Nested PCR assay for detection of Corynespora leaf fall disease caused by Corynespora cassiicola

Corynespora leaf fall disease of Hevea brasiliensis caused by Corynespora cassiicola has been a major cause of severely reduced rubber latex production in Asia and Africa. The aim of this study was to determine whether physiological races of C. cassiicola exist in the Hainan and Yunnan provinces, China, and to accelerate and simplify the process of diagnosis. We conducted pathogenicity tests with 17 C. cassiicola isolates on detached leaves of four different Hevea rubber clones (genotypesPR107, Dafeng 95,RRIM600 and Reyan 7-33-97). Pathogenicity tests showed that 16 of the isolates were pathogenic to RRIM 600, which confirms these isolates as C. cassiicola race 1. An initial PCR was performed to amplify a portion of the ITS1-5.8S-ITS2 region using a C. cassiicola species-specific primer set (CCF and CCR-2).DNA fragments of 272 bp in length indicated the presence of C. cassiicola. To improve the sensitivity, a nested PCR assay was developed to obtain DNA fragments of the expected length (152 bp)using diluted (1 : 100)amplified product from the initial PCR as the template and species-specific oligonucleotides CCF and CCR-1 as primers. The lowest concentration of fungal DNA that could be detected by nested PCR was 168 fg, while its concentration in the initial PCR was 16.8 pg. Therefore, the sensitivity of detection was enhanced 100-fold using nested PCR. In addition, the pathogen could be detected from artificially infected Hevea rubber trees 3 days after inoculation. Due to its high sensitivity, specificity, and reliability, nested PCR is useful for the screening and certification of young C. cassiicola-free Hevea rubber plants for distribution to commercial growers.