Inhibitory Effect Of Extracellular Extract From Bacillus Vallismortis On Brown Rot Of Peach Fruit

Peach fruit is popular with consumers because of its rich nutrients and multifunctional ingredients.However,due to the high temperature and humidity during its harvest season,as well as the possibility of mechanical injury during transportation and storage,peach fruit is extremely susceptible to fungal infection.The brown rot infected by Monilinia fructicola is one of the main diseases that cause the loss of commercial value of postharvest peach fruit.Compared with traditional physical and chemical control,biological control does not have the problems of microbial resistance,pesticide residue and environment pollution.Microbial metabolites used for inhibiting fruit diseases is the most common biological control method.Bacillus vallismortis has the advantages of simple condition of culture,rapid growth and reproduction,tolerance to harsh environment and can produce secondary metabolites that inhibit the growth of a variety of pathogens.Therefore,this study explored the inhibitory effect of the crude extract of the fermentation broth of B.vallismortis on peach fruit brown rot through in vitro and in vivo experiments.The detailed results are as follows:(1)The best culture conditions of B.vallismortis were screened by the method of determining the zone of inhibition.The best fermentation medium was minimal salt medium.The proper culture conditions were determined as the fermentation time is 72 h,the culture temperature is 30℃,p H value is 7.2,the inoculation amount is 1%,the liquid volume is 100 m L,and the optimal rotation speed is 180 rpm/min.The crude extract of B.vallismortis fermentation broth was obtained by centrifugation,adjusting the p H value of fermentation broth to obtain precipitation,freeze-drying the precipitation and methanol resolution.The plate hemolysis test confirmed that there was no hemolytic ring or grass green ring in the plate of B.vallismortis,which preliminarily confirmed that the strain was a safe strain.(2)The effect of the fermentation extract of B.vallismortis on brown rot fungus were conducted by in vitro test.The fermentation extract of B.vallismortis significantly(P < 0.05)inhibited the hypha growth of M.fructicola.Under scanning electron microscopy,it was observed that the mycelium after treatment with the fermentation extract of B.vallismortis had swelled and deformed.Under transmission electron microscopy,it was found that the mycelium had cavities and the structure was damaged.Moreover,the ergosterol content in the mycelium was significantly reduced.After fermentation extract treatment of spores,with the increase of the concentration and treatment time,the germination and elongation of spores were significantly inhibited.Furthermore,the results of PI and DCFH-DA staining showed that the fluorescence ratio of spores increased after the crude extract treatment,which demonstrated that the increase of reactive oxygen species in spores caused spores apoptosis.(3)The effect of fermented extract of B.vallismortis on peach fruit brown rot were conducted in vivo test.Respectively,peach fruit was treated with water,methanol,bacterial liquid,fermentation supernatant,1 mg/m L,10 mg/m L and 100 mg/m L crude extract of B.vallismortis fermentation broth.When the crude extract concentration was 100 mg/m L,the control effect on postharvest disease of peach fruit was best,and the peach fruit disease incidence and lesion diameter were significantly reduced.The enzyme activity and gene expression of chitinase and β-1,3-glucanase were significantly enhanced by the crude extract.Moreover,the expression of disease-resistant genes(ICS1,NPR1,TGA1,PR1,PR10,AOC,LOX,COI1 and MYC2)and the content of salicylic acid were increased,which indicated that the treatment of B.vallismortis fermention broth crude extract might induce disease resistance of peach fruit through salicylic acid pathway and jasmonic acid pathway.(4)After the treatment of peach fruit with B.vallismortis extract,the content of hydrogen peroxide and superoxide anion in the peach fruit was decreased,the content of NO was increased,the activities of antioxidant enzymes such as superoxide dismutase,peroxidase,catalase,phenylalanine ammonia lyase,ascorbate peroxidase and glutathione were increased and the content of total phenols was increased,indicating the crude extract from B.vallismortis fermentation broth induced disease resistance of peach fruit through enhancing the antioxidant capacity of peach fruit.