Poster: Diseases of Plants: Disease Detection & Diagnosis
Specific detection of the root-lesion nematode Pratylenchus scribneri using conventional and real-time PCR
G. YAN (1), D. Huang (2), G. Yan (3) (1) North Dakota State University, U.S.A.; (2) North Dakota State University, Department of Plant Pathology,, U.S.A.; (3) North Dakota State University, Department of Plant Pathology,, U.S.A.
Pratylenchus scribneri is a plant-parasitic root-lesion nematode causing economic damage to various crops worldwide. Identifying root-lesion nematodes to species using traditional morphological method is an arduous task requiring extensive training on nematode taxonomy. Consequently, molecular methods were developed to identify P. scribneri by conventional and real-time PCR using a species-specific primer set that was designed based on the alignment of 20 sequences of internal transcribed spacer of rDNA of seven Pratylenchus spp. The primer set specifically amplified DNA of P. scribneri but not DNA from five other Pratylenchus spp. or ten non-Pratylenchus spp. A single melt curve was observed indicating a high specificity according to real-time PCR analysis. The capability of specific PCR was tested using DNA extracts of root-lesion nematodes from 23 soil samples collected from various fields with five crops in North Dakota and Minnesota. The PCR primers and protocols were successfully used in P. scribneri identification. The conventional PCR could detect DNA of 1:8 dilution of two nematodes equivalent to 1/4 of a nematode. The real-time PCR was more sensitive and able to amplify DNA of serial dilutions (2 nematodes - 1/128 nematode) with Cq values ranging from 25.4±0.0 to 33.6±0.4. The developed PCR assays provide a rapid and reliable identification of P. scribneri, suitable for use in diagnostic laboratories for detection of field infestations with this nematode.