Diversities And Ecological Types Of Rhododendron Root-associated Fungi In Liangshan Yi Autonomous Prefecture, Sichuan

Root-associated fungi (RAF) are indispensable components of the ecological processes involving organic matter decomposition, nutrient cycling, and density dependence in plant community. Thus, RAF may have profound ecological significances for plant community succession, maintenance of biodiversity, as well as habitat restoration and reconstruction.China is the origin and distribution center of Rhododendron, the worldwide famous ornamental plant. Studying the phylogenetic and functional diversity of the RAF of Rhododendron plants in their natural habitats is not only essential for revealing the environmental adaptability of Rhododendron,but also has great implications for conservation and utilization of Rhododendron germplasm resource.In this study, a molecular toolbox was developed for fast and accurate identification of the RAF community of Rododendron species through comparing the influence of different pooling strategies of samples and number of sequence reads to revealing the RAF species composition of Rhododendron plants. This molecular toolbox was used to identify the diversity of RAF of 19 Rhododendron plant species in Liangshan Yi Autonomous Prefecture of Sichuan Provence, China. Both the putative trophic mode and guild of RAF were assigned by linking sequences with metadata obtained by a systematic review of published works on RAF ecology from multiple independent studies, and by functional analysis provided by the FUNGuild software. In addition, Redundancy analysis was used to revealing the effects of host plants and study site for the community structure of RAF.The results are as follows:(1) Pooling strategies have effects on detecting of RAF. Species richness of fungal communities clone library constructed by pooling samples after PCR was higher than that of pooling prior to PCR. Notably, the prior to PCR pooling strategy effectively alleviated the unwanted amplification of host plant sequences when fungal specific primer ITS1F and ITS4 were used. Accumulation curves of fungal species suggested that sequencing at least 50 clones can fully reflect species composition of clone library of Rhododendron RAF community. Clone library constructed by post PCR pooling of samples is better in providing accurate views of Rhododendron RAF diversity and community structure.(2) The phylogenetic diversity of Rhododendron RAF were high. A total of 485 fungal species (OTUs) were detected in hair roots of 224 Rhododendron plant individuals. Of these fungi,312 species fell into Ascomycota,including Leotiomycetes (195 OTUs),Dothideomycetes (25 OTUs), Sordariomycetes (23 OTUs), Eurotiomycetes (23),Lecanoromycetes (7 OTUs), Pezizomycetes (3 OTUs), Geoglossomycetes (2 OTUs) and unknown ascomycetes (34 OTUs); Basidiomycota were representing by 168 fungal species,containing Agaricomycetes (159 OTUs), Malasseziomycetes (2 OTUs),Spiculogloeomycetes (2 OTUs), Tremellomycetes (1 OTU), Microbotryomycetes (1 OTU),Cystobasidiomycetes (1 OTU) and 2 unknown basidiomycetes species. In addition, four,two and one species show close affinity to Glomeromycota, Chytridiomycota, and Zygomycota, respectively. The dominant fungal groups were Helotiales (170 OTUs) and Sebacinales (66 OTUs) in the Rhododendron RAF community.(3) Some Sebacinales fungal species showed host preference to Rhododendron plants grouped by Subgenus and to study sites. Plant identity and study sites showed significant effects on the species composition of fungal communities. However, variances in fungal species composition explained by these two factors were limited according to the results of Redundancy analysis.(4) Rhododendron RAF are functionally diversified. A total of 107 species of RAF can be identified to genus or species levels, and 21% fungal species can be assigned to a certain ecological types. Results of FUNGuild showed that, fungal species associated with roots of Rhododendron plant can be assigned to multiple trophic modes and guilds,including well known ericoid mycorrhizal symbionts of 6 species (i.e., Oidiodendron spl,Oidiodendron sp2, Oidiodendron sp3, Oidiodendron sp4, Oidiodendron sp5and Rhizoscyphus ericae), as well as repeatedly documented endophytes of 11 species (e.g.Phialocephala sp8, Phialocephala fortinii and Leptodontidium sp.), 21 ectomycorrhizal fungal species (e.g. Meliniomyces sp2, Sebacina sp2 and Meliniomyces spl), one symbiotic fungi Pezoloma ericae, and 50 saprotrophs (e.g. Lachnum virgineum, Myceana sp3 and Tetracladium sp.). In addition, 254 species of RAF had more than 97% sequences identity with target fungi in NCBI. The results of systematic review showed that majority of fungil involved a variety of host plants, such as arbuscular mycorrhizal, ectomycorrhizal, ericoid mycorrhizal, orchid mycorrhizal. And a small number of RAF, (e.g. Sebacinales sp13、Helotiales sp13、Helotiaceae sp17) have only been reported in species of Ericaceae,so far.This study has revealed that phylogenetic and functional diversity of Rhododendron RAF community are very high. These findings will not only help to understand how functional diversity of RAF affects ecosystem services, but also provide implications for conservation of Rhododendron plant diversity.