Physiological And Molecular Mechanisms Of Action Of The Yeast Debaryomyces Nepalensis For Control Of The Pathogen Colletotrichum Gloeosporioides In Mango Fruit

Mango(Mangifera indica.L)is a popular tropical fruit with unique flavor and high nutrition.Anthracnose caused by Colletotrichum gloeosporioides is one of the most common postharvest diseases of mangoes.Due to chemical fungicides causing the problems of food safety,consumer pursue the safe and natural products.The demands drive current trend of the preservation technologies toward safer.Biopreservation technology is a hot topic at home and abroad.Among them,the use of antagonists to control post-harvest diseases is in line with the green safety and sustainable agricultural development strategy.The antagonist Debaryomyces nepalensis obtained from the previous laboratory was used to inhibit the postharvest mango anthracnose.The physiological mechanisms of action involved in the inhibition were identified(production of hydrolytic enzymes,parasitism,biofilm production,competition for nutrient and space,induction of resistance,production of volatile antimicrobial compounds).Furthermore,the molecular mechanism of the ?-1,3-glucanase gene of D.nepalensis was studied.The research results are as follows:(1)It was confirmed by Congo red plate staining that D.nepalensis can produce and secrete fungal cell wall lyase(?-1,3-glucanase).(2)Using optical microscopy and scanning electron microscopy,it was found that the parasitic phenomena.It was observed that D.nepalensis adhered to the hyphal walls of the C.gloeosporioides and it produced biofilms on the mango surface and yeast cells attached to each other.(3)The nutrition and space competition of D.nepalensis yeast and pathogens were studied by inoculation of yeast and pathogens.The best antagonistic activity was observed on mangoes inoculated with D.nepalensis 24 h prior to the addition of pathogen,and the reduction in lesion diameter was 93.4%.It showed that the earlier the time of yeast inoculation,the shorter the lesion diameters,indicating that mangoes inoculated with D.nepalensis in advance had significantly improved biocontrol activity to inhibit the pathogen.D.nepalensis competed for nutrients(FeCl3)to inhibit the spore germination and competed for space with the pathogen(p<0.05)which showed significant biological control activity.(4)Studies on enzyme activity related to disease resistance indicate that yeast D.nepalensis can induce the improvement of phenylalanine ammonia-lyase(PAL),peroxidase(POD),chitinase(CHT)and ?-1,3-glucanase(GLU)of mangoes which contributes to the fruit disease resistance.(5)The volatile compounds produced by D.nepalensis were analyzed by solid phase microextraction(SPME)combined with gas chromatography-mass spectrometry(GC-MS).The result reveals that D.nepalensis can produce the volatile antimicrobial compounds,The VOCs produced by D.nepalensis reduced the mycelial growth.The inhibition of C..gloeosporioides,by VOCs was 41.63%and 13 compounds were detected,including four alcohols,two esters,two acids,one phenolic,one ketone,one alkane,one ether and one amide compound,It revealed that phenylethyl alcohol(29.62%relative peak area,RA)as the dominant VOC mainly produced by D.nepalensis,and it had a significant inhibitory effect on fungal mycelia.A pure standard of ?-phenylethyl alcohol was used for in vitro trials against the conidia germination of the C.gloeosporioides in order to determine EC50.The resulting EC50 values 1.99 ?L/mL.(6)The function of the ?-1,3-glucanase gene produced by D.nepalensis yeast was studied for the first time.A gene coding endo-?-1,3-glucanase,named for DnENGyy was cloned from Debaryomyces nepalensis.The length of the gene is 2307 bp.Bioinformatics analysis was carried out to derive a total of 768 amino acids with a predicted molecular mass of 86.61 kDa.An isoelectric point(pI)of 6.10 was characterized.The DnENGyy protein contains a conserved domain of group 81 of glycosyl hydrolase.The recombinant plasmids were linearized and electroporated into Pichia pasroris.The transformed cells were screened on MD plates.Then,the strains were inoculated into the BMMY medium.The strains with high enzyme activity were selected using laminarin plates by Congo red method.The enzyme activity of endo-?-1.3-glucanase secreted into the culture solution was determined to be 38.61 U/mL at 48 hours.The protein expressed by recombinant yeast can inhibit the spore germination of C.gloeosporioides.