The present study aimed at gaining an insight into the abundance and genetic diversity of culturable N-fixing epiphyte bacteria on the phyllosphere of maize in arid and semi-arid regions of Iran.Leaf samples of the maize variety, 'single cross 704' (Zea mays L.) were collected from different locations in Iran. The community of culturable N-fixing epiphyte bacteria present was examined by 16S rRNA sequencing, BOXAIR-polymerase chain reaction (PCR) and restricted fragment length polymorphisms analysis of 16S rRNA gene (16S-RFLP). Approximately, 31·82% of the 242 isolates were identified as N-fixers by cultivation of bacteria in Rennie medium and detection of their nifH gene. The N-fixers were affiliated with four bacterial phyla: Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. 16S rRNA sequencing detected 16 genera and 24 different species in the identified phyla. The most dominant genus was Bacillus and the species identified were B. pumilus, B. amyloliquefaciens, B. subtilis, B. paralicheniformis, B. licheniformis, B. niabensis and B. megaterium. In total, 22 RFLP groups were present among the isolates originally identified as N-fixing bacteria. BOXAIR-PCR showed that there was a low similarity level among the N-fixing bacteria isolates, and genetic differentiation of individual strains was relatively great.Our findings suggest that nitrogen-fixing epiphyte bacteria on the phyllosphere of maize may provide significant nitrogen input into arid and semi-arid ecosystem.This research implies that phyllosphere epiphyte diazotrophs have much to offer in sustainable agriculture and can be an alternative to chemical N-fertilizers for providing nitrogen to crops arid and semi-arid regions.© 2020 The Society for Applied Microbiology.

In a previous study by our group, CH4 oxidation and N2 fixation were simultaneously activated in the roots of wild-type rice plants in a paddy field with no N input; both processes are likely controlled by a rice gene for microbial symbiosis. The present study examined which microorganisms in rice roots were responsible for CH4 oxidation and N2 fixation under the field conditions. Metaproteomic analysis of root-associated bacteria from field-grown rice (Oryza sativa Nipponbare) revealed that nitrogenase complex-containing nitrogenase reductase (NifH) and the alpha subunit (NifD) and beta subunit (NifK) of dinitrogenase were mainly derived from type II methanotrophic bacteria of the family Methylocystaceae, including Methylosinus spp. Minor nitrogenase proteins such as Methylocella, Bradyrhizobium, Rhodopseudomonas, and Anaeromyxobacter were also detected. Methane monooxygenase proteins (PmoCBA and MmoXYZCBG) were detected in the same bacterial group of the Methylocystaceae. Because these results indicated that Methylocystaceae members mediate both CH4 oxidation and N2 fixation, we examined their localization in rice tissues by using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). The methanotrophs were localized around the epidermal cells and vascular cylinder in the root tissues of the field-grown rice plants. Our metaproteomics and CARD-FISH results suggest that CH4 oxidation and N2 fixation are performed mainly by type II methanotrophs of the Methylocystaceae, including Methylosinus spp., inhabiting the vascular bundles and epidermal cells of rice roots. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

There is growing recognition that classifying terrestrial plant species on the basis of their function (into 'functional types') rather than their higher taxonomic identity, is a promising way forward for tackling important ecological questions at the scale of ecosystems, landscapes or biomes. These questions include those on vegetation responses to and vegetation effects on, environmental changes (e.g. changes in climate, atmospheric chemistry, land use or other disturbances). There is also growing consensus about a shortlist of plant traits that should underlie such functional plant classifications, because they have strong predictive power of important ecosystem responses to environmental change and/or they themselves have strong impacts on ecosystem processes. The most favoured traits are those that are also relatively easy and inexpensive to measure for large numbers of plant species. Large international research efforts, promoted by the IGBP–GCTE Programme, are underway to screen predominant plant species in various ecosystems and biomes worldwide for such traits. This paper provides an international methodological protocol aimed at standardising this research effort, based on consensus among a broad group of scientists in this field. It features a practical handbook with step-by-step recipes, with relatively brief information about the ecological context, for 28 functional traits recognised as critical for tackling large-scale ecological questions.

