Genomic plasticity of the Azospirillum genus in a biotechnological context

Genomic plasticity of the Azospirillum genus in a biotechnological context | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Genomic plasticity of the Azospirillum genus in a biotechnological context Sarah Henaut-Jacobs , Felipe F. Rimes-Casais , Gabriel Quintanilha-Peixoto , Gabriela Petroceli-Mota , Bruno da Costa Rodrigues , Isabella de Oliveira Pinheiro , View ORCID Profile Rodrigo Nunes-da-Fonseca , View ORCID Profile Fabio Lopes Olivares , Rampal S. Etienne , View ORCID Profile Thiago M. Venancio doi: https://doi.org/10.1101/2025.10.16.682878 Sarah Henaut-Jacobs 1 Laboratório de Química e Função de Proteínas e Peptídeos (LQFPP), Biology and Biotechnology Center, State University of Northern Rio de Janeiro , Campos dos Goytacazes, Brazil 2 Groningen Institute for Evolutionary Life Sciences, University of Groningen , Groningen, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: s.henaut.jacobs{at}rug.nl thiago.venancio{at}gmail.com Felipe F. Rimes-Casais 1 Laboratório de Química e Função de Proteínas e Peptídeos (LQFPP), Biology and Biotechnology Center, State University of Northern Rio de Janeiro , Campos dos Goytacazes, Brazil Find this author on Google Scholar Find this author on PubMed Search for this author on this site Gabriel Quintanilha-Peixoto 1 Laboratório de Química e Função de Proteínas e Peptídeos (LQFPP), Biology and Biotechnology Center, State University of Northern Rio de Janeiro , Campos dos Goytacazes, Brazil Find this author on Google Scholar Find this author on PubMed Search for this author on this site Gabriela Petroceli-Mota 3 Laboratório de Biologia Celular e Tecidual (LBCT), Biology and Biotechnology Center, State University of Northern Rio de Janeiro , Campos dos Goytacazes, Brazil Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bruno da Costa Rodrigues 4 Laboratório Integrado de Ciências Morfofuncionais (LICM), Instituto de Biodiversidade e Sustentabilidade (NUPEM/UFRJ), Federal University of Rio de Janeiro , Macaé, RJ, Brazil Find this author on Google Scholar Find this author on PubMed Search for this author on this site Isabella de Oliveira Pinheiro Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rodrigo Nunes-da-Fonseca 4 Laboratório Integrado de Ciências Morfofuncionais (LICM), Instituto de Biodiversidade e Sustentabilidade (NUPEM/UFRJ), Federal University of Rio de Janeiro , Macaé, RJ, Brazil Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rodrigo Nunes-da-Fonseca Fabio Lopes Olivares 3 Laboratório de Biologia Celular e Tecidual (LBCT), Biology and Biotechnology Center, State University of Northern Rio de Janeiro , Campos dos Goytacazes, Brazil Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Fabio Lopes Olivares Rampal S. Etienne 2 Groningen Institute for Evolutionary Life Sciences, University of Groningen , Groningen, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Thiago M. Venancio 1 Laboratório de Química e Função de Proteínas e Peptídeos (LQFPP), Biology and Biotechnology Center, State University of Northern Rio de Janeiro , Campos dos Goytacazes, Brazil Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Thiago M. Venancio For correspondence: s.henaut.jacobs{at}rug.nl thiago.venancio{at}gmail.com Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Extensive agriculture and the use of chemical fertilizers cause notable environmental impacts on multiple levels, from reducing soil microbiota diversity to groundwater contamination. In this context, the usage of plant growth-promoting bacteria (PGPB) presents a sustainable alternative to enhance crop production while mitigating these adverse effects. Azospirillum , a bacterial genus renowned for its beneficial capabilities, particularly phytohormone production, is a key component of many commercial inoculants. In this work, we performed a comparative genomic analysis of all publicly available Azospirillum genomes and four novel isolates belonging to our microbial collection. Our analysis identified a species complex within the genus, which we designate the A. brasilense species complex, comprising species already used in commercial bioconsortia. This complex is characterized by a core set of exclusive genes linked to chemotaxis and host-recognition capability. Furthermore, we also validated the biosafety of the A. brasilense