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

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

The Need as Indicated by Stakeholders: Estimates over the last 25 years indicate that plant-parasitic nematodes cause 10-14% average annual yield loss among the world's major crops (Sasser and Freckman, 1987), and losses ranging from minimal in some localities to as high as 15% in other areas in United States major crops (Koenning et al., 1999; McSorley et al., 1987). Indications from initial results of another global survey by the nematology community of nematode associated crop losses are that these estimates remain at similar levels. In economic terms, these estimated annual crop losses translate to at least $8 billion in the United States and $78 billion worldwide (Smiley, 2005). In addition, economic loss associated with increasing material and application costs of nematicides and associated with trade embargos due to actual or suspected quarantine nematode infestations exacerbate nematode problems in agriculture. Increasingly, scientific evidence and public awareness have heightened concerns about environment quality, food quality, and human health and safety relative to pest management in agricultural production. The need for alternative, integrated nematode management has been propelled by the actions triggered by the Montreal Protocol and the Food Quality Protection Act (FQPA) of the 1990s. Phase-out of methyl bromide is in the latter stages where current use has been reduced by about 96% and is allowed only under negotiated Exceptional Use Permit. Based on FQPA requirements, it is likely that widely-used and efficacious nematicides will be unavailable or greatly restricted in the future. For example, Nemacur, a commonly used nematicide on several crops was canceled in 2007, Furadan, commonly used on corn was cancelled in 2009, and Temik, an important potato nematicide was cancelled in 2011. In addition, the soil fumigant nematicide 1,3-dichloropropene (Telone II) is a B2 carcinogen, reviewed under FQPA and must be used under more rigorous restriction and recently supplies have been limited. Together with another fumigant, metam-sodium, the fumigant nematicides have been identified by California EPA as the largest agricultural source VOC (volatile organic compound) contributors to air pollution by ground level ozone formation. Development of methyl iodide, an anticipated replacement for methyl bromide has recently been abandoned. New US EPA Phase 2 labeling for all soil fumigants, implemented December 1, 2012, establishes mandatory buffer zones surrounding treated fields that will further curtail the use of these products. Both locally and nationally, the agricultural production community (our stakeholders) is scrambling to find viable and agro-ecologically sustainable alternatives to chemical-based soil pathogen and nematode control. In addition, world travel and commerce have accelerated the dissemination of pest species, including plant-parasitic nematodes. Development and application of new diagnostic protocols for accurate identification of nematode species is beneficial and required for national and international regulatory and quarantine agencies relative to free trade and economics, as exemplified by recent trade restrictions on movement of potatoes due to findings of quarantine-status cyst or root-knot nematodes in US production areas such as Idaho. The nematology community has repeatedly advocated the need for funding support focused on the basic and applied research required to advance agro-ecologically sustainable alternative management approaches and accurate nematode detection and diagnostics. This proposed project renewal addresses these needs directly for the most important groups of plant parasitic nematodes, by building on advances made in the W-2186 project over the last 5 years. Importance of, and Consequences Without, the Work: Cyst, root-knot and other nematode species included in this project are the most important groups of plant-parasitic nematodes in the United States. Management of these nematodes in United States agriculture during the past fifty years has been largely via the application of broadly efficacious nematicides. Nematicidal activity, especially of soil fumigants, is generally non-discriminating, even between nematode species and genera. Therefore, understanding the genetic variability and adaptation potential among nematodes was not important for effective nematode control. In contrast, desirable alternative nematode management strategies involve combinations of crop rotation, host plant resistance, cultural manipulations, and biological control. All of these tactics may have specific genotype-level interactions with nematodes and are influenced by the production practices and environmental conditions. Hence, variability and adaptation in nematode populations must be considered to successfully develop and deploy alternative management strategies. This multistate project was initiated because the membership recognized the increasing importance of characterizing the genetic variation in nematode populations and its influence on success of alternative nematode management strategies. An example highlights the value of this project: Years of research went into the development of cyst-nematode resistant soybeans but the potential benefits of the resistance were limited due to the rapid selection of resistance-breaking nematode isolates. The biological processes in nematodes that influence the development of effective management strategies are complex, involving changes over multiple generations and interactions between nematode, host plant and environment. Therefore, a renewal of the project as requested herein is critical for continuing and completing the research required to meet the overall project goals. A major shift in nematode-management strategies is occurring, from almost exclusive reliance on soil-applied nematicides, to the use of combinations of alternative strategies such as crop rotation, host plant resistance, cultural manipulations and biological control (Ferris et al., 1992). An important difference from nematicides is that the alternatives are influenced directly by genetic variability existing in target nematode field populations. Hence, the successful use of alternatives requires more information to implement than nematicide-based strategies. Herein lies the logic and raison dêtre of W-2186 and its proposed revision: assessment and characterization of variability and adaptation extant in nematode populations and agro-environmental conditions influencing host-nematode interactions will assist in the successful application of alternative management approaches, in addition to guiding initial development and deployment of new strategies. For example, knowing the frequency of virulence genes in a nematode population will allow deployment of corresponding resistance genes and promotion of resistance durability. Except in the clearly demonstrable instances where resistance-breaking nematode populations are detected, the subtle influence of genetic variability in nematode populations has been considered only to a limited extent. However, the research conducted under the current W-2186 multistate project has provided considerable evidence that this variability is important. Results from current work indicate that genetic variability and adaptation potential in nematode populations are responsible for the aberrant and inconsistent results of many experiments assessing resistance, crop rotations, host ranges, cover/trap cropping, and biological control. The plasticity of nematode responses to abiotic environmental factors such as temperature, moisture, soil conditions, and host nutrient status stems from genetic variability, and such responses require much additional characterization. Greater understanding