Large numbers of bacterial and fungal endophytes have been reported from different plant tissues: roots, nodules, leaves, flowers and sprouts of legumes, with numbers ranging from few to more than 150. Endophytes can accelerate seedling emergence, promote plant establishment under adverse conditions and enhance plant growth. Endophytic microbes promote plant growth by helping plants in acquiring nutrients, e.g. via nitrogen fixation, phosphate solubilization or iron chelation, by preventing pathogen infections via antifungal or antibacterial agents, by outcompeting pathogens for nutrients by siderophore production, or by establishing the plant's systemic resistance. Further growth promotion is affected by producing phytohormones such as auxin or cytokinin, or by producing the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which lowers plant ethylene levels. For establishment of endophytes in different tissues, endophytic microbes must be compatible with the host plants and able to colonize the tissues of the host plants without being recognized as pathogens. A particular bacterium or fungus may affect plant growth and development using one or more of these mechanisms, and they may use different mechanisms at various times. The population density of endophytes is highly variable, depending mainly on the microbial species and host genotypes, developmental stage and environmental conditions. Genotypic and cultivar specific endophytes have also been reported. The quantum benefit derived by plants from an endophyte and vice versa is still not clear. It seems that the endophytic genus or species best adapted for living inside a plant is naturally selected. Here, we concentrate on soil or rhizosphere-derived endophytes recruited out of a large pool of soil or rhizospheric microbes. Some endophytes are more aggressive colonizers and displace others, but seeming lack of strict specificity has been observed. However, the processes of host-microbe signaling and colonization and the mechanisms leading to mutual benefits are less-well characterized. It is still not clear which population of microorganisms (endophytes or rhizospheric) promotes plant growth and the way the interactions among endophytes influence plant productivity. Though attempts to know the molecular ecology and interactions are underway, a high amount of progress is required to fully understand the mechanism of establishment, the way interactions take place in planta, between different microbes and plants and exlusive benefits by endophytes and plants.Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

位于海南岛西南部的尖峰岭是目前我国为数不多、保存得较为完好的具有热带雨林性质的森林类型。在较典型的山地雨林中，设置1 hm2的固定样地，记录到DBH≥5.0 cm的乔木171种，隶属于52科93属，总株数1099株，其中乔木种株数为1024。样地的Shannon-Wiener指数(H′)、均匀度指数(E)和Simpson指数(D)分别为4.11、0.80和0.97，但取样面积和测定个体的起始大小等取样技术对物种多样性指数有显著的影响。随着取样面积的增加，H′值逐渐增加；但超过了4000 m2以后，增加不明显。E和D值在取样面积达到2000 m2后，基本保持不变。随着测定个体起始直径的增加，物种数、科数、H′和D都呈现出明显的减少趋势，而E值增加。径级频度分布的分析表明，该山地雨林的垂直结构可以划分成4个层次，每个层次的个体大小在空间上呈均匀或随机分布。从气候学、科属组成以及与世界热带雨林物种多样性的比较等方面，讨论了尖峰岭山地雨林在世界雨林中的地位。气候学分析表明，尖峰岭山地雨林在温度带上属于亚热带/暖温带范围，但由于其丰沛的降水量，使物种多样性较为丰富，具备了雨林的特点。对科属组成的分析表明，亚热带/暖温带科属占优势，其中典型的亚热带/暖温带科——壳斗科和樟科占总重要值的34％以上，而热带科属成份较少。与世界其他地区的典型热带林相比较，尖峰岭山地雨林的物种丰富度显著偏低，但高于某些降水量少的地区的热带林。因此，我们认为尖峰岭山地雨林雨典型热带雨林有较大差别，具有由热带雨林向亚热带/暖温带雨林过渡的性质。此外，文本提出了平均种群密度(MPD)和种数-个体数关系这两种反映物种多样性测度的指标和表达式，并利用样地资料进行了分析。

Mosses in high-latitude ecosystems harbor diverse bacterial taxa, including N-fixers which are key contributors to nitrogen dynamics in these systems. Yet the relative importance of moss host species, and environmental factors, in structuring these microbial communities and their N-fixing potential remains unclear. We studied 26 boreal and tundra moss species across 24 sites in Alaska, USA, from 61 to 69° N. We used cultivation-independent approaches to characterize the variation in moss-associated bacterial communities as a function of host species identity and site characteristics. We also measured N-fixation rates via N isotopic enrichment and identified potential N-fixing bacteria using available literature and genomic information.Host species identity and host evolutionary history were both highly predictive of moss microbiome composition, highlighting strong phylogenetic coherence in these microbial communities. Although less important, light availability and temperature also influenced composition of the moss microbiome. Finally, we identified putative N-fixing bacteria specific to some moss hosts, including potential N-fixing bacteria outside well-studied cyanobacterial clades.The strong effect of host identity on moss-associated bacterial communities demonstrates mosses' utility for understanding plant-microbe interactions in non-leguminous systems. Our work also highlights the likely importance of novel bacterial taxa to N-fixation in high-latitude ecosystems. Video Abstract.

