Genome sequence-based identification of bacteria nodulating Mimosa pudica growing in Eastern Himalayas and Western Ghats of India and description of Cupriavidus mimosae sp. nov. and Cupriavidus gehlotii sp. nov

Genome sequence-based identification of bacteria nodulating Mimosa pudica growing in Eastern Himalayas and Western Ghats of India and description of Cupriavidus mimosae sp. nov. and Cupriavidus gehlotii sp. nov | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome sequence-based identification of bacteria nodulating Mimosa pudica growing in Eastern Himalayas and Western Ghats of India and description of Cupriavidus mimosae sp. nov. and Cupriavidus gehlotii sp. nov Lekha Sharma, Mitesh Khairnar, Aabeejjeet Pansare, Krishna Mohan Medicherla, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5391658/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The invasive weed Mimosa pudica (wild) is widespread in India’s tropical regions and nodulated by beta-rhizobia, including Cupriavidus and Paraburkholderia . This study investigates the genetic diversity of rhizobia associated with M. pudica in the Eastern Himalayan (EH) and Western Ghat (WG) regions of India. MALDI-TOF-MS and 16S rRNA gene phylogeny identified EH rhizobia as Paraburkholderia , while WG rhizobia belonged to Cupriavidus . Core-gene phylogeny ( bac120 genes) and average nucleotide identity (ANI) values confirmed EH strain SKND8 as Paraburkholderia caribensis . In contrast, WG strains WGtm5 T and WGlv3 T clustered within the Cupriavidus taiwanensis species complex but had ANI values below 95.1% with validly published species, suggesting they are novel species. Distinct clusters in bac120 gene phylogeny and ANI values below 95.8% further indicate that both strains represent different species. Major fatty acids identified in WGtm5 T and WGlv3 T included C 16: 0 , C 16:1 ꙍ7c and/or C 16:1 ꙍ 6c and C 18: 1 ꙍ 7c and/or C 18: 1 ꙍ 6c. Based on ANI values, core-gene phylogeny, and phenotypic data, we proposed two novel species, Cupriavidus mimosae sp. nov for WGtm5 T (MCC4888 T = KACC 22828 T ) and Cupriavidus gehlotii sp. nov for WGlv3 T (MCC4890 T = KACC 22827 T ). The presence of nodulation and nitrogen-fixation genes and nodule formation in Vigna radiata and Vigna unguiculata by strain SKND8, WGtm5 T and WGlv3 T highlighted their nitrogen fixation potential. Additionally, WG strain WGmk3, identified as Herbaspirillum huttiense , lacks nodulation genes and may be an endophyte of M . pudica . MALDI-TOF MS core-gene phylogeny rhizobia Western Ghats Eastern Himalayas Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Nitrogen is an essential element for all living beings, while a small group of organisms known as diazotrophs can fix atmospheric nitrogen. Among the group diazotrophs, rhizobia represent the bacteria which fix nitrogen by entering into symbiosis with host plants mostly legumes by forming specialized structures called nodules. Mimosa pudica is a member of the tribe Mimosaseae in the mimosoid subfamily of legumes ( Leguminosae/Fabaceae ). The legume originates in South America and spread throughout the tropical and semi-tropical regions as a weed (Barneby 1991). This perennial shrub is widely distributed to different regions of India where it mostly grows on non-perturbed environmental conditions. Particularly, in the tropical and semi-tropical rain forests of the Eastern Himalayan (EH) and Western Ghats (WG), which are known for their rich in diversity of flora and fauna. The genetic diversity and host-microbe interactions of Mimosa pudica rhizobia have been extensively studied in different parts of the world, largely due to their ecological contribution to biological nitrogen fixation. Mimosa pudica , like other Mimosa species enter into symbiosis preferentially with Cupriavidus , Paraburkholderia and Trinickia belonging to beta-subclass of phylum Proteobacteria (now Pseudomonadota ) (Klonowska et al. 2012; Gehlot et al. 2013; Dall’Agnol et al. 2017; los Santos et al. 2018; Silva et al. 2018; Liu et al. 2020; Paulitsch et al. 2020; Dias et al. 2021; Mavima et al. 2021). However, members of Rhizobium and Ensifer belonging to alpha-proteobacteria have also been reported to nodulate Mimosa species (Klonowska et al. 2012; Gehlot et al. 2013; Baraúna et al. 2016). On one hand, Mimosa species endemic to a region or country have been reported to form symbiosis often with alpha-rhizobia (Wang et al. 1999; Gehlot et al. 2013; Bontemps et al. 2016). On the other, the invasive species of Mimosa , including M. pudica enter into symbiosis with beta-rhizobia including Paraburkholderia phymatum , Paraburkholderia mimosarum and Cupriavidus taiwanensis (Klonowska et al. 2012; Gehlot et al. 2013; Dall’Agnol et al. 2017; los Santos et al. 2018; Silva et al. 2018; Liu et al. 2020; Paulitsch et al. 2020; Dias et al. 2021; Mavima et al. 2021). Several species of the genus Paraburkholderia have been reported to nodulate Mimosa species, such as P. nodosa (Dall’Agnol et al. 2017), P. symbiotica (Sheu et al. 2012), P. piptadeniae and P. ribeironis (Bournaud et al. 2017), P. youngii and P. atlantica (Mavima et al. 2021), P. tuberum (Mishra et al. 2012), P. phymatum , P. mimosarum , and P. diazotrophica (Sheu et al. 2013). Similarly, multiple species of Cupriavidus including, C. taiwanensis (Chen et al. 2008), C. necator (Poehlein et al. 2011), “C. neocaledonicus” (Klonowska et al. 2020) have been reported to nodulate Mimosa . Cupriavidus taiwanensis was the main symbiotic partner of M. pudica in four different sites in New Caledonia (Klonowska et al. 2012). Furthermore, five different genotypes of Cupriavidus were identified during this study and a new species C. neocaledonicus was proposed (Klonowska et al. 2020). A core-gene-based phylogeny of Mimosa symbionts indicated that C. taiwanensis is a complex of several closely related species (Clerissi et al. 2018). India, with its diverse ecosystems and numerous biodiversity hotspots, offers a rich environment for the exploration of Mimosa rhizobial diversity. Certain regions, such as the EH and WG, may be particularly important for understanding the diversity and distribution of these symbiotic relationships. In the present study, we isolated the Mimosa pudica symbionts from EH and WG, and used tools like MALDI-TOF MS and genome sequencing for their precise identification. We also proposed the description of two new species and resolved the C. taiwanensis species complex based on taxogenomic analyses. Materials and methods Isolation of rhizobia from root nodules of Mimosa pudica Mimosa pudica roots with nodules were collected from ten locations in the Eastern Himalayan (EH) and Western Ghat (WG) regions (Fig S1; Table S1). The root nodules were placed in phosphate buffer with 20% glycerol (v/v) and transported at 4°C in a vaccine box (Naik and Rahi 2022). The vials containing the collected nodules were refrigerated at 4°C until further use. To isolate bacteria from the root nodules, the nodules were surface sterilized using 25% sodium hypochlorite solution and 70% ethanol (Naik and Rahi 2022). The surface sterilization of the nodule was confirmed by rolling the sterilized nodules on yeast extract mannitol agar (YEMA) plate before crushing. Isolation of bacteria was done by crushing the surface sterilized nodules and streaking on YEMA media containing 0.025% congo red (M721, Himedia, India) at 30 ℃ for 24-48 hrs (Vincent 1970). Plates were observed for the growth of glistening-white bacterial colonies and confirm the purity of bacterial colonies by multiple streaking. MALDI-TOF-MS approach for Identification of isolates All strains were screened for MALDI-TOF-MS based identification as described earlier (Rahi et al. 2016). In brief, the strains were grown in YEMA plates for 36 h, and a smear of freshly grown culture was applied onto the MALDI target plate followed by the addition of one µl of alpha-cyano-4- hydroxycinnamic acid (HCCA) matrix solution. Mass spectra were generated in the mass range 2 to 20 KDa in a linear positive mode in the MALDI-TOF MS instrument (autoflex, Bruker Daltonics, Germany) (Kurli et al. 2018). The acquired protein spectral profiles were compared with the MALDI Biotyper database (Bruker Daltonik). The criteria of microbial identification was based on the biotyper score values; strain with value ≥2.0 confirmed species level identity and if it within the range 1.7 to 1.99 the strain was identified only at genus-level. Phylogenetic analysis of 16S rRNA genes The genomic DNA was extracted using standard CTAB method (Minas et al. 2011) described in JGI catalogue version 3. Amplification of 16S rRNA genes was performed using the universal primer pair 27F-1492R (Weisburg 1991) and the amplicons were sequenced using ABI PRISM Big Dye Terminator v3.1 Cycle Sequencing kit on a 3730xl Genetic Analyzer (Applied BioSystems, Thermo Scientific, USA). Obtained sequences were searched as query sequence to find the similar sequences in the EzBioCloud database of 16S rRNA genes of the type strains of prokaryotic species (Yoon et al. 2017). All the query and closely related sequences were aligned using CLUSTAL W program (Larkin et al. 2007). The neighbour joining (NJ) phylogenetic trees were inferred for the aligned nucleotide sequences. MEGA 11 was used to determine best-fit models of nucleotide substitution and for performing NJ analysis (Tamura et al. 2021). Using 1000 bootstrap replications, the Tamura 3-parameter model was applied to the 16S rRNA dataset to construct a phylogenetic tree. Genome-based analyses The genome sequencing of strains WGtm5 T , WGlv3 T , WGmk3 and SKND8 was performed using Illumina Novaseq platform with a 2×150bp paired-end protocol. Quality of raw data was checked using the FastQC tool (https://www.bioinformatics.babraham.ac.uk/projects/ fastqc). Quality filtered reads were assembled using SPAdes version 3.12.0. (Bankevich et al. 2012). Assembled sequences were annotated using RAST (Rapid Annotation using subsystem technology) (https://rast.nmpdr.org/) pipeline and sequence-based comparisons were performed using the SEED Viewer (Jin et al. 2022). Furthermore, the annotated sequences were analyzed to detect biosynthetic gene clusters (BGCs) responsible for various secondary metabolites using an online genome mining pipeline antiSMASH 5.0 (Blin et al. 2019). The genes responsible for nitrogen fixation and nodulation were detected and sequences of nifH and nodC genes were extracted from the genome sequences. The sequences of nifH and nodC genes were searched as queries to identify the related gene using NCBI BLASTn tool (Altschul et al. 1990). The search was done against the non-redundant nucleotide database with and without a limit to sequences from type material. Phylogenetic analyses was done for nifH and nodC gene sequences as described for 16S rRNA gene phylogeny. Two genome datasets were made, the