Diversity Of Endophytic And Epiphytic Fungi And Allelopathic Potential Of Their Metabolites In Differently Aged Casuarina Equisetifolia Litter

Casuarina equisetifolia is an excellent tree species of coastal protection forest in Hainan Province.In recent years,there has been aging of forest stand and regeneration difficulties mainly due to the allelopathic effects of forest land.C.equisetifolia is rich in endophytic fungi resources,and it has been found that there are some allelopathic substances in the secondary metabolites of its root endophytic fungi,which indicates that microorganisms participate in the allelopathy of C.equisetifolia.As the intermediate of nutrient transfer in forest land,the litter has endophytic and epiphytic fungi,which may also be related to the allelopathy of C.equisetifolia.However there has not been any report on these researches.Therefore,this study selected three C.equisetifolia forest(young-aged forest,middle-aged forest and mature-aged forest)in the Guilinyang sea coast,Haikou City,Hainan Province as the research sites and analyzed the diversity of endophytic and epiphytic fungal communities in the C.equisetifolia litter of different ages by Illumina Miseq high-throughput sequencing to explore the structural characteristics of fungi community in the litter.We combined the classical method and ITS rDNA sequencing to isolate and identify the culturable fungi in the litter.We applied the pure culture fungal fermentation liquid of the litter to C.equisetifolia seeds to explore the fungi's allelopathy potential on the C.equisetifolia seeds.We adopted GC-MS to detect the secondary metabolites of endophytic and epiphytic fungi in the culturable litter with relatively strong allelopathy potential,and compared the common substances between C.equisetifolia litter,soil and root extract.This study provided a foundation for the study of the fungal ecological function of C.equisetifolia litter,and a theoretical basis for further study on the synthesis mechanism and allelopathy mechanism of the allelochemicals in C.equisetifolia forest,and a theoretical support for solving the regeneration difficulties of C.equisetifolia forest.The main research results were as follows:1.A total of 669,476 effective sequences were obtained from the endophytic and epiphytic fungi in litter of C.equisetifolia of different ages,from which 225 endophytic fungi and 293 epiphytic fungi were detected.The diversity of epiphytic fungi was higher than that of endophytic fungi,which was the most obvious in middle-aged forest,then the young-aged growth forest,and the least obvious in mature-aged forest.The endophytic and epiphytic fungi had a lot in common in the community composition.Ascomycota was their common dominant phylum,and basidiomycetes accounted for a high proportion in both fungi.But their dominant fungal communities were different,the Hydnodontaceae and unclassifiedXylariales were dominant in the endophytic fungi,while the unnamed Capnodiales,unclassified Pleosporales and Mycosphaerellaceae were dominant in the epiphytic fungi.pH value and organic carbon of C.equisetifolia litter significantly affected the community structure of endophytic fungi,while volume-weight of soil and ammonium nitrogen significantly affected the community structure of epiphytic fungi.2.58 species of litter fungi were isolated and identified,including 27 species of endophytic fungi,41 species of epiphytic fungi and 10 mutual fungal species of endophytic fungi and epiphytic fungi,such as Pestalotiopsis sp.,Fungal endophyte and Mycosphaerella sp.Among the endophytic fungi,there are 11 species in young-aged forest,15 species in middle-aged forest and 4 species in mature-aged forest;and Aspergillus,Cladosporium and Mycosphaerella were the dominant genera of endophytic fungi.Among the epiphytic fungi,there were 23 species in young-aged forest,15 species in middle-aged forest and 9 species in mature-aged forest;and Cladosporium,Pestalotiopsis,Trichoderma and Didymosphaeriaeae were the dominant genera of epiphytic fungi.3.The fermentation liquid of most litter fungi inhibited the germination of C.equisetifolia seeds,while a few species showed the promotion effect.Fungal endophyte,Pestalotiopsis sp.,Mycosphaerella sp.And Citrus maxima showed the strongest inhibition effect;and Coniochaetaceae sp.and Asordaria conoidea showed the strongest promotion effect;and Lentinus squarrosulus had neither promotion effect nor inhibition effect.4.The secondary metabolites of endophytic and epiphytic fungi of C.equisetifolia litter mainly include alcohols,phenols,aldehydes,esters,ethers,ketones,alkanes,pyridines and organic acids.n-Hexadecanoic acid,octadecanoic acid,2,4-bis(1,1-dimethylethyl)-,Phenol,2,2'-methylenebis [6-(1,1-dimethylethyl)-4-methyl-were the mutual allelopathic substances of Fungal endophyte and Pestalotiopsis sp..1,2-Benzenediol was the specific allelopathic substances of Pestalotiopsis sp..The allelopathic substances Phenol,2,4-bis(1,1-dimethylethyl)-and Phenol,2,2'-methylenebis[6-(1,1-dimethylethyl)-4-methylwere detected in the fermentation liquid of litter fungi and the extract liquid of litter,soil and root.The results showed that the community composition of endophytic and epiphytic fungi in C.equisetifolia litter was affected by the age of C.equisetifolia forest and the physicochemical properties of living environment.The secondary metabolites of most species of litter fungi community had allelopathic potential for C.equisetifolia seeds and allelopathic substances were detected in them.Some of the allelopathic substances were mutual shared by C.equisetifolia litter fungi,litter,soil and root,indicating that litter fungi participated in theallelopathy of C.equisetifolia,and the allelopathic substances it produced may diffuse in soil and root with the degradation of litter,leading to the accumulation of allelopathic substances in forest land.