Preparation and application of liquid bioformulation of Metschnikowia citriensis on citrus fruits

Abstract: Antagonistic yeast has widely expected to replace chemical fungicides, serving as an effective biological agent to control postharvest diseases of citrus. Metschnikowia citriensis, a new species of Metschnikowia spp, isolated from citrus leaves and identified laboratory, was efficient to control postharvest green mold and sour rot caused by Penicillium digitatum and Geotrichum citri-aurantii on citrus fruit, indicating great application and development value. However, the unstable effect of biocontrol agents and the unclear mechanism of biocontrol have led to a low success rate of commercial application of antagonistic yeast. Therefore, it is highly urgent to develop the antagonistic yeast biological agents with long shelf life and stable control effect for the commercialization of M. citriensis. In this study, an attempt was made to explore the preparation of liquid bioformulation with M. citriensis as the main active ingredient. Single factor and response surface tests were carried out to optimize the formulation of the protective agent for liquid bioformulation. An in vitro and in vivo test was also conducted to further evaluate the control effect of formulation on the main postharvest diseases of citrus fruits, particularly for the application of liquid preparation. The results showed that four protective agents were screened via a single factor test, including trehalose, sodium glutamate, Tween 80, and proline. The formula of the protective agent was selected: trehalose 19.74%, sodium glutamate 1.05%, Tween 80 4.24%, proline 1.11%, where the best protective effect on M. citriensis, and the survival rate of yeast increased from 5.02% to 54.96%. The storage stability of liquid bioformulation showed that the survival rate of M. citriensis was still more than 60% after 30 days of storage when the temperature was lower than 10 ℃. More importantly, the shelf life of preparation reached more than 90 days, when the storage temperature was -20 ℃. As such, a relatively low storage temperature was beneficial to the longer shelf life of bioformulation. In vitro and fruit tests were carried out to verify the application effect of preparation. It was found that there was no significant change in the inhibitory effect of liquid bioformulation on postharvest pathogen and pigment production capacity of M. citriensis, compared with fresh yeast, where the inhibition zone reached more than 8 mm. There was also no significant difference in the number of fresh and liquid yeast at the fruit wound, both of which grew well at the wound. Fresh yeast performed remarkable control effects on blue and green mold, sour rot, and anthrax on citrus fruits, while liquid preparation presented no significant difference, compared with fresh yeast. Specifically, the formulation reduced the incidence of four citrus postharvest diseases by 25.00%-48.33%. Consequently, the preparation of M. citriensis can widely be expected to effectively retain cell viability and biocontrol efficacy. The liquid bioformulation with M. citriensis as the main active ingredient demonstrated an excellent control effect on postharvest citrus diseases. The finding can provide strong theoretical and practical support to the application of M. citriensis in the biological control of postharvest citrus diseases.