Studies On The Diversity Of Endosphere And Rhizosphere Fungi On The Cypripedium Japonicum Thunb.

Orchid seeds are tiny and lack of endosperm for storing nutrients,and the seeds germinate successfully only when they are infected by the appropriate symbiotic fungi in the natural environment.That is one of the reasons for the orchids being endangered.Cypripedium japonicum Thunb.,which is endemic endangered orchid in East Asia,has decreased dramatically in recent years,and has been listed in appendix I of the Convention on International Trade in Endangered Species of Wild Fauna and Flora(CITES).However,its studies of this species are limited and the diversity and specificity,as well as dynamic change of its mycorrhizal fungi at different times and habitats are still inefficiently understood.Here,we investigated the roots and rhizosphere soil fungi communities of four wild populations of C.japonicum in the Tianmu mountains(Hongshedong,Laodian,Baojianshi)and Qingliang peak with high-throughput sequencing of Illumina MiSeq,and processed bioinformatics and statistical analyses to explore the diversity of rhizosphere and endosphere fungi,as well as the influence of site,growth stage and taxonomic factors on the symbiotic fungal community of C.japonicum.The main results were as follows.1.From all the samples(17 plants)of C.japonicum,we obtained 896 OTUs(518,688 sequences)of endosphere fungi,belonging to 7 phyla,25 classes,64 orders,142 families and 246 genera,and 5,442 OTUs(1,116,937 sequences)of the rhizosphere fungi,belonging to 9 phyla,34 classes,115 orders,296 families and 695 genera.2.The typical mycorrhizal fungi that symbiosis with C.japonicum are Ceratobasidiaceae(Cantharellales)and Tulasnellaceae(Cantharellales).In addition,the dominant genera include Aspergillus,Cladosporium,Entorrhiza and Ilyonectria.At the meantime,the typical mycorrhizal fungi in rhizosphere were Serendipitaceae and Sebacinaceae(Sebacinales),and the dominant groups were Mortierella,Ilyonectoria,Saitozyma and Solicoccozyma.There were significant differences between endosphere and rhizosphere fungi diversity of C.japonicum,which indicated that endosphere fungal community and rhizosphere fungal community were relatively independent.3.Comparing and analyzing the endosphere and rhizosphere fungal communities of C.japonicum in four populations,we found that the endosphere and rhizosphere fungal communities of C.japonicum would change with the different sites.There were significant differences abundance among the fungal communities in different sites,as well as the dominant groups.In the three endosphere fungal communities of Tianmu Mountain,Aspergillus was dominant,while in Qingliang peak was Entorrhiza.4.Analyzing the endosphere and rhizosphere fungal communities of C.japonicum in flowering and fruiting stage in Hongshedong,we found that the symbiotic fungi OTUs in the flowering stages was 1.5 times higher than that in the fruiting stage,as well as the diversity and abundance of endosphere fungi.The main dominant groups were also significantly different,the flowering stage was Entorrhiza,and the fruiting stage was Aspergillus,while there was no significant difference between the rhizosphere fungal communities.The results showed that C.japonicum could meet nutrition demand at different growth stages and adapt to the changes of external environment by changing the abundance and dominant groups of its fungal community.5.By investigating and comparing the endosphere and rhizosphere fungal communities of C.japonicum,Goodyera biflora and Ligularia stenocephala in the Hongshedong population,it was found that the three species living in the same habitat would choose different fungi from the environment to symbiosis and provide nutrition for growth.Moreover,although the C.japonicum and G.biffora are Orchids and L.stenocephala belongs to Compositae,the differences of the symbiotic fungal communities among the three species is not necessarily related to their taxonomic relationships.Generally speaking,the symbiotic fungal community of C.japonicum will change with the environment and other conditions,and the fungi specificity is not obvious.