Diversity Of Endophytes In Oxytropis Glacialis On The Qinghai-Tibet Plateau And Its Adaptability To The Environment

Endophytes play an important role in the survival and diffusion of host plants.Oxytropis glacialis are endemic and widespread in the Qinghai-Tibet Plateau,and the whole grass is poisonous.It is one of the poisonous plants of Locoweed that affects the development of animal husbandry in Tibet,and the toxin-swainsonine is produced by its endophytic fungi.At present,research on endophyte of Oxytropis glacialis is mostly focused on the isolation and identification of endophytic fungi,and the study on the diversity of endophyte of Oxytropis glacialis has not been reported.In this study,Illumina MiSeq high-throughput sequencing and bioinformatics methods were used to analyze the diversity,microbiome structure and adaptive relationship between endophytes in different ecological environments and different plant tissues,and to reveal the network interaction model among endophytes of Oxytropis glacialis,so as to provide a basic reference for the scientific management and utilization of locoweed poisonous plants in Qinghai-Tibet Plateau.The main results and conclusions are as follows.1.The results of endophytic bacteria diversity analysis of Oxytropis glacialis based on 16S rRNA sequencing of TAS and TAM samples from Tibet showed that total 236205 sequences and 476 OTUs were obtained,which are belonging to 17 phyla and 281 genera.There were no significant differences between the TAM and the TAS samples.Composition analysis and difference analysis showed that the dominant phyla in different samples were Proteobacteria.Pseudomonas,Lachnoclostridium 5,Pantoea and other 13 genera were predominant genera.According to the environmental analysis,soil conductivity?EC?,air CO₂ concentration?CC?and soil temperature?ST?have different degrees of significant effects on the differences of bacterial community structure,and 56%of dominant bacteria are significantly related to environmental factors.Functional prediction analysis suggested that the abundance of genes related to each function of KEGG metabolic pathway and COG were similar among samples in different regions,and there were genes related to secondary metabolites biosynthesis and catabolism,which provided the possibility for other metabolic pathways and biological functions to play a role.In different ecological environments,the structure and function of bacterial communities are relatively stable,the functions of endophytic bacteria to participate in the functional regulation of host plants diverse metabolic networks,and the abundance of dominant bacteria varies with the environment.This is conducive to the survival and diffusion of plant-endophytic bacteria in a more severe ecological environment.2.The results of endophytetic fungi diversity analysis of Oxytropis glacialis based on 16S rRNA sequencing of TAS and TAM samples from Tibet showed that total 1715403 sequences and 710 OTUs were obtained,which are belonging to 7 phyla and 241 genera.The results of Diversity analysis showed that the endophytic fungi structures of TAS and TAM samples were significantly different.Composition analysis and difference analysis showed that the advantage phylum of both TAS and TAM samples are Ascomycota.Gibberella,Pleosporaceae unclassified genus?and Chaetosphaeronema ect.are core microbiom,Cladophialophora,Lachnum,ect.were significantly differet?P<0.05?between TAS and TAM.FUNGuild analysis showed that the proportion of main nutrient modes of endophytic fungi of Oxytropis glacialis was PA>UN>SA>SY,PA-SA,PA-SY,SA and SY had significant differences between different regions?P<0.05?.CCA analysis showed among all the environmental factors that significantly affected the fungi structure between samples,soil p H?R²=0.8358?and soil conductivity?R²=0.7449?have the higher explanatory rate than CO₂ concentration?R²=0.2834?.The regional specificity of microbiome structure and function of endophytic fungi of Oxytropis glacialis is greater than that of conservatism in different ecological environments.The nutrition mode is adapted to microbiome structure,and the microbiome structure is closely related to environmental factors.Significant differences in nutritional patterns and microbial community structure of plant endophytic fungi may contribute to survival and diffusion in different environments.3.The diversity of endophyte in leaf and root tissues of Oxytropis glacialis was studied,and the adaptive relationship between the structure and function differences of endophytic community and the environmental factors was analyzed.In this study,175,791 effective bacterial sequences were sequenced,and 428 OTUs belonged to 19 genera and267 