Development and testing of oligonucleotide probes for detection and identification of some fungal pathogens and endophytes of conifers

Conifers do not display symptoms of foliage disease for up to 2 years after infection by pathogenic fungi. Symptomless latent infections of nursery stocks are responsible for epidemic and economic losses caused by needle cast and needle blight of conifer seedlings. Conifer seedlings sold to Christmas tree growers or foresters pass inspection even though they carry symptomless infections. Rapid, easy, and accurate detection of latent infections will help to develop a certification system for nursery stocks as pathogen free prior to shipment for outplanting. In this study, we developed molecular methods based on PCR and dot-blots assays for detection and identification of some of the most serious and damaging diseases of certain conifer trees. Internal transcribed spacers (ITS) of ribosomal DNA were sequenced and species-speck oligonucleotide probes were developed for more than 20 pathogenic and endophtic fungi of conifer trees: Douglas fir, spruce, Fraser fir, juniper and pine. The primer pair for each species was tested in both direct and nested PCR assays of DNA from mycelium, fruiting bodies, and needles. Almost all primer pairs amplified species-specific PCR products of specific sizes from their target DNA in direct PCR of mycelium or fruiting bodies of fungi. Cross-reactions were only observed between the two species of the same Genus that did not have any or sufficient variability in the ITS region. Also each primer pair detected the intended fungus directly in infected needles with or without symptoms. Especially, primer pairs developed for detection of Rhabdocline and Swiss needle cast of Douglas fir, Phomopsis tip blights of juniper, Cyclaneusma, Lophodermium, Dothistroma and Brown spot needle cast of pine were highly sensitive and accurate in detections from symptomless needles of current year growth. Nested PCR with primer pairs increased the sensitivity of detection and allowed the detection of latent, low level infections at species-specific optimum annealing temperatures. The technique was useful for amplification of endophytes from symptomless, pathogenic fungi-free current year's needles as they are present at very low incidences. The primers amplified PCR products of expected size from target sequences in all assays. RFLP analysis of PCR products amplified by the primers from needles in both nested and direct PCR amplifications confirmed the identities of amplicons to their respective target DNA sequences.; In dot-blot assays, the probes tested specifically hybridized to their intended target DNA's at determined optimum hybridization temperatures. The probes differentiated the fungi even at subspecies level as achieved with probes of Rhabdocline taxa. As a result, the molecular assays developed in this study could be used for inspection and certification of nursery stocks or seedlings prior to sale.