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Development of a multiplex PCR assay for rapid and simultaneous detection of rubber tree pathogens Phytophthora spp. and P. palmivora

Nion Chirapongsatonkul^a,*, Kittichon U-taynapun^b, Thitikorn Chanwun^c, Nunta Churngchow^a

 
ABSTRACT:     Phytophthora spp. cause devastating effects on crops and natural ecosystems worldwide. In Thailand, P. palmivora is an important pathogenic species of the rubber plant (Hevea brasiliensis), reducing latex production. This study aimed to develop a PCR-based method to rapidly and simultaneously detect the genus Phytophthora and P. palmivora. A single round nested multiplex PCR with 4 primers, Phy1s/Phy2a/Pal1s/Pal2a, and a single round semi-nested multiplex PCR using 3 primers, Phy1s/Phy2a/Pal2a, were established. The obtained PCR products of 1025 bp and 650 bp represent the ITS1-5.8S-ITS2-28S region of the genus Phytophthora and P. palmivora, respectively. By using various amounts of DNA of a positive strain P. palmivora KBNM 9 and the vector containing ITS region as templates, the sensitivity of these methods was compared to the simple PCR. The single round semi-nested multiplex PCR was applied for the detection of P. palmivora in infected rubber leaves and soil as well as the leaves and bark collected from planted areas. The results of application and the sensitivity test using the DNA of P. palmivora mycelium are in agreement. Only one band of 650 bp representing the genus Phytophthora and P. palmivora was detected at low template number. The DNA from severe symptom-exhibiting and dehydrated leaves, however, showed negative results comparable to the classical isolation method that showed no Phytophthora and P. palmivora growth on selective media. This technique may be used to detect and manage Phytophthora infection in important crops.

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* Corresponding author, E-mail: nion.c@psu.ac.th

Received 29 Jan 2015, Accepted 20 Jul 2015