Community Diversity And Promotion Of Diazotrophic Methane-oxidizing Bacteria In The Root Zone Of Rice Field In Northern China

Methane(CH₄)is the second most important greenhouse gas in the atmosphere next to carbon dioxide,and rice fields are one of the main sources of CH₄emissions,accounting for 5%-19%of the global CH₄budget.Root zone methane oxidizing bacteria(MOB)plays an important role in reducing CH₄emission from paddy fields.Studies in rice fields in temperate regions have shown that rice root type II MOB play a dominant role in CH₄oxidation and N₂fixation.The north of China has cold temperatures and mostly saline-alkali land.It has been reported that as the global warming northern regions are becoming more suitable for rice cultivation.However,little is known about methane oxidation depending N₂fixation,key player and growth promotion by MOB of rice root zone of different varieties of saline soil in northern region.In this study,four kinds of rice rhizosphere samples from two rice varieties with different nitrogen application rates(low nitrogen and high nitrogen)from the breeding base of high-end rice varieties in saline-alkali land(Hohhot)were studied,using stable isotope ¹⁵N feeding,DNA and RNA based sequencing,green fluorescent protein(GFP,Green fluorescent protein)labeling technique to investigated the CH₄-depending nitrogen-fixing activity,community diversity and growth-promoting effect of diazotrophic MOB of rice root zone in paddy field in northern alpine area were studied.The results are as follows:(1)The stable isotope ¹⁵N-N₂feeding experiment showed that the presence of CH₄significantly increased the N₂fixation activity of rice roots and rhizosphere soil,and the CH₄-dependent N₂fixation activity and nitrogen fixation rate of J3rhizosphere were stronger than those of 131varieties.The N₂fixation activity and N₂fixation rate of root system of the same variety of rice plant were much higher than those of rhizosphere soil.(2)The results of PCoA and cluster analysis based on pmoA and nifH gene amplicon sequencing showed that the effect of nitrogen fertilizer on the community structure of MOB and nitrogen-fixing bacteria in rice rhizosphere(rhizosphere soil and root)was greater than that of rice varieties.(3)The community composition of MOB in rhizosphere soil was dominated by type I MOB Methylococcaceae(54.3-87.7%),and nitrogen fertilizer had a great effect on the genus level of the two rice varieties.Methylomonas(42.1-66.9%)was dominant in both varieties at high nitrogen levels.At the DNA level,all root samples were dominated by Methylomonas(24.1-80.3%),except for the LNJ3 roots,which were dominated by Methylocystis(4.5-58.4%).At the transcriptional level,Methylomonas(46.8-80.3%)was dominant in all root samples,which suggested that it played a key role in methane oxidation in rhizosphere.(4)Diazotrophic MOB(13-27%)occupied the dominant position in the community composition of nitrogen-fixing bacteria in rhizosphere soil,in which type I Methylobacter(9.8-17.1%)was the most dominant genus,which played an important role in nitrogen fixation in methane-dependent rhizosphere soil.In addition to diazotrophic MOB,other heterotrophic nitrogen-fixing bacteria such as Azoarcus,Zoogloea and unclassified-Rhodocyclale play a key role in heterotrophic nitrogen-fixing activity.(5)The community composition of root-associated nitrogen-fixing bacteria was different at the level of DNA and RNA.Compared with DNA level,rice root Azoarcus(29-54%)was the dominant genus at cDNA level,followed by type I MOB Methylomonas(21-35%).At the same time,nitrogen fertilizer had a great effect on heterotrophic nitrogen-fixing bacteria.With the increase of nitrogen fertilizer,the abundance of Azoarcus bacteria decreased by 12.2-15.9%,while the abundance of Azotobacter and Azonexus showed an upward trend.This shows that Methylomonas play an important role in methane-dependent root nitrogen fixation activity,while Azoarcus is the dominant heterotrophic nitrogen fixation activity.(6)GFP-labeled MOB strain PRM1 had good growth-promoting effect on 131and J3 rice plants,and was easy to colonize the root.The results further prove that MOB is green emission reduction and growth promoting bacteria with application potential.This study provides a good idea for using functional flora to solve the problem of nitrogen fertilizer reduction and methane emission mitigation in paddy field,and provides a scientific basis and strain resources for the improvement of sustainable and green development of paddy ecosystem.