Selection And Control Effect Of Antagonistic Bacteria Against Peach Brown Rot

Peach fruit is valued for its flesh and enjoyed for its taste and nutritional value,this fruit exhibits a short postharvest life at room temperature due to its high susceptibility to decay pathogens and rapid quality deterioration.Monilinia fructicola is one of the most destructive pathogens causing considerable production losses during postharvest handling of the fruit.Current management of this disease still relies on the use of synthetic chemical fungicides.Nevertheless,the long-term use of chemical fungicides will lead to the emergence of drug resistance,drug residues and environmental pollution.Biological control has been a hot spot in the field of disease control because of its safety and efficient characteristics.Recently,antagonistic microorganism,as one way of biological control,has been considered as the most promising alternative to chemical fungicides.Recently,volatile organic compounds?VOCs?produced by microorganisms have been evaluated as a new approach for postharvest disease control.However,limited research information is available related to the efficacy of biofumigation volatiles for the control of peach brown rot.The objective of this study was to select effective antagonistic bacteria against M.fructicola as the potential biological agents,evaluate the effects of these strains on peach brown rot in vitro and in vivo.A solid phase micro-extraction syringe was used to trap volatiles,and identified them by the GC-MS process.The key volatiles that could be used to suppress pathogens were identified.And evaluate the effects of antagonistic bacteria combined with fungicides on peach brown rot in order to lay the theoretical foundation for its practical application.Main results of this research are as follows:?1?Fifty-seven strains of bacteria were isolated from the peach rhizosphere soil using serial dilution method.Preliminary screening showed that four bacterial strains,namely,12a,14b,15k,and 16d,presented remarkable inhibitory activity on M.fructicola mycelial growth.In vitro,M.fructicola showed 70.6%and 56.5%of mycelia growth inhibitions in presence of12a and 14b,respectively.12a and 14b were identified as Bacillus vallismortis and Bacillus altitudinis respectively by phenotypic,physiological,biochemical and 16S rRNA gene sequence analysis.?2?In vitro evaluation of antagonistic activity,the sterile fermentation filtrate and volatile metabolites of strain 12a and 14b both showed a strong inhibition on the mycelia growth of M.fructicola.Especially,the inhibitory effect of the volatile metabolites of strain 12a was more significant?p<0.05?.The ultrastructure of M.fructicola treated with volatiles from 12a or 14b was severely damaged;transmission electron microscopy analysis also showed its empty cavity.For further study,in vivo tests indicated that fermentation broth and volatile metabolites of strain 12a and 14b also delayed the onset of brown rot and significantly?p<0.05?inhibited the development of lesion diameter of peach fruit.?3?The volatile compounds produced by 12a and 14b were identified according to the gas chromatography–mass spectrometry analysis.These VOCs originated from several chemical classes,including ketones,acids,alkanes,alcohols,esters,phenols,and many others.The four volatiles,6-methyl-2-heptanone,2-pentylfuran,isodecyl methacrylate and cedrol showed remarkable inhibitory effects on pathogens.The inhibition rate of the 50?L/L artificial mixture of these four volatiles was 59.3%in the plate test.Moreover,the mycelial growth was completely inhibited when the tested concentration of the mixture reached 100?L/L.?4?Those fungicides which can well combine with 12a and 14b were selected and then the sensitivity of M.fructicola to fungicides was verified.And the fruit body was used to validate the control effect of combining processing to postharvest diseases of peach fruits.The in vitro experiment showed that midazolam,fipronil and low doses of thiophanate-methyl,when combined,did not inhibit the growth of strains 12a and 14b and M.fructicola showed different sensitivities to midazolam,fipronil and thiophanate-methyl,but the EC₅₀ values all were less than 1%of those of the recommended dosage.The fruit in vivo experiment showed that,12a or 14b combined with low dosage of midazolam,fipronil and thiophanate-methyl had a better control efficacy on peach brown rot.?5?The broad-spectrum antibacterial effect of selected antagonists,namely,12a and 14b,against several frequent postharvest fungal pathogens of fruits and vegetables were tested using dual-culture inhibition on agar plate;these pathogens included Ceratocystis fimbriata,Botrytis cinerea,Gloeosporium musarum,Valsa mali.Strain 12a on NA medium inhibited a broad range of pathogens with inhibition rate over 60%.Strain 14b also showed broad-spectrum antifungal effect,but its antifungal activity was slightly lower than that of12a.