first one consisted the genome sequences from the type strains of species closely related to the root nodulating bacteria, belonging to Paraburkholderia , Cupriavidus and Herbaspirillum were downloaded in FASTA format from the NCBI genome assembly database (https://www.ncbi.nlm.nih.gov/assembly). The second one was made to resolve the Cupriavidus taiwanensis species complex, and consists of 178 genome sequences of Cupriavidus species (https://www.ncbi.nlm.nih.gov/assembly accessed on 20 September 2023). Both genome datasets were used to construct independent phylogenies using bac120 core-genes, by using GTDBTk 2.1.1 pipeline (Chaumeil et al. 2020) and also to calculate ANI values using fastANI 1.33 (Jain et al. 2018). The obtained aligned gene sequences were used to build a maximum likelihood phylogenetic tree using IQ-TREE 2.2.2.2 (Minh et al. 2020), with 1000 ultrafast bootstrap replications and selection of the best method by ModelFinder (Kalyaanamoorthy et al. 2017). All trees were displayed using iTOL (Letunic et al. 2019). Pairwise ANI was calculated with the kmer = 16, fragment length = 3000, minimum shared fraction = 0.2 using fastANI 1.33 (Jain et al. 2018). The ANI values were displayed in the core-gene phylogenies by inserting coloured ribbon strips. Digital DNA-DNA hybridization (DDH) values and confidence intervals were calculated using the recommended settings of the Genome-to-Genome Distance Calculator (GGDC) 2.1 (Meier-Kolthoff et al. 2013). Physiology and chemotaxonomy Morphological characteristics of all the strains were observed by streaking them on YEMA plates incubated under aerobic conditions at 30 ℃ for 24-48 h. Cell morphology of the strains was determined by microscopic sight of Gram-stained culture and reaction was carried out by following Gram’s protocol (Aneja 2007). The cell motility was examined following the technique described by Arora (2003). The strains were also tested for growth at different temperatures (10°C, 20°C, 30°C, 40℃ and 50°C), NaCl concentrations [0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5% and 4% (w/v) at 0.5% intervals] and pH values ranging (4 - 9 at 1.0 pH unit intervals) was examined in Yeast Mannitol broth at 30 °C in OSI at 180rpm for 7 days. The nitrate reduction test was performed using α-nephathylamin reagents in broth culture and the catalase activity was determined by observing immediate effervescence in a 3% (v/v) Hydrogen peroxide solution (Cappucino and Sherman 2014). Reduction of carbon sources viz. glucose, fructose, dextrose, mannitol, sucrose, lactose, Sodium Acetate, Sodium Succinate, Sodium Propionate, Sodium Citrate and Sodium Pyruvate was performed using the protocol developed by Reiner (2012). To estimate the whole-cell fatty acids, the strains were incubated on YEMA at 30℃ for 48 hrs. Preparation and extraction of fatty acid methyl esters were performed by following Sasser’ s protocol (2001) using the Microbial Identification System (MIS; MIDI). Cellular fatty acids were identified using the Microbial Identification software package (Sherlock version 6.1; MIDI database, TSBA6). Determination of host range of representative rhizobial isolates Four representative strains were examined for their nodulation ability using different hosts including Vigna radiata, Pisum sativum, Vigna aconitifolia, Cicer arietinum, Vigna unguiculata, Arachis hypogaea, Trigonella foenum-graecum, Phaseolus vulgaris, Cyamopsis tetragonoloba and Lens culinaris (Hui., 2014). The seeds of all legumes were surface sterilized with sodium hypochlorite (4% available chlorine) and allowed to germinate at 28 ℃. The seedlings were then transferred to polypropylene pots containing moist sterile vermiculite (3 seeds per pot, 10 pots of test plants per each strain treatment). Each seedling was received supplemented with 20 ml of the nutrient solution (g/l): K2HPO4 1.0, KH2PO4 0.25, MgSO4 1.0, Ca3(PO4)2 0.2, FeSO4 0.02, H3BO3 0.005, (NH4)2MoO4 0.005, ZnSO4 × 7 H2O 0.005, MnSO4 0.002 (Novikova and Safronova, 1992) at an interval of 10 days. The seedlings were inoculated with 10 9 cells per pot of strains WGlv3 T , WGtm5 T , WGmk3 and SKND8 separately. The uninoculated plants were served as negative control. The plant growth experiment was terminated after 45 days of inoculation and data was recorded for nodule number, root and shoot length and dry weight. The experiment was carried out in a completely randomised design in a glasshouse and each treatment was replicated three times. Statistical analysis and data availability Statistical analysis was conducted using one-way analysis of variance (ANOVA) and means were separated by Duncan’s multiple range test at p ≤ 0.05 using SPSS statistics 27. The assembled genomes and the raw datasets were submitted using the NCBI Sequence Read Archive tool kit by uploading on SRA FTP platform. The GenBank/EMBL/DDBJ accession numbers for the strain SKND8 genome (JAKUCO000000000), Bioproject No (PRJNA802963), raw reads sequences SRA ID (SRR17868289); for the strain WGmk3 genome (JAKYWT000000000), Bioproject No (PRJNA809344), raw reads sequences SRA ID (SRR18106144); for the strain WGlv3 T genome (JALHRX000000000), Bioproject No (PRJNA809334), raw reads sequences SRA ID (SRR18106231); and for the strain WGtm5 T genome (JALHRY000000000), Bioproject No (PRJNA809348), raw reads sequences SRA ID (SRR18106221). Sequences of 16S rRNA gene of root-nodulating bacteria have been deposited with NCBI Genbank under accession numbers: OK336461-OK336464, OM281129-OM281133. Results and Discussions Isolation and MALDI-TOF-MS identification of isolates Isolation of bacteria from the healthy Mimosa pudica root nodules collected from EH and WG of regions of India, resulted in 29 bacterial isolates with typical rhizobia-like cultural characteristics on the YEMA-CR medium. Good quality mass spectra ranging from 2000-20000 Da were generated for 72.5% strains, of which 20.6% were identified as Cupriavidus nector and 13.7% as Paraburkholderia caribensis with score value ≥ 2.00 indicated species level identification (Table S2). Furthermore, 17.2% strains (three isolates as Cupriavidus and two as Paraburkholderia ) were identified at the genus level with score value between 1.700 to 1.999. Three (10.3%) strains could not be identified and the database search resulted non-reliable identity with score value < 1.7. MALDI-TOF MS based identification is highly dependent on the group of bacteria, as some of the bacterial groups are underrepresented in the database, leading to only genus-level identification or no identification (Rahi et al. 2016). Several studies on plant associated bacteria have suggested that, development of in-house database improve the identification results (Ferreira et al. 2011; Khairnar et al. 2022). Phylogenetic analysis based on the 16S rRNA gene The 16S rRNA gene sequences of all strains were obtained with a length ranging from 1100- 1,600 bp (Table S3). The search results of 16S rRNA gene sequences were in corroboration with the MALDI-TOF MS based identification. However, 16S rRNA gene sequences lead to the identification of all strains, at least up to the genus-level. All EH strains showed high sequence similarity (≥99%) to Paraburkholderia caribensis strain MWAP64 T . On the other hand, two WG strains, were closely related to Cupriavidus oxalaticus strain Ox1 T with ≥99% similarity, and the strain WGmk3 showed the highest (99.80%) similarity to Herbaspirillum huttiense strain ATCC 14670 T . Notably, WG strains WGlv3 T , WGtm5 T and WGmk3 showed more than 99% sequence similarity to two or more species. The phylogenetic tree reconstructed based on 16S rRNA gene sequences placed the root-nodulating bacteria into three clades including, Cupriavidus , Herbaspirillum and Paraburkholderia (Fig.1). The EH strains were placed in Paraburkholderia clade, with Paraburkholderia caribensis as their nearest neighbour. However, the WG strains were placed in two clades, the strain WGmk3 was nested within the Herbaspirillum clade, sitting close to the type strains of Herbaspirillum huttiense and Herbaspirillum aquaticum . On the other hand, the strains WGtm5 T and WGlv3 T were placed in Cupriavidus clade, with Cupriavidus oxalaticus as their closest neighbour.Although the strains showed close evolutionary relationships with the described species, it was difficult to assign the strains to any of these species. Several studies have highlighted the low species-level resolution of the 16S rRNA gene sequence data, particularly among the members of group rhizobia (Rahi et al. 2020; Young et al. 2021). Genome features: Genome sequencing and assembly of strains SKND8, WGmk3, WGtm5 T and WGlv3 T yielded genome sizes of 9.2, 5.3, 6.7 and 6.8 Mbp, respectively with the DNA G + C content ranging from 62.4 to 66.9 mol%. The quality features of genome sequences generated in this study (Table S4) are in corroboration to the minimal genome standards proposed for new taxa description (Chun et al. 2018). The phylogenetic tree constructed based on bac120 core genes, placed the root-nodulating bacteria in three major groups similar to 16S rRNA gene phylogeny (Fig. 2). The placement of strain SKND8 close to Paraburkholderia caribensis in the core gene phylogeny and high ANI (>95-96%) and dDDH (>70%) values confirm that the strain belongs to P. caribensis . Paraburkholderia is the most dominant genus among the bacterial nodulating Mimosa species across the globe (Dall´Agnol et al. 2017; Dias et al. 2021; Paulitsch et al. 2021). Initially isolated from vertisol microaggregates P. caribensis , has also been reported to nodulate Mimosa pudica in Southern China (Chen et al. 2008; Liu et al. 2020). However, the distribution of P. caribensis was far less in comparison to P. mimosarum and P. phymatum , which are widely distributed in both the invaded and the original regions of the Mimosa species (Liu et al. 2020). Additionally, several other species of Paraburkholderia including P. bannesnsis , P. nodosa , P. phenoliruptrix , P. diazotrophica ,and P. symbiotica have been reported nodulating Mimosa spp. mostly from Brazil (Sheu et al. 2012; Paulitsch et al. 2021; Dias et al. 2021). The strain WGmk3 was placed close to Herbaspirillum huttiense subsp. putei and also shared high ANI and dDDH values, confirming affiliation to H. huttiense (Fig. 2). Strain of Herbaspirillum huttiense has often been reported as plant endophytes (Yan et al. 2018; Jia et al. 2022), and to colonize plants like rice (Andreozzi et al. 2019) and tea (Jia et al. 2022). Additionally, Herbaspirillum species have exhibited multiple plant growth-promoting attributes including nitrogen fixation (Gulati et al. 2011; Yan et al. 2018; Andreozzi et al. 2019; Jia et al. 2022). However, the members of Herbaspirillum were never isolated from root nodules. In this study, we did not detect any symbiotic genes in the genome of the strain WGmk3, indicating