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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
2010 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
Molecular Diagnostics of Nematodes. Root-knot nematode (RKN) species are parasitic on a wide range of host plants, including alfalfa, turfgrasses, and numerous other crops. The anatomical features of many RKNs are similar, confounding accurate species identification. Molecular characterizations were conducted for several RKN populations from turfgrass in Arizona, California, Oregon, and Washington and compared with populations of known turfgrass RKN species from the U.S. and the U.K. This research provides the first comprehensive molecular analysis of root-knot nematodes on turf in the western U.S. In addition, comprehensive molecular characterizations were completed for sting nematode on soybean in Delaware and corn cyst nematode from Greece.


4.Accomplishments
1. Rapid, sensitive molecular test for potato cyst nematodes. 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 developed a highly sensitive molecular method for identification and detection of potato cyst and lookalike tobacco cyst nematodes. This research is significant because the method is highly sensitive, specific, and faster than conventional molecular methods of identification. Because the pale cyst nematode is regulated as a quarantine pest by many countries and causes economic damage to potato worldwide, scientists, regulators, and extension agencies will use this research to better identify and prevent the spread of this nematode.


Review Publications
Nakhla, M.K., Owens, K.J., Li, W., Wei, G., Skantar, A.M., Levy, L. 2010. Multiplex real-time PCR Assays for the identification of the potato cyst and tobacco cyst nematodes. Plant Disease. 94(8):959-965.

Last Modified: 12/22/2014
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