Symbiotic Fungi Diversity Of Germination Stage And Symbiotic Germination Mechanism Of Gymnadenia Conopsea Seeds

Gymnadenia conopsea is one of widely distributed terrestrial orchids.It is often used in traditional Chinese medicine,Tibetan medicine and Mongolian medicine.It has the effect of tonifying kidney and strengthening essence,relieves pain.However,it has not realized artificial cultivation and the wild population of G.conopsea has declined sharply.Thus,it was listed in the National Key Protected Wild Plant(Category II)due to the increasing market demand and the low natural reproduction rate.Therefore,it is of great significance to explore seed symbiotic germination of G.conopsea with fungi for the protection of germplasm resources and the recovery of the population.Orchids have a natural symbiotic relationship with mycorrhizal fungi.Mycorrhizal fungi not only provide nutrients for orchids,improve their stress resistance,but also promote seed germination.In previous study,we found that Ceratobasidium GS2 could effectively promote the seed germination of G.conopsea.In this study,germinated G.conopsea seeds with GS2 were used as research material,Illumina MiSeq high-throughput sequencing technology was used to determine the diversity of symbiotic fungi and bacteria in different germination stages of G.conopsea seeds,then the culturable symbiotic fungi were isolated and their germination promoting activities were screened.TMT labeled quantitative protein technique was used to study the molecular mechanism of fungi promoting seed germination of G.conopsea at proteome level.The main research results are as follows:1.Symbiotic germination of G.conopsea seeds with GS2 was successfully achieved under natural conditions for the first time.The germination rate of embryos break through testa was 11.96±2.65%,early seedlings stage was 3.16±1.63%.Under indoor conditions,a breeding system for indoor G.conopsea seedlings was initially established.These results laid a good foundation for the restoration of the population of G.conopsea and the largescale imitation of wild cultivation to provide a source of seedlings.2.There were significant differences in fungal community composition among seeds of G.conopsea at different germination stages(R2=0.568,p=0.001).A total of 116 OTUs were detected from seeds of different germination stages,Ceratobasidiaceae was dominant group accounting for 96.46%in all OTUs,43 OTUs shared by four germination stages were fungal core OTUs.Similarly,there were significant differences in bacterial community composition among seeds of G.conopsea at different germination stages(R2=0.314,P=0.001).A total of 162 OTUs of bacterial community were detected,Pseudomonadaceae and Burkholderaceae were the absolute dominant groups,73 OTUs shared by four germination stages were bacterial core OTUs.High diversity of symbiotic fungi and bacteria was detected at different germination stages of G.conopsea seeds,it indicates that in addition to GS2,there were also a large number of indigenous fungi and bacteria involved in seed germination and development.3.A total of 31 strains were isolated from seeds of different germination stages,among which 5 strains,including GS2,had the activity of promoting seed germination of G.conopsea.These results verified the effectiveness of GS2 in seed germination of G.conopsea under natural conditions.The other strains did not promote seed germination,and their ecological functions need to be further clarified.4.A total of 98 differential proteins were obtained from pure cultured mycelia and symbiotic mycelia with G.conopsea seeds,including 68 up-regulated and 30 downregulated proteins.These differential proteins were significantly enriched in nutrient metabolism and signaling pathway.It mainly contains starch and sucrose metabolism,amino sugar and nucleotide sugar metabolism,galactose metabolism,MAPK signaling pathway.These results showed that GS2 mycelium not only provided nutrients for seed germination,but also obtained nutrients such as sugars and lipids from germinating seeds.In this study,we first achieved seeds symbiotic germination of G.conopsea with fungi under natural conditions.The diversity of symbiotic fungi in different germination stages was determined.GS2 was isolated from germinated seeds again,which showed its effectiveness in promoting seed germination under natural conditions.Other Ceratobasidiaceae fungi which can promote seed germination can also be used in the future cultivation of G.conopsea.The differential proteome in mycelia of GS2 strain in promoting seed germination of G.conopsea was preliminarily analyzed,the interaction mechanism between mycelium and seed at proteome level was preliminarily speculated.The results of this study will further realize the imitation wild scale cultivation of G.conopsea and analyze the molecular mechanism of seed germination promoted by fungi.