity groups, agricultural industry representatives, extension educators, and homeowners. The Technical Committee will continue to sponsor a web page with project reports and links to other nematode management web sites. Participants from MA and RI have extensive extension responsibilities and operate nematode diagnostic laboratories that service numerous grower groups. These labs will utilize data generated from this project to make more precise nematode control recommendations to growers. Results obtained in this project will be presented at commodity group meetings and annual extension meetings and will also be published in extension bulletins, fact sheets, newspaper articles, and posted on web pages. In addition, field days for growers and extension educators will be held at the field trials sites as appropriate. Finally, several extension educators and growers will be collaborating in the field trials, thus demonstrating a direct outreach effort.

Organization/Governance

The Technical Committee will consist of at least one voting member from each of the participating states (Attachment A), the administrative advisor, and the CSRS representative. Each year the technical committee will elect a chairperson, secretary, and at least one member-at-large to serve as an executive committee. The regional Technical Committee will meet annually to report on the research results obtained, discuss and exchange information and ideas and to plan and coordinate next years work relating to the objectives of this proposal. A coordinator for each of the objectives may be designated to facilitate the coordination and reporting of the research being conducted by the collaborators. The technical committee may invite other scientists with experience in biological control, crop production systems, integrated pest management, sustainable agricultural practices, and others to participate in the annual meeting to provide specific information and strengthen the discussion.

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Attachments

Land Grant Participating States/Institutions

AL, CT, FL, GA, MA, MI, NY, OH, OR, PA, RI, TN, TX, VT, WV

Non Land Grant Participating States/Institutions

USDA-ARS, USDA-ARS Beltsville Agricultural Resarch Center