Study On The Relationship Between Citrus Fruit Drop And Endophytic Fungi

Citrus,one of the most important fruit crops in the world,is widely cultivated in tropical and subtropical regions.China has more than 4,000 years of citrus cultivation history.It not only has accumulated abundant germplasm resources but also has developed a perfect cultivation management system.There are fruit drop phenomena from post-flowering to harvest.The two physiological fruit drops that occurred in late April and early June are serious.At present,the research on the factors that cause citrus fruit drop mainly focuses on fruit tree cultivation and weather condition.In this study,weintended to explore the mechanism of citrus physiological fruit drop from the perspective of endophytic fungi.In this study,the ITS high-throughput sequencing technology was performed to analyze the microbes inside the dropping fruits and healthy fruits of E'gan No.1 Ponkan(Citrus reticulata Blanc.cv.ponkan)and Newhall Navel Orange(Citrus sinensis [L.]Osbeck).The results showed that 296 and 369 OTUs(operational taxonomic units)were identified in the healthy fruits and dropping fruits of E'gan No.1,respectively.Between the two groups of fruits,the alpha diversity indicated no significant difference in the richness of the endophyte community,while the beta diversity showed difference in the community structure of the endophyte.In all,261 and 359 OTUs were detected in the healthy fruit and dropping fruit of Newhall,respectively.Between the two groups of fruits,alpha diversity indicated a significant difference in the richness of the endophyte community,and beta diversity showed a difference in the community structure of endophyte.It suggests that the richness of the fungal community and the difference in composition and structure may be related to fruit drop.The cluster analysis of the top 20 abundance(genus level)fungi in the fruit demonstrated that the fungi from 5 genera of Sclerotinia,Paraphaeosphaeria,Trichoderma,Rhizopus,and Mucor were enriched in the healthy fruits of two citrus varieties compared with the dropping fruits.While Fusarium spp.,Alternaria spp.,Aureobasidium spp.,Erythrobasidium spp.,Curvularia spp.,Cercospora spp.,Papiliotrema spp.,Rhodosporidiobolus spp.,Curvibasidium spp.,Piskurozyma spp.,Sclerostagonospora spp.,Cystofilobasidium spp.,Zasmidium spp.were accumulated in the dropping fruits of two citrus varieties.The number of Colletotrichum spp.and Epicoccum spp.accumulated in the healthy fruit of E'gan No.1 was higher than that in the dropping fruits,while in Newhall the number of Colletotrichum spp.and Epicoccum spp.identified was higher than that in the healthy fruits than in the fruit drop.At the same time,we found that there were differences in the types and numbers of common fungal species in healthy fruits and fruit drop.The analysis of the endophyte compositions of citrus dropping fruits and healthy fruits of E'gan No.1 and Newhall laid the foundation for a comprehensive understanding of the diversity of endophytic fungi,the subsequent analysis of the microbial causes of fruit drop,and the study of the interaction between endophytes and host.To further determine the relationship between endophytes and citrus fruit drop,we inoculated the endophytic fungal isolated from the dropping fruit samples onto the immature citurs fruits and detected the changes of the content of abscisic acid and ethylene in the inoculated fruits.For each citrus variety,23 fungal strains were isolated from the dropping fruits.These isolates were inoculated onto the immature fruits of corresponding citrus varieties.The results of the pathogenicity test showed that most strains did not cause obvious lesions on the citrus fruits.While after the inoculation of the endophytes,the fruits became yellow around the inoculation site.The content of abscisic acid and ethylene in the citrus fruits inoculated with fungal strains was determined by chromatography.The results showed that the contents of abscisic acid in both E'gan No.1and Newhall green fruit of inoculated with Alternaria tenuissima(EG-13?NHE-3)and Fusarium incarnatum(EG-15?NHE-6)strains were significantly higher than those of the control fruits.The ethylene released by E'gan No.1 and Newhall green fruits inoculated with Fusarium incarnatum(EG-10)were significantly more than that by the control groups.This demonstrates that some spicies of Alternaria tenuissima and Fusarium incarnatum isolated in this study could cause an increase of abscisic acid content in citrus green fruit,and Fusarium incarnatum could also cause an increase in ethylene content.Combining the results of ITS sequencing and hormone content determination,we found that the accumulation of Fusarium spp.and Alternaria spp.was higher in thedropping fruit than in the healthy fruit.It suggested that some spicies of Alternaria spp.and Fusarium spp.in the dropping fruit might lead to early yellowing and shedding of the fruit by increasing in the abscisic acid and ethylene content.This study explores the mechanism of citrus physiological fruit drop caused by endophytic fungi and provides new clues for further understanding of citrus cultivation.