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

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Modern agriculture continues to rely on technological advances and off-farm inputs to increase yields and profitability. In particular, most crops rely heavily on fertilizers and pesticides to maintain acceptable production levels.

Plant-parasitic nematodes are important pathogens on most food and fiber crops and without appropriate controls will cause loss of yield and quality. Chemical nematicides have been the primary management tool for over fifty years but many products have now been removed from the market or are under review. There is an urgent need for the development of alternative control options. Such work is necessary to develop environmentally sound agricultural systems that minimize chemical use while maintaining high production standards.

Related, Current and Previous Work

Crop rotation, soil amendments, genetic resistance:

Nematode control tactics such as rotation, cover cropping, green manuring, organic amendments and plant resistance have been evaluated as alternatives to chemical nematicides. Crop rotation is perhaps the oldest and most effective cultural practice for controlling plantparasitic nematodes (Good, 1968). Rotation crops that reduce nematode populations have been shown to serve as either nonhosts, less suitable hosts, or as antagonistic plants (Johnson, 1982; Trivedi and Barker, 1986; Merwin and Stiles, 1989; Halbreridt, 1996). Nonhosts may simply not be parasitized and have an effect similar to fallow. Antagonistic plants may produce compounds which stimulate hatch without allowing reproduction or actively reduce nematode populations by the production of nematicidal or toxic compounds (Halbrendt, 1996). Nematode-suppressive plants which produce these compounds may be utilized to deliver them by means of rotation, inter-cropping, cover cropping, or soil amendment with either green manure or dry plant residue.

A number of rotation or cover crops which reduce nematodes more than fallow have been identified (Halbrendt, 1996; Ko and Schmitt, 1996; LaMondia, 1997; Rodriguez-Kabana, 1992). Crop rotations which include these antagonistic or allelopathic plants may be thought of as active and those that are simply non-hosts as passive (Rodriguez-Kabana, 1992). The list of plants antagonistic to nematodes has grown but research on the mechanism of allelopathy, resistance or antagonism has not kept pace. While an understanding of the mechanism involved is not necessary for control by either resistance or allelopathy, this information would be important for selection and development of breeding lines with increased efficacy.

Two promising grain crops for nematode reduction by rotation are diploid `Saia' oats (Avena strigosa) and sorgho-sudangrass (Sorghum bicolor x S. sudanense). These crops have been reported to be poor hosts of lesion and root-knot nematodes (Colbran, 1979, Fay and Duke, 1977). Grain or grass rotations and companion crops conserve soil, reduce compaction and increase water infiltration into soil (Newenhouse and Dana, 1989). The use of oats and sorghum as allelopathic rotation or companion crops can suppress a number of weed species (Neustruyeva and Dobretsova, 1972, Putnam and DeFrank, 1983). Some oat accessions exuded up to three times as much scopoletin, a root growth inhibiting compound, as standard 'Garry' oats (Fay and Duke, 1977). Oats and sorghum also have been shown to produce fungicidal root exudates toxic to soilborne fungi such as Fusarium and Gaumannomyces (Crombie and Crombie, 1986, Nimbal et al., 1996). Recently, resistance to lesion nematodes has been associated with greater production of avenocin in 'Saia' oats than in susceptible oats (B. B. Brodie, pers. comet.).

Because of the importance of developing non-chemical nematode controls, a number of antagonistic plants have recently been bred for increased efficacy against nematodes and other soilborne pathogens. These include selected varieties of Brassica spp and Tagetes spp. The nematode-suppressive effects of marigold (Tagetes spp.) have been documented for over 50 years (Tyler, 1938; Steiner 1941). The antagonistic effect is due to toxicity of metabolites in the root exudates (Siddiqui and Alarm 1987a, b; 1988a). Although the presence of nematicidal compounds has been well documented, there are conflicting reports concerning the efficacy for suppression of a number of nematodes and the compounds responsible (Gommers et al., 1980; Gommers and Bakker, 1988). The differential ability of Tagetes species to suppress nematodes may be due to variation in species, cultivars, edaphic factors, nematode populations, and the active nematicidal compounds (Eisenback, 1987; Arevalo and Ko, 1989; Alarn et al., 1990). Recently, marigold cultivars have been specifically selected for increased nematicidal efficacy.

A number of leguminous crops also have been reported to have nematode suppressive properties. Vicente and Acosta (1987) reported that velvetbean root exudates were nematicidal and Rodriguez-Kabana (1992) suggested that bacteria in the velvetbean rhizosphere were antagonistic to cyst and root-knot nematodes.

