Project Number: 1275-22000-254-00
Project Type: Appropriated

Start Date: Mar 18, 2008
End Date: Mar 17, 2013

Objective:
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.

Approach:
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.