Rapid and sensitive detection of Phytophthora colocasiae associated with leaf blight of taro by species-specific polymerase chain reaction assay

摘要

The failure to adequately identify plant pathogens from culture-based morphological techniques has led to the development of culture-independent molecular approaches. The timely and accurate detection of pathogens is a critical aid in the study of epidemiology and biology of plant diseases. In the case of regulated organisms, the availability of sensitive and reliable assay is essential when trying to achieve early detection of pathogens. We developed and tested the PCR assay for detection of Phytophthora colocasiae, an oomycetes pathogen of leaf blight of taro and of rotting of taro tubers. The method described here is specific for P. colocasiae when tested across fungal, bacterial, and other Phytophthora species. In conventional (single-round) PCR, the limit of detection was 20 pg DNA for both primer sets, whereas in nested PCR the detection limit for both was 0.2 pg. In sampling studies, P. colocasiae-specific primers were used to detect leaf blight in infected leaves and tubers of taro cultivar. The causal pathogen P. colocasiae was detected by PCR from artificially infected tubers after 16 h of post inoculation, before any visible symptoms were present. The method was also tested to detect fungal DNA in infected leaves and infested soils. The PCR assay and direct tissue extraction methods provide tools which may be used to detect P. colocasiae pathogens in taro planting material and thus limit the transmission and spread of new, aggressive strains of P. colocasiae in taro-growing regions.