Other crops such as canola (rapeseed, Brassica napus L. and B. campetris L), also have been implicated in reducing soil densities of nematodes (Davis et al. 1993; Mojtahedi et al. 1993). These plants synthesize large quantities of sulfur-containing glucosinolates in all tissues (Sang et al. 1984). When these plants are incorporated into the soil as a green manure, the glucosinolates hydrolyze to fungicidal and nematicidal isothiocyanates (Ettlinger et al. 1968). The level of nematode control has been correlated with glucosinolate concentration in plant extracts (Jing and Halbrendt, 1994). The incorporation of canola shoots to soils heavily infested with Meloidog)ne chitwoodi reduced nematode populations more than fallow treatments (Mojtahedi et al. 1993), and the application of canola meal significantly reduced populations of P. neglectus and subsequent Verticillium wilt in potato (Davis et al. 1993).

Additional alternative practices include soil amendments and mulches. Green manure treatments with certain nematode antagonistic plants may provide even greater nematode control than when used solely as a cover or rotation crop. (Siddiqui and Alarm 1987c, 1988b). Composts have been widely used as peat substitutes in the nursery industry, resulting in disease suppression (Hoitink and Grebus, 1994). The mechanisms put forth for this suppression (Alam and Jairajpuri, 1990) have included: changes in the physical and chemical properties of soil, including nutrition and the production of nematotoxic substances released directly or by microbial breakdown; changes in the microbial ecology of soil that affect antagonistic organisms or the release of antagonistic microbial metabolites; and the induction of plant resistance or tolerance to nematodes as well as the induction of systemic acquired resistance (Zhang et al, 1996; Zhou and Paulitz, 1994). Lail~fondia et al. (in press) demonstrated over 4 years in field microplot studies that soil amendment with spent mushroom compost reduced potato early dying severity by 24% (AUDPC for symptomatic leaves), increased marketable tuber yields by 94%, increased leaf photosynthesis by 43%, and reduced lesion nematode densities in subsequent rye cover crops by 48%.

Plant resistance is the most economical and environmentally safe means of controlling plant-parasitic nematodes. Host plant resistance and tolerance are important when crops must be grown in the presence of potentially damaging populations of nematodes. Resistance refers to the suppressive effects of plant genes on nematode development and/or reproduction (Trudgill, 1991). Tolerance refers to the ability of a plant to grow and produce an acceptable yield while supporting moderately large numbers of plant parasitic nematodes. Ideally, plant resistance and tolerance could be combined.

Most host plant resistance has been identified for specialized host-plant relationships, such as the sedentary endoparasitic cyst and root-knot nematodes, but resistance to other nematodes such as the migratory endoparasite Pratylerrchus, also has been identified (Potter and Dale, 1994). Mechanisms of resistance to nematodes include both constitutive plant compounds and factors induced by nematode infection. Resistant plants may contain toxic compounds such as alpha terthienyl, present in marigolds (Veech, 1981), compounds which reduce egg hatch (Gapasin, 1988), or lectins which may affect host-finding ability (Martian-Mendoza et al., 1992). Incompatible host response to nematodes ranges from nonspecific tissue necrosis to necrosis around a feeding site or a hypersensitive response that prevents nematode development (Kaplan and Keen, 1980).

Genes conferring resistance have been identified and incorporated into many crop cultivars. Resistance to root-knot and cyst nematodes in vegetable crops and tobacco have been identified and developed as a part of this project (Thies et al. 1997; LaMondia, 1991). While resistance has been durable in many situations, selection by resistance genes has often resulted in a population shift to nematode races able to overcome plant resistance. It will be important to integrate resistance with other control tactics to maintain nematode control and reduce selection pressure against resistance gene(s).

Biological control agents:

Suppression of plant-parasitic nematodes with nematode predators, parasites or disease agents is a desirable alternative to chemicals. Deploying and managing biocontrols will likely become increasingly important components of integrated pest management programs and sustainable agricultural systems. Biological control agents occur in diverse taxa. and include nematode trapping or endoparasitic fungi, predatory nematodes, arthropods (e.g. mites and collembola), bacterial parasites, and predatory protozoa. Understanding this diversity will be a critical step in adapting management practices to realize the full potential of biological control. However, because of the large number of potential biocontrol agents it is desirable and beneficial to focus efforts on one organism.

Pasteuria penetrans is a promising biological control agent against root-knot nematodes in the southeastern United States and has been selected for detailed studies. The role of P. penetrans in the northern tier of states has not been evaluated. The use of this bacterium to suppress plant-parasitic nematodes has been tested on many crops, mostly in greenhouse pots (Chen and Dickson, 1998). Pasteuria penetrans suppressed Meloidogyne spp. on bean, brinjal, chickpea, cucumber, eggplant, gape, hairy vetch, kiwi, mung, okra, peanut, pepper, rye, soybean, tobacco, tomato, and wheat (Chen and Dickson, 1998). Some isolates of Pasteuria spp. have been reported to suppress Belonolaimus longicaudatus on bermudagrass (Giblin-Davis, 1990), Heterodera avenae and H. zeae on unspecified crops (Bhattacharya and Swarup, 1988), H. cajani on cowpea (Singh and Dhawan, 1994), H. elachista on rice (Nishizawa, 1987), and Xiphinema diversicaudatum on peach (Ciancio, 1995b).

