Abstract:Black-rotten disease in Psidium guajava, caused by Botryosphaeria rhodina, is one of the most destructive plant diseases. A rapid and accurate method for the specific detection of B.rhodina is essential to prevent widespread devastation in mainland of China. Based on differences in internal transcribed spacer (ITS) sequences of B.rhodina and other Botryosphaeria spp., a pair of species-specific primers BF1/BR1 was designed. The primer pairs amplified a single 287 bp product from all isolates of B.rhodina that was not amplified from any other isolates tested. The sensitivity increased by 1 000-fold to 1 pg by developing a nested PCR procedure that used ITS1/ITS4 as the first-round primers combined with BF1/BR1 specificity was confirmed by using the PCR assay to detect B.rhodina in plant tissues infected by the pathogen. The PCR-based detection methods developed here could simplify both plant disease diagnosis and pathogen detection, as well as guide plant disease management. In addition, the classification implication of ITS sequence homology was found in fungi by comparing sequences from Botryosphaeria spp.