Development of specific PCR primers for identification and detection of Rhizopycnis vagum

Rhizopycnis vagum is a recently described coelomycete known to belong to the complex of root rot pathogens contributing to vine decline of cucurbits in several parts of the world. However, the fungus has also been reported to infect tomato, and as an endophytic associate of mycorrhizal roots of wild, asymptomatic Pinus halepensis and Rosmarinus officinalis plants in Italy. To accelerate epidemiological and ecological investigations on this fungus, a PCR primer pair was developed. Primers Rv1-F and Rv1-R were designed, based on alignment of internal transcribed spacer (ITS) sequences (ITS1-5.8S-ITS2), which amplified a 396-bp fragment from all R. vagum isolates tested, including isolates pathogenic to melons and endophytic isolates from mycorrhizae. Specificity of the primer pair was verified both in silico (BLAST searches using each primer string as a query) and in PCR assays, where the primers failed to amplify DNA from any isolate of fungi taxonomically related to R. vagum (e.g. Massarina walkeri and Stagonospora spp.) and other vine decline and common soilborne pathogens (e.g. Monasporascus cannonballus, Acremonium cucurbitacearum, Fusarium spp. and Rhizoctonia solani). Under optimum conditions, detectable amplification of the specific sequence required 0.05 pg of target DNA. Amplification of the expected 369-bp fragment was also obtained from DNA root extracts of nearly asymptomatic Cucumis melo plants inoculated with R. vagum under greenhouse conditions.