Reduced fertilizer usage is one of the objectives of field management in the pursuit of sustainable agriculture. Here, we report on shifts of bacterial communities in paddy rice ecosystems with low (LN), standard (SN), and high (HN) levels of N fertilizer application (0, 30, and 300 kg N ha(-1), respectively). The LN field had received no N fertilizer for 5 years prior to the experiment. The LN and HN plants showed a 50% decrease and a 60% increase in biomass compared with the SN plant biomass, respectively. Analyses of 16S rRNA genes suggested shifts of bacterial communities between the LN and SN root microbiomes, which were statistically confirmed by metagenome analyses. The relative abundances of Burkholderia, Bradyrhizobium and Methylosinus were significantly increased in root microbiome of the LN field relative to the SN field. Conversely, the abundance of methanogenic archaea was reduced in the LN field relative to the SN field. The functional genes for methane oxidation (pmo and mmo) and plant association (acdS and iaaMH) were significantly abundant in the LN root microbiome. Quantitative PCR of pmoA/mcrA genes and a (13)C methane experiment provided evidence of more active methane oxidation in the rice roots of the LN field. In addition, functional genes for the metabolism of N, S, Fe, and aromatic compounds were more abundant in the LN root microbiome. These results suggest that low-N-fertilizer management is an important factor in shaping the microbial community structure containing key microbes for plant associations and biogeochemical processes in paddy rice ecosystems.

We investigated the dynamics of the bacterial composition and metabolic function within Akashiwo sanguinea bloom using a 100-L indoor microcosm and metagenomic next-generation sequencing. We found that the bacterial community was classified into three groups at 54% similarity. Group I was associated with “during the A. sanguinea bloom stage” and mainly consisted of Alphaproteobacteria, Flavobacteriia and Gammaproteobacteria. Meanwhile, groups II and III were associated with the “late bloom/decline stage to post-bloom stage” with decreased Flavobacteriia and Gammaproteobacteria in these stages. Upon the termination of the A. sanguinea bloom, the concentrations of inorganic nutrients (particularly PO43−, NH4+ and dissolved organic carbon) increased rapidly and then decreased. From the network analysis, we found that the A. sanguinea node is associated with certain bacteria. After the bloom, the specific increases in NH4+ and PO43− nodes are associated with other bacterial taxa. The changes in the functional groups of the bacterial community from chemoheterotrophy to nitrogen association metabolisms were consistent with the environmental impacts during and after A. sanguinea bloom. Consequently, certain bacterial communities and the environments dynamically changed during and after harmful algal blooms and a rapid turnover within the bacterial community and their function can respond to ecological interactions.

Recent work has suggested that in temperate and subtropical trees, leaf surface bacterial communities are distinctive to each individual tree species and dominated by Alpha- and Gammaproteobacteria. In order to understand how general this pattern is, we studied the phyllosphere bacterial community on leaves of six species of tropical trees at a rainforest arboretum in Malaysia. This represents the first detailed study of 'true' tropical lowland tree phyllosphere communities. Leaf surface DNA was extracted and pyrosequenced targeting the V1-V3 region of 16S rRNA gene. As was previously found in temperate and subtropical trees, each tree species had a distinctive bacterial community on its leaves, clustering separately from other tree species in an ordination analysis. Bacterial communities in the phyllosphere were unique to plant leaves in that very few operational taxonomic units (0.5%) co-occurred in the surrounding soil environment. A novel and distinctive aspect of tropical phyllosphere communities is that Acidobacteria were one of the most abundant phyla across all samples (on average, 17%), a pattern not previously recognized. Sequences belonging to Acidobacteria were classified into subgroups 1-6 among known 24 subdivisions, and subgroup 1 (84%) was the most abundant group, followed by subgroup 3 (15%). The high abundance of Acidobacteria on leaves of tropical trees indicates that there is a strong relationship between host plants and Acidobacteria in tropical rain forest, which needs to be investigated further. The similarity of phyllosphere bacterial communities amongst the tree species sampled shows a significant tendency to follow host plant phylogeny, with more similar communities on more closely related hosts.

