Cultural-morphology and molecular analysis of Botryodiplodia theobromae, a pathogen of coconut fruit

Methods

Eight isolates of B. theobromae used in this study were collected from diseased coconut fruit samples (showing signs of rot and freshly fallen nuts) in two major coconut producing areas in Nigeria. Pure culture of isolates were obtained using potato dextrose agar (PDA) medium. The culture medium and microscopy were used to also examine the cultural and morphological characters of isolates.

For molecular characters of isolates, DNA was extracted from each isolate and amplified with a polymerase chain reaction (PCR) using a universal primer (ITS1F/4R) and three specific primers (Lt347-F/R, Bt2aF/bR, and EF1-728F/EF2-728R). Furthermore, PCR amplicons obtained from the ITS1F/4R primers were sequenced and analyzed using bioinformatics and phylogenetic methods. The genetic similarity and variation of isolates were also determined.

Results: The colony texture/color, ITS1/4 gene sequence information and phylogenetic analysis clustered the eight isolates of B. theobromae into five categories. The ITS1/4 sequence information using a blast search in the NCBI database, confirmed all eight fungal isolates as B. theobromae. Conclusion: This study has provided a guide for proper categorization of B. theobromae, that is a prerequisite for early diagnosis and management of this pathogen in coconut producing areas.