species complex and confirmed the plant growth-promoting potential of our novel isolates, highlighting their suitability for developing new biofertilizers. Introduction Global agriculture faces the pressing challenge of ensuring food security without harming the environment and human health ( FAO, 2017 ). Although critical since the Green Revolution, the historical over-reliance on chemical fertilizers and pesticides has led to severe consequences, including ecosystem contamination and a decline in biodiversity. Thus, the adoption of sustainable agricultural strategies has gained traction over the years, including the use of microbial inoculants containing Plant Growth-Promoting Bacteria (PGPB) ( Shahwar et al ., 2023 ). These microorganisms promote plant growth through various mechanisms, including biofertilization, production of growth-regulating hormones, countering pathogens and pests, and enhancing plant stress tolerance ( Kumar et al ., 2022 ; Bittencourt et al ., 2023 ). Central to this field is Azospirillum , a genus of Gram-negative, diazotrophic Alphaproteobacteria. First identified in 1925 and gaining prominence in the 1970s ( Cassán et al ., 2020 ), these microbes are known for their metabolic versatility and wide distribution ( Cassán et al ., 2020 ); species of Azospirillum are commonly found in the root zones of critical crops such as maize and rice, and have been discovered in a vast range of environments, from high-salinity soils and peat bogs ( Reinhold et al ., 1987 ; Doroshenko et al ., 2007 ) to contaminated industrial sites ( Ojeda-Morales et al ., 2015 ), demonstrating the genus exceptional adaptability and potential. A combination of direct and indirect plant-growth-promoting mechanisms drives the beneficial effects of Azospirillum . While Biological Nitrogen Fixation (BNF) was the first major trait studied in Azospirillum , the production of phytohormones, particularly Indole-3-Acetic Acid (IAA), is now recognized as the key mechanism underlying its beneficial effects, primarily through the enhancement of root system architecture ( Bashan and Levanony, 1990 ). Furthermore, these bacteria employ a suite of other strategies, including phosphate solubilization, production of iron-scavenging siderophores, and synthesis of various other growth regulators that work together to boost plant health and resilience ( Bottini et al ., 1989 ; Cohen et al ., 2009 ; Kusajima et al ., 2018 ). The agricultural significance of Azospirillum is underscored by its extensive use in commercial inoculants, especially within the large-scale farming systems in South America ( Cassán et al ., 2020 ). These products play a vital role in moving towards more sustainable practices by reducing the need for synthetic fertilizers. However, the current market is surprisingly limited in taxonomic diversity. The majority of the available inoculants are formulated using just two closely related species: A. brasilense and A. argentinense , with a single strain, Az39, dominating commercial products ( Cassán et al ., 2020 ). This reveals a remarkable contrast between the genus diversity (which has a total of 31 species identified in NCBI databases) and its phylogenetically narrow application in agriculture. While the capabilities of important agricultural strains of Azospirillum have been revealed through sequencing and genomic inference, commercial interest has largely remained focused on nitrogen fixation. A broader, systematic genomic exploration of the entire genus would open opportunities to investigate novel strains and uncover additional mechanisms of plant growth promotion. In this work, we report a comprehensive comparative genomic analysis of publicly available Azospirillum genomes, describing their pangenome and genetic repertoire. In addition, we sequenced and analyzed four novel Azospirillum isolates obtained from a large local collection of microorganisms of biotechnological interest. These strains isolated from plant sources were selected based on their potential as candidates for bioconsortia. Our analyses reveal an Azospirillum species cluster containing species commonly associated with plant growth promotion. This cluster presents a large set of exclusive genes, mainly linked to chemotactic capability, which appear to define the cluster’s lifestyle. Together, these findings point to the existence of novel species with