The nitrate regulates soybean nodulation and nitrogen fixation systemically, mainly in inhibiting nodule growth and reducing nodule nitrogenase activity, but the reason for its inhibition is still inconclusive.

Microbial metabolism powers biogeochemical cycling in Earth's ecosystems. The taxonomic composition of microbial communities varies substantially between environments, but the ecological causes of this variation remain largely unknown. We analyzed taxonomic and functional community profiles to determine the factors that shape marine bacterial and archaeal communities across the global ocean. By classifying >30,000 marine microorganisms into metabolic functional groups, we were able to disentangle functional from taxonomic community variation. We find that environmental conditions strongly influence the distribution of functional groups in marine microbial communities by shaping metabolic niches, but only weakly influence taxonomic composition within individual functional groups. Hence, functional structure and composition within functional groups constitute complementary and roughly independent "axes of variation" shaped by markedly different processes.Copyright © 2016, American Association for the Advancement of Science.

Forest management with N‐fixing trees can improve soil fertility and tree productivity, but have little information regarding belowground carbon processes and microbial properties. We aimed to evaluate the effects of three forest management regimes, which were Erythrophleum fordii (N‐fixing tree), Pinus massoniana (non‐N‐fixing tree), and their mixed forest, on soil respiration and microbial community composition in subtropical China, using Barometric Process Separation and phospholipid fatty acid profiles, respectively. We found that the inclusions of N‐fixing species in forests significantly increased the soil respiration, but have no effects on SOC and ecosystem total C stock. In addition, soil microbial communities were obviously different among the three forest management regimes. For instance, total and bacterial PLFAs were higher in the E. fordii and mixed forest than in the P. massoniana forest. Conversely, fungal PLFAs in the P. massoniana forest were elevated versus the other two forests. Soil total N, nitrate‐N and pH were the key determinants shaping the microbial community composition. Our study suggests that variations in soil respiration in the studied forests could be primarily explained by the differences of root biomass and soil microbial biomass, but not soil organic carbon. Although soil fertility and microbial biomass were promoted, N‐fixing plantings also brought on increased CO2 emissions in laboratory assays. The future decision of tree species selection for forest management in subtropical China therefore needs to consider the potential influences of tree species on CO2 emissions.

Elucidating the relationship between the variation of plant leaf functional traits and the environment is necessary for understanding the adaptation mechanism of plants and predicting changes in ecosystem structure. In this study, the leaf traits of desert plants in Ebinur Lake National Wetland Nature Reserve in Xinjiang, China were studied from the aspects of plant life forms (annuals, perennials and shrubs), phylogenetic signals, and relation to soil properties, using the principal component analysis, variance decomposition, and one-way analysis of variance. The results showed that: (1) There were significant differences in leaf carbon concentration (annuals>shrubs>perennials), leaf nitrogen concentration (shrubs ≥ perennials ≥ annuals), and leaf moisture content (perennials ≥ annuals ≥ shrubs) among the life forms, but there was no significant difference in leaf phosphorus concentration. Besides, soil nitrogen and phosphorus were significantly positively correlated with leaf carbon concentration and leaf nitrogen concentration. (2) There were significant differences in leaf carbon concentration, leaf nitrogen concentration, specific leaf area, and leaf moisture content between C3 and C4 plants, while the differences in P and leaf dry matter content were not significant. Besides, there were significant differences in leaf carbon concentration, leaf nitrogen concentration, specific leaf area, and leaf moisture content between leguminous and non-leguminous plants. Leguminous plants had higher leaf carbon concentration, leaf nitrogen concentration, and specific leaf area than non-leguminous plants, while non-leguminous plants had higher leaf moisture content than leguminous plants. (3) One way ANOVA analysis showed that taxonomy had a more significant effects on leaf carbon concentration, leaf nitrogen concentration, specific leaf area, and leaf moisture content than soil properties, and the coefficient of variation of leaf carbon concentration was greater than 50%. The phylogenetically independent contrasts analysis showed that the phylogenetic signal of all leaf traits was detected in all species and low (K value < 1, p > 0.05), indicating that the functional traits were weakly affected by phylogenetics. Therefore, desert plants in the Ebinur Lake Basin evolved to adapt to arid environments, and leaf traits showed convergent variation.