of nematode genetic response and adaptation to abiotic factors will be important in optimizing the design of cultural management tactics such as manipulations of planting and harvest times, wet or dry fallow, and soil solarization. The potential for invasive nematode pests to establish in our agricultural production systems also can be better determined from studies on genetic response and adaptation to local environments. Without the proposed work continuing in a coordinated manner, the participants believe that effective nematode management alternatives will be developed more slowly, with success coming more on an ad hoc basis and with economic inefficiencies and a high likelihood of short-term failure of new products or management approaches. Knowledge gained from our main focus on cyst, root-knot and other significant nematodes will be applied and tested between nematode groups within the project matrix (see Table 1 in Attachments). This will strengthen the overall scientific scope of the research activities and will broaden the impact of the findings to benefit agriculture in multiple states. Technical Feasibility of the Research: Recent advances in molecular and genetic methodologies and knowledge will facilitate the study of nematode genetic variability and adaptation and promote diagnostic protocols with much greater resolution than has been possible. Some of these protocols have been developed and tested under the current project. For example, shared root-knot and cyst populations led to characterization of a mitochondrial cytochrome oxidase I (COI) gene that promises to alleviate current ambiguities in molecular species identification within these difficulty-to-identify genera. Addition of these and other new sequence information generated by the current project to on-line databases and knowledge-based systems will assist in information transfer to user groups in the relevant agricultural communities. The root-knot and cyst nematodes are distributed throughout the United States and are damaging pathogens, parasitizing a wide range of important crops. Three groups of nematodes are the primary focus for this project: Group I - The warm-temperature root-knot species (Meloidogyne incognita, M. javanica, M. arenaria); Group II - The temperate root-knot species (M. chitwoodi and M. hapla); Group III - The cyst species (Heterodera schachtii, H. cruciferae, H. glycines, Globodera pallida, and the newly discovered species G. ellingtonae). These nematodes are the subject of research efforts in the designated participating states. Current research is addressing many areas of management for these three groups, including: development and deployment of nematode-resistant plants; rotation to reduce population densities of these pathogens; cover crops, trap crops and soil amendments including green manures to reduce population densities; the role of weed hosts in bringing about phenotypic changes in nematode populations; characterization of resistance genes and resistance responses; the development of molecular diagnostic protocols for nematode identification and the reference databases necessary for their implementation. Thus, the project participants share a strong common interest that will provide the central focus for both project members and other collaborators. In addition, parallel studies will be made by some participants on other endoparasitic nematodes, reniform nematode (Rotylenchulus reniformis), lesion nematodes (Pratylenchus spp.), stem and bulb nematodes (Ditylenchus spp.), important ectoparasitic nematodes including stubby root (Paratrichodorus spp.), dagger (Xiphinema spp.), and ring (Mesocriconema) nematodes; and outgroups including Mermithidae and C. elegans that provide genetic models for assessing variability and underlying mechanisms. This will maximize both the scientific scope of the project and its multi-state impact in agriculture. Characterizing genetic variability  requisite for novel management strategies: The unifying theme of this proposal is that genetic variability is a critical biological feature that complicates management and enhances the pest status of species and populations of the highly specialized plant parasitic nematodes. W-2186 participants and others have begun to document the extent of genetic variability within populations, and the agro-environmental factors that influence it. Rapid developments in molecular biology techniques and their application through this project will continue to increase our understanding of the genetic processes involved. As understanding of genetic variability in nematode populations increases, it has become clear that the race concept and other means of characterizing nematode population differences are inadequate. Failure of current nematode management, such as breakdown of resistance, and successful development of novel approaches can be resolved through greater understanding of the underlying genetic and biological processes in parasitic nematode populations vis a vis management. For example, the importance of mutation compared to maintained variability in field populations is unclear, and this is a research area that will be pursued. Genetic variability can impact both the effectiveness and longevity of alternative nematode-management strategies based on host plant resistance, crop rotation, cultural manipulations and biological control. Therefore, continuing the knowledge development in these systems should provide rational guidance for the design and development of management strategies. The project focus is on understanding nematode variability and adaptation, such that it can be identified, characterized, and managed or manipulated to benefit agricultural production systems. This requires research on the phenotypic and genotypic characterization of variability and gene frequencies, including aspects of stability and adaptability, of host range, response to resistance, response to environmental conditions, biological processes (e.g. fecundity) and morphology. This approach is being complemented and aided by development of markers to identify variability by molecular, histochemical, and morphological polymorphisms. The development of molecular techniques with greater efficiency, predictability and ease of use will expedite the nematode genetic analyses and design of management systems. Current and previous work under W-2186 has allowed participants to make advances on these research goals. However, this work cannot be considered complete and pressure for alternatives to nematicides has increased. Relative to our objectives, it is exciting that the arsenal of established and new tools used to address our applied research questions is increasing rapidly (e.g., Atkinson et al., 2001; Brito et al., 2004; Blok and Powers, 2009; Gleason et al., 2008; Handoo et al., 2012; Karl and Avise, 1993; McClure et al, 2012; Powers, 2004; Powers et al., 2005; Sambrook and Russell, 2001; Skantar et al., 2007; Sukno et al., 2007; Vos et al., 1995; Webster, 2004). Four key considerations based on nematode genetic variability are central to development and deployment of alternative management strategies as proposed under this multistate project: 1. Host plant resistance  The genetic composition of nematode populations is changed by the selection pressure imposed by growing resistant cultivars. The changes include shifts in species composition, and shifts in presence and frequency of nematode virulence alleles matching specific resistance genes in crop cultivars (Petrillo et al., 2006). Similar potential shifts may occur in response to nematode-resistant trap crops. Little is known of the existing frequency of virulence alleles, the frequency with which new alleles are generated, or the underlying mechanisms that regulate changes in genetic variability in root-knot, cyst and other nematode populations. As more sources of resistance are bred into cultivars, knowledge of gene frequency and stability effects assumes greater importance in determining the direction and requirements of breeding for nematode resistance, and the effective long-term deployment of available resistant cultivars (Starr et al., 2002; 2009). 