| Maize（Zea Mays L.）is grown worldwide and is of great importance for food production.Several studies have shown that microbiomes have essential functions in maize performance,such as promoting nutrient uptake,improving the resistance of plants to stressful environments and regulating plant growth.However,the assembly mechanism of maize bacterial community and the key functional taxa associated with host fitness under different nutritional conditions have not been systematically understood yet.In the present study,we investigated the microbiome of seven plant compartments（bulk soil;rhizosphere,root endosphere,stem endosphere,xylem endosphere,leaf endosphere and phylloplane）in maize genotypes grown across a range of environmental conditions（i.e.,different fertilization regimes,soil types,climate zones）.Furthermore,targeted screening for diazotrophic bacteria and designed synthetic communities to explore and verify the nitrogen-fixation capacity of maize core bacterial communities.The main findings were as follows:1.Soil microbial community structure and function under long-term fertilization.Long-term fertilization resulted in significant changes in soil p H,total carbon,total nitrogen and nitrate nitrogen of red soil,aquic soil and black soil,and these soil properties explained 92.83%and88.61%of the variation in bulk soil and rhizosphere bacterial community,respectively.Bacterial communities were most sensitive to fertilization in red soils compared to fluvo-aquic soils and black soils.Fertilization-induced changes in soil p H were the main factor driving the community variation.Additionally,we found that rhizospheric community was dominated by deterministic processes.Plant rhizosphere selects more bacteria involved in nutrient cycling under nutrient-poor conditions,while bacterial communities recruited under nutrient-enriched conditions present an over representation of functions involved in lipid metabolism and biosynthesis of other secondary metabolites.These results point towards a potential strategy,i.e.,maize selects specific bacterial taxa to rhizosphere based on functional traits beneficial to its own performance,rather than selecting particular species.2.Maize microbial community structure and function under long-term fertilization.Maize microbiome assembly along the soil-plant continuum was shaped predominantly by plant compartments,followed by sites and fertilization treatments.A FEAST analysis showed that maize endophytic bacterial communities were mainly derived from bulk soils.From bulk soils to epiphytes to endophytes,host selection pressure sequentially increased,and xylem selectively recruits highly conserved microbes dominated by Gammaproteobacteria.Compared with the root and leaf interface,which is in closer contact with the soil,xylem endosphere is hardly affected by geographical and climatic distances.Moreover,FAPROTAX analysis suggested that the number of OTUs involved in N-cycling was approximately 2 times higher in xylem than in leaf,and 1.5 times higher than in stem endosphere.And the ratio of nif H to 16S r RNA in xylem was about twice that in the root endosphere and four times that in the leaf endosphere,especially in control soils.These results suggested that a higher proportion of diazotrophic bacteria was enriched in xylem under low nitrogen stress.Untargeted metabolomic analysis revealed that enriched bacterial taxa may be associated with a significant increase in compounds such as flavonoids in xylem sap.3.Design and validation of synthetic community with nitrogen-fixation potential inhabiting the maize xylem sap.Abundance-occupancy analyses identified 25 OTUs that were persistent in xylem in all samples.We then cross-referenced the V5-V7 region of the 16S r RNA gene of the 109isolated strains against the highly abundant OTUs（>0.01%）in xylem.We found that these isolated strains shared sequence identity with 14 of the 25 core OTUs in xylem.The two of the 14 strains（Klebsiella variicola MNAZ1050 and Citrobacter sp.MNAZ1397）displayed remarkably high nitrogenase activity,1179.60 and 1214.48 nmol C2H4/mg protein hour,respectively;2 core non-N-fixers（Acinetobacter sp.ACZLY512 and Rosenbergiella epipactidis YCCK550）with the potential to assist in N-fixation,and they significantly increased the nitrogenase activity by93.90%and 85.16%,respectively.The genome analysis of these strains showed that they may have strong respiratory metabolism and ammonia assimilation ability,therefore creating a micro-oxygen and low-ammonia environment conducive to biological N-fixation.On this basis,we established synthetic communities（Syn Coms）consisting of two core diazotrophs and two helpers.GFP-tagged strains and 15N isotopic dilution method demonstrated that these Syn Coms do thrive and contribute,through biological nitrogen fixation,11.8%of the total N accumulated in maize stems.The Syn Coms also increased root dry biomass,suggesting that there is a multi-mechanism synergistic interaction between the core endophytes and their host.In conclusion,the highly conserved bacterial taxa in xylem sap represent an important and neglected plant functional‘gut’microbiome.By assessing the assembly and functions of maize microbiomes across soil types,climate zones,and genotypes,this study confirmed the nitrogen-fixing and growth-promoting potential of xylem microbiome,which has important reference significance and value for future research on plant growth-promoting bacteria and the development of nitrogen-fixing microbial product. |