While many strains of Pasteuria are nematode species-specific, cross-genetic suppression of nematodes also has been observed (Mankau and Prasad, 1972; Bhattacharya and Swarup, 1988). Pasteuria penetrans simultaneously reduced population densities of Pratylenchus scribneri and root galls induced by M. javanica and M. incognita in tomato (Mankau and Prasad, 1972). An Indian isolate of P. penetrans parasitized both Heterodera spp. and M. incognita (Bhattacharya and Swarup, 1988). Endospores of P. penetrans were mass-produced on M. incognita and when endospores were incorporated into soil, numbers of cysts of H. avenae on wheat roots were reduced.

A successful example of the biological control potential of P. penetrans for management of root-knot nematodes on peanut was reported recently (Chen, 1996; Chen et al., 1996). Endospores of P. penetrans were incorporated into field microplots in the first year only at 0, 1,000, 3,000, 10,000, or 100,000 endospores/g of soil. Root galls and pod galls were significantly reduced at 100,000 endospores/g of soil in the first year. In the second year, root galls and pod galls were reduced at 10,000 and 100,000 endospores/g of soil. Pod yields increased 58% and 94% at 10,000 and 100,000 endospores/g of soil, respectively (Chen et al., 1996). In the third year, root galls and pod galls were nil at 100,000 endospores/g of soil, and were reduced at 1,000, 3,000, and 10,000 endospores/g of soil. Pod yields were increased 180%, 291%, 221%, and 272% at 1,000, 3,000, 10,000, and 100,000 endospores/g of soil, respectively (Chen et al., unpubl.). Population densities of J2 in soil at harvest also were significantly reduced at 10,000 and 100,000 endospores/g of soil in the third year. Apparently, the establishment and amplification of P. penetrans in the field microplots played an important role in the increased suppression of root-knot nematodes over the 3-year period.

Isolates of Pasteuria spp. failed to suppress populations ofMeloidogyne spp. on sugarcane (Spaull, 1984), Helicotylenchus lobus on turfgrass (Ciancio et al., 1992), and Tylenchulus semipenetrans (Ciancio and Roccuzzo, 1992). A survey in sugarcane fields in South Africa revealed that population densities of Meloidogyne spp. were generally higher in fields infested with P. penetrans and that the level of nematode parasitism was greater at higher nematode densities (Spaull, 1984). On turfgrass, there was no correlation between the population density of Helicotylenchus lobus and the percentage of nematodes with endospores (Ciancio et al., 1992). However, an increase in parasitism was observed 2 months after a 10-fold nematode population growth (Ciancio et al., 1992).

Mode of action: Pasteuria penetrans reduced the number of JZ penetrating roots (Brown and Smart, 1985; Davies et al., 1988a; 1988b; Sekhar and Gill, 1990), number of females in roots (Davies et al., 1991), female fecundity (Bird, 1986; Bird and Brisbane, 1988), number of J2 in soil (Chen et al., 1997c; Davies et al., 1988a; 1988b), and number of eggs on roots (Ahmed and Gowen, 1991; Bird and Brisbane, 1988; Chen et al., 1997c; Weibelzahl-Fulton et al., 1996). Movement and mobility of J2 were reduced and their ability to locate host roots was affected when J2 were encumbered with endospores (Davies et al., 1991; Mankau and Prasad, 1977).

Pasteuria species are gram-positive, dichotomously branched, endospore-forming bacteria with septate mycelium (Mankau and Imbriani, 1975). Most of the species identified to date show great promise as biological control agents of several of the most important plant-pathogenic nematodes. To date four species of the bacterium have been described. These were differentiated by their host preference, developmental characteristics, and size and shape of sporangia and endospores (Sayre and Starr, 1989). Pasteuria ramosa, which parasitizes water fleas of the genera Daphnia, is the type species of the genus (Ebert et al., 1996). The other three species of Pasteuria are parasites of plant-parasitic nematodes: P. penetrans on Meloidogyne spp., P. thornei on Pratylenchus spp., and P. nishizawae on cyst nematodes of the genera Heterodera and Globodera (Sayre and Starr, 1989). The terminal hyphae of the mycelium elongate to form sporangia and eventually endospores. Endospores are nonmotile and resistant to desiccation.

