Identification Of A Novel Antagonistic Yeast Species And Its Mechanisms In Controlling The Major Postharvest Diseases Of Citrus Fruit

Penicillium digitatum and Penicillium italicum,the causal agent of green and blue mold,are responsible for significant economic damage of citrus fruits.Using chemical fungicides to control these pathogens is not considered sustainable,because of the increasing public concern about their hazards for human and environmental health,as well as the emergence of fungicide-resistant strains.Biological control with antagonistic yeasts has become a promising alternative.However,the antagonistic yeasts that have been reported to effectively control citrus postharvest diseases remain rare and show limited efficacy.Given that the biocontrol mechanisms of antagonistic yeasts vary greatly among different species,isolation and screening novel yeast species with biocontrol potential and clarifying their mechanisms of action are crucial to the product development of excellent biocontrol agents.In addition,since various severe stresses encountered in the process of production and application could cause the intracellular reactive oxygen species(ROS)accumulation,resulting in cell damage and loss of vitality,biocontrol yeasts cannot achieve the same effect compared with synthetic fungicides,which is the main factor hindering their commercial application.Therefore,developing strategies to enhance their oxidative stress tolerance is necessary to improve their efficacy.In this study,morphological,physiological and biochemical characteristics and phylogenetic analyses were used for taxonomic identification of a novel species of antagonistic yeast.The biocontrol mechanisms of this novel species against P.digitatum and P.italicum were investigated by testing the yeast antagonist-fruit interaction and the effect of yeast cells and their extracellular metabolites on the growth of pathogens,and its key mechanisms were evaluated by comparison with other excellent biocontrol yeasts that have different morphological characteristics.Moreover,proline was utilized to improve oxidative stress tolerance and biocontrol efficacy of this novel species,and possible mechanisms involved were also investigated.The main research findings were summarized as follows:(1)The chromosomal DNA similarity of the strain FL01~T and FL02 was analyzed by DNA-DNA hybridization,and the result of 82.2%similarity indicated that they are conspecific.The cells are globose to ovoidal,no pseudohyphae is formed.The maroon pigment(pulcherrimin)was detected in cultures of the strain FL01~T and FL02.Spherical,heavy-walled chlamydospore-like“pulcherrima”cells are common,and asci arise from chlamydospores.Quick maturing asci rupture and many smooth,spheroidal ascospores were rapidly released from the asci,and free spores tended to agglutinate.Regarding the sequences of the D1/D2 domains of 26S rDNA,this species differs from Metschnikowia koreensis by seven(1.5%)substitutions,and by>100 mismatches in the internal transcribed spacer(ITS)region.What's more,this species has differential physiological and biochemical characteristics and can be distinguished from its closest species M.koreensis by its ability to ferment galactose,assimilate raffinose,melibiose,L-rhamnose,nitrate and nitrite,by its growth at 37°C and in a vitamin-free medium,and by its failure to hydrolyze urea.Therefore,the strain FL01~T and FL02 were described as a new species of Metschnikowia and we named it Metschnikowia citriensis sp.nov.The strain FL01~T with a smaller distance from M.koreensis is used as the type strain of this novel species.(2)The pathogenicity potential of M.citriensis sp.nov.(FL01~T and FL02)was evaluated in vitro,and results showed that these yeasts were relatively safe.The comparison between M.citriensis sp.nov.and other excellent biocontrol yeasts(Candida oleophila FL14 and Pseudozyma antarctica FL17)with different morphological characteristics indicated that yeasts varied greatly in their key mechanisms of action against P.digitatum and P.italicum.The adhesion to mycelia,the biofilm formation,and the iron depletion were hypothesized as the key action mechanisms of M.citriensis sp.nov.FL01~T and FL02.(3)Proline could function as a cell protectant and inducer of biofilm formation of M.citriensis sp.nov.Addition of 1 mM proline not only induced the maroon pigment production that directly reduced the intracellular iron content and indirectly diminished the intracellular ROS levels of strain FL01~T,but also regulated catalase(CAT)and superoxide dismutase(SOD)activities,and hence inhibited ROS-and iron-induced apoptosis.In addition,pigment production also enhanced the capability of biofilm formation of strain FL01~T.The abilities of proline to scavenge intracellular ROS and inhibit apoptosis,increase pigment production,and promote biofilm formation contribute to the improvements in oxidative stress tolerance and biocontrol efficacy of M.citriensis sp.nov.