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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Mycology and Nematology Genetic Diversity and Biology Laboratory » Research » Research Project #436138

Research Project: Improved Identification and Diagnostics of Plant Nematodes for Sustainable Grass and Forage Production Systems

Location: Mycology and Nematology Genetic Diversity and Biology Laboratory

2020 Annual Report

1a. Objectives (from AD-416):
Objective 1: Identify and describe invasive and emerging plant-parasitic nematodes from forage and biomass legumes, turf, grasses, and associated rotation crops - including species of quarantine significance - to enable specific and timely management of these pests. (NP215 1B.4, 1B.5) Objective 2: Improve molecular methodologies for the identification and classification of nematodes from forage legumes and grasses in order to better predict and respond to nematode outbreaks and to improve detection and diagnostic methodologies to distinguish closely related or morphologically similar species. (NP215 1B.4, 1B.5, 3B) Subobjective 2A: Barcodes and markers for improved molecular phylogenies. Subobjective 2B: New molecular diagnostic assays. Subobjective 2C: DNA recovery and barcoding from formalin-fixed nematodes.

1b. 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 cyst, root-knot, lesion, and stem nematodes or others affecting these plants. 3. Molecular phylogenetic analysis will be performed using DNA sequence. alignment programs such as CLUSTAL and MAFFT and tree building methods based on distance, parsimony, and maximum likelihood methods. 4. 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. 5. Methods to neutralize or repair DNA damage caused by specimen fixation will be combined with PCR and DNA sequencing in order to identify ways of recovering DNA information from degraded specimens.

3. Progress Report:
Progress was made on both objectives which fall under National Program 215, Pasture, Forage, and Rangeland Systems Component 1, Provide fundamental knowledge and understanding of interacting ecological components of pastures, rangelands, forages and turf systems. Progress relating to Objective 1 Milestone 1 (identification of root-knot nematodes, Meloidogyne spp.) included molecular identification and characterization of barley root-knot nematode from turfgrass in Idaho; guava root-knot nematode from Thailand; Javanese root-knot nematode on hoya from Guam; and Seville root-knot nematode on corn from South Carolina. Progress on Objective 2, milestones 2A and 2B was achieved by molecular identification and phylogenetic analysis of new species of cyst nematodes from the genera Punctodera and Cactodera and molecular characterization of lesser known nematode species from the genus Coslenchus from Maryland and Russia. Moreover, a team of ARS and University of Idaho scientists used morphological and molecular means to identify the amaryllis lesion nematode from a vineyard in Kern County, CA. Subobjective 2A was met through the investigation of the heat shock protein 90 (Hsp90) and cytochrome oxidase I (COI) barcode markers to improve molecular phylogenies for the cyst and root knot nematodes described above and under Accomplishments. These markers were also used to resolve a possible species complex found within the family of spiral nematodes. Under 2C, preliminary experiments were performed with formalin-fixed root knot nematodes (Meloidogyne enterolobii) from the USDA Nematode Collection to repair DNA damage that would lead to successful amplification of target genes.

4. Accomplishments
1. Alfalfa cyst nematode is identified as an emerging pest. Annual crop losses due to nematodes and diseases are estimated at 10% of the $9.3 billion alfalfa crop in the United States, with nematodes posing a significant emerging threat. ARS scientists from Beltsville, Maryland, and a university scientist from Idaho used anatomical and molecular features to identify juvenile and adult nematodes collected from alfalfa fields in Utah as the alfalfa cyst nematode. This finding represents the first record of alfalfa cyst nematode from this state and is further evidence of a possible new threat to alfalfa, which is grown in all 50 states and is the third most valuable field crop. This information will be used by plant pest management specialists and regulatory officials to manage and potentially contain this nematode to prevent inadvertent movement to additional areas.

2. Cactus cyst nematode was identified from Idaho. Cyst nematodes damage the roots of many kinds of plants throughout the world, including cactus plants, which serve as drought-tolerant forage for livestock and protect against soil erosion in rangeland systems. In this study, a team of ARS scientists and a colleague from Idaho analyzed anatomical and molecular traits to identify the cactus cyst nematode for the first time from a cactus in Idaho. This discovery is significant because new molecular information obtained for this species will facilitate future identifications and guide researchers and diagnosticians who need to distinguish among important cyst nematodes that occur in arid soils.

3. Goosegrass cyst nematode identified from corn in Indiana. Cyst nematodes cause significant economic and yield losses on $50 billion corn crop each year. In this study, ARS researchers from Beltsville, Maryland, along with university scientists in Indiana identified and characterized the goosegrass cyst nematode (Vittatidera zeaphila) from a corn field in Indiana using anatomical features and DNA markers. This work significantly expanded the molecular information useful for identifying this species and is important because it documents the second state in which this nematode was found. Scientists, extension agents and action agencies engaged in nematode research and control will use this research to track the distribution of this nematode on corn.

4. Guava root-knot nematode identified from cotton in Brazil. The guava root-knot nematode (Meloidogyne enterolobii) is an extremely virulent species of root-knot nematode, which has been steadily spreading into new crops and locations around the world and poses a significant threat to both vegetable and agronomic crops. A team of researchers including ARS scientists from Beltsville, Maryland, and Tifton, Georgia, with scientists from Brazil, used molecular diagnostics to identify this nematode for the first time from cotton in Brazil. This research also showed that the nematode could reproduce successfully on seven genotypes of cotton, several of which contained resistance to other root-knot nematodes. This nematode poses a major threat to the $6 billion cotton crop in the United States due to the total lack of known resistance to this nematode. This discovery will enhance the ability of first responders, and research scientists track the spread of this damaging pest and develop and deploy new management methods to areas where they are most needed.

5. Record of Any Impact of Maximized Teleworking Requirement:
Maximized telework suspended our ability to perform molecular diagnostics of important, but non-urgent nematode specimens. Routine culture transfers, preparation, and long-term storage of nematodes for our frozen specimen collection were disrupted, which may impact availability of specimens for future research. Data analysis was delayed due to downsized computer resources and more limited access to office files and notebooks. Spontaneous interactions with colleagues that spur collaboration and creative solutions to problems were significantly diminished.

Review Publications
Skantar, A.M., Handoo, Z.A., Kantor, M.R., Carta, L.K., Faghihi, J., Ferris, V. 2020. Characterization of Vittatidera zeaphila (Nematoda: Heteroderidae) from Indiana with molecular phylogenetic analysis of the genus. Journal of Nematology. 52:1-8. https:/doi:10.21307/jofnem-2020-024.
Skantar, A.M., Handoo, Z.A., Kantor, M.R., Hult, M.N., Hafez, S.A. 2019. First report of the cactus cyst nematode, Cactodera cacti, from a cactus garden in Idaho. Journal of Nematology. 51:1-6.
Handoo, Z.A., Skantar, A.M., Hafez, S.A., Kantor, M.R., Hult, M.N., Rogers, S.T. 2020. Molecular and morphological characterization of the alfalfa cyst nematode, Heterodera medicaginis, from Utah. Journal of Nematology. 19(52):1065-1081.
Galbieri, R., Davis, R.F., Scoz, L., Belot, J., Skantar, A.M. 2020. First report of Meloidogyne enterolobii on cotton in Brazil. Plant Disease.