The apparent utility of P. penetrans for biological control of root-knot nematodes has prompted several efforts to produce the organism in culture (Bishop and Ellar, 1991 ; Riese et al., 1988; Previc and Cox, 1992). At present, only limited production of Pasteuria has been attained in vitro. Major hurdles to the development of Pasteuria spp. as a biological agent include the definition and control of events necessary for the formation of infective spores. In the absence of protocols to mass produce spores of Pasteuria spp. for direct application, the development of strategies for their amplification via agronomic practice still holds great promise for the biocontrol of target nematode species (Dickson, unpublished). In order for spores to attach to juvenile root-knot nematodes, they must bear appropriate surface molecules that recognize and bind to receptors on the cuticle of the nematode host. Interference with spore attachment by lectins specific for N-acetylglucosamine and/or alpha-glucoside, alpha-mannoside residues suggests these ligands may participate in the recognition process (Bird et al., 1989). Proteins isolated from P. penetrans spores have been shown to react with wheat-germ agglutinin (WGA) and concanvalin A (ConA), which indicates the presence of potential ligand receptors on the spores. These findings have provided the basis for a model in which glycopeptides on the surface of the spores, designated as spore adhesins, are recognized by lectins on the cuticle of the nematode (Davies and Danks, 1993; Persidis et al., 1991). Antibodies directed against these adhesion proteins as well as the proteins themselves were able to inhibit attachment of the spores to the nematode cuticle (Charnecki et al., 1996; Davies et al, 1992; Davies and Danks, 1993, Davies and Redden, 1997; Persidis et al., 1991).

The surface coat of plant-parasitic nematodes carries a net negative charge (Spiegel and McClure, 1995). The spores of P. penetrans also bear an electronegative charge that is affected by pH and ionic strength, as well as ion valency (Afolabi et al., 1995). The pH and ionic composition of a medium will be considered as variables in performing attachment assays to characterize potential adhesions. More recent studies have implicated that hydrophobic forces also participate in the overall adhesion process (Davies et al., 1996). It is important to note that this overall process may be complex, with several different types of chemical interactions participating. The interaction of specific polypeptides with specific ligands may be most important in the recognition process as an initial step in the overall adhesion process.

Polyclonal antibodies have been prepared that react with several spore-specific proteins and which block the attachment of spores to the host cuticle (Charnecki et al., 1996; Chen et al., 1997b). These will be used to identify peptide and/or carbohydrate epitopes in specific proteins involved in the recognition processes. Also, two mouse monoclonal antibodies have been produced, one (2A4 IgM) which detects an epitope shared by several peptides resolved by SDS-PAGE, and the other (5F1) which detects an epitope on a single polypeptide. Both of these monoclonal antibodies are able to discriminate different spore isolates with respect to native surface antigens (detected by ELISA) and to denatured polypeptides (detected by Western Blots following SDS-PAGE).

The ZA4 IgM antibody was produced in ascites and purified by gel filtration. It detects a number of bands ranging in size from 23 to 205 KDa by Western blots. These were resolved by SDS-PAGE of endospore extracts, and it is probable that it recognizes a glycan epitope. The ability of the lectin, wheat-germ agglutinin (WGA), to detect these same bands indicates that they bear glycan moieties including either b(1-4)-finked N-acetyl gluosamine or sialic acid residues. The inability of the 2A4 monoclonal antibody to detect fetuin, ovalbumen or pancreatic ribonuclease B, all of which are readily detected with WGA, indicates that this antibody recognizes an epitope that includes but is not restricted to the glycans recognized by WGA. (Charnecki et al., 1998). Using ELISA with plated endospores from different isolates (P20, P 120, and B4) indicate that these epitopes are on the surface of the spores. This same antibody failed to react with plated Bacillus subtilus endospores (evaluated by ELISA) or with UDC extracts of these spores (evaluated by Western blot), which indicates that they are relatively unique to endospores of Pasteuria spp. (Harrison and Preston, unpublished).

Spores have been developed that can be lluorescently labeled with fluorescein isothiocyanate with no detectable loss of their ability to attach to second-stage juveniles (J2) of M. incognita (Charnecki et al., 1996). The labeling involves the conjugation to spore coat proteins different from most of those detected with antibodies that block the attachment of spores to nematodes. These labeled spores also are able to infect nematodes to provide progeny spores. These have been particularly useful in providing quantitative data on the ability of various ligands to block attachment of endospores to nematodes. With this approach, the 2A4 monoclonal antibody was shown to effect 50% inhibition of the attachment of P20 spores to M. arenaria race 1 at an IgM concentration of 1.3 x 10-I0 M. This antibody and its (Fab')2 and Fab fragments will be those first used for the isolation and characterization of the adhesin epitope(s).

The primary approaches to biological control of nematodes have been augmentation of indigenous control agents and inundation with specific organisms. Long rotations or monoculture of susceptible hosts can induce microflora suppressive to specific nematode pests that maintains the pest population at levels below economic thresholds. The best known example is the widespread control of Heterodera avenae in Europe by the fungi Nematophthora gynophila and herticillium chlamydosporium. In the US, suppression of Meloidogyne spp. by the bacterium Pasteuria penetrans has been reported (Weibelzahl-Fulton, et al., 1996). Augmentation of indigenous agents also has been proposed as a mode of action for a variety of organic soil amendments (Rodriguez-Kabana, 1986; Kaplan and Noe, 1993).

