Progress 10/01/16 to 09/30/17

Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop molecular methodologies for the identification and classification of alfalfa nematodes, needed to support diagnosticians and growers in making timely and accurate management decisions in response to nematode outbreaks, and to aid plant breeders in developing new nematode resistant cultivars. Objective 2: Identify and characterize nematodes found with endophytes of alfalfa, forage grasses, and weeds in order to discover novel associations with increased crop productivity or improved adaptability to arid sites. Approach (from AD-416): 1. Nematodes that infect alfalfa and other forage legumes, grasses, rotation crops, or potential weed hosts will be identified using molecular markers including ribosomal, mitochondrial, Hsp90, and other nuclear genes. 2. New diagnostic assays, including RFLPs, and conventional or real-time PCR assays will be developed for Meloidogyne spp., Pratylenchus spp., Ditylenchus spp., or others affecting these plants. 3. Molecular information from the diagnostic work will be integrated with morphological data and information regarding biogeography, pathogenicity, and host range to generate new and improved phylogenetic schemes. 4. Molecular diagnostic tools to aid in screening resistant alfalfa germplasm will be developed to detect and quantify nematodes from infected alfalfa tissue. 5. Fungal-feeding nematodes and their associated fungal endophytes will be isolated from alfalfa, forage grasses, or weeds and identified and characterized by morphological and molecular methods. 6. Fungal-feeding nematodes will be cultured with fungal symbionts in lab cultures to determine suitability of endophytes to support nematode growth and reproduction. Progress towards Objective 1 was focused on development of molecular diagnostic tests for identification of plant-parasitic nematodes of alfalfa cropping systems. The lesion nematode Pratylenchus penetrans is one of the most widespread and economically important nematodes impacting alfalfa and many other crops throughout the U.S. This nematode can be difficult to distinguish from other lesion nematodes by traditional microscopic methods. Research was performed with scientists from North Dakota State University to develop a molecular diagnostic assay for the lesion nematode using DNA extracted directly from soil samples. This assay was used to quantify P. penetrans in soil and accurately distinguish this species from other lesion nematodes present in mixed nematode populations. Other progress relevant to Objective 1 included the identification and molecular characterization of lesion, cyst, and stem nematodes determined through phylogenetic analysis of mitochondrial cytochrome oxidase, nuclear Hsp90, and ribosomal molecular markers. Molecular identification and phylogenetic analysis were conducted for cyst and ring nematodes from Greece, further expanding the known distribution of some species. With MNGDBL and other ARS colleagues, new state records for two cyst nematodes were confirmed through molecular analysis of ribosomal and Hsp90 markers; this includes the soybean cyst nematode Heterodera glycines in New York and the corn nematode Vittatidera zeaphila from Indiana. Progress relevant to Objective 2 focused on phylogenetic studies of fungal feeding nematodes from alfalfa, forage grasses, and weeds. This included construction of gene trees from new populations of alfalfa stem, seed gall, ring, and root-knot nematodes that were used to clarify species boundaries and relationships, and may form the basis for design of new molecular diagnostics for those species. Accomplishments 01 New sensitive molecular diagnostic for stubby root nematodes. Plant- parasitic nematodes are microscopic worms that attack plant roots and cause an estimated ten billion dollars of crop loss each year in the United States and 100 billion dollars globally. The stubby root nematode Paratrichodorus allius has been reported on alfalfa in California, a major state for alfalfa production. This nematode is most significant for its ability to transmit the tobacco rattle virus that causes corky ringspot disease, resulting in significant economic losses to the potato industry. In this research, scientists from North Dakota State University and an ARS scientist from Beltsville, Maryland, developed highly sensitive molecular diagnostic assays to detect P. allius using DNA extracted directly from soil samples. The new diagnostic tests will be used by university, state, federal, or commercial diagnostic laboratories, other scientists, action agencies, and extension agencies to accurately quantify this stubby root nematode to better implement effective management decisions in the field. 02 Identification of low numbers of soybean cyst nematodes in soil. The soybean cyst nematode (SCN) is a major pathogen of soybean throughout the world. It is difficult to distinguish SCN from closely related cyst nematodes based solely on the anatomical features of juveniles, many of which appear very similar among species. In order to improve existing diagnostic capabilities, a team of scientists from ARS, North Dakota State University, and the University of Minnesota developed a molecular assay to detect and identify low densities of SCN from field soils. This research developed a unique method for removing contaminants and inhibitors from soil-extracted DNA, improving sensitivity and reliability without requiring nematodes to be isolated from soil samples. This research will be used by scientists and diagnostic laboratories to accurately detect and quantify SCN from infested fields. 03 Identification of endophytic nematodes. While most nematodes are found below ground, some inhabit above-ground plant parts where they feed on fungi that may be good or bad for crops. A major problem with determining the role of these fungal-feeding nematodes is that the anatomical descriptions and keys used for their identification are outdated and unreliable. Therefore, ARS scientists from Beltsville, Maryland, and an ecologist from the University of Idaho in Moscow, identified and described from poplar tree leaves two nematodes that feed on fungi, some of which cause plant disease. Information on anatomical features was compiled for new populations of two previously described nematode species, and a family tree based on DNA markers was constructed for this group of nematodes. Now ecologists can confidently identify nematodes of different species wherever they may occur in the world. This information will be used by scientists, extension agents and pest managers engaged in nematode and fungi research and control.