plant growth-promoting potential, broadening biotechnological applications of the genus Azospirillum . Methods Genome sequencing Four bacterial isolates collected between August and October 2022 were selected from the Collection of Microorganisms of Biotechnological Interest at LBCT-UENF ( Table 1 ). Before genome sequencing, isolates were cultivated in Nutrient Broth (NB) medium to ensure viability and growth. Preliminary identification through 16S rRNA gene sequencing was conducted in a sequencing facility (NGS - Piracicaba, Brazil). Bacterial DNA samples were extracted using the Wizard Genomic DNA Purification kit (Promega - Madison, WI, USA) and sequenced on the PromethION 2 Solo system at the Institute of Biodiversity and Sustainability (UFRJ, Macaé, Brazil). Sequencing quality was assessed using FastQC v0.12.0. Low-quality reads were trimmed using Trimmomatic v0.39 ( Bolger, Lohse and Usadel, 2014 ), and genomes were assembled with SPAdes v4.2.0 ( Prjibelski et al ., 2020 ). View this table: View inline View popup Download powerpoint Table 1: Newly sequenced Azospirillum isolates from the UENF collection and their sources of isolation. Collection and curation of publicly available genomes A total of 296 Azospirillaceae genomes were downloaded from the GenBank database using NCBI Datasets v2 ( https://www.ncbi.nlm.nih.gov/datasets/ ) in July 2024 (Table S1). Genome quality was assessed using BUSCO v5.8.2 ( Seppey, Manni and Zdobnov, 2019 ). Genomes exhibiting less than 95% completeness, more than 5% duplication, or more than 500 contigs were discarded. Pairwise genomic identity was estimated with Mash v2.3 ( Ondov et al ., 2016 ) to estimate pairwise genomic identity (1 - Mash distance). Also, genomes showing more than 99% identity were considered redundant; in such cases, the assembly with superior quality metrics (contig number, L50, and N50) was retained for downstream analyses. Genus delimitation and phylogenetic analysis To resolve the structure of the Azospirillum genus within our dataset, we performed Average Nucleotide Identity (ANI) analysis (PyAni v0.3.0-alpha). ANI values were calculated for the 119 curated genomes (115 public genomes, plus our four newly sequenced isolates). Rhodospirillum rubrum (GCA_000013085.1) was included as an outgroup to root the phylogeny. Genomes forming a coherent cluster identified as Azospirillum in the ANI analysis were retained for downstream analyses (Table S1). Additionally, Azospirillum groups were delineated using a graph-based approach derived from Mash distances, as previously described ( Passarelli-Araujo et al ., 2021 ) and ( Henaut-Jacobs, Passarelli-Araujo and Venancio, 2023 ). For this analysis, we used the genomes from the Azospirillum cluster identified in the ANI step. Mash distances (1−Mash values) were used to estimate pairwise genomic relationships, with genomes represented as vertices in the resulting graph. A genus-level phylogeny was also inferred from a species tree constructed with orthologous genes identified by Orthofinder v3.0.1 b1 ( Emms and Kelly, 2019 ). A maximum-likelihood phylogenetic tree was then generated using IQTree2 v2.3.6 ( Minh et al ., 2020 ), with R. rubrum serving as the outgroup. Pangenome analysis Azospirillum genomes were annotated with Prokka v1.14.6 ( Seemann, 2014 ). Pangenome analysis was then carried out using Roary v3.13.0 ( Page et al ., 2015 ) with an 80% identity threshold for orthology assignment. Genome clustering was guided by the original genus phylogeny established in the previous session. The pangenome was partitioned into four categories based on gene prevalence across genomes: Core (>99%), Soft core (99%-95%), Shell (95%-15%), and Cloud (<15%) ( Matthews et al ., 2024 ). Accessory pangenome functionality After analyzing the clustering of the accessory pangenome across Azospirillum genomes, we selected the cluster containing the most agriculturally relevant genomes, which formed a single community (See “ A. brasilense is part of a great Azospirillum species complex” in the Results). A reference genome for this community (GCA_022023855.1) was chosen based on assembly quality metrics (completeness and N50). This reference genome was submitted to STRING for functional annotation of exclusive genes in the complex. Proteins annotated as hypothetical or unknown were manually curated and renamed using UniProt