2. Host range for rotations and cover-cropping - The host ranges of important nematode species have been defined within general limits, but the extent of variability in host range among populations within species is not well-characterized. Thus, although most cyst nematodes have narrow host ranges and are amenable to control by non-host rotation programs, less is known about the extent of reported hosts outside the typical host taxa, the likelihood of shifts in host range, or the host ranges of new species. For example, although sugarbeet cyst nematode hosts are found almost entirely within the Brassicaceae and Chenopodiaceae, tomato (Solanaceae) has been reported to host this nematode in California and Utah, with potentially serious consequences for rotation planning in western sugarbeet production areas. For the new cyst species Globodera ellingtonae in Oregon, little host range data are available. Evidence for genetic adaptations and modification of nematode host range has also been presented whereby local nematode populations are better adapted to local weed populations. The processes involved in these interactions are poorly understood. In contrast to cyst nematodes, root-knot nematode species have broad host ranges of more than 2000 plant species from diverse plant families. Much of this host range information has been compiled from numerous tests and observations based on non-standardized host testing procedures, and in most cases with only one or a few isolates per species. Standardized conditions are needed to determine whether differences are due to variability in nematode populations or to differences in susceptibility in the plant lines used. For example, there is evidence that local weed populations influence the behavior of root-knot nematode populations on subsequent crops (Trojan et al., 2007). Resolving the true levels and stability of host range relationships will be critical to development and implementation of non-host crops in rotation and cover-cropping programs, and for determining the role that host weed species play in maintaining nematode population levels. 3. Cultural controls - Many are based on manipulating abiotic effects on nematode populations to suppress nematode activity or infection. Examples include wet or dry fallowing so nematodes starve while active (wet fallow) or die from extreme moisture stress (dry fallow). Soil solarization involves natural heating of soil under plastic cover to attain the thermal death point of nematodes. Avoidance may include changes in planting and harvest dates, such as delaying planting in the fall to avoid infection activity, and early planting or late harvest of crops to avoid additional nematode generations. Genetic variability in nematodes for response to temperature and moisture has been demonstrated, but it is not known how quickly or how stable such adaptive changes are, nor their frequency. Soil amendments such as green manures and various bioproducts show promise for nematode suppression in some systems and require further study. 4. Biological controls - Some potential biological control agents of cyst and root-knot nematodes are known to have specific host ranges among target nematode species, such as the host specificity of the bacterium Pasteuria penetrans among root-knot nematode species and populations. Such specificity may be controlled genetically, through surface protein binding and recognition between bacterium and nematode, suggesting that genetic variability in Meloidogyne may influence the potential of the bacterium and similar organisms as useful biological control agents. Advantages of a Multistate Effort: Under the current project, the membership effectively initiated research to apply emerging methodologies to obtain knowledge of the variability and adaptation potential in nematode populations. The W-2186 membership proposes to continue and extend these efforts in this regard, to identify and characterize the variability in cyst, root-knot, and other important nematodes. The participants share research interests on primary nematode pathogens and bring complementary expertise to the project. In this iteration of the project, we will determine gene frequencies, genetic stability, and adaptation and fitness, such that genetic variability can be managed and manipulated in agricultural production systems by appropriate alternative management strategies. The cyst and root-knot species are of primary importance as major nematode pathogens and as actual or potential invasive nematodes in most agricultural production areas and cropping systems throughout the United States. This is reflected in the proposed contributions from participating states across the country. The diversity in cropping systems and rank of importance of nematode groups among participating states clearly provides opportunities for conducting meaningful collaborative research on major nematode pathogens exposed to similarities and variations in crop (host), resistance, environmental and agro-ecological conditions. The participants utilize the opportunity to collaborate in ways that enhance the benefits accrued from the research, as opposed to what might be gleaned if the researchers were to simply pursue individual projects within limited geographic boundaries. For example, the warm climate root-knot species will be studied by participants from 10 of the 15 participating institutions (see Attachment Table 1)  a group effort that will pay large dividends in understanding nematode variability relative to management. Based on benefits of the current project we believe the similarities in the target nematode groups and the problems for nematode management imposed by variability and adaptation can be researched most efficiently through this coordinated multistate project. This team approach enables a pooling of scientific expertise and resources to maximize the amount and quality of the information that can be generated. The resources available to researchers working within the Agricultural Experiment Stations have been continually declining in recent years, and are particularly limited for nematology programs at this time. Conversely, the demands and expectations for new, environmentally friendly management tactics have never been greater. Accordingly, the membership has experienced a do more with less environment. This multistate project can provide some relief, as a necessary forum for rapid scientific advancement in aspects of both basic and applied research directed toward nematode diagnostics and development of alternative management strategies. For example, it is unlikely that all participating states will have programs devoted to molecular research on nematodes, and yet the need for molecular-level approaches for diagnosis and to assess genetic variability is required to address significant problems. This project has ongoing molecular-based programs in a few states (e.g. California, Hawaii, Mississippi, Nebraska, New Mexico, Washington) that can act to facilitate research by other participant states. In turn, those states focusing on phenotypic differences in nematode populations can provide nematode populations and isolates for molecular analysis. This coordinated approach minimizes unnecessary duplication of research programs, and provides fertile opportunities for a seamless, interactive approach to development of integrated nematode management. Most importantly, the project also enhances the quality and applicability of the research findings across geographic locations and agricultural production systems.