that this strain was an endophytic bacterium. Cupriavidus clade includes two strains WGtm5 T and WGlv3 T placed close to the members of Cupriavidus taiwanensis species complex, consisting of C. alkaliphilus , C. nantongensis , C. taiwanensis and “C. neocaledonicus” (Fig. 2) (Poehlein et al. 2011; los Santos et al. 2012; Sun et al. 2016; Feng et al. 2019; Klonowska et al. 2020). Cupriavidus (formerly Ralstonia ) taiwanensis have been reported to nodulate Mimosa pudica in different parts of the world, including India (Verma et al. 2004; Barrett et al. 2006; Amadou et al. 2008; Klonowska et al. 2012). The ANI values for the newly isolated strains WGtm5 T and WGlv3 T were below 96% (species delineation value) to the type strains of all valid species of Cupriavidus (Table S4). Similarly, dDDH values below 70%, indicated that the strains represent new species of Cupriavidus . Detailed genome-based analyses including core-gene phylogeny (Fig. 3), and ANI values exhibited abundant polymorphism among C . taiwanensis strains and highlighted that C . taiwanensis is a complex of several closely related species (Clerissi et al. 2018). A core-gene phylogeny and pairwise ANI between all Cupriavidus genomes, indicated that the C . taiwanensis species complex has 12 genospecies (Fig. 3). Four of these genospecies already have valid species names including, C . taiwanensis , C. nantongensis , C . alkaliphilus and “C. neocaledonicus” . The genospecies A-D are represented by single strains. The type strain of C . taiwanensis was placed in a cluster with eight strains, and share less than 96% ANI values with the other members of C . taiwanensis species complex and represents genospecies E. The type strain of C. nantongensis was alone in representing the genospecies G, while two strains LMG 19430 and MLR2-44 representing genospecies H were placed close to it. Three strains including the type strain of “C. neocaledonicus” represented the genospecies I. The type strain of C . alkaliphilus was placed in a cluster with five strains with ANI values more than 98%, representing genospecies L. Additionally, a subcluster of nine strains and an independent strain cmp52 are also within the genospecies L, sharing more than 96% ANI values with the type strain of C . alkaliphilus . The strain WGlv3 T placed independently with less than 96% values, with all the type strains of described species and strains of genospecies within the C . taiwanensis species complex, representing genospecies J. Similarly, strain WGtm5 T was placed in a cluster, which further divide into two subcluster represent the genospecies K. The presence of subclusters and sharing ANI values 96-97% in genospecies K and L, indicate the presence of subspecies. In addition to the circumscription of C . taiwanensis species complex into 12 genospecies, the results of core-gene phylogeny and ANI calculation confirmed that the strains WGlv3 T and WGtm5 T represent two new species of Cupriavidus genus. Monophyletic grouping in core-gene phylogeny and ANI values below 96% have been used to delineate genospecies boundaries in the case of Rhizobium legumonosarum species complex (Young et al. 2021). Genes involved in nitrogen fixation, nodulation, secondary metabolism We analyzed the pangenome of strains WGmk3, WGlv3 T , WGtm5 T and SKND8 to identify the accessory genomic features of the newly isolated species. Here, genome sequences were examined to identify the symbiosis-related genes and secondary metabolite regions. A subset of nod , nif and fix genes were predicted in the genome SKND8, WGlv3 T and WGtm5 T . The genome SKND8 contained a cluster of nine nod genes: nodXSHD1ABC including two copies of nodIJU genes. A set of genes involved in nitrogen fixation, nifABXENQVWHDKZTU and fixABCUL , have been located and dispersed throughout the genome. Both strains WGtm5 T and WGlv3 T showed similar nod genes organisation with the presence of a cluster of nodTWIDASB genes. Additionally, a putative nodJ gene was identified for strain WGlv3 T . Notably, common sets of nifABENWHDK genes and fixBCX genes were observed for both strains. Additionally, secondary metabolite-producing genes and pathways have been identified in the genome sequences of the root-nodulating bacteria by using anti-SMASH online server search. The strains SKND8 exhibited the presence of seven putative secondary metabolite gene clusters including, arylpolyene, phosphonate, terpene, redox-cofactor, hserlactone, exopolysaccharides related NRPS and TfuA-related RiPPs. Member of beta rhizobia have been shown to produce antioxidant exopolysaccharide(Xia et al. 2021). While genome WGmk3 contained only three regions: arylpolyene, terpene and serobactin C related siderophore. Previously, serobactin C (siderophores) produced by Herbaspirillum seropedicae was reported by (Rosconi et al. 2013). In strain WGlv3 T gene clusters related to lankacidin C and kirromycin biosynthesis observed, overall, six secondary metabolites regions including siderophore (taiwachelin) were identified. Earlier, Kreutzer et al. (2012) reported taiwachelin type of siderophore in the genomesequence of Cupriavidus taiwanensis LMG19424. Six secondary metabolites regions including arylpolyene, lankacidin C and bacillomycin D biosynthesis cluster, Xanthoferrin related siderophore were predicted in the genome of WGtm5 T . Xanthoferrin is the α-hydroxycarboxylate type siderophore which is required for optimum growth inside cabbage (Pandey et al. 2017). Previously, serobactin C, taiwachelin and xanthoferrin types of siderophore reported from rhizobia in independent studies (Kreutzer and Nett 2012; Rosconi et al. 2013; Pandey et al. 2017). Phylogenetic analysis based on symbiotic (nodC and nifH) genes Symbiosis genes of rhizobia, including nodulation and nitrogen fixation genes, are part of the accessory and mobile genome. Several rhizobia share specific symbiosis genes, which allow them nodulate specific hosts. Rhizobia with specific symbiosis genes can be divided into several symbiovars. To identify the symbiovars within the rhizobial strains isolated from Mimosa pudica , nodC and nifH genes were extracted from the genomes. Blastn search of the nifH gene exhibited that strain SKND8 belongs to sv. tropicalis , with 99.9% sequence identity with Paraburkholderia phymatum STM815 T (Table S5), isolated from Machaerium lunatum in French Guiana, that formed ineffective nodules on the promiscuous host Macroptilium atropurpureum (Moulin et al., 2001). Later, this symbiovar was found in different Paraburkholderia spp. nodulating plants such as Mimosa flocculosa , Mimosa candollei , Mimosa caesalpiniifolia , Mimosa diplotricha , Mimosa pudica , and Piptadenia gonoacantha (Paulitsch et al. 2020; Martinez-Romero et al. 2024). Furthermore, the nifH gene of strains WGlv3 T and WGtm5 T was 100% identical to that of Cupriavidus taiwanensis strains and “Cupriavidus neocaledonicus” STM 6160 isolated from Mimosa pudica from different parts of world including French Guiana, New Caledonia, China, and Taiwan (Amadou et al. 2008; Mishra et al. 2012; Clerissi et al. 2018; Liu et al. 2020). Notably, similar results were found when we used nodC gene sequences for the Blastn search (see Table S6), indicating that Paraburkholderia caribensis strain SKND8 belong to sv. tropicalis . Similarly, strains belonging to the new species of Cupriavidus share the undescribed symbiovar found in case of Cupriavidus taiwanensis strains and “Cupriavidus neocaledonicus” STM 6160 isolated from Mimosa pudica . Symbiotic genes-based phylogeny resulted in the unique clustering of M pudica rhizobia. Contrary to the 16S rRNA gene and core gene phylogenies, the nifH and nodC gene phylogenetic tree placed the strain SKND8 close to Paraburkholderia diazotrophica JUA2-8 T and Paraburkholderia sp . LEh15 T , respectively. The nodC gene sequences of SKND8 strain were identical to the strains of Paraburkholderia isolated from the hosts like Brazil originating M . pudica (Dall’Agnol et al. 2017) , M. claussenii (de Pires et al. 2019), M . caesalpiniifoli (Martins et al. 2015; de Oliveira et al. 2019) and other mimosoid legumes (Bournaud et al. 2013; Silva et al. 2018) . While symbiotic genes phylogeny placed the strains WGtm5 T and WGlv3 T close to the members of Cupriavidus taiwanensis species complex, consisting of Cupriavidus taiwanensis SWF 66322, “Cupriavidus neocaledonicus” STM 6160, Cupriavidus taiwanensis STM 6041and Cupriavidus taiwanensis SWF66294 strains (Fig. S2 and S3) Determination of host range and Symbiotic efficiency of isolates Inoculation of surface sterilized seeds of Vigna radiata, Pisum sativum, Vigna aconitifolia, Cicer arietinum, Vigna unguiculata, Arachis hypogaea, Trigonella foenum-graecum, Phaseolus vulgaris, Cyamopsis tetragonoloba and Lens culinaris seeds with four test strains significantly influenced the shoot and root length, nodule number and plant dry weight compared to the control. The uninoculated controls did not show nodulation in none of the species, which confirmed the aseptic conditions of the experiment. The shoot length varied from 5.3 to 63.6 per plant, with strain WGtm5 T exhibited the highest efficient shoot length for Vigna unguiculata test plant in comparison to uninoculated control, which was 57.6 cm. The difference between the test plant shoot length and control plant shoot length was 28.8 cm indicated 100% efficiency in term of shoot height enhancer of WGtm5 strain on cowpea plant. While WGlv3 T strain found to be least potent shoot height inducer (4%) as recorded efficient shoot length was 13.86 cm for groundnut test plant (Fig. 4). The plant dry weight of all test plants was measured on a digital weighing balance and compared with the uninoculated control of each species. The mean values of test plant dry weight ranged from 13.76±0.30 to 30.76±0.85 (mean ± SD) mg per plant . Vigna unguiculata species inoculated with strain WGtm5 T recorded the highest efficient increased dry weight which was increased by 8.7 mg when compared with control (Fig. 5). In contrast, Mat bean inoculated with the strain SKND8 recorded the lowest plant dry weight which was decreased by 0.04 mg; however, no significance difference was found in this treatment. Except few treatments such as SKND8 with Vigna aconitifolia and WGlv3 T with Arachis hypogaea , all the representative isolates found to be effective on all the test plants. In this study, there was a significant variation in the number of nodules that formed on the roots of the test plants, including the host plants (Fig. 6). Three out of four strains effectively nodulated M. pudica , while strain WGmk3 failed to nodulate. Additionally, strains SKND8, WGtm5 T and WGlv3 T were able to form nodules in the roots of Vigna radiata , Vigna unguiculata , and Pisum sativum . The mean nodule number per plant varied from 2.33±0.57 for treatment WGtm5 T with Vigna radiata