Development of a multiplex classical polymerase chain reaction technique for detection of Didymella bryoniae in infected cucumber tissues and greenhouse air samples

Abstract

Two sets of primer pairs were evaluated for their usefulness in detecting and identifying Didymella bryoniae (anamorph Phoma cucurbitacearum), causal agent of gummy stem blight, in infected cucumber plant tissues and air samples, using polymerase chain reaction. One primer in each pair was a universal primer that has been used for amplification of septate fungi. The other primer in each pair was an oligonucleotide designed from sequence information in the GenBank database to be specific to D. bryoniae. Each primer pair on its own was specific to genus level, but gave some nonspecific results for other Phoma or Didymella species. However, when these primer pairs were combined in a multiplex PCR, a unique result for D. bryoniae was obtained. The multiplex PCR gave positive results for cucumber tissue infected with D. bryoniae, and negative results for uninfected tissue and for tissue infected with Botrytis cinerea. Positive results were obtained from air samples collected with a rotation impaction sampler situated in a greenhouse containing cucumber plants infected with gummy stem blight. This method is rapid, sensitive, and accurate for detecting and identifying D. bryoniae in pure culture and plant tissue and could be applied to the detection of this organism in air samples. This method, therefore, could be a valuable technique for use in disease prediction.