Related, Current and Previous Work

Likely Impacts of Work: The application of the project findings should expedite the development of new, environmentally benign management strategies to minimize economic losses from nematodes. This in turn should help boost the international competitiveness of our agricultural production systems, at a time when competitive advantage is being eroded by the loss, and potential further loss, of nematicides. The project will also benefit the diagnosis and response process required when invasive nematode pests are suspected or found in production fields or in traded agricultural products. Related, Current and Previous Work: Accomplishments (2008-2013): A full listing of the research publications of the W-2186 project from 2008-2013 is given in the attached Appendices, and Annual Progress Reports for W-2186 are available through the NIMSS site for more in-depth descriptions of related work progress. Following is a summary of the findings that highlights the areas of significant impact in addressing the project goals, with comments concerning the need for additional research. Overall, the project scientists have been very productive in efforts to develop improved techniques for nematode diagnostics, understanding of nematode diversity and genetic variability, processes of nematode fitness and adaptation, and the incorporation of this knowledge into the design and analysis of improved nematode management strategies. These findings directly benefit the 12 participating states and more broadly other states whose crop production systems are compromised by cyst, root-knot and other nematode infestations and invasions. Moreover, many of the accomplishments have resulted from our close collaboration within W-2186, through which shared knowledge, techniques and materials have provided important synergies. Under Objective 1 (characterize genetic and biological variation in nematodes relevant to crop production and trade), nematode (a)virulence and plant resistance gene interactions were elucidated genetically in several important nematode crop combinations. These included root-knot nematode interactions with resistance gene(s) and nonhost determinants in coffee, cotton, cowpea, common and Lima beans, potato, tomato, wheat, and chile pepper, e.g., Roberts et al. (2008) for Lima bean, plus cyst nematode interactions for (a)virulence matching host resistance defined to varying levels in sugarbeet, soybean, wheat,and more recently potato. Within these interactions, new resistance specificities were identified, such as the Rkn genes in cotton and new root-knot resistance in chile pepper, together with the existence and frequency of matching virulence in nematode populations and their geographic distribution, as determined in cowpea. These studies have been used to guide plant breeders,for planning crop rotations, and for cultivar selection. Variation was found in H. glycines isolates used for resistance screening and additional studies are needed to determine the extent to which this affects routine resistance screening procedures in breeding programs. Molecular approaches to nematode identification within and between species have also been advanced under this objective. Molecular techniques have been applied to or developed for identifying species of both the warm and cool climate root-knot nematode species, races and populations after crops and weeds (Blok and Powers, 2009; Powers and Ramírez-Suárez, 2012), and also sugarbeet, soybean and cereal cyst nematodes and their populations, including a molecular barcoding approach within the project (Powers, 2004). These same techniques were instrumental in discovering a new species of Globodera (G. ellingtonae) associated with potato in Oregon and Idaho (Skantar et al., 2011; Handoo et al., 2012). In this example, when potato seed grown at the Oregon State University (OSU) farm was to be sent to Canada the Oregon Department of Agriculture (ODA) sampled the field for potato cyst nematodes in April 2008 and recovered six cysts. The cysts were sent to the USDA-ARS Nematology Laboratory in Beltsville, MD for identification. Morphological and molecular tests confirmed that the cysts belonged to the genus Globodera but were distinct from major potato cyst nematodes G. pallida and G. rostochiensis and from G. tabacum (Skantar et al., 2011). Later in 2008, cysts that resembled the Oregon population were recovered from two fields in two counties in Idaho. Subsequently, this nematode was described as a new species, Globodera ellingtonae (Handoo et al., 2012). Project results confirmed that G. ellingtonae can reproduce on some potato varieties but some evidence of reduced plant growth or reduced yield must be demonstrated before this nematode can be listed as a pathogen of potato. Project scientists also documented variability at the molecular level in some related nematode groups including lesion and spiral nematodes (Subbotin et al., 2008; Subbotin et al., 2011) which affect many cropping systems, for example small grain-potato cropping systems. The understanding of these systems indicates knowledge gaps in additional nematode-crop interactions that need to be filled, and that application of variability and adaptation knowledge must be integrated for developing and optimizing management strategies. Studies on genetic variability in mitochondrial genomes of the nematode family Mermithidae in the current W-2186 have provided important insights into nematode phylogenetic relationships and the dynamics of mitochondrial variation, especially when analyzed comparatively with target phytoparasitic groups such as Meloidogyne and Globodera (Hyman B.C. and Lewis, 2011; Tang and Hyman, 2005). It is anticipated that Mermithidae can serve as a paradigm for parasitic nematodes, and the processes of genome amplification and gene order rearrangement. Under W-2186 Objective 2 (determine nematode adaptation processes to hosts, agro-ecosystems and environments), several systems have been analyzed, revealing high levels of nematode adaptation to parasitic ability on resistant host plants, alternative hosts (weeds), seasonal climatic differences, co-infection of plants by fungal pathogens, and soil conditions. Variation in parasitic ability among root-knot nematodes was described for species parasitizing grain legumes such as cowpea and Lima bean (Roberts et al., 2008), common beans, cotton, tomato, and potato cultivars and wild relatives. These studies demonstrated the need for broad-based forms of resistance for use in crop cultivars and cover and trap crops, for example for M. incognita in cotton (Ulloa et al., 2009) as reviewed by Starr et al, (2008). Earlier, we found that M. incognita populations virulent to resistance in cowpea had reduced fitness (lowered fecundity and increased extinction rates) (Petrillo et al., 2006), contributing to our understanding of nematode adaptation rates and guiding the breeding efforts for Blackeye beans in California and other states (Ehlers and Roberts, 2012). In chile pepper production systems, the reproduction levels by root-knot nematode populations were shown to be influenced by the presence of yellow and purple nutsedge species, demonstrating that previous or alternative hosts can change the parasitic ability of populations on crop plants. In addition, project scientists identified local fitness and adaptation variations in M. chitwoodi and M. hapla populations to temperature regimes during the potato season in the Pacific Northwest, demonstrating the need to adjust predictive models for completion of nematode generations that influence decisions on time of nematicide applications. Studies in Michigan showed that soil texture influences soybean cyst nematode population density in the field and also M. hapla reproductive potential in which adaptation to mineral or muck soils was found, the consequences of which for nematode management require further investigation. Plant-parasitic nematodes can be introduced into new areas and identifying such infestations is critical; potato cyst nematodes Globodera