Preliminary Study On The Specificity Of Symbiotic Germination Between Medicinal Dendrobium Seeds And Mycorrhizal Fungi And Their Mechanism Of Action

As the second largest family of angiosperms,Orchidaceae has the highest degree of evolution,contains many medicinal plants and precious flowers,and has high commercial value.Orchid seeds have no endosperm,so they establish a symbiotic relationship with suitable fungi in order to complete seed germination under natural conditions.Some orchids must depend on mycorrhizal fungi to provide nutrients to complete the entire life history.Orchid mycorrhiza is one of the most concerned mycorrhizal type among seven major mycorrhizal types.Studies have shown that there is a certain specific relationship between orchids and mycorrhizal fungi,but the specific symbiosis mechanism is still unclear.This article starts with the specificity of symbiotic germination between Dendrobium sp.seed and different fungi,and then to explore the seed symbiotic specificity mechanism between Dendrobium officinale with two different mycorrhizal fungi.The research results of this paper are summarized as follows:1.Isolation and identification the endophytic fungi of D.officinale and D.huoshanense358 strains of endophytic fungi from the roots of D.officinale were obtained by the method of tissue block and single hyphae cluster,and 292 strains were identified;23 strains of endophytic fungi from the roots of D.huoshanense were isolated and 20 strains were identified.The identified fungi belong to 65 genera in taxonomy,of which Fusarium spp.(25.00%)accounts for the most.A total of 46 strains of typical orchid mycorrhizal fungi such as Tulasnella fungi were divided into 29 strains after morphological and molecular identification.After constructing a phylogenetic tree,they were divided into 9 clades;A total of 20 strains of typical orchid mycorrhizal fungi such as Sebacina fungi were divided into 11 strains after morphological and molecular identification.After constructing a phylogenetic tree,they were divided into 4 clades.2.Evaluation of mycorrhizal fungi in promoting the germination of medicinal Dendrobium seedsCombining the classification status of mycorrhizal fungi,16 mycorrhizal fungi(11 strains of Tulasnella and 3 strains of Sebacina,1 strain of Athelia and 1 strain identified)were selected for co-cultivation with D.officinale seeds,and it was found that 11 mycorrhizal fungi had better seed promotion,and the germination-promoting activities of those fungi were different.5 strains of mycorrhizal fungi could not promote seeds germination.At the level of species-species interaction,the seeds of D.officinale symbiotic germinate with a variety of mycorrhizal fungi,and the degree of specificity between them is low.20 mycorrhizal fungi(13 strains of Tulasnella,7 strains of Sebacina)co-cultured with seeds of D.huoshanense respectively,and germination-promoting activities of those fungi were different.5 mycorrhizal fungi could not promote seed germination,and the mycorrhizal fungi 12825 isolated from D.moniliforme has the best effect on promoting germination,and the non-mycorrhizal fungus does not promote the germination of seeds.At the level of species-species interaction,the seeds of D.huoshanense can be symbiotic germinated with a variety of mycorrhizal fungi,and the degree of specificity between them is low.This experiment successfully realized the symbiotic germination of D.huoshanense seeds and reached the seedling stage in the laboratory condition for the first time.3.Comparative analysis of plant cell wall degrading enzyme activities of mycorrhizal fungi with different seed germination activitiesPreliminary qualitative analysis of the extracellular hydrolase activity of the abovementioned strains with different promoting seed germination activities was carried out by the plate method.The results showed that the orchid mycorrhizal fungi Sebacina sp.can secrete laccase,while the Tulasnella fungi do not secrete laccase There is no significant difference in the ability of secreting pectinase,protease between most mycorrhizal fungi and non-mycorrhizal fungi.Under the same culture conditions,there is no direct relationship between the ability of produceing enzyme of fungi(laccase,pectinase,and protease)on the specific media with obligate induction of extracellular enzyme secretion and their activities to promote the germination of Dendrobium seeds.It is speculated that the symbiotic germination of D.officinale seeds probabaly depend many kinds of degrading enzymes of many fungi under natural conditions,or the ability of fungi secreting plant cell wall degrading enzymes in a pure culture state is different from the ability to secrete related enzymes in a symbiotic state.4.Comparative transcriptomic analysis the symbiotic germination between D.officinale seeds and two kind of different fungi.By comparative transcriptomic analysis of symbiotic germination of D.officinale seeds inoculated with two different fungal species,the co-expressed and differentially expressed genes of the fungi were screened.We focus on the gene expression encoding small secretory proteins,lipases,proteases,plant cell wall degrading enzymes of different fungi during the process of symbiosis with D.officinale.The results showed that,1003 genes were differentially expressed across all symbiotic samples after inoculated two fungi.The subsequent qRT-PCR verification will be carried out to verify the expression of these genes.In this study,we have preliminarily clarified the specific relationship between the seed germination between D.officinale and mycorrhizal fungi under laboratory conditions;A number of strains with promoting germination were isolated from the roots of D.officinale.Moreover,a set of key candidate genes for germination of D.officinale seeds have been identified,which provides reference data for further study of the symbiosis mechanism between orchid and mycorrhiza fungi.