Inundative release of biocontrol agents has met with success in small plots (Hewlett, et al. 1998), but commercial successes in larger field-scale trials have not yet been achieved (Duncan and Noting, 1998). Major constraints include limited understanding of the ecological niche requirements for introducing organisms to soil and the cost of producing large quantities of the most fastidious parasites of nematodes. In most cases, the agents being considered for release are highly specialized for effectiveness against particular nematode species. For example, the bacterium Pasteuria penetrans occurs in a variety of races that differ in their ability to attack different species of nematodes. Attachment of the bacterium to the host appears to be a key step in regulating this specificity, with different strains attaching preferentially to different nematode species (Oostendorp, et al., 1990). Research into this and other aspects of the activity of this agent is difficult because of the obligate parasitic nature of Pasteuria, and little success has been achieved in attempts to grow the bacterium in pure culture.

One tactic to suppress nematodes with the potential for low-cost, broad application is the use of endophytic fungi in grasses. Endophyte-infected fescue significantly reduced reproduction ofMeloidogyne marylandii and Pratylenclnss scribrreri (Kimmons, et al., 1990) while an endophyte-infected ryegrass supported significantly fewer Xiphinema americanum than an endophyte-free cultivar (Dernoeden, et al., 1990). Because this agent is seed-borne, its use may be easily integrated into cropping systems using grasses as cover or rotation crops, however, a better understanding of the mode of action and the conditions promoting maximum efficacy is needed.

Integrated Pest Management:

Integrated Pest Management systems are a subset of cropping systems practices. These include integration of crop rotations, cover crops, organic amendments, tillage practices, crop protection practices (physical, chemical and biological), and economic and environmental considerations.

At its simplest, IPM programs for nematodes involve evaluating pest density relative to a damage or treatment threshold. Integration of genetic resistance may occur through adjusting the damage threshold to account for specific differences in tolerance among varieties or by suppressing nematode reproduction to maintain populations below the damage threshold. Integration of biological and cultural controls may involve either some measure of the biological control efficacy against the target pest, or implementation of recommended management practices that are known to reduce pest densities while simultaneously providing other cropping system benefits (e.g., rotations, organic soil amendments). Evaluation of these recommended management practices usually involves monitoring changes in nematode population densities. Although this empirical approach lacks the theoretical value of a mechanistic understanding, in many cases, it is the only practicable approach for assessing highly complex soil systems.

Crop rotations can reduce nematode population densities, but may increase populations of other nematode pests, particularly in mufti-species nematode communities (Noe, 1998). Integration must also consider economic returns of rotations crops, additional capital and labor requirements, and grower and market acceptance of the rotation crop. In the Northeast, 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), Meloidogyne hapla on carrot (Kotcon, et al., 1985) and Xiphinema spp. on peach. While crop rotations alone are unlikely to provide nematode control in all nematode-crop systems, their potential as one component of IPM systems has not yet been filly realized. This potential, when integrated with other non-chemical management measures, is likely to become increasingly important as traditional nematicide options are lost.

Biocontrol agents span a continuum between generalist polyphagous organisms and specialists adapted to a single nematode host species or even a limited number of nematode host races. Augmentation of indigenous communities often implicitly relies on a broad diversity of biocontrol agents, of which each is adapted to differing seasons, host life stages, and environmental conditions in the soil. This high biodiversity is needed to assure that at least some of the agents present in a field will be active against the right nematode stages at the right time to promote adequate levels of population suppression. Because of this complexity, understanding the roles and relative importance of individual agents in any particular field is difficult and more progress has been made in systems dominated by one or two highly effective specialists. An ideal agent for commercialization would have a relatively broad spectrum of activity against plant-parasitic nematodes and would be able to successfully colonize a variety of soil environments and cropping systems, but would simultaneously avoid environmental risks from significant non-target effects (Simberloff and Stiling 1996). The indigenous nature of agents such as Pasteuria penetrans, as well as the limited dispersal ability of both these agents and their host nematodes, are factors that demonstrate the inherent ecological safety of biological control as a nematode control strategy.

The patchy distribution and soil-borne nature of nematodes, coupled with the high cost of monitoring and the need to make nematode management decisions well in advance of planting, are special constraints to nematode IPM which emphasize the importance of additional research efforts to support nematode IPM (Duncan and Noling, 1998). Improvements in biological monitoring methods are needed to better assess the spatial distribution of plant-parasitic nematodes as well as the presence of biocontrol agents that may influence nematode population dynamics and yield loss relationships (Sikora, 1992). Long-term sustainability based on a rigid management system is difficult in the face of a rapidly changing economic environment, yet long-term commitments are needed to achieve the benefits of many cultural and biological control practices (Noe, 1998, Duncan and Noling, 1998). The solution to this paradox requires a range of management options that can be implemented as short-term economic conditions dictate, while still integrating the long-term agroecological "costs and benefits" to the cropping system so that the true worth of particular management practices is adequately considered. Mufti-year modeling efforts that integrate population dynamics, environmental variables, yield loss relationships, efficacy of management measures and economic factors have been touted as essential to optimize nematode management, but the research base to support such predictive models is lacking in all but a few cropping systems (Duncan and Noling, 1998). Thus, the challenge of sustainable nematode IPM programs is to understand the multifaceted impacts and interactions of multiple management tactics in order to allow growers to make optimum decisions for long-term nematode control while maximizing economic returns.