rostochiensis and G. pallida are significant economic threats to food production, and a comprehensive detection survey documented absence of G. rostochiensis in Idaho but confirmed the presence of G. pallida in Idaho, and also the absence of these species in Oregon but the presence of a new species, G. ellingtonae. Project members described a new genus and species of cyst nematode, Vittatidera zeaphila (Bernard et al., 2010).This species was discovered on corn in Tennessee and has a host range centered on corn with little to no reproduction on other grasses or dicots tested. Description of the new cyst nematode G. ellingtonae in Oregon is an important project outcome with implications for potato production and trade. Project members have also discovered new infestations of described species expanding known distributions such as soybean cyst nematode (H. glycines) identified on Kauai in Hawaii. These discoveries require further survey and study to determine their origin and adaptation potential. Under Objective 3 (develop and assess nematode management strategies in agricultural production systems), root-knot, cyst, and other nematode management systems have been studied, developed or improved for several cropping systems and production areas. Several examples highlight nematode-cropping systems that have been and will continue to be a project focus, targeting the most damaging nematode problems. In Arkansas rotations that include resistant soybean are being optimized for H. glycines control. Likewise, sugarbeet production systems in California and Idaho have been adjusted based on W-2186 studies to utilize trap cropping, green manures and modified rotation sequences for cyst nematode management. Various soil amendments and solarization have been assessed in Hawaiian cropping systems relative to impacts on soil nematode communities (Marahatta et al., 2012). In California, annual field and vegetable crop systems have been enhanced for root-knot nematode management utilizing host plant resistance in tomato, cotton, carrot and grain legume crops. Similarly, chile pepper, alfalfa and cotton production systems in New Mexico have been modified for root-knot nematode and nutsedge management, incorporating agronomic practices for integrated weed/nematode control using resistant alfalfa cultivars (Schroeder et al., 2009, 2010). In the Pacific Northwest, potato production systems have been modified to optimize the choice of rotation crops and timing of nematicide applications for managing the root-knot species M. hapla and M. chitwoodi. A four-year crop rotation study demonstrated that M. chitwoodi could be managed with crops currently grown in the Columbia Basin of Oregon and Washington when the host crops were grown early rather than late in the rotation. Studies on green manures showed that radish crops can be successful but require different management strategies in different climatic environments. Additional work in the Pacific Northwest potato production system showed that hot water and irradiation treatments were effective ways to eliminate root-knot infestations in harvested tubers, an important consideration for potato exports. Screens for resistance to M. chitwoodi and nematode vectored corky ringspot disease also provided options for potato growers to choose less susceptible varieties, although these results require additional confirmation in field studies. The regional utility of these management approaches will be tested on both cyst and root-knot nematode targets in different cropping systems. Developing predictive tools to aid in nematode management has also formed part of the project effort, including improving a fertilizer-use efficiency model by incorporation of nematode population density data, and use of nutsedge plant counts as a visual indicator and predictor of M. incognita population densities in alfalfa-chile pepper rotations (Schroeder et al., 2010). These are selected examples of many advances in nematode management being made under W-2186 participation in which the collective knowledge gained in one system is being applied to other systems as a real strength of this multistate effort. These nematode management systems are at various stages of development that require additional study and modification under W-3186. Under the W-2186 objective to implement rapid information transfer of project results to stakeholders, the host plant and nematode database called NEMABASE was further developed and expanded to incorporate project findings, and project findings were also incorporated into updates of the online resource NEMAPLEX (Ferris, 2012). Our findings have been presented at national meetings attended by producers, scientists, and professionals. Presentations were made to state and federal regulators and at field days and extension meetings. Information was extended to smaller groups through facility tours, email, telephone conversations, and traditional hard copy. Several project participants have extension appointments and activities. As an example of translating our work into stakeholder benefit, rejections of Oregon grass seed exported to Korea due to inceptions of nematodes associated with the seed were resolved when it was determined that the nematodes in question did not pose a risk of plant disease to Korean Agriculture. Annual value of the contract of the impacted stakeholders alone was $2 million. Had these nematodes been found in seed from other farms in Oregon, annual exports of $5 million could have been in jeopardy. Areas Needing Further Investigation: Many of the cropping systems we are studying involve complex, multi-year rotations requiring several years of experimentation to test the various permutations of cropping sequences, the durability of resistance relative to nematode selection for virulence, and nematode adaptation to environment and to other control measures. Efforts under W-2186 have laid the foundations for integrating new approaches to nematode management that consider the genetic variability extant in nematode species. Additional study and modification of these systems for managing nematodes is necessary in order to design and optimize new integrated strategies. Coupled with these efforts, new DNA sequence based techniques have developed rapidly in the last five years and their versatility and knowledge value has increased tremendously, while at the same time their costs of application to nematode diagnostics and research into nematode variation has dropped considerably. These advances in molecular protocols are also applicable for selection of nematode resistance traits in crop plants for plant breeding. We must take advantage of these new resources and apply them to the nematode-crop systems and invasive nematode threats that the project members are familiar with and for which representative samples and populations are available. The continuing reduction in nematicide usage, availability and overall desirability and cost effectiveness, plus the increasing actual or potential introduction of invasive nematode species places added pressure on our need to develop alternative nematode management strategies coupled with efficient diagnostics, a primary goal of the proposed project. Other Regional Projects: The only current multistate project with potential overlap to this proposal is S-1046: Improved management of plant-parasitic nematodes through modern diagnostic tools and increased use of host resistance, which focuses primarily on cotton, peanut and soybean cropping systems of the southeastern U.S. It emphasizes resistant variety development and emphasizes soybean cyst nematode and especially reniform nematode, in contrast to the current proposal. Thus, while portions of its goals are similar to the current proposal, it focuses primarily on host plant resistance and a different set of cropping systems to the ones proposed herein. Recently (November 2012) a joint meeting of W-2186 and S-1046 was held for sharing and coordination of the relevant research interests and we plan to do this again with the S-1046 group during the next phase.

