| Wax apple fruit(Syzygium samarangense)is a kind of tropical fruits,which is very popular for consumers because of the beautiful appearance and rich nutrition content.However,wax apple fruit is difficult to storage,it is very easy to be decay and damaged by the pathogenic microorganisms on the condition of higher temperature and humidity.Due to the negative influence of chemical fungicides on food safety,the technology of green and environmental protection is the current development trend,and biological control has become a hot topic in the postharvest preservation of fruits and vegetables.In this experiment,a soft rot pathogen was isolated from the decay wax apple fruit,and three antagonistic strains were isolated from the soil with the pathogen as the target.The effects of three antagonistic strains on the inhibition of soft rot and storage quality of‘Daye'fruit were studied.Moreover,the mechanism of antagonistic strains on controlling pathogen was also discussed.At the same time,the activity of disease resistant enzymes were analyzed,and the cloning and expression of phenylalanine ammonia lyase(PAL)gene were conducted too.The main results are as follows:(1)A pathogen was isolated from wax apple fruit with typical soft rot disease harvested from the Qionghai plant base of Hainan province,and it was identified as Pestalotiopsis microsporaby by the methods of morphology and molecular biology.A total of 143 strains were isolated from the root soil at the same garden,and three antagonistic strains called G-1,HP-1,D-1 were obtained by using the flat-stand method,and they were identified as Bacillus velezensis,Bacillus atrophaeus and Bacillus safensisrespectively.The inhibitory rates of three antagonistic strainson the growth of Pestalotiopsis microspora were 68.79%,58.00%and55.25%,respectively.(2)Research on nutrition and space competition:By changing the inoculation time,the spatial competition between pathogen and antagonistic strains was studied.The results demonstrated that the earlier time it was inoculated,the sooner it occupied a favorable space,and the smaller lesion diameter was,and the inhibitory effect was the better.By measuring the number of antagonistic strains growth,it was found that the presence of pathogen did not affect the growth of antagonistic strains when they were inoculated with the pathogen in the same wound.Instead,they promoted the growth of antagonistic strains,it indicated that the antagonistic strains could consume the interstitial nutrients earlier and inhibite the survival conditions of pathogen.(3)By the method of double-plate,it was found that three antagonistic strains produced a large amount of non-volatile antibacterial substances,and the inhibition rate reached 87.5%,83.83%and 75%.By the methods of flat buckle and GC-MS,it was found that three antagonistic strains could produce a small amount of volatile antimicrobial substances,and most of the volatile substances were ketones,alkanes,alcohols,acids and esters.(4)The inhibitory effect of three antagonistic strains on Pestalotiopsis microspora was studied in vivo.The results showed that after inoculating antagonistic strains,all of three antagonistic strains could effectively control the soft rot pathogen and reduce the incidence rate.By soaking fruit with suspension of the antagonistic strains,on the tenth day of storage at 25?,the inhibition rates on pathogen reached 40.0%,45.5%and 45.4%,respectively,which were significantly higher than those of the control group.The concentration of suspension at 10~8 CFU/m L was found to be most effective to maintain of the contents of soluble solids,titratable acid,total phenol and flavonoid,but there was no significant difference in weight loss rate.In addition,all of three antagonistic strains could significantly induce the activities of chitinase,?-1,3-glucanase,phenylalanine ammonia-lyase,peroxidase and polyphenol oxidase of fruits,which contributed to the disease resistance of fruit.(5)Using RT-PCR technique,the c DNA segments of PAL gene were cloned from wax apple fruit.Homology analysis showed that PAL1 encoded 293 amino acid residues,the molecular weight of this protein was 32.7KD,p I was 9.58.PAL3 encoded 240 amino acid residues,the molecular weight of this protein was 26.6KD,p I was 5.29.PAL1 and PAL3were highly homologous to other PAL nucleotide sequences from different plant species,and they were a number of PAL superfamily.Throughq RT-PCR method,it was observed that PAL1 and PAL3 genes were expressed throughout the whole storage period of fruit,and the expression level of PAL1 and PAL3 genes exhibited the trend of increasing then decling,and the maximum of expression level of PAL were presented on the eighth day. |
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