A PCR-based method to detect species of Gondwanamyces and Ophiostoma on surfaces of insects colonizing Protea flowers

Flower heads of economically important members of the genus Protea L.amature into conspicuous, often long-lived infructescences, which in South Africa are commonly colonized by species of the ophiostomatoid fungi Gondwanamyces G.J.Marais & M.J. Wingfield and Ophiostoma Syd. & P. Syd. It is suspected that these fungi are transported between infructescences by insects. To develop techniques that would enable detection of ophiostomatoid fungi on insects, primers GPR1 and OSP1 were designed based on unique 28S ribosomal DNA sequences of Gondwanamyces and Ophiostoma from Protea. Multiplex polymerase chain reaction of these primers, combined with universal primer LR6, yielded fragment lengths of 885 and 637 bp. Positive amplification was achieved from as little as 30 and 45 pg of fungal genomic DNA for Gondwanamyces and Ophiostoma, respectively, and fragments of identical lengths were amplified from insects artificially inoculated with these fungi. No other tested fungal species showed amplification with GPR1 or OSP1 and LR6. Using these primers two insect species (Genuchus hottentottus Fabricius and Oxycarenus maculates Stal.) collected from Protea repens L.ainfructescences were confirmed as carriers of Gondwanamyces proteae (M.J. Wingfield et al.) G.J. Marais & M.J. Wingfield and Ophiostoma splendens G.J. Marais & M.J. Wingfield, respectively. The method developed in this study represents a rapid detection system that can be used to understand the relationship between insects and ophiostomatoid fungi found associated with flowers of South African species of Protea.