Objectives

1. Characterize genetic and biological variation in nematodes relevant to crop production and trade.
   
2. Determine nematode adaptation processes to hosts, agro-ecosystems and environments.
   
3. Develop and assess nematode management strategies in agricultural production systems.
   

Methods

The research focus of each participating state is given as a matrix in Table 1. The nematode group and main crop areas are indicated, together with the procedural research emphasis, as covered under the three objectives. Research coordination will ensure that standardized procedures are generally followed and research findings can be compared within and across nematode, plant and subject area categories. Plant germplasm (accessions, breeding lines, cultivars), nematode isolates of representative species and populations with GPS location data, and DNA primers for markers will be available among the participants. Experimental protocols and procedures are structured according to the main target nematode groups under the three objectives. All objectives have a common focus of addressing critical aspects of nematode variability, adaptation, and management, with the application and extension of the findings tailored to meet individual state and sub-regional needs, in addition to those at the project-wide level. While findings under all objectives are considered important to each state, duplication will be avoided by partitioning individual state research activities. This structure will also ensure that all phases of the objectives are being met, while addressing local state needs, a structure that has worked well in the W-2186 project. Following are examples: Heterodera schachtii will be considered in California, Hawaii, Nebraska, and Idaho; Heterodera glycines will be considered in Alabama, Arkansas, Michigan, Mississippi, Nebraska, and Tennessee, while the Group II cool-climate root-knot species (Meloidogyne chitwoodi and M. hapla) will be studied in detail in California, Michigan, Nebraska, New Mexico, Oregon, Idaho, and Washington. However, Oregon, Idaho, and Washington will focus on potato and (or) small grain interactions, New Mexico will focus on specialty crop interactions, while Washington and California will focus on host-plant resistance. Oregon, Idaho, and New Mexico will address host range and rotations, while California, Idaho, and Washington will address cover-cropping, and green manure treatments. Similar partitioning of research activity will be made for the other nematode groups. Objective 1: Phenotypic assessments will be made on isolates of nematode populations that are collected following survey and documentation of habitat, locality, and agronomic or cropping history of the collection site. This information will provide important background considerations for the level and nature of any phenotypic differences detected in comparative experiments. In most programs, investigators either have a partial or nearly complete collection of live cultures for genetic comparisons. For example, for Group I warm-climate root-knot populations, 30 isolates have been assembled at California-Riverside and a group of isolates is being assembled in Arkansas, while collections of group II cool-climate root-knot nematodes M. chitwoodi and M. hapla isolates are maintained and expanded in Oregon and Washington. For cyst nematodes, numerous populations of H. glycines are being cultured in Arkansas, Michigan, Nebraska and Tennessee, and a collection of H. schachtii and H. cruciferae geographic and host-selected isolates is under culture in Hawaii. Assays of (a)virulence response to resistant lines and cultivars and of host range will be made under greenhouse and controlled environment conditions using well-established experimental procedures. The host range and virulence testing for the root-knot species (Groups I and II) will include standard sets of host differential plants and also differential cultivars and crop plants applicable to their local cropping systems, e.g., on turf and pecan in Arkansas. The biotype scheme for root-knot nematode virulence will be expanded using known accessions, breeding lines, and cultivars of critical crops. For cyst nematodes, H. glycines will be examined on resistant soybean differentials and H. schachtii on sugarbeet breeding lines with resistance, while the endoparasitic nematode Rotylenchulus reniformis will be examined on pineapple lines in Hawaii and on grain legumes or cotton in Alabama, Arkansas, Hawaii and Mississippi. The cyst nematodes are excellent "model" nematode systems for genetic studies because they reproduce sexually. Conversely, many root-knot species are parthenogenetic (asexual). Their genetics will be studied using isofemale or single descent lineages to track inheritance, variability and adaptation, and via mendelian approaches using species that can reproduce sexually, such as M. hapla and M. chitwoodi. This work was developed under the current W-2186 project and several valuable segregating populations are now available. This work will take advantage of both established and new molecular techniques. Variability in outgroups including Mermithidae and C. elegans will be included by California (Hyman, UC Riverside) for comparative molecular genetics where appropriate. Understanding the replication-mediated foundations of mtDNA divergence using these outgroups will positively impact how we view the durability of mtDNA-based identification, phylogenetic inference, and molecular diagnostics. Techniques for multilocus genomic and mtDNA analysis are well established in several participants' laboratories (Oliveira et al., 2011; Powers and Ramírez-Suárez, 2012). PCR and sequencing based techniques will be combined with transmission genetics of phenotypes of interest to conduct analyses of markers and associated traits. Meloidogyne, Heterodera, Globodera and other nematode populations will be typed phenotypically for (a)virulence with respect to numerous host-plant resistance genes from different crop plant species and close wild relatives, for host range, and for biological traits including responses to abiotic soil conditions. Molecular marker analyses of these populations will lead to stable marker systems for nematode (a)virulence phenotypes, host range determinants (including changes in phenotypic response associated with prior parasitism of weeds), and geographical variants for diagnostic purposes, using mtDNA, ribosomal DNA, and nuclear DNA sequence and markers (Powers and Ramírez-Suárez, 2012). They also will provide stronger and more accurate diagnostic protocols. This will also allow us to monitor changes in gene frequency in fields, as we subject field populations to different cropping sequences. From a perspective of diagnostics, high resolution DNA markers will facilitate standardization of identifications and aid in analysis of hypothesized pathways for dispersal that may explain current species or haplotype distributions. Members of the proposed W3186 are in a unique position to address the question of Meloidogyne haplotype distribution across the western and central regions of North America. High resolution markers are available for the mitochondrial genome of Meloidogyne and most nematology laboratories in W3186 participating states are PCR capable. University of California-Riverside and University of Arkansas both have relatively low cost DNA sequencing facilities. In addition to GenBank, DNA sequences of COI barcodes can be deposited in the Barcode of Life Database (BOLD) together with associated metadata as well as the international database of quarantine organisms (QBOL). For the first time, a standardized geographic map of Meloidogyne haplotypes, ranked the most economically important genus of nematodes in the world, can be constructed and made available online. This approach will allow the monitoring of changes in haplotype compositions with changing agricultural practices or climate changes, enable comparisons of haplotype diversity within fields, and also allow detection of new emerging pathogens or recent invasive nematodes. For instance, if M. enterolobi were introduced to California, it could readily be detected in routine molecular assays. Excitingly, we are on the threshold of developing diagnostic systems that feature the simultaneous analysis of multiple species based on