Real-time-PCR for detection and identification of Anguina funesta, A. agrostis, A. tritici, and A. pacificae

Authors: LI, WENBIN - Animal And Plant Health Inspection Service (APHIS); NAKHLA, MARK - Animal And Plant Health Inspection Service (APHIS); Skantar, Andrea; ZONGHE, YAN - Animal And Plant Health Inspection Service (APHIS)

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/30/2014
Publication Date: 5/18/2015
Publication URL: http://handle.nal.usda.gov/10113/62030
Citation: Li, W., Zonghe, Y., Nakhla, M.K., and Skantar, A.M. 2015. Real-time-PCR for detection and identification of Anguina funesta, A. agrostis, A. tritici, and A. pacificae. Plant Disease. DOI:10.1094/PDIS-09-14-0959-RE.

Interpretive Summary: Plant-parasitic nematodes are microscopic worms that attack plant roots and cause an estimated ten billion dollars of crop loss each year in the United States and 100 billion dollars globally. Species of seed gall nematodes are regulated as quarantine pests by many countries due to economic damage to wheat or grasses or the ability to carry bacteria that are toxic to livestock. Growers and regulatory officials face an enormous problem in that the existing molecular methods for distinguishing these nematodes are slow and occasionally inaccurate. In the present study, scientists from APHIS and ARS designed a new molecular diagnostic test that can rapidly detect and distinguish four different seed gall nematodes of wheat and grasses. This research is significant because the new assay is species-specific, highly sensitive, and faster than existing molecular methods for identification of seed gall nematodes. This test will be useful for federal and state diagnostic labs, domestic and international research scientists, regulatory personnel, or extension agencies for identifying and preventing further damage caused by seed gall nematodes.

Technical Abstract: A number of seed, leaf, and stem gall nematodes are of significance to the forage and landscape grass and livestock industries. In North America, the bentgrass nematode, Anguina agrostis, reduces seed production on Agrostis tenuis and several other grass species. Anguina funesta is a seed-gall nematode that is most significant for its association with the toxigenic bacteria Rathayibacter toxicus. The wheat seed gall nematode A. tritici causes significant damage to wheat and other cereals; while it has been found in many countries worldwide, it has not been detected in the United States since 1975. Molecular methods based upon sequence variation in the ribosomal internal spacer region are useful for accurate identification of Anguina species. Described herein are new species-specific primers and TaqMan probes for real-time PCR identification of Anguina agrostis, A. funesta, A. tritici, and A. pacificae. Primer and probe combinations were each specific for the intended species and were sensitive enough to detect 10 copies of nematode ribosomal DNA. PCR was also specific and sensitive in multiplex assays that included genus-specific as well as species-specific primers and probes. These standardized real-time PCR protocols should facilitate fast and accurate identification of Anguina species by diagnostic laboratories.