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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

2013 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:
This is the final report for this project. Accomplished milestones included descriptions of new nematode species and populations new to the U.S., diagnostics for nematodes of regulatory importance, and detailed family trees that clarified species relationships. Root-knot nematodes (RKNs) are the most economically destructive nematodes in the world. Some parasites of alfalfa, grasses, and other crops share anatomical similarities that confound identification. Research with colleagues from Arizona, Utah, and California yielded molecular identifications and family trees for nematodes from a survey of turfgrass from the western U.S. New state records were reported for several known species. A new test was developed for identification of two closely related RKN; an unusual RKN from ornamental palm was also described. This research increases diagnostic accuracy of grass nematodes, enabling diagnosticians to make accurate recommendations for control. Cyst nematodes damage alfalfa rotation crops such as potato. During the project, pale potato cyst nematode (PCN) was described from the U.S. for the first time. ARS and APHIS scientists from Beltsville designed an assay for PCN that that is sensitive, specific, and faster than previous diagnostics. This test is now the APHIS protocol and has been adopted by researchers throughout the U.S. and Canada. A new PCN distinct from known species was subsequently discovered in Idaho and Oregon and described as a novel third PCN. This work supported regulatory action on infested Idaho fields to prevent further spread of this PCN, protecting trade of potatoes worth an estimated $1 billion. With Oregon colleagues, novel methods were developed for identification of lesion nematodes using soil extracted DNA. These assays distinguished anatomically similar species that are widespread on wheat. This approach greatly increased diagnostic capacity, and broadened grower awareness of the impact of the lesion nematode on wheat profitability ($51 million annual loss in the Pacific Northwest alone). Lesion nematodes affecting alfalfa may be similarly identified. One approach for broadening plant resistance to nematodes is to interfere with genes whose functions are essential for completion of nematode life cycles. This project included research to characterize the Hsp90 target gene from several plant-parasitic species, including cyst, root-knot, lesion, sting, stunt, and spiral nematodes. Comparison of genetic information revealed amino acids critical for gene function and DNA sequences unique to plant parasites. Hsp90-specific inhibitory molecules expressed in plants may increase their resistance to nematodes. Some nematodes consume fungi as well as plants, but old keys for nematode identifications have hampered their accurate diagnosis. Identification was provided for nematodes discovered on cheatgrass, a major weed in western wheat and alfalfa crop production. The weed stem contained a fungus on which the nematode was cultured in the lab. These findings demonstrated the need for further research on nematode associates of endophytes, providing a foundation for research included in the new project plan.


4.Accomplishments
1. New molecular diagnostic test for root-lesion nematode. Root-lesion nematode is one of the most widespread and economically important species restricting crop productivity in the Pacific Northwest. This nematode is difficult to quantify from plant roots for studies that require large-scale sampling, such as evaluation of wheat cultivars, rotation crops, and other management practices. Therefore, scientists from Oregon State University and ARS scientists from Beltsville, MD, Pullman, WA, and Pendleton, OR developed a highly sensitive molecular diagnostic assay to detect root-lesion nematode using DNA extracted directly from soil samples. Quantification of nematodes from soil using this test was shown to be comparable to manual counting methods. The results are significant because this method of quantifying lesion nematodes from soil is comparable to the commonly used but laborious methods currently in use, while being faster and more sensitive to perform. Commercial diagnostic laboratories, other scientists, action agencies, and extension agencies engaged in nematode research and control will use this research.

2. Identification of seed gall nematode from fescue. Some species of leaf or seed gall nematode are of concern to the forage grass and livestock industries for their association with bacteria that produce a harmful toxin. ARS scientists in Beltsville, MD performed urgent species identifications for seed gall nematodes originating from annual ryegrass in Oregon, determining the species to be one of regulatory importance, although scientists from Ft. Detrick, MD found that no toxic bacteria were present. Additional gene sequences were determined for seed gall nematodes of grasses and related nematodes and used for the design and implementation of highly sensitive diagnostic molecular tests. This research is significant because it established the presence of this regulated seed gall nematode in the United States for the first time and provided an assay capable of rapidly distinguishing regulated seed gall nematodes from related but unregulated species. This assay will be used by diagnosticians during future surveys to determine the extent of infestation of these bacteria-carrying seed gall nematodes.

3. Identification of corn cyst nematode from Greece. The corn cyst nematodes are important pests of corn, a crop of major importance for human consumption, as feed for livestock, and as a source of bioenergy. A team of ARS scientists from Beltsville, MD and colleagues from Greece analyzed anatomical and molecular traits to identify the corn cyst nematode isolated from a cornfield in Greece, the first report of this nematode from that country. This discovery is significant because new molecular information obtained for this unusual European population will facilitate future identification of corn cyst nematode. This anatomical and molecular description will guide researchers and diagnosticians identifying economically important cyst nematodes of cereal and bioenergy crops.


Review Publications
Yan, G., Smiley, R.W., Okubara, P.A., Skantar, A.M., Reardon, C.L. 2013. Developing a real-time PCR assay for detection and quantification of Pratylenchus neglectus in soil. Plant Disease. 97(6):757-764.

Skantar, A.M., Handoo, Z.A., Zanakis, G.N., Tzortzakakis, E.A. 2012. Molecular and morphological characterization of the corn cyst nematode, Heterodera zeae, from Greece. Journal of Nematology. 44(1):58-66.

Last Modified: 4/23/2014
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