Discovery And Utilization Of Orchid Mycorrhizal Fungi Resources In Beijing Area

Orchid plants are of a natural symbiotic relationship with mycorrhizal fungi.Mycorrhizal fungi play an important role in seed germination and plant growth,and are also considered to be important ecological factors for the survival,richness,and spatial distribution of orchids.The research on orchid mycorrhizal fungi is of great significance for the conservation of orchids and the sustainable use of resources.In this study,10 species of Orchidaceae belonging to 9 genus in Beijing region were collected,including Gymnadenia conopsea,Platanthera chlorantha,Tulotis fuscescens,Cypripedium macranthum,Cypripedium guttatum,Dactylorhiza viridis,Herminium monorchis,Malaxis monophyllos,Spiranthes sinensis,Neottia acuminata.The methods of paraffin section,Illumina MiSeq high-throughput sequencing,plating individual carefully separated fungal pelotons and symbiotic seed germination were used to study their mycorrhizal structures,the mycorrhizal fungal community composition,the isolation and culture of mycorrhizal fungi and the ecological function of mycorrhizal fungi respectively,which contribute to comprehensive understanding of the interaction between mycorrhizal fungi and orchids in Beijing,provide references for the breeding and protection of endangered wild orchids in Beijing and promote the ecological restoration of the population.The main results are as follows:1.Microscopic observations of eight species of orchids in Beijing suggested that the velamen was thin and consisted of only one layer of cells.Hyphae destroyed the velamen,invaded and colonized cortical parenchyma cells.And the fungus mostly present loose hyphae or pelotons which showed typical mycorrhizal characteristics.2.The high-throughput sequencing of orchid mycorrhizal fungi in Beijing produced a total of 486,344 sequences,among which basidiomycete sequences was 392,188,accounting for 80.64%of the total sequences.The others were small amounts of ascomycetes and unidentified fungal species.The basidiomycetes sequences were classified into 194 OTUs,among which 83 OTUs with a number of 387162 sequences accounting for 98.72%of the basidiomycete group,belonged to common mycorrhizal fungi,including Ceratobasidiaceae,Tulasnellaceae,Sebacinales,Thelephoraceae,Russulaceae,Mycenaceae and Cortinariaceae.The member of Ceratobasidiaceae(20 OTUs),Tulasnellaceae(9 OTUs)and Sebacinales(34 OTUs)were the main mycorrhizal fungi with a number of 378386 sequences,accounting for 94.33%?99.97%of the common mycorrhizal fungi in different orchid species.The mycorrhizal specificity of different species of orchids was distinct.The variation of the composition of mycorrhizal fungi in orchids was observed in different regions.3.Eight strains of fungi were obtained from the roots of Orchidaceae in Beijing by using plating individual carefully separated fungal pelotons.From the perspective of morphological characteristics and molecular biological identification,four strains of fungi belonged to Tulasnellaceae,and three strains belonged to Ceratobasidiaceae,only one strain belonged to Sebacinales.4.The eight isolated mycorrhizal fungi were screened out by symbiotic germination experiments to examine their effect on seed germination of four medicinal Orchidaceae species,that is,Gymnadenia conopsea,Herminium monorchis,Spiranthes sinensis and Liparis japonica.Two fungal strains with evident effect on germination and seedling establishment were screened out.Only strain GS2 can effectively promotting the seed germination of Gymnadenia conopsea,which showed higher specificity between the seed and germination mycobiont.The strain HH1 can effectively promotting the seed germination of Herminium monorchis,Spianthes sinensis and Liparis japonica,which showed higher germination activity.The process of symbiotic germination of four orchid species was observed and recorded.The microstructure also showed that the fingus established a symbiotic relationship with the protocorm or seedlings.Through the research in this paper,we have defined the mycorrhizal fungal community composition of orchid plants in Beijing.The active strains found to promote seed germination can be further applied in artificial cultivation of orchid plants and ecological restoration of wild populations.