Nitrogenase Activity And Diazotrophic Community Composition Of Different Lichens

Nitrogen,as an essential nutrient for the growth of organisms,is a major limiting factor for the primary productivity of terrestrial ecosystems.The biological nitrogen fixation of lichens is one of the important sources of nitrogen in nature.However,the mechanisms by which lichens construct their diazotrophic communities are still unclear,and the nitrogen fixation capacity of different lichens and their relationship with environmental factors and diazotrophic community structure still need to be studied systematically.In this study,the nitrogenase activity and diazotrophic community characteristics of different types of lichens and their substrates(soils)were studied by acetylene reduction assay,real-time fluorescence quantitative PCR and high-throughput sequencing technology targeting nifH gene,and the influencing factors were discussed.At the same time,diazotrophs were isolated and cultured in Ashby nitrogen-free medium,and the community characteristics of culturable diazotrophs were analyzed by combining phenotypic characteristics,physiological and biochemical characteristics and 16S rRNA gene identification.The main results are shown as follows:(1)The nitrogenase activity and diazotrophic community composition of different lichen species and different photosynthetic types of lichens were different.The nitrogenase activity and nifH gene abundance of diazotrophic community in cyanolichens were significantly higher than those of chlorolichens at the same site,while the α-diversity of diazotrophic community was significantly lower than that of chlorolichens.Principal component analysis showed that the diazotrophic community composition of different lichen species at the same site was significantly different,indicating that species may play an important role in the construction of diazotrophic community of thalli in the same habitat.(2)The nitrogenase activity of thalli from three terricolous lichens was significantly higher than that of the corresponding substrate soils and control soils without lichen,and the nifH gene abundance of diazotrophic community was also significantly higher than that of the control soils without lichen,respectively.And principal component analysis showed that the diazotrophic community composition of thalli,substrate soils and control soils without lichen was significantly different in the three terricolous lichens.(3)The nitrogenase activity and diazotrophic community composition of thalli and soils were affected by different environmental factors.The correlation analysis showed that the nitrogenase activity of thalli was significantly positively correlated with the content of total nitrogen and total phosphorus,and significantly negatively correlated with α-diversity of diazotrophic community.And the nitrogenase activity of soils was significantly positively correlated with the total nitrogen,conductivity and the abundance of diazotrophic community.Canonical correspondence analysis showed that organic carbon was the main factor affecting the diazotrophic community composition in thalli,while carbon to nitrogen ratio and ammonium nitrogen were the main factors determining the diazotrophic community composition in soils.(4)A total of 127 strains of diazotrophs were isolated from thalli and soils,88.98% of which were gram-negative bacteria,90% of which showed positive catalase reaction,while most of them were negative for methyl red reaction,acetylmethyl methanol reaction,oxidase reaction and starch hydrolase reaction,accounting for 96.06%,98.43%,85.04% and 70.87% of all culturable diazotrophs,respectively.16S rDNA sequence analysis showed that the 23 typical culturable diazotrophs belonged to 11 genera,including Streptomyces,Microbacterium,Novosphingobium,Bacillus,Rhodococcus,Arthrobacter,Caulobacter,Pseudomonas,Acinetobacter,Paenibacillus and Sphingomonas.In conclusion,species and symbiotic photosynthetic organisms have significant effects on the nitrogenase activity and diazotrophic community composition in thalli.The nitrogenase activity in both thalli and soils is significantly positively correlated with the total nitrogen content,while the environmental factors affecting the diazotrophic community composition in thalli and soils were different.The results are helpful to further understand the biological nitrogen fixation function of lichens and the relationship between the formation of diazotrophic community in thalli and their substrates,provide strong evidence for understanding the contribution of widely distributed lichens to nitrogen storage in terrestrial ecosystems,and also provide a basis for understanding and utilizing diazotrophic community associated with lichens.