Impacts
(N/A)

Publications

• Huang, D., Yan, G., Skantar, A.M. 2017. Development of real-time and conventional PCR assays for identifying stubby root nematode Paratrichodorus allius. Plant Disease. 101(6):964-972. doi: 10.1094/PDIS- 10-16-1431-RE.
• Baidoo, R., Yan, G., Nelson, B., Skantar, A.M., Chen, S. 2017. Use of chemical flocculation and nested PCR for Heterodera glycines detection in DNA extracts from field soils with low population densities. Plant Disease. doi: 10.1094/PDIS-08-16-1163-RE.
• Carta, L.K., Li, S., Skantar, A.M., Newcombe, G. 2016. Morphological and molecular characterization of two Aphelenchoides from poplar leaves. Journal of Nematology. 48(1):28-33.
• Skantar, A.M., Prior, T., Fleming, C. 2017. DP 18: Anguina spp. In: International Standards for Phytosanitary Measures IPSM 27, Diagnostic Protocols for Regulated Pests. Rome: Secretariat of the International Plant Protection Convention (IPPC). 34 pp.

Progress 10/01/15 to 09/30/16

Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop molecular methodologies for the identification and classification of alfalfa nematodes, needed to support diagnosticians and growers in making timely and accurate management decisions in response to nematode outbreaks, and to aid plant breeders in developing new nematode resistant cultivars. Objective 2: Identify and characterize nematodes found with endophytes of alfalfa, forage grasses, and weeds in order to discover novel associations with increased crop productivity or improved adaptability to arid sites. Approach (from AD-416): 1. Nematodes that infect alfalfa and other forage legumes, grasses, rotation crops, or potential weed hosts will be identified using molecular markers including ribosomal, mitochondrial, Hsp90, and other nuclear genes. 2. New diagnostic assays, including RFLPs, and conventional or real-time PCR assays will be developed for Meloidogyne spp., Pratylenchus spp., Ditylenchus spp., or others affecting these plants. 3. Molecular information from the diagnostic work will be integrated with morphological data and information regarding biogeography, pathogenicity, and host range to generate new and improved phylogenetic schemes. 4. Molecular diagnostic tools to aid in screening resistant alfalfa germplasm will be developed to detect and quantify nematodes from infected alfalfa tissue. 5. Fungal-feeding nematodes and their associated fungal endophytes will be isolated from alfalfa, forage grasses, or weeds and identified and characterized by morphological and molecular methods. 6. Fungal-feeding nematodes will be cultured with fungal symbionts in lab cultures to determine suitability of endophytes to support nematode growth and reproduction. Under Objective 1, intra- and inter-specific diversity of stem nematode (Ditylenchus spp.) from alfalfa and other crops was determined through phylogenetic analysis of nuclear and ribosomal molecular markers. A new species of root-knot nematode isolated from the invasive weed, garlic mustard in Oregon was identified on the basis of multiple molecular sequences including nuclear heat shock protein 90, ribosomal, and mitochondrial genes in combination with morphological analysis performed by colleagues within the Nematology Laboratory. Phylogenetic analysis further clarified species boundaries relative to overlapping boundaries inferred from ribosomal markers for some tropical root-knot nematode species. Molecular identification and phylogenetic analysis were conducted for cyst and ring nematodes from Greece, further expanding the known distribution of some species. The veracity of a study which proposed a mitochondrial marker as able to distinguish lineages of tropical root-knot nematodes was tested on populations in our collection. Heat shock protein 90 primers previously published for molecular diagnosis of potato cyst nematodes were re-analyzed in comparison to our unpublished data, revealing possible problems with assay specificity. This information will be useful to diagnosticians who may be considering this approach for diagnosis of potato cyst nematode. Progress relevant to Objective 2 included phylogenetic studies of nematodes isolated from alfalfa foliage, roots, and soil. Analysis of multiple molecular markers was performed to clarify relationships among these understudied species, providing a basis for further understanding of the relationships among nematodes, endophytes (fungi living within plant tissues), and alfalfa health. Accomplishments 01 Molecular diagnostics of root-knot nematodes. Nematodes are microscopic worms that cause billions of dollars in crop losses worldwide each year. Root-knot nematodes are an important group of nematodes that damage many kinds of plants, including carrot, by invading the roots and interfering with nutrient uptake. Identifying these nematodes from soil can be very labor intensive and anatomical features can be very similar across species. In the present study, an ARS scientist at Beltsville, Maryland, in collaboration with scientists from Denmark developed a new molecular test to detect the northern root knot nematode. The results are significant because the new test was sensitive, highly specific, and able to detect this species directly from infested soil. This research will be used by researchers and diagnosticians to accurately detect and quantify northern root knot nematodes from infested fields and for directing management decisions in carrot cultivation. 02 Identification of dagger nematode. Plant-parasitic nematodes are microscopic worms that attack plant roots and cause billions of dollars of damage to forest and ornamental trees. One problem with determining the extent of nematode damage is that the nematodes present in many areas are not known. In this study, ARS scientists at Beltsville, Maryland, together with scientists from the California Department of Food and Agriculture and the U.S. Forest Service, using DNA and microscopic features, characterize a species of economically important dagger nematode from soil around the roots of live oak trees in Jekyll Island, Georgia. The results are significant because this discovery represents a new location for this nematode. The necessary information for scientists to correctly identify this potential virus-vectoring species wherever it may occur, is provided. This research will be used by scientists, action agencies, and extension agencies to further define the species variation, host range and geographic distribution of this nematode.