to 9.33±0.57 for treatment SKND8 with Pisum sativum. The strain SKND8 induced 4.66±0.57,8.33±0.57 and 9.33±0.57 nodules in Vigna radiata , Vigna unguiculata and P. sativum , respectively. Both the strains WGtm5 T and WGlv3 T were able to nodulate Vigna radiata and Vigna unguiculata . However, these nodules might likely not be effective or may have some other bacteria, as in this study we did not perform the re-isolation of root-nodulating bacteria from these nodules. Noteworthy, Cupriavidus sp. isolated from Mimosa pudica exhibited nodulation in V. unguiculata (Silva et al. 2012). Furthermore, beta-rhizobia including Paraburkholderia have been reported to nodulate V. unguiculata (Castro et al. 2017; Ramrez et al. 2020; Muindi et al. 2021). Morphology, Physiology, and chemotaxonomy of new taxa The colonies of strains WGtm5 T and WGlv3 T were circular, translucent, gummy, cream-white, smooth and round with entire margin on YEMA-Cr media. Excessive amount of extracellular polysaccharides were observed around the colonies of both strains when grown on YMA plates. Microscopic study inferenced that the cells of strains WGtm5 T and WGlv3 T were short rod-shaped, motile and Gram-stain negative. Both strains can grow at temperatures ranging from 20 ℃ to 40 ℃ but not at 10 or 50 ℃. The optimum temperature for their growth was 30 ℃. In terms of pH, strains WGtm5 T and WGlv3 T displayed growth across a range of 5–8, with the best growth observed at pH 7.0. Both the strains were able to tolerate 1% of NaCl (w/v), but fail to grow in 2% NaCl (w/v). In enzyme detection, strains WGtm5 T and WGlv3 T tested positive for nitrate reductase enzyme and catalase enzyme test. Furthermore, a comparison of the physiological and biochemical traits of the newly discovered strains in this investigation with the closely related members C. taiwanensis species complex was carried out (Table 1). The growth ranges for temperature and pH in the cases of Cupriavidus taiwanensis LMG 19424 T , Cupriavidus necator LMG 8453 T and Cupriavidus nantongensis X1 T were consistent with the ranges reported earlier (Makkar et al. 1987; Estrada-de los Santos et al. 2012; Sun et al. 2016). It is worth noting that, both strains WGtm5 T and WGlv3 T could grow at 1% (w/v) NaCl, while fail to grow at 2% (w/v) NaCl, which is contrary to the type strains of other species of Cupriavidus (Sun et al. 2016). A total of 17 different cellular fatty acids, in different quantities, were detected in strains WGtm5 T and WGlv3 T (Table 2). It was noted that the fatty acid compositions of the strains WGtm5 T and WGlv3 T were identical to the type strains of Cupriavidus taiwanensis , Cupriavidus necator , Cupriavidus nantongensis and Cupriavidus alkaliphilus with differences in their proportions (Makkar et al. 1987; Chen, et al. 2001; Estrada-de los Santos et al. 2012; Sun et al. 2016). The major fatty acids detected were C 14:0 , C 14:0 2OH, C 15:1 ꙍ6c, C 16:0 , C 16:1 ꙍ5c, C 16:0 2OH, C 16:1 2OH, C 17:0 , C 17:0 cyclo, C 18:0 and C 18:1 2-OH for strains WGtm5 T and WGlv3 T . Noticeably, C 19:0 fatty acid was present in strain WGlv3 T in comparison to WGtm5 T and other reference type strains. Conclusions The microsymbionts of Mimosa pudica in India exhibit a distinct geographic distribution, with Paraburkholderia caribensis being prevalent in the Eastern Himalayan region and two new species of Cupriavidus identified in the Western Ghats. Moreover, Paraburkholderia caribensis strain SKND8 was placed within the sv. tropicalis on the basis of nifH and nodC genes sequence analyses. Additionally, a non-rhizobial strain belonging to Herbaspirillum huttiense was isolated from the root nodules of Mimosa pudica in the Western Ghat. Based on the genomic features such as phylogenetic grouping, ANI and dDDH, and phenotypic characteristics, two Western Ghat strain represent two new species within the genus Cupriavidus . We propose the names Cupriavidus mimosa sp. nov. for the type strain WGtm5 T and Cupriavidus gehlotii sp. nov for the type strain WGlv3 T . Our analyses indicate that these new species are part of the Cupriavidus taiwanensis species complex, which includes 12 genospecies. Of these, four are already named, while the others await taxonomic descriptions. Notably, seven genomes, including that of the strain WGtm5 T , were placed with in the newly proposed Cupriavidus mimosa , while Cupriavidus gehlotii is currently represented only by the strain WGlv3 T . The cross-nodulation tests demonstrated that the test strains could elicit nodules on the roots of Vigna radiata , Vigna unguiculata , and Pisum sativum . Description of Cupriavidus mimosae sp. nov. Cupriavidus mimosa (mi.mo'sae. N.L. gen. n. mimosae . of the plant genus Mimosa, from which the strain was isolated). Cells are Gram-negative, aerobic, short rod-shaped and motile. Colonies are round, smooth, raised, translucent, gummy, small and cream-white and grow 0.1 to 0.2 cm in diameter after 48 hours of growth on YMA medium at 30℃. Optimum growth was observed at a pH of 7.0, a temperature of 30℃ and a NaCl concentration of 0.25% (w/v). Strains do not grow at temperatures 10℃ and below 10℃ or at 50℃ and above 50℃, at pH values of below 5.0 and above 8.0, or at NaCl concentrations above 1.0% (w/v). The major fatty acids detected were C 14:0 , C 14:0 2OH, C 15:1 ꙍ6c, C 16:1 ꙍ5c, C 16:0 2OH, C 16:1 2OH, C 17:0 , C 17:0 cyclo, C 18:0 , C 18:1 2-OH, with a high amount of C 16:0 fatty acid Positive for catalase, oxidase and nitrate reductase. Assimilates D-fructose, dextrose, sodium acetate and sodium citrate, but does not assimilate the sucrose, lactose, D-mannitol, sodium succinate, sodium propionate and sodium pyruvate. The genome sequence has a DNA G + C content of 67.0%, spanning 6,703,278 bp. The NCBI accession numbers for the 16S rRNA (OM281132), atpD (OM574816), recA (OM574813), gyrB (OM574814), dnaK (OM574815), nodC (OM574818) and nifH (OM574817) genes, and genome sequence GCA_023952485.1 (JALHRY000000000) of strain WGtm5 T . The type strain is WGtm5 T (MCC4888 T = KACC 22828 T ). Description of Cupriavidus gehlotii sp. nov. Cupriavidus gehlotii (geh.lo'ti.i. N.L. gen. n. gehlotii, of Gehlot, in the name of Professor Hukum Singh Gehlot (Dept. of Botany, JNV University, Jodhpur, India), to honour his contribution to the research of indigenous nitrogen-fixing bacteria from different region of India). Cells are Gram-negative, aerobic, short rod-shaped and motile. Colonies are round, smooth, raised, translucent, gummy, small and cream-white and grow 0.1 to 0.2 cm in diameter after 48 hours of growth on YMA medium at 30℃. Optimum growth was observed at a pH of 7.0, a temperature of 30℃ and a NaCl concentration of 0.25% (w/v). Strains do not grow at temperatures 10℃ and below 10℃ or at 50℃ and above 50℃, at pH values of 4.0 or above 8.0, or at NaCl concentrations above 1.0% (w/v). The major fatty acids detected were C 14 : 0 , C 14 : 0 2OH, C 16 : 1 ꙍ5c, C 17 : 0 , C 17 : 0 cyclo, C 18 : 0 , C 18 : 1 2-OH, with a high amount of C 16 : 0 fatty acid. Positive for catalase, oxidase and nitrate reductase. Assimilates D-fructose, dextrose, sodium acetate, D-mannitol (weakly) and sodium citrate, but does not assimilate the sucrose, lactose, sodium succinate, sodium propionate and sodium pyruvate. The genome sequence has a DNA G + C content of 67.10%, spanning 6,755,320 bp. The NCBI accession numbers for the 16S rRNA (OM281131), atpD (OM574810), recA (OM574807), gyrB (OM574808), dnaK (OM574809), nodC (OM574812) and nifH (OM574811) genes, and genome sequence GCA_023952475.1 (JALHRX000000000) of strain WGlv3 T . The type strain is WGlv3 T (MCC4890 T = KACC 22827 T ). Declarations Acknowledgements: We are grateful to Prof. Arnab Sen, Department of Botany, University of North Bengal, Siliguri, for providing access to his laboratory for the isolation of Mimosa pudica root-nodulating bacteria from the Eastern Himalayan region. We thank the National Centre for Microbial Resource, Pune for various research facilities. This work used the computational and storage services (TARS cluster) provided by the IT department at Institut Pasteur, Paris. The authors would like to thank Prof. H. S. Gehlot of JNV University, Jodhpur, India, for granting us permission to propose a new species named in his honour. 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Paulitsch, F., Delamuta, J.R.M., Ribeiro, R.A., da Batista, J.S.S., Hungria, M. (2020) Phylogeny of symbiotic genes reveals symbiovars within legume-nodulating Paraburkholderia species. Syst. Appl. Microbiol. 43, (6). https://doi.org/ 10.1016/j.syapm.2020.126151 126151. Paulitsch, F., dos Reis Jr, F. B., & Hungria, M. (2021). Twenty years of paradigm-breaking studies of taxonomy and symbiotic nitrogen fixation by beta-rhizobia, and indication of Brazil as a hotspot of Paraburkholderia diversity. Archives of Microbiology , 203 (8), 4785-4803. Poehlein, A., Kusian, B., Friedrich, B., Daniel, R., & Bowien, B. (2011). Complete genome sequence of the type strain Cupriavidus necator N-1. Rahi, P., Giram, P., Chaudhari, D., Kiran, S., Khullar, A., Chandel, M., ... & Mahajan, B. (2020). Rhizobium indicum sp. nov., isolated from root nodules of pea (Pisum sativum) cultivated in the Indian trans-Himalayas. Systematic and Applied Microbiology, 43(5), 126127. Rahi, P., Prakash, O., & Shouche, Y. S. (2016). Matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF MS) based microbial identifications: challenges and scopes for microbial ecologists. Frontiers in Microbiology, 7, 1359. Ramírez, M. D. A., España, M., Lewandowska, S., Yuan, K., Okazaki, S., Ohkama-Ohtsu, N., & Yokoyama, T. (2020). Phylogenetic analysis of symbiotic bacteria associated with two Vigna species under different agro-ecological conditions in Venezuela. Microbes and Environments , 35 (1), ME19120. Reiner, K. (2012). Carbohydrate fermentation protocol. energy , 11 , 12. Rosconi, F., Davyt, D., Martínez, V., Martínez, M., Abin‐Carriquiry, J. A., Zane, H., ... & Fabiano, E. (2013). Identification and structural characterization of serobactins, a suite of lipopeptide siderophores produced by the grass endophyte Herbaspirillum seropedicae . Environmental Microbiology , 15 (3), 916-927. Sasser, M. (2001). Tracking” a strain using the sherlock microbial identification system (MIS). 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A manual for the practical study of the root-nodule bacteria. Weisburg, W. G., Barns, S. M., Pelletier, D. A., & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology , 173 (2), 697-703 Xia, M., Zhang, S., Shen, L., Yu, R., Liu, Y., Li, J., ... & Zeng, W. (2021). Optimization and characterization of an antioxidant exopolysaccharide produced by Cupriavidus pauculus 1490. Journal of Polymers and the Environment , 1-10. Yan, X., Wang, Z., Mei, Y., Wang, L., Wang, X., Xu, Q., ... & Wei, C. (2018). Isolation, diversity, and growth-promoting activities of endophytic bacteria from tea cultivars of Zijuan and Yunkang-10. Frontiers in Microbiology , 9 , 1848. Yoon, S. H., Ha, S. M., Kwon, S., Lim, J., Kim, Y., Seo, H., & Chun, J. (2017). Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. International Journal of Systematic and Evolutionary Microbiology , 67 (5), 1613. Young, J. P. W., Moeskjær, S., Afonin, A., Rahi, P., Maluk, M., James, E. K., ... & Tian, C. F. (2021). Defining the Rhizobium leguminosarum species complex. Genes , 12 (1), 111. Tables Table 1: Differentiating characteristics of strain 1, WGtm5 T ; 2, WGlv3 T ; 3, Cupriavidus taiwanensis LMG 19424 T ; 4, Cupriavidus necator LMG 8453 T ; 5, Cupriavidus nantongensis X1 T ; 6, Cupriavidus alkaliphilus LMG 26294 T in comparison to its closest phylogenetic neighbours. *+, positive; -, negative; W, weakly positive; ND, Not defined Characteristics 1 2 3 4 5 6 Source of isolation Root nodules of Mimosa pudica Root nodules of Mimosa pudica Root nodules of Mimosa pudica Root nodules of Mimosa pudica Sludge sample Rhizosphere in alkaline soils pH range for growth (optimum) 5-8 (7) 5-8 (7) 6-9 (7) 6-9 (7) 6-9 (7) 6-10 (7.5) Temperature range for growth (◦C) (optimum) 20-40 (30) 20-40 (30) 15-42 (30) 15-40 (30) 20-42 (30) 15-42 (30) 1% NaCl (w/v) + + + + + + 2% NaCl (w/v) _ _ + + w + Motility + + + _ + + Catalase + + + + + + Nitrate Reductase + + + + + + d-fructose + + _ + _ + d-glucose _ _ _ _ _ _ sucrose _ _ _ _ _ _ dextrose _ _ _ + _ _ lactose _ _ _ _ _ _ d-mannitol _ + _ + _ _ citrate + + w + w _ *Data for strain 3 from Chen, et al. (2001); 4 from Makkar et al. (1987); 5 from Sun et al. (2016) and for 6 from Estrada-de los Santos et al. (2012). Table 2: Cellular fatty acid compositions (%) of strains WGtm5 T and WGlv3 T and related species of the genus Cupriavidus Strains: 1, WGtm5 T ; 2, WGlv3 T ; 3, Cupriavidus taiwanensis LMG 19424 T ; 4, Cupriavidus necator LMG 8453 T ; 5, Cupriavidus nantongensis X1 T ; 6, Cupriavidus alkaliphilus LMG 26294 T . Fatty acids 1 2 3 4 5 6 C 14 : 0 3.21 3.29 3.1 2.2 3.8 3.1 C 14 : 0 2OH 3.04 2.81 1.3 1.7 1.6 1.5 C 15 : 1 ꙍ6c 0.20 - - - - - C 16 : 0 23.43 26.28 28.3 27.4 28.9 31.6 C 16 : 1 ꙍ5c 0.23 0.24 - - - - C 16 : 0 2OH 0.20 - - - - - C 16 : 1 2OH 0.44 - - - - - C 17 : 0 0.34 0.39 - - - - C 17 : 0 cyclo 1.20 1.56 6.2 4.7 11.0 12.0 C 18 : 0 0.69 0.83 0.5 - 0.5 - C 18 : 1 2-OH 1.01 0.66 - - - - C 19:0 - 0.26 - - - - C 19 : 0 cyclo ꙍ8c - - - - 1.8 - Summed features* 2 9.91 9.36 7.4 7.3 5.2 6.8 3 33.39 33.14 32.1 33.6 27.9 27.9 5 0.15 0.39 - - - - 8 22.51 20.74 20.5 21.8 18.4 15.4 *Summed features are combinations of fatty acids that cannot be separated by the MIDI system. Summed feature 2 = C 12 : 0 aldehyde and/ or unknown ECL 10.9525; summed feature 3 = C 16:1 ꙍ7c and/or C 16:1 ꙍ6c; summed feature 5= C 18:0 ante and/or C 18:2 ꙍ6,9c; summed feature 8 = C 18 : 1 ꙍ7c and/or C 18 : 1 ꙍ6c. a Data for strain 3 from Chen, et al. (2001); 4 from Makkar et al (1987); 5 from Sun et al. (2016) and 6 from Estrada-de los Santos et al. (2012). Additional Declarations No competing interests reported. Supplementary Files Supdatav0.3.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5391658","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":379010730,"identity":"e84c8605-3c0e-410e-b83d-4a2b0f9c874c","order_by":0,"name":"Lekha Sharma","email":"","orcid":"","institution":"C U Shah University","correspondingAuthor":false,"prefix":"","firstName":"Lekha","middleName":"","lastName":"Sharma","suffix":""},{"id":379010731,"identity":"0b68aeea-e731-437e-9edd-1bfa9bdf20db","order_by":1,"name":"Mitesh Khairnar","email":"","orcid":"","institution":"National Centre for Cell Science","correspondingAuthor":false,"prefix":"","firstName":"Mitesh","middleName":"","lastName":"Khairnar","suffix":""},{"id":379010733,"identity":"430d3ba2-4694-487e-9f70-2b60f49ff6cc","order_by":2,"name":"Aabeejjeet Pansare","email":"","orcid":"","institution":"National Centre for Cell Science","correspondingAuthor":false,"prefix":"","firstName":"Aabeejjeet","middleName":"","lastName":"Pansare","suffix":""},{"id":379010734,"identity":"88aacc51-063d-4178-8eaf-14a0150f3720","order_by":3,"name":"Krishna Mohan Medicherla","email":"","orcid":"","institution":"Birla Institute of Scientific Research","correspondingAuthor":false,"prefix":"","firstName":"Krishna","middleName":"Mohan","lastName":"Medicherla","suffix":""},{"id":379010735,"identity":"1238044d-9d2a-4ebf-8d33-136e8d83d599","order_by":4,"name":"Girish K Goswami","email":"","orcid":"","institution":"Regional Science Centre Bhavnagar, GUJCOST, DST, Govt of Gujarat","correspondingAuthor":false,"prefix":"","firstName":"Girish","middleName":"K","lastName":"Goswami","suffix":""},{"id":379010736,"identity":"ef1d3b22-db27-4664-89ff-73bad051e72c","order_by":5,"name":"Praveen Rahi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYHACNoaECjhHjlgtZ8AUCBgTqYWxjRQt5tKHnz14OO+OvPz85mPSFQwG+QS1WPalmRskbntmuOEYW5rkGQYDywZCWgzOMJhJJG47zLiBjcdMsoHhjwFBWwzOsH+TSJxz2H5+G/83oBYDYrTwAG1pOJzYcIyHjTgtlj08ZRIJx54lbziWZmzZYECEFnMe9m2SP2ru2M5vPvzwZkMFMQ6DUAdQuaRoGQWjYBSMglGABQAAwWI3wiVjDagAAAAASUVORK5CYII=","orcid":"","institution":"Institut Pasteur, Université Paris Cité","correspondingAuthor":true,"prefix":"","firstName":"Praveen","middleName":"","lastName":"Rahi","suffix":""}],"badges":[],"createdAt":"2024-11-05 03:08:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5391658/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5391658/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69336125,"identity":"1fb3c251-c440-459b-94bb-03c0c1a23c9b","added_by":"auto","created_at":"2024-11-19 10:01:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":60764,"visible":true,"origin":"","legend":"\u003cp\u003eA neighbour-joining (NJ) tree inferred from 16S rRNA sequence analysis of a \u003cem\u003eMimosa pudica\u003c/em\u003e root-nodulating bacteria and their related taxa. The tree topologies were determined by bootstrap values 1000 replicationsand the scale bar represents the number of nucleotide substitutions.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5391658/v1/4d98aba549680d53ff589ba3.png"},{"id":69336122,"identity":"13d3c773-1491-4d14-90f1-91fa926046da","added_by":"auto","created_at":"2024-11-19 10:01:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":94513,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree inferred using the likelihood method in IQ-TREE pipeline using the concatenated alignment of \u003cem\u003ebac120\u003c/em\u003egenes of the newly isolated bacterial strains from root-nodules of \u003cem\u003eMimosa pudica\u003c/em\u003e and their closely related taxa. Values shown next to the branches are the percentage of replicate trees with associated taxa clustered together in the bootstrap test (1000 replicates).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5391658/v1/c5d9d63b347263fba28d18bb.png"},{"id":69336128,"identity":"10b1c9db-d61f-4e39-8264-efbe139a60ac","added_by":"auto","created_at":"2024-11-19 10:01:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":171586,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree constructed based on \u003cem\u003ebac120\u003c/em\u003e genes using IQ-TREE for \u003cem\u003eCupriavidus \u003c/em\u003especies strains isolated from the root-nodules of \u003cem\u003eMimosa pudica \u003c/em\u003eand members of \u003cem\u003eCupriavidus taiwanensis \u003c/em\u003especies complex. Dots at branching points indicate the percentage of bootstrap values.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5391658/v1/d305f1f5af506d1425102bdf.png"},{"id":69336123,"identity":"53add799-5b0a-41e7-9f73-8e0ad0dc88a5","added_by":"auto","created_at":"2024-11-19 10:01:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":65632,"visible":true,"origin":"","legend":"\u003cp\u003eBox plot depicting the effect of \u003cem\u003eMimosa pudica\u003c/em\u003eroot-nodulation bacteria inoculation on the plant growth derived as a measure of plant length.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5391658/v1/68b948af7311557abc5ca1cd.png"},{"id":69336124,"identity":"f4eb6595-11b2-4d8f-aefb-384e42e0fb79","added_by":"auto","created_at":"2024-11-19 10:01:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":67185,"visible":true,"origin":"","legend":"\u003cp\u003eBox plot depicting the effect of \u003cem\u003eMimosa pudica\u003c/em\u003e root-nodulation bacteria inoculation on the plant growth derived as a measure of plant dry weight.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5391658/v1/9ba7f2a61456de43fee9eb8b.png"},{"id":69336972,"identity":"57f09862-f0c6-413f-b306-c19a2ef65f3e","added_by":"auto","created_at":"2024-11-19 10:09:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":54623,"visible":true,"origin":"","legend":"\u003cp\u003eBox plot depicting the effect of \u003cem\u003eMimosa pudica\u003c/em\u003e root-nodulation bacteria inoculation on the number of root nodules.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5391658/v1/e7327c2288cd519b51fd5c6c.png"},{"id":80589138,"identity":"62f4384a-912c-4c18-b8f8-02697d9e16f0","added_by":"auto","created_at":"2025-04-15 02:01:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2166357,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5391658/v1/76f6543c-0711-4be5-906f-80959d992a6c.pdf"},{"id":69336127,"identity":"ed79f377-c3d7-4939-97a5-d9270b179173","added_by":"auto","created_at":"2024-11-19 10:01:49","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":831810,"visible":true,"origin":"","legend":"","description":"","filename":"Supdatav0.3.docx","url":"https://assets-eu.researchsquare.com/files/rs-5391658/v1/c83c02792523afd1f882f848.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome sequence-based identification of bacteria nodulating Mimosa pudica growing in Eastern Himalayas and Western Ghats of India and description of Cupriavidus mimosae sp. nov. and Cupriavidus gehlotii sp. nov","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNitrogen is an essential element for all living beings, while a small group of organisms known as diazotrophs can fix atmospheric nitrogen. Among the group diazotrophs, rhizobia represent the bacteria which fix nitrogen by entering into symbiosis with host plants mostly legumes by forming specialized structures called nodules.\u0026nbsp;\u003cem\u003eMimosa pudica\u003c/em\u003e is a member of the tribe \u003cem\u003eMimosaseae\u003c/em\u003e in the mimosoid subfamily of legumes (\u003cem\u003eLeguminosae/Fabaceae\u003c/em\u003e). \u0026nbsp;The legume originates in South America and spread throughout the tropical and semi-tropical regions as a weed (Barneby 1991).\u0026nbsp;This perennial shrub is\u0026nbsp;widely distributed to different regions of India where it mostly grows on non-perturbed environmental conditions. Particularly, in the tropical and semi-tropical rain forests of the Eastern Himalayan (EH) and Western Ghats (WG), which are\u0026nbsp;known for their rich in diversity of flora and fauna.