genera.There were 757113 fungal effective sequences,and 391OTUs belonged to 7 phyla and 149 genera.Diversity analysis showed that there was no significant difference in endophytic community richness in different tissues.The diversity of the endophytic fungi community in the root was significantly greater than that of the leaf,and the diversity of the endophytic community in the root was less than that of the leaf but there was no significant difference.Composition analysis and difference analysis showed showed that the dominant endophytic bacteria in different tissues was Proteobacteria,and the dominant endophytic bacteria was Ascomycota.The total genus of endophytic bacteria and endophytic fungi among tissues accounts for more than 90%of the total abundance.The 29 genus were enriched in different tissues and had significant contributions to the formation of inter-organizational differences?P<0.05?.CCA typical analysis and linear regression analysis showed that soil water content SM significantly affected the endophytic bacteria?R²=0.241,P=0.024?and endophytic fungi?R²=0.223,P=0.031?.Beta diversity and soil p H?R²=0.571,P<0.001?mainly affects the endophytic fungal Alpha diversity richness index.In the functional analysis,the functional analysis of the endophytic bacteria PICRUSt showed that the functional composition of COG was dominated by unknown functions,and there were certain differences between the functional structures.But the correlation between environmental factors and functions is not significant.Funguild trophotype analysis of endophytic fungi indicated that unknown UN was the dominant nutritional mode of Oxytropis glacialis endophytic fungi.There were significant differences between tissues of PA-SA-SY and SA-SY?P<0.05?.However,environmental factors only have significant explanatory significance for the structure of PA and PA-SA trophic structure.Relatively conservative endophytic bacteria and relatively specific endophytic fungal community structure were formed in different tissues of Oxytropis glacialis.Environmental factors have a significant impact on endophyte diversity,but the explanation of the differential function of endophytes between tissues is not significant.The diversity and difference of functions provide a guarantee for the realization of tissue-specific functions,and these characteristics may promote the adaptation and spread of Oxytropis glacialis on the Qinghai-Tibet Plateau.4.Correlation network analysis was carried out on Oxytropis glacialis endophytes,and the interaction pattern with swainsonine-associated endophytes was analyzed as an example.Venn analysis showed that the number of core endophytic bacteria in root and leaf tissue?54,62?was higher than that of core endophytic fungi?22,13?,and the species of core endophyte in root tissue was similar to that in leaf tissue?76,75?.Phylogenetic tree analysis showed that Alternaria,a potential endophytic fungus producing swainsonine,and Brevundimonas,an endophytic bacteria that degraded swainsonine,existed stably in the host plant as core microbiome in Oxytropis glacialis.The correlation network analysis showed that the modularization index among endophytic fungi was higher,the node connectivity?average degree?between endophytic bacteria was closer,and the overall synergism was greater than antagonism.The overall interaction among endophytic fungi was higher than that among endophytic fungi and lower than that among endophytic bacteria,but the interaction related to core bacteria accounted for less than 15%of the total interaction network.The core microbiome may transfer the influence to the microbial community mainly through indirect interaction.Among them,Alternaria and Brevundimonas as the core genera participate in the network interaction among Oxytropis glacialis microbiome with significant indirect correlation?|?|>0.6,P<0.01?,and this regulation may be affected by both the host and the environment.The interaction network relationship between endophytic community of Oxytropis glacialis is mainly positive feedback.The tight connection of endophytic bacteria in the core and the relatively high modular characteristics of endophytic fungi provide the possibility for the realization and regulation of endophytic function.Core microbiome transfer their important ecological functions to the whole microbial community through indirect interaction.Among them,the endophytic bacteria that degrade swainsonine and the endophytic fungi that produce swainsonine work together to maintain the dynamic balance of swainsonine in Oxytropis glacialis,which provides a possible way for the biotreatment of Oxytropis glacialis.However,its internal signal transduction mechanism needs to be further studied.