Isolation And Identification Of The Antagonistic Bacteria Against Postharvest Papaya Anthracnose And The Mechanisms Involved

Papaya(Carica papaya L.)is a kind of famous tropical fruits,which is very popular among consumers.However,it is susceptible to infection by pathogens during postharvest storage and transportation.The anthracnose is one of the most serious diseases in postharvest papaya fruit.In this study,the ‘Risheng' papaya produced in Hainan was used as the research material,a pathogenic strain was isolated from the lesion of papaya anthracnose and its characteristics were studied.At the same time,this pathogen was used as a target to screen for antagonistic bacteria,and we have obtained antagonistic yeast JM-1 and antagonistic bacteria W-2,which have good antagonistic effects relatively.Then,the effect of JM-1 on the the storage quality and control effect on anthracnose of papaya fruit were studied.Meanwhile,the molecular mechanism of the antagonist against anthracnose of papaya was explored.The main results are as follows:(1)The anthracnose pathogen WT-1 was isolated from the infected papaya fruit.Through pathogenicity test,morphological identification and r DNA-ITS sequence analysis,it was identified as a pathogenic strain of Glomerella magna.The results of biological characteristics showed that the optimal growth temperature and sporulation temperature of WT-1 was 30 ?.There was no significant difference in the diameter of hyphae under the conditions of full light and dark alternating,however,the spore production was highest under the condition of 12 h light and dark alternating.The optimum p H range of 6 to 8 for mycelium growth was observed,and a large number of spores could be produced when the p H is 7 to 8.The most suitable carbon source is glucose,followed by fructose and sucrose,and the most suitable nitrogen source is peptone.The conidia lethal temperature of the pathogens was 60 ? for 10 min.(2)225 strains were isolated from the soil,leaves and peels of different orchards in Hainan.19 strains with good antagonistic effect on WT-1 were obtained through primary screening and double screening.Among them,W-2 and JM-1 had the strongest antagonistic effects on anthracnose,and their antagonistic indexes against WT-1 reached 3.23 and 2.78 respectively.Results showed that the W-2 was Bacillus atrophaeus and the JM-1 was Hanseniaspora opuntiae.(3)Enlarged mycelia and deformed spores were observed in vitro by the action JM-1.Fruits treated with JM-1 at a concentration of 1×108 CFU/m L had the best relative control effect on papaya anthracnose under storage at 25 ?.There is spatial competition between JM-1 and WT-1.JM-1 was able to colonize and form biofilms in the wounds of papaya fruits.JM-1 competed for nutrients(Fe Cl3)to inhibit the spore germination.The volatile gas produced by JM-1 inhibited WT-1 by 49.32% in vitro conditions.JM-1 can induce ?-1,3-glucanase(GLU),chitinase(CHI),phenylalanine ammonia lyase(PAL),and peroxidase(POD)activities in papaya fruits,improving fruit disease resistance.Meanwhile,the study found that JM-1 has the ability to secrete chitinase,which can degrade fungal cell walls,and the size of the chitinase gene encoded in JM-1 by PCR amplification was about 2000 bp.(4)A gene coding ?-1,3-glucanase,named for GLU-A02 was cloned from papaya fruit.Bioinformatics analysis was carried out to derive a total of 323 amino acids with a predicted molecular mass of 34733.10 Da.An isoelectric point(p I)of 5.18 was characterized.The instability index of 37.09 indicates that the protein is stable.The GLU-A02 protein contains a conserved sequence of glycosyl hydrolase family 81.Through q RT-PCR,the expression levels of key defense enzymes--GLU and POD genes in postharvest papaya fruits were found to be increased significantly by treating with JM-1,and expression levels GLU were 2.45,1.22,and 1.09 times of the control group on the 3rd,6th,and 12 th days,respectively.The expression levels of POD were 1.44 times and 2.02 times of the control group on the 9th and 12 th days,respectively.