A nested multiplex PCR for species-specific identification and detection of Botryosphaeriaceae species on mango

Lasiodiplodia theobromae (Pat.) Griff. & Maubl, Neofusicoccum parvum Pennycook & Samuels, N. mangiferae Syd. & P. Syd., and Fusicoccum aesculi Corda, all anamorphs of Botryosphaeriaceae species, are the causal agents of mango stem-end rot and fruit rot in Taiwan. Identification of these fungal species based on morphology has not been easy due to their extensive plasticity for some of the morphological characters. To aid reliable identification of Botryosphaeriaceae species associated with mango fruits, four pairs of species-specific primers were designed according to sequences of the ribosomal internal transcribed spacers (ITS), and a rapid method was established based on nested multiplex polymerase chain reaction (PCR) in this study. To perform the analysis, PCR was first run with ITS1 and ITS4 as the primers, followed by a second PCR with the addition of all four sets of species-specific primers. With this method, a low limit of 100 fg(-1) pg of purified fungal DNA was detectable. It could also successfully detect L. theobromae, N. parvum, N. mangiferae and F. aesculi in total DNA extracted from inoculated mango fruits. This assay provides a rapid and sensitive method for the identification of Botryosphaeriaceae species and diagnosis of mango fruit rot and stem-end rot as well.