Impacts
(N/A)

Publications

• Sapkota, R., Skantar, A.M., Nicolaisen, M. 2015. A Taqman real-time PCR assay for detection of Meloidogyne hapla in root galls and in soil. Nematology. 18(1):147-154.
• Handoo, Z.A., Carta, L.K., Skantar, A.M., Subbotin, S.A., Fraedrich, S.S. 2016. Molecular and morphological characterization of a Xiphinema chambersi population from live oak trees in Jekyll Island, Georgia, with a redescription of the species and comments on its morphometric variations. Journal of Nematology. 48(1):20-27.
• Akinbad, S.A., Mojtahedi, H., Guerra, L., Eastwell, K.C., Villamor, D.E., Handoo, Z.A., Skantar, A.M. 2013. First report of Xiphinema rivesi (Nematoda, Longidoridae) in Washington State. Plant Disease. 91(7):770. DOI: 10.1094/PDIS-09-13-0961-PDN.

Progress 10/01/14 to 09/30/15

Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop molecular methodologies for the identification and classification of alfalfa nematodes, needed to support diagnosticians and growers in making timely and accurate management decisions in response to nematode outbreaks, and to aid plant breeders in developing new nematode resistant cultivars. Objective 2: Identify and characterize nematodes found with endophytes of alfalfa, forage grasses, and weeds in order to discover novel associations with increased crop productivity or improved adaptability to arid sites. Approach (from AD-416): 1. Nematodes that infect alfalfa and other forage legumes, grasses, rotation crops, or potential weed hosts will be identified using molecular markers including ribosomal, mitochondrial, Hsp90, and other nuclear genes. 2. New diagnostic assays, including RFLPs, and conventional or real-time PCR assays will be developed for Meloidogyne spp., Pratylenchus spp., Ditylenchus spp., or others affecting these plants. 3. Molecular information from the diagnostic work will be integrated with morphological data and information regarding biogeography, pathogenicity, and host range to generate new and improved phylogenetic schemes. 4. Molecular diagnostic tools to aid in screening resistant alfalfa germplasm will be developed to detect and quantify nematodes from infected alfalfa tissue. 5. Fungal-feeding nematodes and their associated fungal endophytes will be isolated from alfalfa, forage grasses, or weeds and identified and characterized by morphological and molecular methods. 6. Fungal-feeding nematodes will be cultured with fungal symbionts in lab cultures to determine suitability of endophytes to support nematode growth and reproduction. Progress relevant to Objective 1 includes identification of nematode species from two of the leading alfalfa-growing states in the U.S. (Idaho and Montana). A probable new species of root-knot nematode (RKN) from garlic mustard weed in Oregon was identified through analysis of multiple molecular markers, thereby distinguishing it from suspected northern RKN and other known species. A population of potato cyst nematode from Idaho was molecularly characterized in order to establish its genetic relatedness to the original isolation of that species from elsewhere in the state. The most closely related DNA sequences were from a population in the UK, and the within species variation found in the Idaho population was similar to other European and South American isolates. Molecular and morphological studies were conducted to characterize a population of cereal cyst nematode from Montana, representing the first occurrence of that species in the state. A spiral nematode isolate from vineyards was characterized using multiple molecular markers to clarify its genetic relationship to similar species. Each of these species identifications is significant for its impact on management or quarantine decisions, with the results serving the immediate needs of regulatory agencies and extension personnel. Significant progress relevant to Objective 2 included the molecular identification of fungal-feeding nematodes isolated from alfalfa foliage, roots, and soil collected