With the assistance of Mr. Alan Moore of USDA/CRIS, a search of the USDA Research (CRISTEL database) has been conducted for the field of plant nematology using the following key words and phrases: field crops (potatoes, carrots, lettuce, onions), fruits (peaches and strawberries), and integrated pest management. One hundred and forty-five studies were identified. There are other mufti-state research projects (W-186, 5-282 and NC-215) in the United States that address plant nematode problems, but these projects have different objectives and focus on other crop systems (e.g., alfalfa, field corn, sweetpotato, okra, cotton, wheat, soybean and melon). The proposed NE-171 project outline does not duplicate the efforts of other research projects to date. Comments by peer reviewers support this conclusion.

Objectives

1. Evaluate the effects of rotational crops, organic amendments and host crop genetics on nematode community structure.
   
2. Characterization of biological control agents for suppression of plant-parasitic nematodes.
   
3. Comparison and evaluation of IPM system management of plant-parasitic nematodes based on crop rotation, organic amendments, host crop resistance and biological control agents.
   
4. 
   

Methods

All state and federal laws will be followed in securing the required permits for interstate movement of pathogens, nematodes, soil or other regulated items. Objective l: Evaluate the effects of rotational crops, organic amendments and host crop genetics on nematode community structure. Agricultural systems in the Northeast 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. Scientists in eight states will conduct complementary or collaborative studies to assess the impact of rotation and cover crops (CT, MA, MD, MI, NY, PA, RI, and WV) on plant-parasitic nematode populations and nematode community structure. Results from our current project have identified several nematode-antagonistic crops. These crops include rotation and cover crops such as marigold, velvetbean, sesame, crotalaria, Avena strigosa, sudangrass and sorgho-sudangrass. These crops will be evaluated for nematode suppressive effects against root-knot, cyst, lesion or dagger nematodes in parallel (CT, PA) or complementary (NY, WV) studies. The mechanism of antagonism of cover and rotation crops, which reduce populations of these nematode genera during growth or as a green manure, will be determined in the greenhouse, field microplots, and in the field by scientists in CT, NY, and PA. The toxicity of root exudates or plant breakdown products such as glucosinolates released by the breakdown of 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 nematodes. 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. While juveniles were unaffected, egg hatch was reduced by exposure to plant infusions. 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. The effects of organic soil amendments on nematodes will be investigated in CT, MA, MI, NY, PA, RI, and WV. The mechanism of suppression will be evaluated by determining the direct effects of amendments against nematodes (CT, MI, NY, PA, and WV) in soil assays as well as the indirect effects of amendments on microbial populations such as the growth of bacterial species (RI), which produce fatty acids toxic to nematodes. Genetic plant resistance developed in CT, MD and ARS-SC as well as heritage varieties with resistance or tolerance (WV), will be evaluated for potential integration with nematode antagonistic crops. The effects of these treatments on nematode diversity and community structure will be assessed and evaluated as a potential indicator of soil health. Diversity and maturity indices will be developed in WV and MI to assess the effects of nematode control tactics on community structure. Objective 2. Characterization of biological control agents for suppression of plant-parasitic nematodes. Obj. 2.1) Survey plant-pathogenic nematodes for occurrence of Pasteuria spp. A workshop will be conducted during the first multi-state meeting of the new project to teach members how to recognize Pasteuria spp. on different species of plant-parasitic nematodes. A pamphlet will be prepared and distributed to each member of the technical committee for use as a guide for proper handling of Pasteuria spp. The workshop and pamphlet will ensure that all members are following prescribed methods in our goal to document the occurrence of Pasteuria on plant-parasitic nematodes in northern regions of the United States. The following protocol will be followed. Sites chosen for sampling will be based on former cropping history and the importance of plant-parasitic nematode diseases. Extension personnel will be contacted for input regarding the sites chosen. Priority will be given to identifying important agricultural sites that were formerly conducive for nematode disease, but have become or are becoming suppressive for the nematode disease. If conducive-suppressive sites can not be identified, then sites will be chosen that have a known high density of plant-parasitic nematodes. Soil and root samples will be collected from the chosen field sites based on a sampling pattern described by Barker et al., 1986. The samples will be stored at 10 C until they are processed (Barker et al., 1986). Nematodes will be extracted from soil and roots by methods described by Barker et al., 1986). The presence or absence of Pasteuria spp. will be documented, the nematode host identified, the soil classified, and the crop plant, and degree of crop damage associated with the nematode recorded. If Pasteuria species are present, the bacterium will be stored in a sand medium for further study. Cooperating states are FL, NM, MI, NY, and PA. Obj. 2.2) Determine the survivability and host preference of isolates of Pasteuria spp. from different geographic regions. Numerous isolates of Pasteuria spp. have been collected from field sites in Florida that have been identified as being suppressive to root-knot, lesion, ring, and sting nematodes. Also, an isolate of Pasteuria spp. was recently reported on the soybean cyst nematode, Heterodera glycines, in Illinois (Noel and Stanger, 1994). Selected isolates from Florida and those identified under objective 2.1 from northern states will be compared for their survivability, host preference, and biocontrol potential. Florida isolates that attach to Meloidogyne incognita, M javanica, and M. arenaria will be tested on M. hapla, the northern root-knot nematode and indigenous populations of M. incognita. Preliminary trials have shown that one Florida isolate of P. penetrans attached to MD populations ofM. incognita and H. glycines. Selected isolates of