| Orchidaceae is a group of angiosperms with extremely wide distribution in nature,and has important values in ornamental,medicine and scientific research.The Trib.Neottieae Lindl clade is a clade under the family Orchidaceae,including Neottia,Holopogon,Epipactis,and Listera,covering photosynthetic autotrophic,mixotrophic,and fully fungal heterotrophic types.Due to the deterioration of living environment,loss of pollinating insects and low seed germination rate,many species of this clade are on the verge of extinction,so the conservation of the plants of this clade is urgent.Almost all orchids are mycorrhizal-dependent plants,and mycorrhizal fungi play an indispensable role in orchid seed germination and the whole growth stage of the plants.Therefore,it is extremely important to conduct in-depth research on mycorrhizal fungi of orchids for orchid conservation.However,our knowledge of mycorrhizal fungi of the Orchidaceae Trib.Neottieae Lindl plants is still limited,and we know little about the symbiotic relationship between plants of this family and mycorrhizal fungi,which limits the conservation of biodiversity and the development of conservation measures for this family of plants.In order to understand the diversity of root fungi in Trib.Neottieae Lindl plants,we conducted a root fungal isolation test on the Cephalathera longibracteata collected from the Yunvfeng area of Jilin,mainly by surface sterilization of tissue blocks,and successfully isolated six fungal strains from the roots of the C.longibracteata,which are isolated,purified and ITS sequencing for molecular identification.Subsequently,in order to clarify the factors affecting the diversity of mycorrhizal fungi of Trib.Neottieae Lindl plants,we set three factors based on the sample:habitat,nutritional relationship,and affinity.The sequences of the ITS region of the fungal pairs of Trib.Neottieae Lindl plants were selected to obtain genomic information by high-throughput sequencing,and the fungal sequences were analyzed bioinformatically using R language,IQtree and QIIME software,so as to investigate the factors affecting the diversity of mycorrhizal fungal groups of Trib.Neottieae Lindl plants.The results are as follows.(1)The six fungal strains isolated from the C.longibracteata belonged to Mortierellaceae,Polyporaceae,Diaporthaceae,and Schizophyllaceae families of fungi,among which the Mortierellaceae and Schizophyllaceae families of fungi have been reported as orchid mycorrhizal fungi.The remaining two species have not been reported in the literature as mycorrhizal fungi.Performing subsequent seed germination experiments showed that the six fungal species isolated could not germinate the seeds of C.longibracteata.(2)High-throughput sequencing of the roots of Neottia camtschatea,Neottia acuminata and Holopogon pekinensis collected in Beijing.The dominant mycorrhizal fungi of all three plants belong to the family Sebacinaceae.The mycorrhizal fungalα-diversity was higher in the N.camtschatea than the other two species,and the differences in mycorrhizal fungi among the three Trib.Neottieae Lindl plants in Beijing were highly significant(PERMANOVA:R~2=0.86,p<0.01);In N.camtschatea,N.divaricata,N.pinetorum and N.fugongensis collected in Yunnan.After sequencing,the dominant mycorrhizal fungi of the four species differed,with the dominant mycorrhizal fungus of N.camtschatea being from the Sebacinaceae family,while the rest of the dominant mycorrhizal fungi were from the Serendipitaceae family.Theα-diversity of mycorrhizal fungi was the lowest and the highest in N.camtschatea and N.divaricata,respectively,and the difference of mycorrhizal fungi among the four plants was significant(R~2=0.68,p<0.05);The dominant mycorrhizal fungi of N.camtschatea in Beijing and Yunnan were all from the family Sebacinaceae,Theα-diversity analysis revealed that the mycorrhizal fungi of N.camtschatea in Beijing were more abundant,and the uniformity and diversity of mycorrhizal fungi of N.camtschatea in Yunnan were higher.The differences between the two regions were not significant(PERMANOVA:R~2=0.87,p>0.05),while the significance tests for geographic distance and phylogenetic group differences of mycorrhizal fungi PCD and fungal OTU numbers between samples found p-values higher than 0.05,indicating no significant correlation between geographic distance and mycorrhizal zone differences(PCD:p=0.3,PCDc:p=0.8,PCDp:p=0.4;R~2=0.47,p>0.05).(3)We collected N.camtschatea,N.acuminata,H.pekinensis,N.divaricata,N.fugongensis,N.pinetorum,N.mucronat,Corybas taliensis,Epipactis veratrifolia and Epipactis xanthophaea were divided into photosynthetic types with leaves and heterotrophic types without leaves according to whether they had leaves(whether they could photosynthesize or not).The results showed that the heterotrophic orchid plants tended to have fungal symbiosis in the Sebacinaceae family,while the dominant fungus in the photosynthetic orchid plants was in the Serendipitaceae family.α-diversity analysis andβ-diversity analysis revealed that the mycorrhizal fungal diversity as well as richness of the photosynthetic type were higher than that of the heterotrophic type,and the difference between the two types was extremely significant(PERMANOVA:R~2=0.21,p<0.01).In the cluster analysis it can be seen that almost all heterotrophic plants in the Trib.Neottieae Lindl have the Sebacinaceae family as the dominant mycorrhizal fungal specific symbiosis,and photosynthetic plants in addition to the Sebacinaceae family are also able to interact with Serendipitanceae,Mortierellaceae,Ceratobasidiaceae Russulaceae,Thelephoraceae and other families of mycorrhizal fungi widely symbiotic,but the heterotrophic and photosynthetic plants were almost clustered together,indicating that the trophic relationship has some influence on the diversity of mycorrhizal fungi.(4)Mycorrhizal fungal diversity was calculated among the sample groups of N.camtschatea,N.acuminata,N.divaricata,N.fugongensis,N.pinetorum,N.mucronata,C.taliensis and E.veratrifolia.The results revealed a weak phylogenetic signal between host and symbiotic fungi(K<1),and similarly,there was no significant symbiotic signal(Para Fit Global=0.3705,p-value=0.713),presumably due to the influence of the complex environment in the field,which also requires subsequent in-depth experiments to remove environmental factors.Through the above study,we found that trophic relationship was the most significant factor affecting mycorrhizal fungal diversity,and habitat as well as affinity had less influence on mycorrhizal fungi.The study on the influencing factors and coevolutionary relationships of mycorrhizal fungal diversity in the Trib.Neottieae Lindl is important to reveal the mechanism of action of orchids and symbiotic mycorrhizal fungi,and also provides a suitable theoretical basis for the biodiversity conservation of endangered orchids. |
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