Diversity Of Denitrifying Methantrophs And Their Distribution In Root Zone Of Three Emergent Macrophytes In Eutrophic Wuliangsuhai Wetland

Emergent macrophytes play important roles in biogeochemical nitrogen and carbon cycle in wetland.And aerobic methanotrophs in rhizospheres of emergent plants consume up to 18%-90%of methane produced in the vegetated wetland,but it remains unclear their distribution and whether they participate in denitrification in wetlands.In this thesis,Wuliangsuhai Lake,a eutrophic lake wetland in the Mongolian plateau,was taken as the study site.Three common emergent macrophytes,Phragmites australis?PA?,Typha angustifolia?TA?,and Scirpus triqueter?ST?,containing roots and rhizosphere sediments,and unvegetated sediment as control samples,were collected from the littoral wetland of the lake.Using high-throughput sequencing,clone library,real-time PCR,catalyzed reporter deposition fluorescent in situ hybridization?CARD-FISH?,the abundances,diversities and microhabitat distribution of bacteria,aerobic methanotrophs,methanotrophic denitrifiers and other heterotrophic denitrifying bacteria in root-zone of the three plants and sediment were analyzed.Further,the biological localization of aerobic methanotrophs in root tissues of three plants were observed.The research results were as follows:?1?High-throughput sequencing analysis based on 16S rRNA genes,type I methanotrophs?belonging to Methylococcaceae?accounted for between 0.22%and0.68%of the total bacteria communities,significantly higher than type II methanotrophs?belonging to Methylocystaceae?with 0.02%to 0.17%.And the relative abundance of the two kinds of methanotrophs in roots were higher than sediments?rhizosphere sediment and unvegetated sediments?;?2?Sequencing analysis of functional genes indicated that 95.2%pmoA gene?encoding particulate methane monooxygenase?sequences were phylogenetically affiliated with type I methanotrophs,which indicated the root-associated methanotrophs was dominated with type I methanotrophs.Among them,Methylomonas denitrificans–like organisms?denitrifying methanotrophs?were the most main groups,accounting for 44.4%of the total communities.This was further substantiated by sequencing analysis of nirS genes?encoding nitrite reductase?with97.6%sequences belonging to type I methanotrophs;31.2%sequences of nirS genes were also closest to Methylomonas denitrificans;?3?The three genera—Methylomonas,Methylobacter and Methylovulum of type I aerobic methanotrophs had genetic potential for denitrification.Methylomonas showed an increasing trend from sediment to rhizosphere sediment to root,whereas Methylobacter showed the opposite trend.Compared to Methylomonas and Methylobacter,Methylovulum was detected in minor numbers;?4?Real-time PCR analysis demonstrated that the abundances of pmoA,nirS and nirK genes in roots were all significantly higher than those in rhizosphere sediments and unvegetated sediments.In the same sample,the copy numbers of the nirS gene were significantly higher than the nirK.In addition,the copy numbers of both pmoA and nirS genes in roots were higher in PA and ST than those in TA;?5?Further,type I methanotrophs were localized in root tissues by CARD-FISH.Signals of type I methanotrophs were clearly observed in the vascular cylinder,around the aerenchyma of PA and TA roots tissues,and also in the epithelial cells in vertical sections of ST root tissues;?6?The abundance of nosZ gene?terminal denitrification functional gene,encoding N₂O reductase?in root zone of three plants,was slightly lower than that in unvegetated sediments.The total nosZ-type dinitrifiers in unvegetated sediment was highly diverse,but Novel Halomonas,Hydrogenophaga and Thiobacillus were dominant completed denitrifiers in the root zone of three plants.This study emphasized that the roots of emergent macrophytes enriched for type I methanotrophs,especially acted selectively on Methylomonas.We provided the first molecular evidence that the presence of nitrite-reducing genes?nirS?in type I methanotrophs in natural wetland.This indicated that type I methanotrophs had genetic potential for denitrification.These results expand the metabolism spectrum of root-associated type I methanotrophs.Type I methanotrophs with nirS genes as incomplete denitrifiers dominated in root zone,together with the nosZ-type complete denitrifiers in sediment cooperated to regulate methane emission and denitrification process,showing the response and feedback of denitrifying type I methanotrophs to saline-alkaline stress,high nitrogen input and high Cu and root conditions in Wuliangsuhai wetland.Therefore,the aquatic plant and microbial symbiosis system in eutrophic wetland has the dual functions for the reduction of the greenhouse gas and purification of water quality,providing guiding significance to construct constructed wetland system.