information. Protein-protein interactions were further explored in Cytoscape. Genes exclusive to the A. brasilense species complex were also screened for their orthologs hosted in STRING ( Szklarczyk et al ., 2025 ). The corresponding accession codes were retrieved using the STRING plugin of Cytoscape v3.10.2 ( Kohl, Wiese and Warscheid, 2011 ), applying a confidence score threshold of 0.9. Singlets and clusters containing fewer than three genes were discarded. The remaining genes were reannotated with InterProScan ( Jones et al ., 2014 ). Specific traits profiling Given the substantial agricultural relevance of the Azospirillum genus, we investigated key traits related to antibiotic resistance and plant growth-promotion potential. Antibiotic resistance profiles were inferred for all genomes using The Comprehensive Antibiotic Resistance Database (CARD) v3.1.3 ( Alcock et al ., 2023 ), a reference database for antibiotic resistance-associated genes. Usearch v11.0.667 ( Zhou, Liu and Li, 2024 ) was employed to align Azospirillum genomes to CARD with a 60% identity cutoff. Simultaneously, plant growth-promotion potential was assessed by screening genomes against an in-house curated database, also utilizing Usearch. This database comprises genes associated with direct plant-growth promotion, such as those involved in nitrogen fixation, phosphate solubilization, and phytohormone biosynthesis. To reduce phylogenetic bias, we incorporated diverse reference sequences for each gene, ensuring robust inference of gene presence regardless of evolutionary distance from the reference sources. Results and Discussion The Azospirillum genus is well established within the Azospirilaceae family ANI analysis and graph-based Mash distance clustering allowed us to precisely define the boundaries of the Azospirillum genus within our dataset. The vast majority of genomes previously classified as Azospirillum formed a dense and cohesive cluster, while a small subset had weak relationships with this core group and were excluded from the downstream analyses due to their affiliation with closely related taxa ( Figure 1 ). The genomes of our four newly sequenced isolates exhibited a strong relationship with other Azospirillum genomes, corroborating their preliminary 16S rRNA-based classification ( Table 1 , Figure S1). Download figure Open in new tab Figure 1. Heatmap using ANI values between every pair of genomes (represented as 1-Mash). Colors in the left annotation bar represent species classification in GenBank. Genomic clustering further revealed two major groups within the genus. One of these clusters notably encompassed numerous genomes with well-established plant growth-promoting capacities, particularly A. brasilense and A. argentinense . This clear pattern of relatedness, highlighting agriculturally relevant species, provided the framework for our subsequent in-depth analyses of Azospirillum taxonomic classification and functional potential. A. brasilense is part of a greater Azospirillum species complex The precise definition of bacterial species remains a challenge in microbial ecology, often debated as to whether genetic diversity exists on a continuum or in discrete groups ( Caro-Quintero and Konstantinidis, 2012 ; Cohan, 2019 ). Genetic discontinuity, representing abrupt breaks in genomic identity, is a key concept for delineating species boundaries. While a 95% ANI is a widely accepted threshold for species classification ( Konstantinidis, Ramette and Tiedje, 2006 ; Barghouthi, 2011 ), recent research quantifies this discontinuity, termed δ ( Passarelli-Araujo, Venancio and Hanage, 2025 ), by measuring the steepest change in genomic identity within a genomic dataset. Further, this quantitative approach reveals a significant ( Brockhurst et al ., 2019 ; Dewar et al ., 2024 ) association between genetic discontinuity and a species’ lifestyle, primarily reflected in its pangenome features. Species with high genetic discontinuity often exhibit closed pangenomes, suggesting specialized lifestyles with limited gene exchange ( Hollensteiner et al ., 2023 ; Dewar et al ., 2024 ). Conversely, species with more open pangenomes and versatile environmental adaptations tend to show less pronounced, yet still discernible, genetic breaks ( Brockhurst et al ., 2019 ). This spectrum of discontinuity, where clear divisions are sometimes evident and