from Utah. Phylogenetic studies based on multiple molecular markers were performed to clarify relationships among these understudied species, providing a basis for further understanding of the relationship between nematodes, endophytes (fungi living within plant tissues), and alfalfa health. Molecular markers were also characterized from two different fungal feeding nematodes found on poplar leaves. Identification of these nematodes is significant because they can affect the balance of endophytic fungi that are important determinants of plant health. Accomplishments 01 Molecular diagnostics of seed gall nematodes. Certain species of seed gall nematodes are regulated as quarantine pests by many countries due to economic damage to wheat or grasses or for the ability to carry bacteria that are toxic to livestock. Growers and regulatory officials face an enormous problem in that the existing molecular methods for distinguishing these nematodes are time consuming and occasionally inaccurate. Scientists from ARS and APHIS in Beltsville, Maryland, designed a new molecular diagnostic test that can rapidly detect and distinguish four different seed gall nematodes of wheat and grasses. This research is significant because the new assay is species-specific, highly sensitive, and faster to perform than existing molecular methods for identification of seed gall nematodes. This test will be useful for federal and state diagnostic labs, domestic and international research scientists, regulatory personnel, or extension agencies for identifying and preventing further damage caused by seed gall nematodes. 02 Identification of genes expressed in response to root-knot nematode infection of alfalfa. Root knot nematodes (RKN) are widely distributed and economically important sedentary nematodes that inflict significant damage on alfalfa. Breeding alfalfa for resistance to nematodes has been hampered by a lack of understanding of the plant-parasite interaction. ARS scientists from Beltsville, Maryland, used high throughput sequencing technology to identify nematode molecular markers associated with RKN parasitism of alfalfa roots and alfalfa genes that are either highly expressed or suppressed in response to RKN infection. Determining the function of individual genes involved in the alfalfa defense responses to RKN provides critical insights for alfalfa breeding programs by unraveling mechanisms of resistance to these pests, allowing greater precision in screening alfalfa germplasm for nematode resistance. These results will be used by alfalfa breeders, nematologists in academia and government organizations, extension personnel, alfalfa producers and representatives of the plant biotechnology industry. 03 Identification of cyst nematodes on wheat. The cereal cyst nematodes are a widespread and important group of microscopic, plant parasitic soil worms that limit production of cereal crops throughout the world. High populations of the cyst nematode Heterodera avenae are estimated to reduce profits from wheat production by at least $3.4 million annually in the Pacific Northwest (PNW). A second wheat-damaging cyst nematode, Heterodera filipjevi, was discovered in the PNW in 2008, and its distribution there is currently unknown. Distinguishing these two nematodes from each other by microscopic methods is laborious and sometimes inconclusive. In the present study, scientists from Oregon State University and ARS scientists from Beltsville, Maryland, and Pullman, Washington, developed a highly specific molecular diagnostic assay to detect and distinguish these two species. The results are significant because the assay can be used to identify these nematodes and distinguish them from similar nematodes, thus providing accurate identifications needed for selection of appropriate wheat cultivars and management practices. Commercial diagnostic laboratories, other scientists, action agencies, and extension agencies engaged in nematode research and control will use this research.