\u003c/p\u003e\n\u003cp\u003eThe genetic diversity and host-microbe interactions of \u003cem\u003eMimosa pudica\u003c/em\u003e rhizobia have been extensively studied in different parts of the world, largely due to their ecological contribution to biological nitrogen fixation. \u003cem\u003eMimosa pudica\u003c/em\u003e, like other \u003cem\u003eMimosa\u003c/em\u003e species enter into symbiosis preferentially with \u003cem\u003eCupriavidus\u003c/em\u003e, \u003cem\u003eParaburkholderia\u003c/em\u003e and \u003cem\u003eTrinickia\u003c/em\u003e belonging to beta-subclass of phylum \u003cem\u003eProteobacteria\u003c/em\u003e (now \u003cem\u003ePseudomonadota\u003c/em\u003e)\u0026nbsp;(Klonowska et al. 2012; Gehlot et al. 2013; Dall\u0026rsquo;Agnol et al. 2017; los Santos et al. 2018; Silva et al. 2018; Liu et al. 2020; Paulitsch et al. 2020; Dias et al. 2021; Mavima et al. 2021).\u0026nbsp;However, members of \u003cem\u003eRhizobium\u003c/em\u003e and \u003cem\u003eEnsifer\u003c/em\u003e belonging to alpha-proteobacteria have also been reported to nodulate \u003cem\u003eMimosa\u003c/em\u003e species (Klonowska et al. 2012; Gehlot et al. 2013; Bara\u0026uacute;na et al. 2016). On one hand, \u003cem\u003eMimosa\u003c/em\u003e species endemic to a region or country have been reported to form symbiosis often with alpha-rhizobia (Wang et al. 1999; Gehlot et al. 2013; Bontemps et al. 2016). On the other, the invasive species of \u003cem\u003eMimosa\u003c/em\u003e, including \u003cem\u003eM. pudica\u003c/em\u003e enter into symbiosis with beta-rhizobia including \u003cem\u003eParaburkholderia phymatum\u003c/em\u003e, \u003cem\u003eParaburkholderia mimosarum\u003c/em\u003e and \u003cem\u003eCupriavidus taiwanensis\u003c/em\u003e (Klonowska et al. 2012; Gehlot et al. 2013; Dall\u0026rsquo;Agnol et al. 2017; los Santos et al. 2018; Silva et al. 2018; Liu et al. 2020; Paulitsch et al. 2020; Dias et al. 2021; Mavima et al. 2021).\u003c/p\u003e\n\u003cp\u003eSeveral species of the genus \u003cem\u003eParaburkholderia\u003c/em\u003e have been reported to nodulate \u003cem\u003eMimosa\u003c/em\u003e species, such as \u003cem\u003eP. nodosa\u003c/em\u003e (Dall\u0026rsquo;Agnol et al. 2017), \u003cem\u003eP. symbiotica\u003c/em\u003e (Sheu et al. 2012), \u003cem\u003eP. piptadeniae\u003c/em\u003e and \u003cem\u003eP. ribeironis\u003c/em\u003e (Bournaud et al. \u0026nbsp;2017), \u003cem\u003eP. youngii\u003c/em\u003e and \u003cem\u003eP. atlantica\u003c/em\u003e (Mavima et al. 2021), \u003cem\u003eP. tuberum\u003c/em\u003e (Mishra et al. 2012), \u003cem\u003eP. phymatum\u003c/em\u003e, \u003cem\u003eP. mimosarum\u003c/em\u003e, and \u003cem\u003eP. diazotrophica\u003c/em\u003e (Sheu et al. 2013). Similarly, multiple species of \u003cem\u003eCupriavidus\u0026nbsp;\u003c/em\u003eincluding, \u003cem\u003eC. taiwanensis\u003c/em\u003e (Chen et al. 2008), \u003cem\u003eC. necator\u003c/em\u003e (Poehlein et al. 2011), \u003cem\u003e\u0026ldquo;C. neocaledonicus\u0026rdquo;\u003c/em\u003e (Klonowska et al. 2020) have been reported to nodulate \u003cem\u003eMimosa\u003c/em\u003e. \u003cem\u003eCupriavidus taiwanensis\u003c/em\u003e was the main symbiotic partner of \u003cem\u003eM. pudica\u003c/em\u003e in four different sites in New Caledonia (Klonowska et al. 2012). Furthermore, five different genotypes of \u003cem\u003eCupriavidus\u003c/em\u003e were identified during this study and a new species \u003cem\u003eC. neocaledonicus\u003c/em\u003e was proposed (Klonowska et al. 2020). A core-gene-based phylogeny of \u003cem\u003eMimosa\u003c/em\u003e symbionts indicated that \u003cem\u003eC. taiwanensis\u003c/em\u003e is a complex of several closely related species (Clerissi et al. 2018). India, with its diverse ecosystems and numerous biodiversity hotspots, offers a rich environment for the exploration of \u003cem\u003eMimosa\u003c/em\u003e rhizobial diversity. Certain regions, such as the EH and WG, may be particularly important for understanding the diversity and distribution of these symbiotic relationships. In the present study, we isolated the \u003cem\u003eMimosa pudica\u003c/em\u003e symbionts from EH and WG, and used tools like MALDI-TOF MS and genome sequencing for their precise identification. We also proposed the description of two new species and resolved the \u003cem\u003eC. taiwanensis\u003c/em\u003e species complex based on taxogenomic analyses.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIsolation of rhizobia from root nodules of Mimosa pudica\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMimosa pudica\u003c/em\u003e roots with nodules were collected from ten locations in the Eastern Himalayan (EH) and Western Ghat (WG) regions (Fig S1; Table S1). The root nodules were placed in phosphate buffer with 20% glycerol (v/v) and transported at 4°C in a vaccine box (Naik and Rahi 2022). The vials containing the collected nodules were refrigerated at 4°C until further use. To isolate bacteria from the root nodules, the nodules were surface sterilized using 25% sodium hypochlorite solution and 70% ethanol (Naik and Rahi 2022). The surface sterilization of the nodule was confirmed by rolling the sterilized nodules on yeast extract mannitol agar (YEMA) plate before crushing. Isolation of bacteria was done by crushing the surface sterilized nodules and streaking on YEMA media containing 0.025% congo red (M721, Himedia, India) at 30 ℃ for 24-48 hrs (Vincent 1970). Plates were observed for the growth of glistening-white bacterial colonies and confirm the purity of bacterial colonies by multiple streaking.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMALDI-TOF-MS approach for Identification of isolates\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll strains were screened for MALDI-TOF-MS based identification as described earlier (Rahi et al. 2016). In brief, the strains were grown in YEMA plates for 36 h, and a smear of freshly grown culture was applied onto the MALDI target plate followed by the addition of one µl of alpha-cyano-4- hydroxycinnamic acid (HCCA) matrix solution. Mass spectra were generated in the mass range 2 to 20 KDa in a linear positive mode in the MALDI-TOF MS instrument (autoflex, Bruker Daltonics, Germany) (Kurli et al. 2018). The acquired protein spectral profiles were compared with the MALDI Biotyper database (Bruker Daltonik). The criteria of microbial identification was based on the biotyper score values; strain with value ≥2.0 confirmed species level identity and if it within the range 1.7 to 1.99 the strain was identified only at genus-level.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePhylogenetic analysis of 16S rRNA genes\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genomic DNA was extracted using standard CTAB method (Minas et al. 2011) described in JGI catalogue version 3. Amplification of 16S rRNA genes was performed using the universal primer pair 27F-1492R (Weisburg 1991) and the amplicons were sequenced using ABI PRISM Big Dye Terminator v3.1 Cycle Sequencing kit on a 3730xl Genetic Analyzer (Applied BioSystems, Thermo Scientific, USA). Obtained sequences were searched as query sequence to find the similar sequences in the EzBioCloud database of 16S rRNA genes of the type strains of prokaryotic species (Yoon et al. 2017). All the query and closely related sequences were aligned using CLUSTAL W program (Larkin et al. 2007). The neighbour joining (NJ) phylogenetic trees were inferred for the aligned nucleotide sequences. MEGA 11 was used to determine best-fit models of nucleotide substitution and for performing NJ analysis (Tamura et al. 2021). Using 1000 bootstrap replications, the Tamura 3-parameter model was applied to the 16S rRNA dataset to construct a phylogenetic tree.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGenome-based analyses\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genome sequencing of strains WGtm5\u003csup\u003eT\u003c/sup\u003e, WGlv3\u003csup\u003eT\u003c/sup\u003e, WGmk3 and SKND8 was performed using Illumina Novaseq platform with a 2×150bp paired-end protocol. Quality of raw data was checked using the FastQC tool (https://www.bioinformatics.babraham.ac.uk/projects/ fastqc). Quality filtered reads were assembled using SPAdes version 3.12.0. (Bankevich et al. 2012). Assembled sequences were annotated using RAST (Rapid Annotation using subsystem technology) (https://rast.nmpdr.org/) pipeline and sequence-based comparisons were performed using the SEED Viewer (Jin et al. 2022). Furthermore, the annotated sequences were analyzed to detect biosynthetic gene clusters (BGCs) responsible for various secondary metabolites using an online genome mining pipeline antiSMASH 5.0 (Blin et al. 2019). The genes responsible for nitrogen fixation and nodulation were detected and sequences of \u003cem\u003enifH\u003c/em\u003e and \u003cem\u003enodC\u003c/em\u003e genes were extracted from the genome sequences. The sequences of \u003cem\u003enifH\u003c/em\u003e and \u003cem\u003enodC\u003c/em\u003e genes were searched as queries to identify the related gene using NCBI BLASTn tool (Altschul et al. 1990). The search was done against the non-redundant nucleotide database with and without a limit to sequences from type material. Phylogenetic analyses was done for \u003cem\u003enifH\u003c/em\u003e and \u003cem\u003enodC\u003c/em\u003e gene sequences as described for 16S rRNA gene phylogeny.\u003c/p\u003e\n\u003cp\u003eTwo genome datasets were made, the first one consisted the genome sequences from the type strains of species closely related to the root nodulating bacteria, belonging to \u003cem\u003eParaburkholderia\u003c/em\u003e, \u003cem\u003eCupriavidus\u003c/em\u003e and \u003cem\u003eHerbaspirillum\u003c/em\u003e were downloaded in FASTA format from the NCBI genome assembly database (https://www.ncbi.nlm.nih.gov/assembly). The second one was made to resolve the \u003cem\u003eCupriavidus taiwanensis\u003c/em\u003e species complex, and consists of 178 genome sequences of \u003cem\u003eCupriavidus\u003c/em\u003e species (https://www.ncbi.nlm.nih.gov/assembly accessed on 20 September 2023). Both genome datasets were used to construct independent phylogenies using bac120 core-genes, by using GTDBTk 2.1.1 pipeline (Chaumeil et al. 2020) and also to calculate ANI values using fastANI 1.33 (Jain et al. 2018). The obtained aligned gene sequences were used to build a maximum likelihood phylogenetic tree using IQ-TREE 2.2.2.2 (Minh et al. 2020), with 1000 ultrafast bootstrap replications and selection of the best method by ModelFinder (Kalyaanamoorthy et al. 2017). All trees were displayed using iTOL (Letunic et al. 2019). Pairwise ANI was calculated with the kmer = 16, fragment length = 3000, minimum shared fraction = 0.2 using fastANI 1.33 (Jain et al. 2018). The ANI values were displayed in the core-gene phylogenies by inserting coloured ribbon strips. Digital DNA-DNA hybridization (DDH) values and confidence intervals were calculated using the recommended settings of the Genome-to-Genome Distance Calculator (GGDC) 2.1 (Meier-Kolthoff et al. 2013).