at other times ambiguous, parallels the challenges encountered in defining “ring species” in ecology, where gradual changes across populations eventually lead to distinct forms without clear points of separation ( Alcaide et al ., 2014 ). Understanding these varying degrees of genetic discontinuity is vital for reassessing traditional bacterial species classifications and appreciating the fluidity of microbial evolution. In our investigation of Azospirillum species delineation, we detected a notable species complex within broader patterns of genetic discontinuity. Most well-characterized species, particularly those already used in commercial inoculants ( Cassán and Diaz-Zorita, 2016 ; Dos Santos Ferreira et al ., 2022 ; Maroniche et al ., 2024 ), were found to be closely related, forming a concise cluster. The canonical 95% ANI threshold proved too stringent, often fragmenting well-supported taxa ( Figure 2 ). Reducing the threshold slightly to 94% effectively uncovered a cohesive species complex that encompasses A. brasilense, A. argentinense, A. baldaniorum, A. formosense, A. aestuarii , and A. tabaci ( Figure 2B ). Importantly, two of our novel isolates (C and D) were also placed within this complex. We hereafter refer to this group as the A. brasilense complex. Using this 94% identity cutoff, we identified a total of 25 genomic communities (Table S2). Download figure Open in new tab Figure 2. Graph-based approach to species delineation. Purple vertices represent type genomes that passed through quality and redundancy analysis, green vertices represent the new Azospirillum isolates introduced in this study, and yellow vertices represent all other genomes. A) Clustering using a 95% identity threshold; B) Clustering using a 94% identity threshold, the highlighted cluster is the A. brasilense species complex. Phylogenetic analysis based on the species tree of orthologous genes confirmed the strong genetic relationships within the A. brasilense complex ( Figure 3 ). This tree also reflected the broader clustering patterns observed in our genus-wide classification, with a clear separation of the A. brasilense complex. Beyond this group, the lower cluster of the tree included additional Azospirillum groups. In contrast, the other large cluster consisted of multiple independent species, comprising twelve previously categorized reference genomes and 16 uncharacterized strains. Download figure Open in new tab Figure 3. Phylogenetic tree based on single-copy orthologs from each genome. The red cluster represents the A. brasilense species complex defined in this study. Bootstrap values of 1 were omitted for clarity. Despite the cladogram topology within the A. brasilense complex suggesting that these genomes form a single species, evidence from the literature indicates otherwise. Phenotypic differentiation among strains—such as variation in preferred pH and carbon sources (e.g., D-fructose, maltose)—supports their recognition as distinct species ( Xu et al ., 2023 ). Nonetheless, these same taxa exhibit very limited genomic differentiation, as revealed by multiple clustering approaches, including both ANI values and phylogenetic analyses of orthologous genes. This phylogenetic classification also allowed us to infer the taxonomic position of A. brasilense UENF 111221 and A. argentinense UENF 211521 , both of which were placed in small, distinct monophyletic clades. Pangenome analysis revealed an exclusive gene cluster in the A. brasilense species complex At first glance, our dataset revealed two distinct clades of closely related genomes. Despite this high genetic similarity, the scientific literature consistently classifies these genomes as separate species. To further explore this separation, we conducted a super-pangenome analysis to identify the genetic determinants underlying the division between the two clades, with particular emphasis on the gene sets associated with the agriculturally important A. brasilense species complex. Our analysis showed that the Azospirillum genus harbors a large and diverse pangenome, characterized by a very small set of conserved core genes ( Figure 4 ). In total, we identified 866 core, 320 soft-core, and 9,163 shell gene families. The vast majority of the repertoire consisted of 56,921 cloud genes, representing 84.6% of the total pangenome and highlighting the remarkable genome plasticity