Impacts
(N/A)

Publications

• Li, W., Zonghe, Y., Nakhla, M.K., and Skantar, A.M. 2015. Real-time-PCR for detection and identification of Anguina funesta, A. agrostis, A. tritici, and A. pacificae. Plant Disease. DOI:10.1094/PDIS-09-14-0959-RE.
• Postnikova, O., Hult, M.N., Shao, J.Y., Skantar, A.M., Nemchinov, L.G. 2015. Transcriptome analysis of resistant and susceptible alfalfa cultivars infected with root-knot nematode Meloidogyne incognita. PLoS One. 10(3):e0123157.
• Yan, G., Smiley, R.W., Okubara, P.A., Skantar, A.M. 2013. Development of a species-specific PCR assay for differentiation of Heterodera filipjevi and H. avenae. Plant Disease. 97(12):241-246.
• Skantar, A.M. 2015. Diseases caused by nematodes. In: Samac, D.A., Rhodes, L.H., and Lamp, W.O., editors. Compendium of Alfalfa Diseases and Pests, 3rd edition. St. Paul, MN: APS Press. p. 72-77.

Progress 10/01/13 to 09/30/14

Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop molecular methodologies for the identification and classification of alfalfa nematodes, needed to support diagnosticians and growers in making timely and accurate management decisions in response to nematode outbreaks, and to aid plant breeders in developing new nematode resistant cultivars. Objective 2: Identify and characterize nematodes found with endophytes of alfalfa, forage grasses, and weeds in order to discover novel associations with increased crop productivity or improved adaptability to arid sites. Approach (from AD-416): 1. Nematodes that infect alfalfa and other forage legumes, grasses, rotation crops, or potential weed hosts will be identified using molecular markers including ribosomal, mitochondrial, Hsp90, and other nuclear genes. 2. New diagnostic assays, including RFLPs, and conventional or real-time PCR assays will be developed for Meloidogyne spp., Pratylenchus spp., Ditylenchus spp., or others affecting these plants. 3. Molecular information from the diagnostic work will be integrated with morphological data and information regarding biogeography, pathogenicity, and host range to generate new and improved phylogenetic schemes. 4. Molecular diagnostic tools to aid in screening resistant alfalfa germplasm will be developed to detect and quantify nematodes from infected alfalfa tissue. 5. Fungal-feeding nematodes and their associated fungal endophytes will be isolated from alfalfa, forage grasses, or weeds and identified and characterized by morphological and molecular methods. 6. Fungal-feeding nematodes will be cultured with fungal symbionts in lab cultures to determine suitability of endophytes to support nematode growth and reproduction. Objective 1 Goal 1.1 includes the description of nematodes that infect alfalfa and other forage legumes, grasses, rotation crops, or potential weed hosts, and the development of phylogenies (i.e., knowledge of evolutionary relationships) that integrate molecular and anatomical data with available information on geography, pathogenicity, and host range. Significant progress in the past year included molecular identification and phylogenetic analysis of stem and foliar nematodes isolated from alfalfa in Utah. Goal 1.2 relates to the development of molecular markers and diagnostic tools to aid in the identification and control of nematodes that infect alfalfa and other forage legumes, grasses, rotation crops, or potential weed hosts. For this goal, molecular analysis of wheat and seed gall nematodes enabled the design and validation of highly sensitive diagnostic tests needed for distinguishing species of regulatory importance from related, more commonplace nematodes. Goal 1.3 includes the discovery of molecular diagnostic tools to detect and quantify nematodes from infected alfalfa tissue. Along with scientists from the ARS Molecular Plant Pathology Laboratory at Beltsville, alfalfa root cultures were infected with root-knot nematodes, followed by RNA sequencing to identify molecular markers from the nematode that are associated with the penetration and infection of alfalfa roots. Together, these molecular results will support improved identification and control recommendations for nematode species of concern in alfalfa agroecosystems and help to improve methods of screening germplasm for nematode resistance. Objective 2 relates to the identification and characterization of foliar or fungal-feeding nematodes from alfalfa, forage grasses and weeds. Significant progress included the molecular identification of fungal-feeding nematodes isolated from alfalfa foliage, roots, and soil collected from Utah. Phylogenetic studies based on multiple molecular markers were performed to clarify relationships among these understudied species, providing a basis for further understanding of the relationship between nematodes, endophytes (fungi living within plant tissues), and alfalfa health. Accomplishments 01 Molecular characterization of root-knot nematodes new to the United States. Root-knot nematodes (RKN) are parasitic on a wide range of host plants, including alfalfa, turfgrasses, and numerous other crops. In this study, ARS scientists and colleagues from Arizona and Utah used anatomical features and molecular markers to identify and describe two populations of RKN from golf course turfgrass; one was a species not previously found in North America. This research is significant because new molecular information obtained for these populations will facilitate future identification of RKN and prevent possible economic harm due to spread of these nematodes to potato-growing areas. This study will aid researchers and diagnosticians in accurately identifying economically important root-knot nematodes that are difficult to tell apart by comparing anatomical features alone. 02 Development of new diagnostic test for a novel potato cyst nematode species. Cyst nematodes are an important group damaging the roots of many kinds of plants, including crops commonly grown in rotation with alfalfa, such as potato. In the present study, ARS scientists in Beltsville, MD and Ithaca, NY developed a novel sensitive molecular test to identify a new species of cyst nematode that was originally found in Oregon and Idaho. This research is significant because previous molecular tests designed to discriminate regulated species of potato cyst nematodes from the unregulated tobacco cyst nematode cannot be used to detect the new species. Because potato cyst nematodes (Globodera spp.) are regulated as a quarantine pest by many countries and cause economic damage to potato worldwide, scientists, regulators, and extension agencies will use this research to distinguish the new species from the currently regulated species and help prevent further infestations. 03 New molecular diagnostic test to distinguish cyst nematodes of wheat. Cyst nematodes are economically important species restricting the productivity of cereal crops in the Pacific Northwest. In the present study, scientists from Oregon State University and ARS scientists from Beltsville, MD, Pullman, WA, and Pendleton, OR developed a highly sensitive molecular diagnostic assay to distinguish between two species of cyst nematode that infect wheat. The results are significant because the assay can be used to identify these nematodes and distinguish them from other nematode species, thus providing accurate identifications needed for selection of appropriate wheat cultivars and management practices. Commercial diagnostic laboratories, other scientists, action agencies, and extension agencies engaged in nematode research and control will use this research to aid in the selection of appropriate cultivars to maximize yield.

