Cultural and PCR‐based detection of Septoria musiva in inoculated hybrid poplar stems

The fungal pathogen Septoria musiva can be difficult to isolate from cankers that result from its colonization of poplar stems, and its persistence in these cankers has not been well studied. In order to compare cultural and polymerase chain reaction (PCR)‐based assays for detection of S. musiva in cankers, stems of susceptible hybrid poplar clone NC11505 were wounded and inoculated in August 2003. At 8, 16, 24 and 32 weeks after inoculation (October and December 2003, February and April 2004, respectively), 110 inoculated stems (plus controls) were harvested and a semiselective culture medium was used in attempts to detect the pathogen in bark and wood. Six chips of bark and six chips of underlying wood from one half of each canker were incubated on the semiselective medium for 2 weeks until pycnidia and conidia of S. musiva could be identified. The number of positive cankers and positive chips (out of six attempts per tissue per canker) was recorded. The remaining halves of cankers from subsets of 70 inoculated stems (plus controls) of those harvested in October 2003 and April 2004 were tested using a PCR‐based assay. Three chips of bark and three chips of underlying wood were ground, and DNA was extracted and then amplified using S. musiva‐specific primers designed from the internal transcribed spacer (ITS) region of nuclear rDNA repeats. The number of positive cankers and positive chips (out of three attempts per tissue per canker) was recorded. For both assays, the number of positive cankers and the number of positive chips per canker decreased with time. Using either assay, however, the pathogen was still detected from at least 49% of cankers at 32 weeks after inoculation.