Endophytic bacteria from Theobroma cacao L. with antifungal activities against Phytophthora palmivora

Black pod disease caused by the pathogenic fungus Phytophthora palmivora is a serious threat to the cocoa industry causing the destruction of cocoa plants in many plantations across Malaysia. The objectives of this study are to determine the antifungal activities of the endophytic bacteria isolates and to identify the isolates. Four selected endophytic bacteria previously isolated from tissues of healthy Theobroma cacao L. designated as isolate LKM-UL, LKM-PA, LKM-PD, and LKM-BL were assessed for their abilities to inhibit the growth of P. palmivora in vitro. Preliminary tests using dual culture method showed that the isolate LKM-BL had the strongest inhibition towards the growth of P. palmivora after 24 h of incubation compared to the other isolates. The growth of LKM-UL, LKM-PA, LKM-PD, and LKM-BL revealed that the antifungal activities against cocoa pathogen increases as the number of endophytic cells increases. The cell-free supernatant from isolate LKM-BL produced the highest antifungal activity against cocoa pathogen with an inhibition zone of 19.5 +/- 0.50 mm during the highest cell growth at 24 h of incubation. The endophytic bacteria were characterized morphologically and based on biochemical tests. Based on the analysis of the 16S ribosomal DNA sequences, isolate LKM-UL, LKM-PA, LKM-PD, and LKM-BL were identified as Bacillus amyloliquefaciens, Pantoea agglomerans, Bacillus pumilus and Bacillus subtilis respectively. The findings indicate that among the four endophytic bacterial isolates studied, B. subtilis LKM-BL showed the highest antifungal activity and has the potential to be used as a biological control agent towards the cocoa pathogen P. palmivora.