Bacterial communities are essential for the functioning of the Earth's ecosystems'. A key challenge is to quantify the functional roles of bacterial taxa in nature to understand how the properties of ecosystems change over time or under different environmental conditions 2. Such knowledge could be used, for example, to understand how bacteria modulate biogeochemical eyeless, and to engineer bacterial communities to optimize desirable functional processes 4. Communities of bacteria are, however, extraordinarily complex with hundreds of interacting taxa in every gram of soil and every millilitre of pond waters. Little is known about how the tangled interactions within natural bacterial communities mediate ecosystem functioning, but high levels of bacterial diversity have led to the assumption that many taxa are functionally redundant's. Here, we pinpoint the bacterial taxa associated with keystone functional roles, and show that rare and common bacteria are implicated in fundamentally different types of ecosystem functioning. By growing hundreds of bacterial communities collected from a natural aquatic environment (rainwaterfilled tree holes) under the same environmental conditions, we show that negative statistical interactions among abundant phylotypes drive variation in broad functional measures (respiration, metabolic potential, cell yield), whereas positive interactions between rare phylotypes influence narrow functional measures (the capacity of the communities to degrade specific substrates). The results alter our understanding of bacterial ecology by demonstrating that unique components of complex communities are associated with different types of ecosystem functioning.

Contents 40 I. 40 II. 41 III. 44 IV. 48 V. 49 VI. 49 VII. 52 VIII. 53 53 References 53 SUMMARY: In the last decade, analyses of both molecular and morphological characters, including nodulation, have led to major changes in our understanding of legume taxonomy. In parallel there has been an explosion in the number of genera and species of rhizobia known to nodulate legumes. No attempt has been made to link these two sets of data or to consider them in a biogeographical context. This review aims to do this by relating the data to the evolution of the two partners: it highlights both longitudinal and latitudinal trends and considers these in relation to the location of major land masses over geological time. Australia is identified as being a special case and latitudes north of the equator as being pivotal in the evolution of highly specialized systems in which the differentiated rhizobia effectively become ammonia factories. However, there are still many gaps to be filled before legume nodulation is sufficiently understood to be managed for the benefit of a world in which climate change is rife.© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Phyllosphere microbiomes play an essential role in maintaining host health and productivity. Still, the diversity patterns and the drivers for the phyllosphere microbial community of the tropical cash crop Rubber tree (Hevea brasiliensis) - are poorly understood. We sampled the phyllosphere of field-grown rubber trees in South China. We examined the phyllosphere bacterial and fungal composition, diversity and main drivers of these microbes using the Illumina® sequencing and assembly. Fungal communities were distinctly different in different climatic regions (i.e. Xishuangbanna and Hainan Island) and climatic factors, especially mean annual temperature, and they were the main driving factors of foliar fungal communities, indicating fungal communities showed a geographical pattern. Significant differences of phyllosphere bacterial communities were detected in different habitats (i.e. endophytic and epiphytic). Most of the differences in taxa composition came from Firmicutes spp., which have been assigned as nitrogen-fixing bacteria. Since these bacteria cannot penetrate the cuticle like fungi, the abundant epiphytic Firmicutes spp. may supplement the deficiency of nitrogen acquisition. And the main factor influencing endophytic bacteria were internal factors, such as total nitrogen, total phosphorus and water content of leaves. External factors (i.e. climate) were the main driving force for epiphytic bacteria community assembly. Our work provides empirical evidence that the assembly of phyllosphere bacterial and fungal differed, which creates a precedent for preventing and controlling rubber tree diseases and pests and rubber tree yield improvement.© 2022 Society for Applied Microbiology and John Wiley & Sons Ltd.

Legumes provide an essential service to ecosystems by capturing nitrogen from the atmosphere and delivering it to the soil, where it may then be available to other plants. However, this facilitation by legumes has not been widely studied in global tropical forests. Demographic data from 11 large forest plots (16-60 ha) ranging from 5.25° S to 29.25° N latitude show that within forests, leguminous trees have a larger effect on neighbor diversity than non-legumes. Where soil nitrogen is high, most legume species have higher neighbor diversity than non-legumes. Where soil nitrogen is low, most legumes have lower neighbor diversity than non-legumes. No facilitation effect on neighbor basal area was observed in either high or low soil N conditions. The legume-soil nitrogen positive feedback that promotes tree diversity has both theoretical implications for understanding species coexistence in diverse forests, and practical implications for the utilization of legumes in forest restoration.

