A molecular diagnostic method for detecting Nacobbus in soil and in potato tubers

Abstract

AbstractSpecies of the genus Nacobbus have the potential to reduce yields of major food crops such as potato, sugar beet and tomato in many parts of the world, thus warranting a quarantine effort to avoid their introduction. Molecular studies offer a new method for routine quarantine diagnostics for this nematode that will be faster and more sensitive than previous methods. A primer set was designed from Nacobbus ITS sequences and their specificity confirmed. DNA was extracted from nematodes, soil and potato tubers for use in PCR. Optimised PCR conditions were established and the PCR products were separated on 2% agarose gels, showing that specific ITS primers for the detection of Nacobbus generated a single PCR product, although band size varied slightly between species and soil isolates. The product was generated from DNA extracted from all the Nacobbus samples but not from other nematodes tested (Pratylenchus, Radopholus, Meloidogyne, Globodera, Heterodera). No bands were generated from the uninfested control soil and control tuber DNA samples, thus demonstrating the specificity of the primers. For the first time, Nacobbus was detected in soil and tuber samples using molecular approaches. These results have important applications not only in analysing advisory samples but also in the screening of material for quarantine purposes.