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePhysiology and chemotaxonomy\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMorphological characteristics of all the strains were observed by streaking them on YEMA plates incubated under aerobic conditions at 30 ℃ for 24-48 h. Cell morphology of the strains was determined by microscopic sight of Gram-stained culture and reaction was carried out by following Gram’s protocol (Aneja 2007). The cell motility was examined following the technique described by Arora (2003). The strains were also tested for growth at different temperatures (10°C, 20°C, 30°C, 40℃ and 50°C), NaCl concentrations [0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5% and 4% (w/v) at 0.5% intervals] and pH values ranging (4 - 9 at 1.0 pH unit intervals) was examined in Yeast Mannitol broth at 30 °C in OSI at 180rpm for 7 days.\u003c/p\u003e\n\u003cp\u003eThe nitrate reduction test was performed using α-nephathylamin reagents in broth culture and the catalase activity was determined by observing immediate effervescence in a 3% (v/v) Hydrogen peroxide solution (Cappucino and Sherman 2014). Reduction of carbon sources viz. glucose, fructose, dextrose, mannitol, sucrose, lactose, Sodium Acetate, Sodium Succinate, Sodium Propionate, Sodium Citrate and Sodium Pyruvate was performed using the protocol developed by Reiner (2012). To estimate the whole-cell fatty acids, the strains were incubated on YEMA at 30℃ for 48 hrs. Preparation and extraction of fatty acid methyl esters were performed by following Sasser’ s protocol (2001) using the Microbial Identification System (MIS; MIDI). Cellular fatty acids were identified using the Microbial Identification software package (Sherlock version 6.1; MIDI database, TSBA6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDetermination of host range of representative rhizobial isolates\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour representative strains were examined for their nodulation ability using different hosts including \u003cem\u003eVigna radiata, Pisum sativum, Vigna aconitifolia, Cicer arietinum, Vigna unguiculata, Arachis hypogaea, Trigonella foenum-graecum, Phaseolus vulgaris, Cyamopsis tetragonoloba \u003c/em\u003eand\u003cem\u003e Lens culinaris \u003c/em\u003e(Hui., 2014). The seeds of all legumes were surface sterilized with sodium hypochlorite (4% available chlorine) and allowed to germinate at 28 ℃. The seedlings were then transferred to polypropylene pots containing moist sterile vermiculite (3 seeds per pot, 10 pots of test plants per each strain treatment). Each seedling was received supplemented with 20 ml of the nutrient solution (g/l): K2HPO4 1.0, KH2PO4 0.25, MgSO4 1.0, Ca3(PO4)2 0.2, FeSO4 0.02, H3BO3 0.005, (NH4)2MoO4 0.005, ZnSO4 × 7 H2O 0.005, MnSO4 0.002 (Novikova and Safronova, 1992) at an interval of 10 days. The seedlings were inoculated with 10\u003csup\u003e9\u003c/sup\u003e cells per pot of strains WGlv3\u003csup\u003eT\u003c/sup\u003e, WGtm5\u003csup\u003eT\u003c/sup\u003e, WGmk3 and SKND8 separately. The uninoculated plants were served as negative control. The plant growth experiment was terminated after 45 days of inoculation and data was recorded for nodule number, root and shoot length and dry weight. The experiment was carried out in a completely randomised design in a glasshouse and each treatment was replicated three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical analysis and data availability\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was conducted using one-way analysis of variance (ANOVA) and means were separated by Duncan’s multiple range test at \u003cem\u003ep \u003c/em\u003e≤ 0.05 using SPSS statistics 27. \u003c/p\u003e\n\u003cp\u003eThe assembled genomes and the raw datasets were submitted using the NCBI Sequence Read Archive tool kit by uploading on SRA FTP platform. The GenBank/EMBL/DDBJ accession numbers for the strain SKND8 genome (JAKUCO000000000), Bioproject No (PRJNA802963), raw reads sequences SRA ID (SRR17868289); for the strain WGmk3 genome (JAKYWT000000000), Bioproject No (PRJNA809344), raw reads sequences SRA ID (SRR18106144); for the strain WGlv3\u003csup\u003eT\u003c/sup\u003e genome (JALHRX000000000), Bioproject No (PRJNA809334), raw reads sequences SRA ID (SRR18106231); and for the strain WGtm5\u003csup\u003eT\u003c/sup\u003e genome (JALHRY000000000), Bioproject No (PRJNA809348), raw reads sequences SRA ID (SRR18106221). Sequences of 16S rRNA gene of root-nodulating bacteria have been deposited with NCBI Genbank under accession numbers: OK336461-OK336464, OM281129-OM281133.\u003c/p\u003e"},{"header":"Results and Discussions","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIsolation and MALDI-TOF-MS identification of isolates\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIsolation of bacteria from the healthy \u003cem\u003eMimosa pudica\u003c/em\u003e root nodules collected from EH and WG of regions of India, resulted in 29 bacterial isolates with typical rhizobia-like cultural characteristics on the YEMA-CR medium.\u0026nbsp;Good quality mass spectra ranging from 2000-20000 Da were generated for 72.5% strains, of which\u0026nbsp;20.6% were identified as \u003cem\u003eCupriavidus nector\u003c/em\u003e and 13.7% as \u003cem\u003eParaburkholderia caribensis\u003c/em\u003e with score value ≥ 2.00 indicated species level identification (Table S2).\u0026nbsp;Furthermore, 17.2% strains (three isolates as \u003cem\u003eCupriavidus\u003c/em\u003e and two as \u003cem\u003eParaburkholderia\u003c/em\u003e) were identified at the genus level with score value between 1.700 to 1.999.\u0026nbsp;Three (10.3%) strains could not be identified and the database search resulted non-reliable identity with score value \u0026lt; 1.7. MALDI-TOF MS based identification is highly dependent on the group of bacteria, as some of the bacterial groups are underrepresented in the database, leading to only genus-level identification or no identification (Rahi et al. 2016). Several studies on plant associated bacteria have suggested that, development of in-house database improve the identification results (Ferreira et al. 2011; Khairnar et al. 2022).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePhylogenetic analysis based on the 16S rRNA gene\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 16S rRNA gene sequences of all strains were obtained with a length ranging from 1100- 1,600 bp (Table S3). The search results of 16S rRNA gene sequences were in corroboration with the MALDI-TOF MS based identification. However, 16S rRNA gene sequences lead to the identification of all strains, at least up to the genus-level. All EH strains showed high sequence similarity (≥99%) to \u003cem\u003eParaburkholderia caribensis\u003c/em\u003e strain MWAP64\u003csup\u003eT\u003c/sup\u003e. On the other hand, two WG strains, were closely related to\u0026nbsp;\u003cem\u003eCupriavidus oxalaticus\u0026nbsp;\u003c/em\u003estrain Ox1\u003csup\u003eT\u003c/sup\u003ewith ≥99% similarity, and the strain WGmk3 showed the highest (99.80%) similarity to \u003cem\u003eHerbaspirillum huttiense\u003c/em\u003e strain ATCC 14670\u003csup\u003eT\u003c/sup\u003e.\u0026nbsp;Notably, WG strains WGlv3\u003csup\u003eT\u003c/sup\u003e, WGtm5\u003csup\u003eT\u003c/sup\u003e and WGmk3 showed more than 99% sequence similarity to two or more species.\u003c/p\u003e\n\u003cp\u003eThe phylogenetic tree reconstructed based on 16S rRNA gene sequences placed the root-nodulating bacteria into three clades including, \u003cem\u003eCupriavidus\u003c/em\u003e, \u003cem\u003eHerbaspirillum\u003c/em\u003e and \u003cem\u003eParaburkholderia\u003c/em\u003e (Fig.1).\u0026nbsp;The EH strains were placed in \u003cem\u003eParaburkholderia\u003c/em\u003e clade, with \u003cem\u003eParaburkholderia caribensis\u0026nbsp;\u003c/em\u003eas their nearest neighbour.\u0026nbsp;However, the WG strains were placed in two clades, the strain WGmk3 was nested within the \u003cem\u003eHerbaspirillum\u0026nbsp;\u003c/em\u003eclade, sitting close to the type strains of \u003cem\u003eHerbaspirillum huttiense\u003c/em\u003e and \u003cem\u003eHerbaspirillum\u003c/em\u003e \u003cem\u003eaquaticum\u003c/em\u003e. On the other hand, the strains WGtm5\u003csup\u003eT\u003c/sup\u003e and WGlv3\u003csup\u003eT\u003c/sup\u003e were placed in \u003cem\u003eCupriavidus\u003c/em\u003e clade, with \u003cem\u003eCupriavidus oxalaticus\u003c/em\u003e as their closest neighbour.Although the strains showed close evolutionary relationships with the described species, it was difficult to assign the strains to any of these species. Several studies have highlighted the low species-level resolution of the 16S rRNA gene sequence data, particularly among the members of group rhizobia (Rahi et al. 2020;\u0026nbsp;Young et al. 2021).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGenome features:\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenome sequencing and assembly of strains SKND8, WGmk3, WGtm5\u003csup\u003eT\u003c/sup\u003e and WGlv3\u003csup\u003eT\u003c/sup\u003e yielded genome sizes of 9.2, 5.3, 6.7 and 6.8 Mbp, respectively with the DNA G + C content ranging from 62.4 to 66.9 mol%.\u0026nbsp;The quality features of genome sequences generated in this study (Table S4) are in corroboration to the minimal genome standards proposed for new taxa description (Chun et al. 2018).\u003c/p\u003e\n\u003cp\u003eThe phylogenetic tree constructed based on \u003cem\u003ebac120\u003c/em\u003e core genes, placed the root-nodulating bacteria in three major groups similar to 16S rRNA gene phylogeny (Fig. 2).\u0026nbsp;The placement of strain SKND8 close to \u003cem\u003eParaburkholderia caribensis\u003c/em\u003e in the core gene phylogeny and high ANI (\u0026gt;95-96%) and dDDH (\u0026gt;70%) values confirm that the strain belongs to \u003cem\u003eP. caribensis\u003c/em\u003e. \u003cem\u003eParaburkholderia\u003c/em\u003e is the most dominant genus among the bacterial nodulating \u003cem\u003eMimosa\u003c/em\u003e species across the globe (Dall´Agnol et al. 2017; Dias et al. 2021; Paulitsch et al. 2021).\u0026nbsp;Initially isolated from vertisol microaggregates \u003cem\u003eP. caribensis\u003c/em\u003e, has also been reported to nodulate \u003cem\u003eMimosa pudica\u003c/em\u003e in Southern China (Chen et al. 2008; Liu et al. 2020).\u0026nbsp;However, the distribution of \u003cem\u003eP. caribensis\u003c/em\u003e was far less in comparison to \u003cem\u003eP. mimosarum\u003c/em\u003e and \u003cem\u003eP. phymatum\u003c/em\u003e, which are widely distributed in both the invaded and the original regions of the \u003cem\u003eMimosa\u003c/em\u003e species (Liu et al. 2020).