Selection of antagonistic bacteria for controlling fungal fruit rot disease of longan after harvest

Abstract

Postharvest longan fruit rot disease caused by fungi is the important problem that result in a great loss of longan production. Application of chemical to control the disease led to adverse effects on consumer health and environment, also, the chemical residue on fruits and exportation. This study aims to isolate, screen and select the potential antagonist bacterium for controlling and reducing the fruit rot disease of longan then develop into the bio-formulation prototype for using in further extending the shelf life and preserving quality of longan. A total of 34 fruit rot samples were obtained from longan orchard in Chiang Mai, Phrae and Lamphun Provinces. The results showed that the most pathogenic fungus is Lasiodiplodia sp. followed by Pestalotiopsis sp., Colletotrichum sp. and Phomopsis sp. Furthermore, Lasiodiplodia sp. (the most virulent pathogen) was identified according to its morphological and molecular characteristics by analysis of the internal transcribed spacer region (ITS) and translation elongation factor 1-alpha (EF1-α) genes. Therefore, this fungus was identified as Lasiodiplodia pseudotheobromae. To select the effective antagonistic bacteria for controlling L. pseudotheobromae, the endophytic bacteria were isolated from healthy parts of longan and the 1,413 isolates of endophytic bacteria were obtained and tested with dual culture technique. The results showed that the bacterium isolate RFCD306 was the most effective on inhibiting L. pseudotheobromae both mycelial growth and conidial germination. The bacterium isolate RFCD306 were identified according to the morphological, biological, biochemical and molecular characteristics by analysis of 16S rRNA gene and it was identified as Bacillus siamensis. The controlling efficacy of Bacillus siamensis RFCD306 against L. pseudotheobromae showed that mycelial growth was inhibited by creating a clear zone between the fungal pathogen and the antagonistic bacterium which could be calculated as 94.40% of inhibition. The efficacy of B. siamensis isolate RFCD306 on inhibition of conidial germination of L. pseudotheobromae was assayed and observed under light compound microscope and scanning electron microscope (SEM) revealed that conidia abortion at 54.48% and caused abnormal swelling germ tube of certain conidia and hypha. The antibiotic synthesis-related genes consisting of bacillomycin D (bacD), iturin A (ituA), surfactin (srfA) and bacilysin (bymA) genes were detected from B. siamensis RFCD306 using the polymerase chain reaction (PCR) technique. The detection of 4 antibiotic synthesis-related genes supported that one of the important mechanisms of B. siamensis RFCD306 on inhibition of L. pseudotheobromae was the production of antibiotic substances or antibiosis. The development of suitable bio-formulations for B. siamensis RFCD306, from out of 10 formulars, found that rice flour-2 was the most proper formulation with amount of bacterial concentration in final product was 1.75 × 1010 CFU/g. When testing the efficacy of mycelial growth inhibition with dual culture technique, it was found that the inhibitory effect was 80.00%. Though, the concentration of antagonistic bacteria was reduced to 5.80 × 107 CFU/g and the inhibition effect was reduced to 35.33% after stored the bio-formulation at room temperature for 6 months. The bio-formulation, rice flour-2 formulation, was applied on harvested longan then storage at 15°C with relative humidity 75%. The result found that the bio-products could delayed the progress of pericarp browning and decreased fruit rot disease by had significant browning index 1.83 and disease incidence 8.33%, respectively and extend shelf life of fresh longan for 13 days.