Multiplex End-Point PCR for the Detection of Three Species of Ophiosphaerella Causing Spring Dead Spot of Bermudagrass.

A multiplex end-point polymerase chain reaction (PCR) assay was developed for identifying the three-fungal species in the genus Ophiosphaerella that cause spring dead spot (SDS), a devastating disease of bermudagrass. These fungi are difficult to identify by morphology because they seldom produce pseudothecia. To achieve species-specific diagnosis, three pairs of primers were designed to identify fungal isolates and detect the pathogen in infected roots. The internal transcribed spacer region, the translation elongation factor 1-α, and the RNA polymerase II second-largest subunit were selected as targets and served as templates for the design of each primer pair. To achieve uniform melting temperatures, three to five random nucleotide extensions (flaps) were added to the 5' terminus of some of the designed specific primers. Temperature cycling conditions and PCR components were standardized to optimize specificity and sensitivity of the multiplex reaction. Primers were tested in multiplex on DNA extracted from axenic fungal cultures and from field-collected infected and uninfected roots. A distinct amplicon was produced for each Ophiosphaerella sp. tested. The DNA from Ophiosphaerella close relatives and other common bermudagrass pathogens did not amplify during the multiplex assay. Metagenomic DNA from infected bermudagrass produced species-specific amplicons while DNA extracted from noninfected roots did not. This multiplex end-point PCR approach is a sensitive and specific molecular technique that allows for correct identification of SDS-associated Ophiosphaerella spp. from field-collected roots.

Hormetic Effects of Thiophanate-Methyl in Multiple Isolates of Sclerotinia homoeocarpa.

Twenty-eight isolates of Sclerotinia homoeocarpa, causal agent of dollar spot disease in turf, were assessed for fungicide hormesis at sublethal concentrations of thiophanate-methyl (T-methyl). Each isolate was grown in corn meal agar amended with 11 concentrations of T-methyl (30,500 to 0.047 µg/liter), and the area of mycelial growth was determined relative to the control. Three replicates were used per concentration, and the experiment was repeated three to five times for each isolate. Reference isolates (EC50 > 20 µg/liter), with no prior history of T-methyl exposure, were highly sensitive and not stimulated by low doses. Likewise, no stimulation was observed in two highly sensitive isolates (EC50 > 30 µg/liter) that had been preconditioned by exposure to T-methyl, or in four T-methyl-tolerant isolates. Seventeen (81%) preconditioned T-methyl-tolerant isolates (EC50 = 294 to1,550 µg/liter) had statistically significant growth stimulation, in the range of 2.8 to 19.7% relative to the control. These results support that hormesis (low-dose stimulation, high-dose inhibition) is a common dose response in preconditioned S. homoeocarpa, particularly in response to subtoxic doses of T-methyl.