of the genus (Figure S2). This plasticity is likely shaped by frequent horizontal gene transfer within the complex polymicrobial environments that Azospirillum species inhabit ( Georgiades and Raoult, 2010 ). Notably, the shell pangenome (orthologs present in 15–95% of genomes) exhibited a distinct structure, with gene families exclusive to one of the major communities but absent in the other. Download figure Open in new tab Figure 4. Pangenome of the Azospirillum genus. The top top annotation bar indicates the distribution of core, soft-core, shell, and cloud genes. The left annotation bar identifies and labels communities containing more than one genome. To further investigate the genomic basis for the phylogenetic groupings, we searched for clade-specific markers. One clade, in particular, stood out: the A. brasilense species complex (community 24) together with the species corresponding to communities 17 ( A. rugosum ), 18 ( A. soli ), and 19 ( A. canadense ). This group alone contained 2,471 orthologous gene clusters unique to its members. Although many of these orthogroups were annotated as hypothetical proteins (Table S3), we leveraged the STRING database to predict their putative functions and gene interaction networks based on sequence similarity ( Figure 5 ). Download figure Open in new tab Figure 5. STRING network of the genes exclusive to the A. brasilense complex. Cluster content for each numbered group is detailed in Table S4. Edge colors indicate gene relationships (neighborhood, fusion, co-expression, or co-occurrence) with a confidence score >0.9. Node colors represent distinct clusters. Distances are not to scale. The STRING network of genes exclusive to the A. brasilense species complex revealed 13 clusters, each containing at least four genes. Functional annotation indicated a high abundance of duplicated and functionally related genes, including ABC transporters (G3DSA:3.40.50.300:FF:000421, IPR022467, IPR022478, IPR051449, IPR050319, IPR050388, and IPR051120), adenylyl/guanylyl (IPR050697) and diguanylate cyclases (IPR050469), chemotaxis methyl-accepting receptors (IPR004090), and at least 37 genes associated with histidine kinases and their regulation, particularly protein families PF02518, IPR050736, IPR050980, and G3DSA:3.30.565.10:FF:000010. Full details for each cluster are provided in Table S4 and Figure S3. Our findings highlight a highly specialized gene repertoire that is strongly linked to chemotaxis and plant-host interactions, including motility and the ability to sense environmental gradients. We hypothesize that this genomic toolkit confers an evolutionary advantage to the A. brasilense species complex, which may underlie its success as a commercial bioinput and enhance the potential use of other species within the same cluster. Screening for genes associated with plant growth-promotion shows their uniform presence within the genus We assessed the plant growth-promotion (PGP) potential of all Azospirillum genomes using an in-house, literature-based reference dataset. This analysis revealed a conserved core set of PGP genes across the genus, including those involved in nitrogen fixation and phosphate solubilization ( Figure 6 ). Notably, the A. brasilense complex harbored the ipdC gene, a central gene for the production of indoles, primarily IAA, an auxin that regulates root development, nutrient uptake, and overall plant growth ( Sun et al ., 2022 ). This could be linked to the usage of already known PGP species from this complex, and highlights the untapped potential of less explored members, such as A. tabaci and A. formosense , both soil isolates with documented PGP capacity ( Lin et al ., 2012 ; Duan et al ., 2021 ) but not yet commercialised in bioconsortia. Importantly, our novel isolates C and D also share this PGP potential with species already recognized for their beneficial interactions with plants. Download figure Open in new tab Figure 6. Presence/absence heatmap of direct plant growth-promoting genes. The top annotation bar indicates the main functional categories associated with each gene. Furthermore, a subset of the A. brasilense species complex also carried the pqqA gene, which was absent from the rest of the genus ( Figure 6 ). pqqA is important for the biosynthesis of pyrroloquinoline quinone (PQQ), a coenzyme that plays a key role in various metabolic