Location: Nematology Laboratory
Project Number: 1245-22000-275-02
Start Date: Oct 01, 2012
End Date: Apr 07, 2014
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.