Pasteuria spp. will be increased in the greenhouse on their preferred host. Once sufficient inoculum is produced, the parasite will be introduced into root-knot nematode infested microplots in the field. Both the nematode and its parasite will be monitored over time to determine whether the parasite will increase to suppressive levels and whether it will survive the winter season. Cooperating states are FL, MD, and MI. Obj. 2.3) Evaluate different crops and methods for growing Pasteuria penetrans. Various plants will be evaluated for production of P. penetrans endospores. Plants to be evaluated include tomato, squash, eggplant, cucumber, radish, okra, and pumpkin. The plants will receive single or double inoculations with nematodes that have endospores of P. penetrans attached. Characteristics to be evaluated include (but are not limited to) host suitability, ease of culture, ease of endospore recovery and endospore yield. Similarly, novel culture techniques will be evaluated for efficiency of producing large quantities of P. penetrans endospores. Comparisons will be made between standard greenhouse pots, a growth chamber set-up equipped with a unique soil moisture control system recently reported by Sardanelli and Kenworthy (1997), and a hydroponic system located at The Land Pavilion located at Walt Disney World, EPCOT . Cooperating states are FL and VID. Obj. 2.4) Determine the sequence of events required for the formation of P. penetrans endospore-associated proteins (adhesions) required for the attachment of spores to the cuticle of nematode hosts. The biochemical events that occur during the development of P. penetrans are poorly understood. Research on spore adhesion may provide valuable insight into the unique relationship the bacterium has with its nematode host. The sequence of events required for the formation of spore-attachment proteins (adhesions) will be studied. Two P. penetrans isolates will be cultured on Meloidogyne arenaria race 1 and M. incognita, respectively, and increased on tomato. IgM monoclonal antibodies (Mab) directed against spores from these two isolates that specifically recognize spore adhesions will be used to probe their formation as a function of development. Twelve, 16, 24, and 38 day old healthy and P. penetrans infected females will be extracted from roots. Proteins will be extracted with SDS and separated by PAGE. Gels will be electro-blotted on nitrocellulose membranes and proteins probed with the Mab. Cooperating states are FL and NY. Obj. 2.5) Morphological and phylogenetic analysis of a Pasteuria sp. from ring nematode. An isolate of Pasteuria sp. discovered in ring nematode, Mesocriconema ornata will be studied by electron microscopy to determine its morphology and also subjected to analysis by molecular techniques. The phylogenetic position of this species relative to that of P. penetrans and P. ramosa will be determined based on analysis of the 16S rDNA sequence encoding 16S ribosomal RNA. These sequences will be amplified by PCR and compared with sequencing reported for two isolates of P. penetrans (Anderson, et al, 1998). Cooperating states are MI and FL,. Obj. 2.6) Additional studies. The influence of organic amendments as sources of organic acids, as well as specific compounds such as propionic and butyric acids on microbial communities will be characterized in CT and RI. The hypothesis being tested is that bacteria and other microorganisms produce organic acids under anaerobic conditions in which biocontrol activity is stimulated. Population dynamics of predatory nematodes will be compared to plant-parasitic nematode dynamics under various rotation sequences in WV. Several methods to assess the role of nematode trapping fungi will be compared to identify methods that best characterize indigenous biocontrol activity. Another series of experiments will examine the role and mode of action of tall fescue infected with endophytic fungi (e.g., Neotyphodium coenophialum) for its potential as a rotation or cover crop to enhance nematode suppressiveness in orchard crops. Objective 3. Comparison and evaluation of IPM system management of plant-parasitic nematodes based on crop rotation, organic amendments, host crop resistance and biological control agents. Ten states (CT, FL, MA, MD, MI, NY, PA, RI, SC and WV) will be involved in the comparison and evaluation of IPM system management of plant-parasitic nematodes based on crop rotation, organic amendments, host crop resistance, and biological control agents. These studies will range from narrowly focused efforts targeted at reducing pest densities below specific damage thresholds, to broad mufti-disciplinary analyses of whole farming systems. Results of procedures under objective 1 and 2 may, in some cases, be extended directly to this objective, whereas in other cases, additional specific trials will be needed to evaluate promising tactics in the context of overall IPM programs. Integrated management of potatoes for control of root lesion and root knot nematodes and potato early dying using compost, green manure, and crop rotations in nematode IPM systems will be assessed in microplots and field plots (NB, CT, WV). While these studies will largely be conducted in association with other on-going projects, collaboration through use of comparable treatments under this regional project will extend the application of the results more broadly across the Northeast. Integration of nematode-resistance and biological control agents in pepper will be evaluated (SC, MD) using agents screened under Objective 1. Collaboration with NY using Pasteuria penetrans and CT and MD using herticillium lecanii will involve field trials of promising isolates against root knot and cyst nematodes. Farming systems research in Michigan will evaluate nematode community structure in four farming systems (conventional, integrated fertilizer, integrated compost, and organic) as part of the Long Term Ecological Research Site. Similar studies of farming in West Virginia will include organic farming systems evaluations of orchard, vegetable, and field crop production operations. These studies will emphasize non-chemical nematode controls via rotations and soil amendments (animal manures versus green manures). Economic analyses of these practices will be conducted under related projects. Although efficacy of these practices for nematode control can be inferred by changes in nematode population densities, biocontrol agent population dynamics and nematode community structure will be analyzed to provide a better mechanistic understanding of the effects of these systems and the opportunities for their application to IPM programs.

