An Investigation On The Preservative Effect Of Bacillus Subtilis KRS034 Volatiles On Postharvest Grapes And Its Preliminary Mechanism

Fresh grapes(Vitis vinifera L.),the mainstay of China’s grape industry,are rich in nutrients and have important health-beneficial functions.During the postharvest process,grapes are susceptible to pathogen infection in the environment due to the rich nutrition,soft and juicy.As a result,fresh grapes are difficult to store,transport and sell for a long time,resulting in significant economic losses.In recent years,postharvest preservation of fruits and vegetables with the help of microorganisms has become a hot research topic.At present,Bacillus subtilis is one of the most important biocontrol microorganisms,and the produced antimicrobial volatile organic compounds(VOCs)have great advantages,but there are few studies on the application of postharvest grape preservation.Therefore,in this study,the strain KRS034 isolated by the research group was used as the material.Firstly,the strain of KRS034 was identified,and the inhibition and pathogenicity were determined and the inhibition components in VOCs were identified.Secondly,the postharvest preservative and freshness effects,physiological quality,induction of defense mechanisms and differential changes at the molecular level based on transcriptomics were investigated in B.subtilis KRS034 VOCs-treated grapes to investigate the preservative effects of B.subtilis KRS034 VOCs on postharvest grapes and their mechanisms.The main results are as follows:(1)Identification,safety and bacterial inhibitory effect of strain KRS034.The strain KRS034 was identified as Bacillus subtilis by morphological observation,physiological and biochemical characteristics and 16S rDNA and pta/rpoD/tpiA sequence molecular biology identification.The results of hemolytic test showed that B.subtilis KRS034 had hemolytic safety and no pathogenicity to humans and animals.It can be used as a safe strain for postharvest preservation of grapes.The fungal inhibition test proved that B.subtilis KRS034 has a broad-spectrum inhibition effect on a variety of putrefactive pathogenic fungi,and proved that B.subtilis KRS034 VOCs had the best inhibition effect on Botrytis cinerea with 96.58%inhibition rate.Gas chromatography-mass spectrometry(GC-MS)was used to identify 16 VOCs in B.subtilis KRS034 that may have potential bacteriostatic properties,including 7 ketones,5 alcohols,3 acids and 1 pyrazine compound.In vitro antibacterial experiments showed that the above compounds had different degrees of inhibitory effect on B.cinerea.(2)B.subtilis KRS034 VOCs treatment can play a good control effect in the process of postharvest preservation of grapes,and does not affect fruit quality.B.subtilis KRS034 VOCs treatment significantly reduced the decay rate,lesion diameter and fungal biomass of grape inoculated with B.cinerea.In terms of physiological quality,compared with grapes without preservation measures after inoculation with B.cinerea,B.subtilis KRS034 VOCs treatment could maintain lower weight loss rate and higher soluble protein,total flavonoids and anthocyanin content.At the same time,B.subtilis KRS034 VOCs treatment can increase the activity of defense-related enzymes(PPO,POD,SOD,CAT)and decrease the content of malondialdehyde(MDA)in healthy grapes,thereby inducing the defense response of grapes during postharvest.(3)The molecular mechanisms by which B.subtilis KRS034 VOCs treatment induces fruit defense response and triggers defensiveness were explored at the transcriptome level.Based on GO functional classification and KEGG pathway enrichment analysis,indicating that after B.subtilis KRS034 VOCs induction,differentially expressed genes were enriched in functions related to grape fruit signal transduction,environmental stress and life activities,as well as metabolic pathways that play an important role in fruit defense:MAPK signal transduction,suberin and wax biosynthesis,nitrogen metabolism,amino sugar and nucleotide sugar metabolism,α-linolenic acid metabolism and glycerophosphate metabolism.In conclusion,B.subtilis KRS034 VOCs treatment was effective in preserving grapes during postharvest,and it is inferred that its defense mechanism may be related to defense enzymes and regulation of various metabolic pathways.