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

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 seminested 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.

Identification of the Causal Agent of Cocoa Pod Rot Disease from Various Locations

“…It proved that 13 collected isolates from rotting pods of 8 surveyed locations were P. Palmivora, the causal agent of black pod diseases on cocoa. The previous research of Chirapongsatonkul et al (2015) also found that molecular identification of P. palmivora isolates with PCR method using specific primers of Pal1s and Pal2a could amplify the target DNA at 650 bp.…”

“…Molecular identification was aimed to ensure that the obtained isolate was P. palmivora. This analysis was conducted using PCR method with specific primers for P. palmivora (pal1s and pal2a) with target size of 650 bp (Chirapongsatonkul et al, 2015). DNA extraction of P. palmivora isolates was performed using CTAB method (Subandiyah, 2003).…”

Gugur Daun Tanaman Karet Akibat Penyakit Phytophthora Spp. Di Perkebunan Karet Jember, Jawa Timur

“…juga ditemukan dari hasil isolasi pada bagian ranting muda atau dekat dengan tunas yang gejalanya hampir sama dengan di tangkai daun dan ibu tulang daun yaitu bercak coklat kehitaman (Gambar 3c). Menurut Soepadmo ( 1 9 7 8 ) dan Chirapongsatonkul et al (2015) gejala penyakit gugur daun Phytophthora yaitu terdapat bercak coklat tua atau hitam pada tangkai daun dengan bintik putih di tengahnya yang merupakan lateks membeku. Pada umumnya bercak terdapat pada bagian pangkal tangkai daun dan kadang-kadang terdapat di ibu tulang daun maupun di ranting.…”

Phylogenetic analyses and cross‐infection studies of Phytophthora species infecting cacao and durian in South‐Central Mindanao, Philippines

“…Aside from P. palmivora, which is considered as the major Phytophthora pathogen of plantation crops, there are many other species of Phytophthora, which are known to attack these crops as reported worldwide (Chirapongsatonkul et al, 2015;Surujdeo-Maharaj, 2016). However, the ambiguity on the identity at the species level of Phytophthora in the Philippines is still a challenge as few studies have been made especially on molecular characterization.…”