Identification of Anguina funesta from Annual Ryegrass Seed Lots in Oregon

Abstract

In 2010, seed galls containing Anguina sp. were isolated from 14 annual ryegrass (Lolium multiflorum) seed lots submitted for phytosanitary testing. To identify the species present, the ITS1 region of the ribosomal DNA of the nematodes from the seed lots was analyzed using a PCR-RFLP method. All nematodes produced a single 540-bp DNA amplicon, which was digested with three restriction enzymes, HaeIII, HinfI, and TaqI. The resulting RFLP patterns matched those of A. funesta. To confirm these results, 525 bp of the DNA amplicon was analyzed by DNA sequencing and BLAST analysis, which verified the sequence was identical to A. funesta (Genbank Accession nos. AF363095, AF363096, and AF363104). Because of the association of A. funesta with Rathayibacter toxicus, a second PCR test was conducted to determine if the bacterium was present in the seed lots. A 200-bp DNA amplicon was amplified from two seed galls, sequenced, and subjected to BLAST analysis. Analysis of the entire DNA sequence failed to identify the bacterium present, although testing by USDA-APHIS verified the bacterium was not R. toxicus. This is the first report of A. funesta in the US; R. toxicus was not found with this detection. Accepted for publication 14 September 2012. Published 24 October 2012.