Measurement of Progress and Results

Outputs

• Establish a low soil volume bioassay to evaluate nematode antagonistic crops.
• Conduct a workshop for all group members to present protocols for Pasteuria penetrans recognition, research and surveys.
• Conduct collaborative rotation and cover crop system research in multiple states to evaluate the impact of rotation and cover crops and soil amendments on nematode populations and community structure.
• Identify and evaluate nematode resistant or tolerant varieties for inclusion in an integrated management program.
• Survey northern states for the presence of Pasteuria using protocols identified in 2000.
• Evaluate different crops and greenhouse or growth chamber systems for the production of large numbers of Pasteuria penetrans endospores.
• Determine the mechanism of nematode antagonism in rotation and cover crops.
• Compare isolates of Pasteuria penetrans for host preference and biocontrol potential.
• Evaluate the winter survival of Pasteuria penetrans in northern states.
• Determine the sequence of events required for formation of endospore-associated proteins and adhesion to nematodes.
• Determine the morphological and phylogenetic relations among Pasteuria penetrans isolates.
• Develop an economic analysis of rotation and soil amendment tactics for nematode control.

Outcomes or Projected Impacts

• Economically viable alternatives to the use of chemical nematicides.
• More sustainable and environmentally-friendly crop production systems.
• Improved soil health status from the application of amendments.

Milestones

(2000): <LI>Establish a low soil volume bioassay to evaluate nematode antagonistic crops. <LI>Conduct a workshop for all group members to present protocols for Pasteuria penetrans recognition, research and surveys. <LI>Initiate collaborative rotation and cover crop system research in field plots and microplots in multiple states. <LI>Identify and evaluate nematode resistant or tolerant varieties for inclusion in an integrated management program.

(2001): <LI>Evaluate the impact of rotation and cover crops and soil amendments on nematode populations. <LI>Survey northern states for the presence of Pasteuria penetrans using protocols identified in 2000. <LI>Evaluate different crops and greenhouse or growth chamber systems for the production of large numbers of Pasteuria penetrans endospores.

(2002): <LI>Initiate research to determine the mechanism of nematode antagonism in rotation and cover crops. <LI>Compare isolates of Pasteuria penetrans for host preference and biocontrol potential. <LI>Evaluate the impact of rotation and cover crops and soil amendments on nematode populations. (2003): <LI>Evaluate the winter survival of Pasteuria penetrans in northern states. <LI>Determine the sequence of events required for formation of endospore-associated proteins and adhesion to nematodes. <LI>Integrate plant resistance with rotation crops to predict nematode population decline under integrated management systems.

(2004): <LI>Evaluate rotation and cover crop effects on nematode community structure in soil. <LI>Determine the morphological and phylogenetic relations among Pasteuria penetrans isolates. <LI>Develop an economic analysis of rotation and soil amendment tactics for nematode control.

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Projected Participation

View Appendix E: Participation

Outreach Plan

Organization/Governance

standard

Literature Cited

Attachments

Land Grant Participating States/Institutions

AL, CT, FL, MA, MD, MI, NY, PA, RI, WV

Non Land Grant Participating States/Institutions

USDA-ARS/Florida, USDA-ARS/Maryland, USDA-ARS/South Carolina