Study On The Epiphytic Microbiota Of Citrus Fruit And Its Role In Postharvest Disease Control

Citrus is an important crop in China,which has sweet and sour taste,unique fragrance,a long history of cultivation,and a wide variety of fine varieties.It is a precious fruit favored by consumers.Epiphytic microbiota of citrus fruits is closely related to fruit health and storability,but not all microorganisms can be cultured in laboratory conditions,so the ecological microbiota of citrus fruits has not been systematically studied.In recent years,the development of high-throughput sequencing technology has made it possible to study culturable and non-culturable microorganisms at one time,which will provide a wealth of microbial community information for the study of microbial community assembly mechanisms,microbe-microbe and microbe-host interactions.Citrus fruits are easily infected by pathogens in postharvest,which can cause great economic loss.Biological control is an important means to control postharvest diseases.However,single antagonist has limitations in the commercialization process,and its disease control range is narrow and its effect is unstable.The comprehensive application of multiple microorganisms can make more effective use of space and nutrition,or achieve better disease control effect.The core microbiome refers to a group of microorganisms that occupy a large proportion or have important functions in a specific host and are usually closely related to host health.Therefore,in this study,citrus fruits from Xiema Town,Beibei District,Chongqing were used as research materials,and metagenomic shotgun sequencing and amplicon sequencing methods were adopted.The structure and function of citrus epiphytic microbiota were analyzed,and the core epiphytic microbiota was discussed.At the same time,important microbial strains were isolated and purified to construct artificial synthetic community,to clarify the control effect of artificial community on citrus postharvest diseases.The main conclusions are as follows:1.Structure and function of epiphytic microbiota in tangerine fruit:(1)The relative abundance of fungi was much higher than that of bacteria,but the species richness was low in the epiphytic microbiota of tangerine fruit.The relative abundance of the bacteria is low,but its higher species diversity,more widely distributed.On the level of the phylum,Ascomycota(82%)and Basidiomycota(14%)as the fungus advantage bacterium group,Proteobacteria(49%),the Cyanophyta(23%)and Firmcutes(7%)advantage group for bacteria.Most high-abundance fungi were pathogens,while high-abundance bacteria contain many potential biological controls or improve plant health beneficial members.(2)According to the results of GO database comparison,molecular functions related to microbial colonization and nutrient competition,such as ATP binding,are more important,and biological processes related to disease resistance,such as transmembrane transport,also make a greater contribution.According to the results of CAZy database comparison,the relative abundances of CBM18,CBM 50 GH19,GH18 GH13 and GT2 gene families were higher than 3% of the total abundances of identified CAZy families,and they were more related to fruit health and disease control.According to the comparison results of CARD-based database,at the phylum level,the microorganisms with antibiotic resistance were mainly Ascomycetes(56.27%),Proteobacteria(21.49%),Basidiomycetes(8.38%)and Cyanobacteria(6.58%).At the order level,the proportion of Tricholocystis(24.00%),Carnonia(16.09%)and Rhizobium(10.56%)was higher.These microorganisms could better adapt to environmental pressure and have higher stability when used as biological control agents.According to the results of PHI database comparison,a total of 10 genes in 3 pathogens were identified,which were related to the interactions between fruits and microorganisms and could be used as disease resistance targets(3)Based on the results of metagenomic shotgun sequencing of tangerine,the script of species and functional association analysis was established,which could provide a background for functional prediction of citrus fruit epiphytic microbiota.2.Core epiphytic microorganisms in citrus fruits of different varieties:(1)The bacterial diversity was higher in the epiphytic flora of “Fengqi” and “Ponkan”fruits,but the fungal diversity was higher in the epiphytic microbiota of tangerine and lemon fruits.Although high-abundance microorganisms were distributed in all the four citrus varieties,there were significant differences in the epiphytic microbial community structure among different varieties at the phylum level.Cyanobacteria and Proteobacteria and Ascomycetes were the dominant epiphytic microbiota in citrus.(2)Based on Venn diagram analysis,at the family level,bacteria such as Alphaproteobacteria and fungi such as Eurotiomycetes were the common microorganisms with high abundance in the four species.At the genus level,the relative abundance of microorganisms of different citrus varieties varied greatly,and the microbial community structure of “Fengqi” and “ponkan” was more similar.Based on correlation network analysis,at the genus level,bacteria such as Acinetobacter and fungi such as Ceraceosorus were strongly correlated with other microbial members,which played an important role in maintaining the stability of community structure.According to LEf Se difference analysis,at the genus level,a total of 11 bacterial genera and 9 fungal genera were defined as biomarkers in the epiphytic community of specific citrus species,that is,their relative abundance in specific groups was significantly higher than that in other groups.According to the above three criteria,a total of 14 bacterial genera and 10 fungal genera were defined as the core epiphytic microflora of citrus fruits.3.Effects of bacterial synthetic community on control of postharvest green mold and acid rot in citrus fruits:(1)115 strains of bacteria were isolated and identified from citrus peel,and 8 strains were selected to construct the synthetic community according to the core epiphytic microorganism group standard.In the plate test,only one strain had direct inhibition effect on the pathogens of citrus postharvest green mold and acid rot,Penicillium dactyliformis and Geotrichum candidum.(2)The bacterial synthetic community and its members could inhibit the occurrence of disease incidence rate and delay the expansion of the diameter of the disease spot.The single bacterial treatment with the best synthetic community had a weaker control effect on the disease in the later stage.The bacterial synthetic community and its individual microbial members can inhibit the occurrence of acid rot and delay the expansion of spot diameter,but some microbial members acting alone can promote the occurrence of acid rot.Synthetic community treatment had the best control effect on acid rot.(3)The effect of bacterial synthetic community soaking treatment on the postharvest natural morbidity of citrus fruits was like that of low concentration commercial insurance agent treatment,and it had a high commercial application potential.After synthetic community soaking treatment,the bacterial community structure of citrus epiphytes could be kept stable at room temperature for a long time.In summary,the species and functional associations of citrus fruit epiphytic microbiota were studied in this paper,and the core microbial members were screened by amplifiers sequencing analysis of the epiphytic microbiota of four different fruit varieties.The important bacterial species resources were obtained by in vitro isolation and culture.According to the core microbial members,the bacterial synthetic community was constructed,and it was verified that the constructed community had a good control effect on the common postharvest diseases of citrus fruits,green mold,and acid rot.