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United States Department of Agriculture

Agricultural Research Service

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Research Project: Improving Extraction and Identification Methodologies for the Plant Pathogenic Nematodes Bursaphelenchus and Aphelenchoides

Location: Nematology Laboratory

Project Number: 8042-22000-275-02
Project Type: Interagency Reimbursable Agreement

Start Date: Oct 01, 2012
End Date: Apr 07, 2014

Objective:
An overview of the objectives are development of new methods for isolating Bursaphelenchus and Aphelenchoides found in APHIS surveys for nematode parasites of palms including coconut palm, and on the development of new DNA markers for improving the taxonomy of the taxonomic group containing these nematodes. More specifically: 1. Improve extraction procedures for Bursaphelenchus cocophilus (the red ring nematode) and Aphelenchoides sp. 2. Develop nematode-specific ribosomal primers or other molecular markers such as CO1, Hsp90 or calreticulin as marker genes for identification of Bursaphelenchus and Aphelenchoides.

Approach:
The pinewood nematode Bursaphelenchus xylophilus will be collected locally to culture on fungi to use as experimental organisms. Next ARS will obtain palm tissue samples typical of palm weevil-associated samples to be received from the APHIS survey. Extraction methods will be tested first on relatively large numbers of the common B. xylophilus to determine specific gravity to more efficiently detect the targeted B. cocophilus to optimize an extraction protocol such as those detailed for different potato parasitic nematodes. These extraction methods include two-flask extraction, standard sieving, standard 1 M sugar flotation, and specific-gravity-optimized sugar flotation. By determining the specific gravity of these nematodes ARS should be able to more gently isolate the nematodes in a smaller volume of liquid than used by previous methods. Because there are few GenBank sequences of nematodes of the Aphelenchida, the molecular information has sparse coverage among many taxonomic groups and is therefore less useful for identification. In the experience of ARS, ribosomal DNA primers that are available for other nematodes need to be optimized for this group to exclude fungi and commonly associated nematodes of the Rhabditida that feed on bacteria and insects. Therefore, the arsenal of primers that may more efficiently generate reliable sequence from these economically important aphelenchid species will be expanded. These markers include the following: ITS ribosomal DNA (a species-level marker surprisingly not present in GenBank for B. cocophilus), nuclear Hsp 90 DNA, and nuclear calreticulin, for which a sequence of B. xylophilus is available in GenBank. Optimized primers for these genes will be tested on B. cocophilus, B. xylophilus, and various Aphelenchoides species to evaluate their utility for rapid and accurate molecular detection.

Last Modified: 10/20/2014
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