DNA extracted from soil. In order for this approach to be effective, the DNA reference database of plant parasitic nematodes must be established and primers of sufficiently high resolution must be validated, as a goal of the project. In the future, this level of analysis may be required for phytosanitary certification. As costs of regulatory surveys and identifications are increasing with the demands for greater specificity, this high-throughput metagenetic approach to nematode diagnostics which identifies multiple species could eventually counteract those cost increases (Porazinska et al., 2009; Porazinska et al., 2012). W3186 will play a central role in the development of this approach. It almost necessitates a team approach due to the need to identify multiple species/or haplotypes from multiple geographic regions, coupled with the testing of multiple genetic markers. Different states will focus on particular species in a collaborative framework: e.g., M. incognita populations from cotton-producing states, including Alabama, Arkansas, California, Mississippi, New Mexico, and Tennessee; M. chitwoodi and M. hapla in the potato-small-grain-alfalfa systems of Oregon, Washington, and Idaho; H. schachtii, H. glycines, and H. cruciferae from California, Hawaii, Michigan, Nebraska, and Tennessee. Objective 2: Phenotypic characterization and genetic markers developed under Objective 1 will provide the basis for selecting candidate populations that show adaptation to increased virulence and parasitism, and for variants associated with geographic (climatic) and soil abiotic isolation and adaptation. The fitness of isolates virulent for specific resistance genes will be assessed by controlled culturing on susceptible plants for multiple generations. Effects of non-agronomic hosts on fitness will also be examined, such as studies on the effects of nutsedges and other weed hosts on root-knot nematodes in cropping systems, which will be expanded to include weed populations from other geographic areas to better understand the implications of this adaptation. Participants also will include comparisons with invasive root-knot nematode species, such as M. enterolobi (Group I) and M. fallax (group II), which have heightened aggressiveness and broad host range. Adaptation and fitness also will be studied by imposing continuous selection for virulence in wild-type populations. Changes in reproductive capacity will be assessed to measure virulence frequencies and genetic stability of virulence. Tests of selected isolates will be made on resistant or nonhost plants other than those used to impose the original selection pressure. For example, in California studies of a model system of M. incognita (a)virulence matching resistance genes in cowpea will be continued (Petrillo et al., 2006). In the field, changes in virulence in H. glycines towards soybean cultivars carrying specific resistance genes will be monitored. Likewise, in Oregon pathogenicity of lesion and ectoparasitic nematodes on blueberry genotypes as well as pathogenicity of G. ellingtonae on different genotypes of potato will be evaluated. In New Mexico, predictive heat unit models for soil temperature, nutsedge emergence and M. incognita reproduction interaction will be used management implementation. In addition, fitness and adaptability of H. glycines races, M. hapla, M. incognita and Pratylenchus to changes in soil nutrition and soil physio-chemical environments will be tested, focusing on sources of nitrogen and types and levels of nutrients and their affects on host plant status. Quantifying nematode adaptability to changes in soil-nutrient environments will be extended as a major emphasis of work in Michigan (Melakeberhan and Avendano, 2008; Melakeberhan et al., 2012), enhancing understanding of possible factors contributing to nematode adaptations. Changes in response to soil amendments including green manures, animal waste products and other bio-products will be monitored in target cyst and root-knot species (Zazada et al., 2008). Objective 3: A range of factors and approaches will be considered in the design of nematode management strategies under Objective 3 - soil ecology and other soil properties, nematode community structure and function, sampling strategies tied with opportunities afforded by precision agriculture technologies for site-specific management. The integration of host resistance, cover, trap, and green manure crops, tillage practices and impact of biological antagonists in crop rotation sequences will be considered in practical combinations and locations. These field-based studies will utilize standard experimental designs (randomized complete block, split-block, split-plot) with control treatments and 4 to 6-fold replications under current production practices. The development of biotyping schemes for cyst and root-knot nematode populations for reaction to host and nonhost crops and to resistant cultivars, and accompanying practical marker systems under Objective 1 will provide important information for resistance and rotation implementation in annual crops. For example, soybean cyst nematode rotation experiments will be conducted with contemporary highly resistant, resistant and susceptible soybean varieties, because the data we have presently is severely outdated for production areas in Arkansas and neighboring states. Crop rotation utilizing only nonhosts as production or cover crops that are grown in rotation with hosts will be evaluated for success in nematode management and profitability. Knowledge on nematode fitness and adaptability developed under Objective 2 will aid in the optimization of durable nematode management strategies. Climatic and soil conditions, different nematode species, and variability in host ranges among different populations will require unique rotations for different growing regions. Several cropping systems being evaluated currently under W-2186 and described earlier have multi-year horizons and crop sequence combinations that require much additional analysis. New ones, such as lesion nematode in raspberry production in the Pacific Northwest will be addressed through integrated management studies. Rotation schemes will be assessed in microplots or infested field sites within the relevant localities. Techniques will be coordinated among participants to facilitate direct comparisons of results. The rapidly changing demands on crop production present new challenges to the use of crop rotation for nematode management. For example, demand for ethanol has resulted in the shift from wheat to corn in potato rotations. While wheat is a host for M. chitwoodi, it is harvested early in the summer and supports limited reproduction. In some areas it can also be followed by green manure cover crops in the fall that significantly suppress nematode populations. In contrast, corn is grown into the fall, supporting several generations of M. chitwoodi and eliminating the opportunity for suppression of population increases with cover crops. However, corn can vary greatly in its host status to M. chitwoodi and different cultivars need to be screened for resistance to inform growers about which varieties would limit population increases. Grower interest in green manure crops and other cover crops for nematode suppression is increasing but little is known about the host status of many of these plants. This needs to be determined so that effective recommendations can be made. In the case of sugarbeet cyst nematode, H. schachtii, and cereal cyst nematode H. avenae, fallow or planting with a green manure crop incorporated after eight weeks will be tested in rotations with non-host crops such as corn, bean, onion or potato between main crops. To enhance understanding of spatio-temporal relationships among nematode populations, soil conditions, biological antagonists, and production practices, models developed to assess agro-ecological efficiency of nematode management strategies (Melakeberhan, 2006; Melakeberhan et al., 2012) will be utilized to integrate nematode community structure-driven soil biological changes for assessing agricultural and ecological efficiencies of cultural practice-based nematode management strategies. In doing so, we will be developing bridges suitable for multi-disciplinary approaches beyond nematology and critical to solving agricultural problems.

