A multiplex PCR assay for three pathogenic Phytophthora species related to kiwifruit diseases in China

Phytophthora cactorum, P. cinnamomi and P. lateralis were reported to be pathogenic on kiwifruit trees in the main planting areas of China. We attempted to simultaneously detect the three pathogens using a multiplex polymerase chain reaction (PCR) and to survey their occurrence in the main production areas. Because of the need to combine different primer pairs for the multiplex PCR and the low specificity of published specific primers for P. cactorum, P. cinnamomi and P. lateralis, new species-specific primers for the three species were designed based on the ras-related protein gene, Ypt1. The specificity of the designed primers was demonstrated using 52 isolates, including 44 Phytophthora species, three Pythium species, and three other soil-borne pathogens. A multiplex PCR method for the simultaneous detection of P. cactorum, P. cinnamomi and P. lateralis was established, and the three pathogens were detected in artificially and naturally infested soils, indicating that these markers can be used in the diagnosis of kiwifruit Phytophthora diseases. In a survey of these pathogens in the main kiwifruit planting areas of China, 99 soil samples were collected at different locations and in different seasons and subjected to the new method, and the distribution of the three pathogens in the main kiwifruit planting areas of China was determined.