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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR STRATEGIES FOR DETECTION AND IDENTIFICATION OF NEMATODES INFECTING FORAGE LEGUMES AND DEVELOPMENT OF HOST RESISTANCE
2012 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:
Descriptions of new nematode populations isolated from alfalfa and other forage legumes, grasses, or their rotation crops help to distinguish one species from another and also document the geographic spread of nematodes. The integration of molecular and anatomical data from nematodes with available information on host range and disease severity can yield nematode family trees that are useful for predicting disease-causing potential whenever a new species is discovered. New molecular markers and diagnostic tools are urgently needed for identification and control of nematodes of regulatory and economic importance. Significant progress on objective 1 included identification of corn cyst nematode from Greece through analysis of anatomical features and gene markers. The relationship of this population to other cyst nematodes was determined through analysis of family trees based upon several gene markers. Progress was also made on the development of diagnostic tests useful for the identification of lesion and cyst nematodes common on cereal crops in the Pacific Northwest. In addition, gene sequences were obtained for seed gall nematodes of grasses and related nematodes in order to provide fundamental knowledge needed for the design and implementation of highly sensitive diagnostic tests needed for identifying species of regulatory importance that may be detected during upcoming field surveys. Related to objective 2, Hsp90, a heat shock protein gene involved in critical life processes in nematodes, was sequenced from several spiral nematode populations from Turkey, Idaho, and Hawaii. Together, these molecular results will support improved identification and control recommendations for nematode species of concern in alfalfa and other forage agroecosystems.


4.Accomplishments
1. Molecular characterization of root-knot nematodes from turfgrass. Root-knot nematodes (RKN) are parasitic on a wide range of host plants, including alfalfa, turfgrasses, and numerous other crops. The anatomical features of many root-knot nematode species are very similar, which complicates accurate species identification based on microscopic examination alone. In this study, an ARS scientist and colleagues from Arizona, California, and Utah used multiple DNA markers to identify and establish relationships among dozens of RKN populations that were isolated from turfgrass throughout the western United States, and developed a new molecular test to discriminate two closely related species. This research is significant because new molecular information obtained for these populations will facilitate future identification of root knot nematodes. This report will aid researchers and diagnosticians in accurately identifying economically important root-knot nematodes that are difficult to tell apart by comparing anatomical features alone.


Review Publications
McClure, M.A., Nischwitz, C., Skantar, A.M., Schmitt, M.E., Subbotin, S.A. 2012. Root-knot nematodes in golf course greens of the western United States. Plant Disease. 96(5):635-647.

Last Modified: 11/22/2014
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