Measurement of Progress and Results

Outputs

• Basic knowledge on biological, ecological and genetic processes underlying the success of cyst, root-knot, and other nematodes as parasites of crop plants.
• New or improved, and safer tactics for the management of nematodes that affect crop production and trade in US agriculture.
• Holistic approaches for integrating different management strategies into IPM systems for nematode control programs.
• A diagnostic DNA barcoding reference data base of major plant parasitic nematodes that impact US agriculture.
• Economic impact and ecological assessment of management approaches for control of plant parasitic nematodes of major agricultural significance.

Outcomes or Projected Impacts

• Increased knowledge base in plant-nematode biology, ecology and genetics for use in identifying novel targets for nematode control.
• Implementation of nematode management tactics that complement reduced pesticide usage, and thereby benefit human health and the environment.
• Promotion of sustainable farm management practices through new nematode management tactics.
• More efficient and effective response and mitigation capabilities for invasive and trade product contamination issues.
• Economic benefits to producers and consumers through reduction in nematode management costs and food production.

Milestones

(2015): Development of new research-based knowledge on nematode variability and adaptation relative to abiotic and biotic parameters in target cropping systems.

(2016): Development and validation of new nematode diagnostic and detection protocols.

(2017): Evaluation of new nematode management tactics under appropriate experimental conditions.

(2018): Transfer of knowledge to stakeholders on new nematode management tactics and new diagnostic and detection protocols.

(0):cerning the year-related milestones indicated above, the research will provide information necessary to implement nematode management alternatives to nematicides. Research in each nematode - cropping system combination included is at various stages of maturity along a sequence of progressive steps based on the order of the objectives. These include developing new basic knowledge, from which a control strategy or diagnostic protocol is developed, that in turn is advanced toward implementation by field experimentation and demonstration, and transfer to stakeholders. For example, identification of resistance in a crop, advancement of the trait into commercial varieties and testing its effectiveness in a cropping system rotation will be made. Each nematode-crop plant combination and cropping system will have its own timeline for completion, but typically will require completion of the objectives in the order presented; thus, Objectives 3 will require completion of Objectives 1 and 2 for a given nematode-crop system.

Projected Participation

View Appendix E: Participation

Outreach Plan

Outreach Plan: The project has a successful track record of disseminating the new knowledge and information created by participants and co-operating colleagues. It is planned that the traditional outlets for transferring project results will continue to be utilized, including peer-reviewed journals, annual progress reports, scientific meeting presentations, and websites. Extension presentations and publications also will be made; both by participants with extension appointments, and by AES and ARS participants, most of whom have a strong applied research component to their programs and who routinely participate in extension-based activities (meetings, presentations, publications) with our agricultural stakeholders. The project members will use web-based delivery vehicles to reach the broad stakeholder base that will benefit from objective, science-based knowledge and information about important plant-parasitic nematode biology, diagnosis, management and regulation. Examples include GenBank for depositing most DNA sequences, the Barcode of Life Database (BOLD) for DNA sequences of COI barcodes together with associated metadata, and the international database of quarantine organisms (QBOL). Among our stakeholders we have identified growers and food processors, national and international trade partners, USDA-APHIS and state regulatory agencies, national and state legislators, fellow scientists, and K -16 teachers and students.

Organization/Governance

The organization and governance of this Multistate Research Project will conform to the guidelines presented in the United States Department of Agriculture's publication "Manual for Cooperative Regional Research". Committee officers include a Chair, a Vice-Chair, and a Secretary. A new secretary will be elected at each annual meeting of the Technical Committee with the current secretary assuming the position of Vice-Chair. The Vice-Chair assumes the position of Chair at the end of the annual meeting. In the event that an Executive Committee is needed, the officers are authorized to serve in that role. For organization of each annual meeting, one member of the Technical Committee will be selected to coordinate and oversee local arrangements for the meeting. The meeting coordinator will be chosen based on the consensus selection of the meeting site, and typically will be the member from the state or institution hosting the meeting. The Technical Committee will rotate the annual meetings between representative Experiment Stations who will be participating in the project. Members will benefit from site visits of research and extension facilities at the host Experiment Station institution. Administrative guidance will be provided by an assigned Administrative Advisor.

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Attachments

Land Grant Participating States/Institutions

AL, AR, CA, HI, ID, MI, MS, NE, NM, OR, WA

Non Land Grant Participating States/Institutions

Department of Biological Sciences, Elizabeth City State University, NIFA