热带山地雨林是海南岛最占优势的植被类型, 以往对热带山地雨林的研究通常基于小尺度, 缺乏大尺度上的长期固定样地监测。作者基于海南岛尖峰岭地区60 ha (1,000 m × 600 m)植被动态监测大样地, 详细描述了该样地所属典型热带山地雨林的群落结构特征, 以期为更深入地揭示我国热带雨林地区的物种多样性及其维持机制提供背景资料。尖峰岭大样地20 m × 20 m样方水平上海拔和坡度分别在866.3-1,016.7 m和1.7°-49.3°间变化。大样地内共记录到439,676株存活的胸径≥1.0 cm的乔灌木植株, 除61个植株未确定种名外, 其他分属于62科155属290种。单位面积植株密度为0.7328株/m² (含萌条和分枝), 20 m × 20 m尺度上单位面积物种数量为32-120种, 平均80种。植物属区系成分以热带性质为主, 共有136属, 占总属数的88.3%; 另有鼠李科鼠李属(Rhamnus)为世界分布, 木兰科拟单性木兰属(Parakmeria)为中国特有分布。按物种重要值排序, 大样地内最具优势(重要值大于2.0)的6个种分别为: 大叶蒲葵(Livistona saribus)、白颜树(Gironniera subaequalis)、厚壳桂(Cryptocarya chinensis)、油丹(Alseodaphne hainanensis)、四蕊三角瓣花(Prismatomeris tetrandra)和海南韶子(Nephelium topengii)。优势科为樟科、壳斗科、茜草科和棕榈科, 重要值均超过了5.0。取样面积达到7.5 ha或取样数量达到2万株时, 计数到的物种数量达到261种, 占总物种数量的90.0%。大样地内稀有种和偶见种各占所有物种的20.7%和37.6%。样地内所有个体平均胸径5.22 cm, 植株径级分布呈明显的倒“J”形。本文为后续尖峰岭大样地的研究提供了背景资料, 尖峰岭大样地的建立也为热带地区生物多样性长期变化监测提供了一个基础平台。

The leaves of plants are colonized by various microorganisms. In comparison to the rhizosphere, less is known about the characteristics and ecological functions of phyllosphere microorganisms. Phyllosphere microorganisms mainly originate from soil, air, and seeds. The composition of phyllosphere microorganisms is mainly affected by ecological and abiotic factors. Phyllosphere microorganisms execute multiple ecological functions by influencing leaf functions and longevity, seed mass, fruit development, and homeostasis of host growth. A plant can respond to phyllosphere microorganisms by secondary metabolite secretion and its immune system. Meanwhile, phyllosphere microorganisms play an important role in ecological stability and environmental safety assessment. However, as a result of the instability of the phyllosphere environment and the poor cultivability of phyllosphere microorganisms in the current research, there are still many limitations, such as the lack of insight into the mechanisms of plant-microorganism interactions, the roles of phyllosphere microorganisms in plant growth processes, the responses of phyllosphere microorganisms to plant metabolites, etc. This review summarizes the latest progress made in the research of the phyllosphere in recent years. This is beneficial for deepening our understanding of phyllosphere microorganisms and promoting the research of plant-atmosphere interactions, plant pathogens, and plant biological control.

Biological nitrogen fixation (BNF) by plants and its bacterial associations represent an important natural system for capturing atmospheric dinitrogen (N2) and processing it into a reactive form of nitrogen through enzymatic reduction. The study of BNF in non-leguminous plants has been difficult compared to nodule-localized BNF in leguminous plants because of the diverse sites of N2 fixation in non-leguminous plants. Identification of the involved N2-fixing bacteria has also been difficult because the major nitrogen fixers were often lost during isolation attempts. The past 20 years of molecular analyses has led to the identification of N2 fixation sites and active nitrogen fixers in tissues and the rhizosphere of non-leguminous plants. Here, we examined BNF hotspots in six reported non-leguminous plants. Novel rhizobia and methanotrophs were found to be abundantly present in the free-living state at sites where carbon and energy sources were predominantly available. In the carbon-rich apoplasts of plant tissues, rhizobia such as Bradyrhizobium spp. microaerobically fix N2. In paddy rice fields, methane molecules generated under anoxia are oxidized by xylem aerenchyma-transported oxygen with the simultaneous fixation of N2 by methane-oxidizing methanotrophs. We discuss the effective functions of the rhizobia and methanotrophs in non-legumes for the acquisition of fixed nitrogen in addition to research perspectives.