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United States Department of Agriculture

Agricultural Research Service

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Research Project: MOLECULAR STRATEGIES FOR DETECTION AND IDENTIFICATION OF NEMATODES INFECTING FORAGE LEGUMES AND DEVELOPMENT OF HOST RESISTANCE

Location: Nematology Laboratory

2011 Annual Report


1a.Objectives (from AD-416)
Objective 1: Discover, refine, and implement improved molecular approaches to modernize classification and aid the identification and control of nematodes in alfalfa cropping systems. Objective 2: Identify and develop novel sources of resistance to nematodes.


1b.Approach (from AD-416)
1. Molecular markers, including ribosomal, mitochondrial, Hsp90, and other nuclear genes will be used to develop new diagnostic assays, including RFLPs, and conventional or real-time PCR assays for Meloidogyne spp., Pratylenchus spp., and Ditylenchus spp. 2. 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. 3. Bioinformatic analysis of EST sequences of selected species will be used to uncover genes that meet selective criteria for use as diagnostic markers and for phylogenetic comparisons. Selected gene sequences will be used to design oligonucleotide primers for amplification,in polymerase chain reactions, of specific gene sequences from populations of phytopathogenic nematodes to evaluate them for genus level, species-wide, and species-specific identification. 4. Novel gene targets from Meloidogyne spp. and Pratylenchus spp. will be advanced through a pipeline of cloning, mRNA expression profiling, and functional characterization, leading to identification of specific genes with the best potential for further development into novel control methods. 5. Based on the information obtained above, selected genes will be targeted for silencing by gene-specific dsRNA; nematodes treated with dsRNA will be monitored for the ability to move, infect host plants, feed and reproduce. Effects on gene expression will be monitored by mRNA extraction and PCR using gene-specific primers directed at the gene to be silenced. Genes that show the most promising phenotypes in the soaking experiments will be transformed into M. truncatula hairy roots using Agrobacterium-mediated transformation methods. Transgenic plants containing target gene RNAi will be infected with root-knot or lesion nematodes and assessed for decreases in nematode infection and reproduction; knock out of target genes will be verified through RT-PCR and in situ detection methods.


3.Progress Report
An urgent need exists for 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. We have molecularly characterized cyst nematodes (species of Cactodera and Punctodera) isolated during surveys for potato cyst nematodes (PCN). These species are sometimes found on weeds or grasses and can be morphologically confused with PCN of quarantine concern. Similarly, dozens of root-knot nematode populations isolated from turfgrass throughout the western U.S. were molecularly compared with DNA sequences generated from known reference populations. The development of molecular diagnostics for these root-knot nematodes (including species that can affect alfalfa, grasses and other hosts) has progressed substantially ahead of schedule and is nearing completion. Finally, we are also developing molecular diagnostics for identification of cyst nematodes common on cereal crops in the Pacific Northwest. All of these molecular methods will enable diagnosticians to more rapidly identify nematode species of concern in alfalfa and other forage agroecosystems.

The development of nematode phylogenies that integrate molecular and morphological data with available information on biogeography, pathogenicity, and host range can yield predictions about disease-causing potential whenever a new species is discovered. Relationships among and within species of root-knot nematodes were established through the construction of phylogenetic trees based on multi-gene molecular data. Bioinformatic analysis localized the position of the heat shock protein gene Hsp90 within two root-knot nematode genomes, confirming the single copy structure predicted in plant-parasitic nematodes. This gene, which is involved in critical life processes in nematodes, was also isolated and characterized in several other root-knot, cyst, and fungal-feeding nematode populations. In addition, molecular characterization and phylogenetic analysis were completed for an above ground plant-feeding nematode associated with fungi inside cheatgrass, a common weed contaminant of alfalfa. The above analyses will aid in the prediction of the potential of nematodes to cause crop losses when new nematode species or populations are discovered.


4.Accomplishments
1. Description of an unusual new potato cyst nematode from Oregon and Idaho. 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. ARS scientists in Beltsville, MD, and Corvallis, OR, and a scientist at Oregon State University described a morphologically and molecularly unusual potato cyst nematode from soil associated with potato fields in Oregon and Idaho. This research is significant because it describes a new population that may become a threat to potato production. 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 population from the currently regulated species and help prevent further infestations.


Review Publications
Castagnone-Sereno, P., Skantar, A.M., Robertson, L. 2011. Molecular tools for diagnostics. In: Jones, J., Fenoll, C., Gheysen, G., editors. Genomics and Molecular Genetics of Plant-Nematode Interactions. New York, NY: Springer Science + Business Media B.V. p. 443-464.

Skantar, A.M., Handoo, Z.A., Carta, L.K., Zasada, I. A., Ingham, R.E., Chitwood, D.J. 2011. Morphological and molecular characterization of Globodera populations from Oregon and Idaho. Phytopathology. 101:(4)480-491.

Last Modified: 4/16/2014
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