processes, such as the oxidation of sugars and alcohols ( Kim et al ., 2003 ). Nonetheless, previous studies have demonstrated that the PQQ operon can function without pqqA ( Matteoli et al ., 2018 ). In the context of phosphate solubilization, PQQ-dependent enzymes—particularly glucose dehydrogenase—play a crucial role in releasing insoluble phosphate from the soil, thereby enhancing plant nutrient acquisition. Although PGP genes are broadly conserved across Azospirillum , two genomes showed a markedly reduced repertoire: Azospirillum sp. C1_MAG_00050 (GCA_027486405.1 (unpublished)) and Azospirillum griseum L-25-5 w-1 (GCA_003966125.1 ( Yang et al ., 2019 )). Both genomes were isolated from lake water samples, with the former being a metagenome-assembled genome (MAG). Their limited PGP gene content may reflect ecological adaptation, as aquatic environments likely impose weaker selective pressure for plant-associated traits. We also analyzed antibiotic resistance genes to infer the biosafety profile of the Azospirillum genomes. No concerning resistance traits were detected in any of the genomes examined. The unique presence of specific plant growth promotion genes ( ipdC, pqqA ) in the A. brasilense species complex ( Figure 7 ), together with this favorable biosafety profile, underscores the potential of these strains for the safe development and application of biofertilizers. Download figure Open in new tab Figure 7. Presence/absence heatmap of antibiotic resistance genes. The top annotation bar indicates the presumed mode of action for each gene. Considering the collective evidence, we propose that the species clustered within the A. brasilense species complex represent up-and-coming candidates for microbial inoculant applications. This conclusion is supported by their consistently favorable biosafety profiles and their shared genetic repertoire of key plant growth-promoting traits. This research expands the scope of potential bioinputs by highlighting less-explored Azospirillum species such as A. formosense and A. baldaniorum . Although A. baldaniorum was only recently described (2020), it already shows a promising genomic toolkit for phytohormone production ( Dos Santos Ferreira et al ., 2020 ). Similarly, A. formosense has demonstrated the ability to engage in synergistic interactions with other beneficial microorganisms, such as Bacillus species ( Yaadesh et al ., 2023 ), but it remains underutilized in agricultural applications. By characterizing these species and reporting novel isolates within this complex, our study underscores their potential as next-generation biofertilizers. Conclusion This comprehensive genomic study of Azospirillum refines current understanding of species boundaries, identifying a major species complex characterized by high genomic identity despite phenotypic distinctiveness. Pangenome analysis revealed unique genetic features within this complex, notably the presence of ipdC and pqqA genes, both central to plant growth promotion. Combined with a favorable biosafety profile and the distinct ecological and physiological traits of the newly defined A. brasilense species complex, these findings underscore the considerable yet underexplored biotechnological potential of Azospirillum species beyond those currently in use. Our work paves the way for the development of more effective and diverse microbial inoculants to advance sustainable agriculture. Author contributions Conceptualization: S.H.-J., I.O.P, and T.M.V.; formal analysis: S.H.-J.; computational methodology: S.H.-J., F.F.R.-C, and G.Q.-P.; wet lab methodology; S.H.-J., G.P.-M., and B.C.R.; writing—original draft: S.H.-J. and T.M.V.; writing—review and editing: S.H.-J., R.S.E. and T.M.V.; funding acquisition: R.N.-F., F.L.O. and T.M.V.; project administration: F.L.O. and T.M.V. All authors have read and agreed to the published version of the manuscript. Acknowledgements This work was supported by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ E-26/210.291/2021), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES; Finance Code 001), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Programa de Apoio à Pesquisa, Inovação e Cultura (PAPIC - UENF). The funding agencies had no role in the design of the study, the collection, analysis, and interpretation of data, or the writing. 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