Studies On Biocontrol Ability, Formulation Production, And The Possible Mechanism Of Antagonistic Yeasts

The antagonistic yeasts in our lab have been proved to be effective biological control agents against the major pathogens of different fruits. In order to enhance the speed of commercializing these biocontrol agents, besides the additional study on mechanism of antagonist, we emphasized the aim of present study on the mass production of cells, development of a shelf-stable formulated product, and response of yeast cells to environmental stress. The main topics of this study were: (1) to investigate the antagonist-pathogen-host interaction in wounds of fruit; (2) to select the optimal nutritional components and the environmental conditions as well as the feasibility of antagonists production at pilot scale in a 120-L bioreactor with the selected conditions; (3) to optimize solid and liquid formulations; (4) to study the mechanism of loss of viability loss in solid and liquid formulations; and (5) to evaluate the presence of inhibitory substances from antagonistic yeast against postharvest pathogens. The results were summarized as follows: 1. In fruits inoculated with Monilinia fructicola alone or combined with Cryptococcus laurentii, an induction of lipid peroxidation and activities of antioxidant enzymes such as SOD, CAT, and POD was observed. Isoenzyme pattern of PPO changed greatly after the symptoms developed and new PPO isoforms were induced. Both M. fructicola and Penicillium expansum could stimulate the growth of C. laurentii in fruit wound following inoculation. The LOX activity and the accumulation of O2·-and H2O2 were significantly suppressed in fruit inoculated with C. laurentii at 24h. In contrast, in fruits inoculated with M. fructicola or P. expansum, both LOX activity and O2·-generation were induced while H2O2 production was significantly suppressed as compared to the control. The results indicated that the accumulation of O2·-combined with the decrease of H2O2 production might account for disease development of peach fruit. 2. The addition of sodium ascorbate provided a significant improvement of the biocontrol activity of C. laurentii against M. fructicola in sweet cherry fruit. The effect of combination of yeasts at 107 cells mL-1 with 200mM sodium ascorbate was similar to that of C. laurentii at 108 cells mL-1 alone. The mechanism by which sodium ascorbate enhanced the biocontrol efficacy of the antagonist might depend on its ability to reduce the growth of M. fructicola and inhibition of antioxidant enzymes in fruit which led to lipid peroxidation in the wound. 3. The results of optimization of growth conditions for Rhodotorula glutinis and C. laurentii cells showed that different antagonists preferred to different nutritional components as well as culture temperature. Growth of R. glutinis and C. laurentii atthe optimized conditions was checked at pilot scale in a 120-L fermenter and population above 1×109 CFU mL-1 was obtained. 4. Protective agent was determined as the most important factor on the viability of the biocontrol yeast R. glutinis and C. laurentii when subjected to freeze-drying. However, the effectiveness of protective medium on the viability of freeze-dried cells was dependent on both protectant concentration and yeast growth stage. Both water and PBS could be used as preservative medium for liquid formulation of C. laurentii, while cells incubated in trisodium citrate (100mM, pH5.8) rapidly lost viability. 5. Stability of freeze-dried biological agents was checked and mass generation of ROS was found to be associated with the rapid loss of viability of two freeze-dried samples, indicating ROS accumulation might mainly account for cell death. The induction of rapid loss of viability by trisodium citrate was highly dependent on the concentration and pH value of the medium. Reactive oxygen species might play an important role during the induction process because rapid ROS production was companied with the cell death. 6. Biocontrol yeasts could produce volatile and non-volatile inhibitory substances against postharvest pathogens. Production of inhibitory substances was highly dependent on the specific antagonist and the tested pathogens showed varying sensitivities to the inhibitors produced. However,specific combinations of yeasts and growth media were essential in determining the production of inhibitory substances against specific tested fungi.