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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
2009 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. DNA markers, including ribosomal, mitochondrial, and nuclear Hsp90, were analyzed from several RKN populations that had been isolated from turfgrass throughout the western United States. A database of sequence information was created for the development of new molecular diagnostic assays for accurate identification of these RKNs. In addition, several populations of potato cyst and tobacco cyst nematodes (Globodera spp.) were molecularly characterized for ribosomal, mitochondrial, and nuclear markers, expanding the sequence databases that will enhance the accuracy of cyst nematode species identifications.


4.Accomplishments
1. Identification of nematodes as ornamentals. Root-knot nematodes are destructive to the roots of many kinds of plants, including alfalfa, grasses, and other forage crops. In the present study, ARS scientists in Beltsville, MD described the anatomical and molecular characteristics of an unusual population of root-knot nematode (Meloidogyne arenaria) isolated from a diseased palm-like ornamental tree (traveler's tree). This research is significant because it helps to further establish the wide range of morphological and molecular variation seen in this common species of root-knot nematode. Because M. arenaria can infect a wide range of forage legumes and grasses and is of economic importance worldwide, scientists, regulators, and extension agencies will use this research to accurately identify this nematode on a variety of crops and recommend appropriate management practices.

2. Molecular identification of sting nematode. Sting nematodes are highly destructive to several plants that grow in sandy soils, including many fruit and vegetable crops, soybeans, turfgrasses, and pine trees. This species causes widespread damage by piercing the plant roots with a long mouth spear or stylet. In the present study, ARS scientists in Beltsville, MD and a University of Delaware extension specialist used anatomical and molecular methods to identify Belonolaimus longicaudatus from soybeans, the first report of this species in the state of Delaware. This research is significant because it expands information regarding the geographical range of this nematode and further describes the morphological and molecular variation present in the species. Because B. longicaudatus can infect a wide range of economically important crops and grasses, scientists, regulators, and extension agencies will use this research to accurately identify this nematode and recommend appropriate management practices.


6.Technology Transfer

Number of Web Sites Managed1
Number of Other Technology Transfer5

Review Publications
Yan, G., Smiley, R., Okubara, P.A., Skantar, A.M., Easley, S.A., Sheedy, J.G., Thompson, A.L. 2008. Detection and Discrimination of Pratylenchus neglectus and P. thornei in DNA Extracts from Soil. Plant Disease. 92(11):1480-1487.

Skantar, A.M., Carta, L.K., Handoo, Z.A. 2008. Molecular and morphological characterization of an unusual Meloidogyne arenaria population from traveler’s tree Ravenala madagascariensis. Journal of Nematology. 40(3):179-189.

Last Modified: 12/28/2014
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