Impacts
(N/A)

Publications

• Nischwitz, C., Skantar, A.M., Handoo, Z.A., Hult, M.N., Schmitt, M.E., McClure, M.A. 2013. Occurrence of Meloidogyne fallax in North America, and molecular characterization of M. fallax and M. minor from U.S. golf course greens. Plant Disease. 97(11):1424-1430.
• Chronis, D.N., Chen, S., Skantar, A.M., Zasada, I.A., Wang, X. 2014. A new chorismate mutase gene identified from Globodera ellingtonae and its utility as a molecular diagnostic marker. European Journal of Plant Pathology. 139(2):239-246. DOI:10.1007/s10658-014-0385-x.

Progress 10/01/12 to 09/30/13

Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop molecular methodologies for the identification and classification of alfalfa nematodes, needed to support diagnosticians and growers in making timely and accurate management decisions in response to nematode outbreaks, and to aid plant breeders in developing new nematode resistant cultivars. Objective 2: Identify and characterize nematodes found with endophytes of alfalfa, forage grasses, and weeds in order to discover novel associations with increased crop productivity or improved adaptability to arid sites. Approach (from AD-416): 1. Nematodes that infect alfalfa and other forage legumes, grasses, rotation crops, or potential weed hosts will be identified using molecular markers including ribosomal, mitochondrial, Hsp90, and other nuclear genes. 2. New diagnostic assays, including RFLPs, and conventional or real-time PCR assays will be developed for Meloidogyne spp., Pratylenchus spp., Ditylenchus spp., or others affecting these plants. 3. Molecular information from the diagnostic work will be integrated with morphological data and information regarding biogeography, pathogenicity, and host range to generate new and improved phylogenetic schemes. 4. Molecular diagnostic tools to aid in screening resistant alfalfa germplasm will be developed to detect and quantify nematodes from infected alfalfa tissue. 5. Fungal-feeding nematodes and their associated fungal endophytes will be isolated from alfalfa, forage grasses, or weeds and identified and characterized by morphological and molecular methods. 6. Fungal-feeding nematodes will be cultured with fungal symbionts in lab cultures to determine suitability of endophytes to support nematode growth and reproduction. This project began on December 14, 2012, as a progression from project number 1275-22000-254-00D. Several nematode specimens needed for initiation of this project have been acquired and processed for DNA analysis, including numerous species that infect alfalfa, forage legumes, grasses, bioenergy crops, rotation crops, or potential weed hosts. Specifically, populations of root-knot, lesion, and stem nematode, and fungal feeding species were obtained for further research.

Impacts
(N/A)

Publications