Antagonistic Action And Bio-Control Effect Of Bacillus Subtilis Strain Fmbj Upon The Postharvest Pathogen Rhizopus Stolonifer

In food production, the main problem caused losses and decay of harvested fruits and vegetables is fungal attack. Soft rot caused by Rhizopus stolonifer is a common disease in Post harvest diseases of peach fruits.Traditionally, synthetic chemical fungicides are the primary means for controlling post harvest disease. However, some fungicides have been removed from the market due to problems related to fungicide toxicity, and potential harm on the environment and human health. Utilization of biological control as an alternative appears to be one of the most promising technology. And how to enhance biocontrol effect is currently a hot topic in the research area.In a screening program performed in our laboratory, we found a strain of B. subtilis named fmbj, which was isolated from soil in China, inhibited many phytopathogenic bacteria and fungi in vitro. The principle objective of the present study was to (i) evaluate inhibitory mechanism of Bacillus subtilis strain fmbj against Rhizopus stolonifer, (ii) to screen the nutrients and develop a microbial community in order to increase the biological control effect. This research will provide theoretical basis for biological control of post harvest disease and peach fruit preservation.The major experimental methods and findings of this study are summarized as follows:1. The Bacillus subtilis fmbj was investigated for its inhibitory effects against postharvest pathogens of peach. The results showed that the fmbj strain successfully inhibit the mycelial growth, sportulation and conidial germination of Rhizopus stolonifer. By means of Scanning electron micrographs (SEM) and Transmission electron micrographs (TEM), strain fmbj is confirmed to strongly induce morphological abnormalities such as mycelial and conidial deviations. These results suggest that Bacillus subtiliss fmbj are considered as potent biological antagonistic bacteria to inhibit growth of Rhizopus stolonifer in the postharvest preservation of peach. 2. Twenty nitrogenous, and sixteen carbohydrates and sixteen mineral ion compounds were evaluated for stimulation of growth of an antagonist Bacillus subtilis strain fmbj and for their effect on germination, and radial growth of the pathogen Rhizopus stolonifer. Compounds that strongly stimulated growth of the antagonist but had little or no stimulatory effect on germination and growth of the pathogen were defined. Then, in accordance with the above principles, has further studied the mixture of three nutrients, screens the most superior mixture nutrients, which were evaluated for their effect on enhancing biocontrol of soft rot disease on peach fruit. In vitro the results indicated that the combination of NaHC03+maltose+histidine hydrochloride strongly stimulated growth of the antagonist but had little stimulatory effect on growth and germination of the pathogen, mixture nutrient FeCl3+rhamnose+threonine was strongly inhibited growth and germination of the pathogen. The results in vitro showed that combining B. subtilis with mixture nutrients show a more effective control on R. stolonifer than applying the antagonistic bacterium or nutrient alone.3. The synergy effect of this antagonistic bacterium in combination with different concentrations of 2-Deoxy-D-Glucose (2-DOG) against pathogenic Rhizopus stolonifer, as well as influence of 2-DOG on growth of both antagonistic bacterium and pathogen were well investigated. The results in vitro showed that only 0.3mg/ml of 2-DOG completely inhibited pathogen growth, while antagonistic bacterium still survived in 20mg/ml of 2-DOG and gathered abundantly in the wound of peach fruit, presenting a high 2-DOG-resistance. In vivo the results indicated that 2-DOG of 4 mg/ml combined with B.subtilis of 3.9×107CFU/ml totally protected peach fruit from decay, but when only antagonistic bacterium of 3.9×10 CFU/ml and 2-DOG was 4mg/ml was used, the disease incidence of peach fruit was 30% and 35%.4. Thirteen yeast strains were tested for its antagonistic activity against soft rot (Rhizopus stolonifer) in PDA medium. The strain (A82-2) was identified as Pichia anomala and Cryptococcus laurentii showed the antagonistic activity against R. stolonifer.The antagonistic action of the strain A82-2 was especially obvious. The study further to coculture of two yeasts with B. subtilis, the results stated that B. subtilis and P. anomala may symbiosis well and develop a stable microbial community.