Development of real-time and conventional PCR assays for identifying stubby root nematode paratrichodorus allius

Abstract

Paratrichodorus allius is an important pest on many crops, particularly on potato due to its ability to transmit Tobacco rattle virus causing corky ringspot disease on tubers. Detection and identification of P. allius are important for effective disease management. In this study, a rapid and reliable molecular diagnosis of this nematode targeting internal transcribed spacer ribosomal DNA was established. The specificity of the designed primers was evaluated using 29 nematode species and results showed that a single amplicon was produced from DNA of P. allius only. Detection sensitivity analysis indicated that a 9.6 × 10−4 ng of DNA template could be detected by conventional PCR and 1.92 × 10−4 ng of DNA by real- time PCR. The PCR assays amplified DNA of stubby root nematodes isolated from 18 soil samples in North Dakota and Minnesota, which were confirmed as P. allius by sequencing. Both conventional PCR and real-time PCR assays amplified target nematodes from complex nematode communities, supporting the success of this molecular diagnosis of P. allius. This is the first report of P. allius identification using the real-time PCR method and from nematode communities with other nematodes using conventional PCR. The new PCR assays provide rapid species identification and are suitable for use in diagnostic laboratories and detection of field infestations with P. allius.