\u0026nbsp;Additionally, several other species of \u003cem\u003eParaburkholderia\u003c/em\u003e including \u003cem\u003eP. bannesnsis\u003c/em\u003e, \u003cem\u003eP. nodosa\u003c/em\u003e, \u003cem\u003eP. phenoliruptrix\u003c/em\u003e, \u003cem\u003eP. diazotrophica\u003c/em\u003e,and \u003cem\u003eP. symbiotica\u003c/em\u003e have been reported nodulating \u003cem\u003eMimosa\u003c/em\u003e spp. mostly from Brazil (Sheu et al. 2012; Paulitsch et al. 2021; Dias et al. 2021). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe strain WGmk3 was placed close to \u003cem\u003eHerbaspirillum huttiense\u0026nbsp;\u003c/em\u003esubsp.\u003cem\u003e\u0026nbsp;putei\u003c/em\u003e and also shared high ANI and dDDH values, confirming affiliation to \u003cem\u003eH. huttiense\u003c/em\u003e (Fig. 2). Strain of \u003cem\u003eHerbaspirillum huttiense\u003c/em\u003e has often been reported as plant endophytes (Yan et al. 2018; Jia et al. 2022), and to colonize plants like rice (Andreozzi et al. 2019) and tea (Jia et al. 2022). Additionally, \u003cem\u003eHerbaspirillum\u003c/em\u003e species have exhibited multiple plant growth-promoting attributes including nitrogen fixation (Gulati et al. 2011; Yan et al. 2018; Andreozzi et al. 2019; Jia et al. 2022). However, the members of \u003cem\u003eHerbaspirillum\u0026nbsp;\u003c/em\u003ewere never isolated from root nodules. In this study, we did not detect any symbiotic genes in the genome of the strain WGmk3, indicating that this strain was an endophytic bacterium.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCupriavidus\u003c/em\u003e clade includes two strains WGtm5\u003csup\u003eT\u003c/sup\u003e and WGlv3\u003csup\u003eT\u003c/sup\u003e placed close to the members of \u003cem\u003eCupriavidus taiwanensis\u003c/em\u003e species complex, consisting of \u003cem\u003eC. alkaliphilus\u003c/em\u003e, \u003cem\u003eC. nantongensis\u003c/em\u003e, \u003cem\u003eC. taiwanensis\u003c/em\u003e and \u003cem\u003e“C. neocaledonicus”\u003c/em\u003e (Fig. 2)\u0026nbsp;(Poehlein et al. 2011; los Santos et al. 2012; Sun et al. 2016; Feng et al. 2019; Klonowska et al. 2020).\u003cem\u003e\u0026nbsp;Cupriavidus\u003c/em\u003e (formerly \u003cem\u003eRalstonia\u003c/em\u003e) \u003cem\u003etaiwanensis\u0026nbsp;\u003c/em\u003ehave been reported to nodulate \u003cem\u003eMimosa pudica\u003c/em\u003e in different parts of the world, including India (Verma et al. 2004;\u0026nbsp;Barrett et al. 2006;\u0026nbsp;Amadou et al. 2008; Klonowska et al. 2012). The ANI values for the newly isolated strains\u0026nbsp;WGtm5\u003csup\u003eT\u003c/sup\u003e and WGlv3\u003csup\u003eT\u003c/sup\u003e were below 96% (species delineation value) to the type strains of all valid species of\u0026nbsp;\u003cem\u003eCupriavidus\u0026nbsp;\u003c/em\u003e(Table S4). Similarly, dDDH values below 70%, indicated that the strains represent new species of \u003cem\u003eCupriavidus\u003c/em\u003e. Detailed genome-based analyses including core-gene phylogeny (Fig. 3), and ANI values exhibited abundant polymorphism among \u003cem\u003eC\u003c/em\u003e. \u003cem\u003etaiwanensis\u003c/em\u003e strains and highlighted that \u003cem\u003eC\u003c/em\u003e. \u003cem\u003etaiwanensis\u003c/em\u003e is a complex of several closely related species (Clerissi et al. 2018). A core-gene phylogeny and pairwise ANI between all \u003cem\u003eCupriavidus\u0026nbsp;\u003c/em\u003egenomes, indicated that the \u003cem\u003eC\u003c/em\u003e. \u003cem\u003etaiwanensis\u0026nbsp;\u003c/em\u003especies complex has 12 genospecies (Fig. 3). Four of these genospecies already have valid species names including, \u003cem\u003eC\u003c/em\u003e. \u003cem\u003etaiwanensis\u003c/em\u003e, \u003cem\u003eC. nantongensis\u003c/em\u003e, \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ealkaliphilus\u003c/em\u003e and \u003cem\u003e“C. neocaledonicus”\u003c/em\u003e. The genospecies A-D are represented by single strains. The type strain of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003etaiwanensis\u003c/em\u003e was placed in a cluster with eight strains, and share less than 96% ANI values with the other members of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003etaiwanensis\u0026nbsp;\u003c/em\u003especies complex and represents genospecies E. The type strain of \u003cem\u003eC. nantongensis\u003c/em\u003e was alone in representing the genospecies G, while two strains LMG 19430 and MLR2-44 representing genospecies H were placed close to it. Three strains including the type strain of \u003cem\u003e“C. neocaledonicus”\u0026nbsp;\u003c/em\u003erepresented the genospecies I. The type strain of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ealkaliphilus\u003c/em\u003e was placed in a cluster with five strains with ANI values more than 98%, representing genospecies L. Additionally, a subcluster of nine strains and an independent strain cmp52 are also within the genospecies L, sharing more than 96% ANI values with the type strain of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003ealkaliphilus\u003c/em\u003e. The strain WGlv3\u003csup\u003eT\u003c/sup\u003e placed independently with less than 96% values, with all the type strains of described species and strains of genospecies within the \u003cem\u003eC\u003c/em\u003e. \u003cem\u003etaiwanensis\u0026nbsp;\u003c/em\u003especies complex, representing genospecies J. Similarly, strain WGtm5\u003csup\u003eT\u003c/sup\u003e was placed in a cluster, which further divide into two subcluster represent the genospecies K. The presence of subclusters and sharing ANI values 96-97% in genospecies K and L, indicate the presence of subspecies. In addition to the circumscription of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003etaiwanensis\u0026nbsp;\u003c/em\u003especies complex into 12 genospecies, the results of core-gene phylogeny and ANI calculation confirmed that the strains WGlv3\u003csup\u003eT\u003c/sup\u003e and WGtm5\u003csup\u003eT\u003c/sup\u003e represent two new species of \u003cem\u003eCupriavidus\u003c/em\u003e genus. Monophyletic grouping in core-gene phylogeny and ANI values below 96% have been used to delineate genospecies boundaries in the case of \u003cem\u003eRhizobium legumonosarum\u003c/em\u003e species complex (Young et al. 2021). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGenes involved in nitrogen fixation, nodulation, secondary metabolism\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe analyzed the pangenome of strains WGmk3, WGlv3\u003csup\u003eT\u003c/sup\u003e, WGtm5\u003csup\u003eT\u003c/sup\u003e and SKND8 to identify the accessory genomic features of the newly isolated species. Here, genome sequences were examined to identify the symbiosis-related genes and secondary metabolite regions. A subset of \u003cem\u003enod\u003c/em\u003e, \u003cem\u003enif\u003c/em\u003e and \u003cem\u003efix\u0026nbsp;\u003c/em\u003egenes were predicted in the genome SKND8, WGlv3\u003csup\u003eT\u003c/sup\u003e and WGtm5\u003csup\u003eT\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The genome SKND8 contained a cluster of nine nod genes: \u003cem\u003enodXSHD1ABC\u003c/em\u003e including two copies of \u003cem\u003enodIJU\u0026nbsp;\u003c/em\u003egenes. A set of genes involved in nitrogen fixation, \u003cem\u003enifABXENQVWHDKZTU\u003c/em\u003e and \u003cem\u003efixABCUL\u003c/em\u003e, have been located and\u0026nbsp;dispersed throughout the genome. Both strains WGtm5\u003csup\u003eT\u003c/sup\u003e and WGlv3\u003csup\u003eT\u003c/sup\u003e showed similar \u003cem\u003enod\u003c/em\u003e genes organisation with the presence of a cluster of \u003cem\u003enodTWIDASB\u0026nbsp;\u003c/em\u003egenes. Additionally, a putative \u003cem\u003enodJ\u003c/em\u003e gene was identified for strain WGlv3\u003csup\u003eT\u003c/sup\u003e. Notably, common sets of \u003cem\u003enifABENWHDK\u0026nbsp;\u003c/em\u003egenes and \u003cem\u003efixBCX\u003c/em\u003e genes were observed for both strains.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdditionally, secondary metabolite-producing genes and pathways have been identified in the genome sequences of the root-nodulating bacteria by using anti-SMASH online server search. The strains SKND8 exhibited the presence of seven putative secondary metabolite gene clusters including, arylpolyene, phosphonate, terpene, redox-cofactor, hserlactone, exopolysaccharides related NRPS and TfuA-related RiPPs. Member of beta rhizobia have been shown to produce antioxidant exopolysaccharide(Xia et al. 2021). While genome WGmk3 contained only three regions: arylpolyene, terpene and serobactin C related siderophore. Previously, serobactin C (siderophores) produced by \u003cem\u003eHerbaspirillum seropedicae\u003c/em\u003e was reported by (Rosconi et al. 2013). In strain WGlv3\u003csup\u003eT\u003c/sup\u003e gene clusters related to lankacidin C and kirromycin biosynthesis observed, overall, six secondary metabolites regions including siderophore (taiwachelin) were identified. Earlier, Kreutzer et al. (2012) reported taiwachelin type of siderophore in the genomesequence of \u003cem\u003eCupriavidus taiwanensis\u003c/em\u003e LMG19424. Six secondary metabolites regions including arylpolyene, lankacidin C and bacillomycin D biosynthesis cluster, Xanthoferrin related siderophore were predicted in the genome of WGtm5\u003csup\u003eT\u003c/sup\u003e. Xanthoferrin is the α-hydroxycarboxylate type siderophore which is required for optimum growth inside cabbage (Pandey et al. 2017). Previously, serobactin C, taiwachelin and xanthoferrin types of siderophore reported from rhizobia in independent studies (Kreutzer and Nett 2012; Rosconi et al. 2013; Pandey et al. 2017).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePhylogenetic analysis based on symbiotic (nodC and nifH) genes\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSymbiosis genes of rhizobia, including nodulation and nitrogen fixation genes, are part of the accessory and mobile genome. Several rhizobia share specific symbiosis genes, which allow them nodulate specific hosts. Rhizobia with specific symbiosis genes can be divided into several symbiovars. To identify the symbiovars within the rhizobial strains isolated from \u003cem\u003eMimosa pudica\u003c/em\u003e,\u0026nbsp;\u003cem\u003enodC\u003c/em\u003e and \u003cem\u003enifH\u0026nbsp;\u003c/em\u003egenes were extracted from the genomes. Blastn search of the \u003cem\u003enifH\u003c/em\u003e gene exhibited that strain SKND8 belongs to sv. \u003cem\u003etropicalis\u003c/em\u003e, with 99.9% sequence identity with \u003cem\u003eParaburkholderia phymatum\u003c/em\u003e STM815\u003csup\u003eT\u003c/sup\u003e (Table S5), isolated from \u003cem\u003eMachaerium lunatum\u003c/em\u003e in French Guiana, that formed ineffective nodules on the promiscuous host \u003cem\u003eMacroptilium atropurpureum\u003c/em\u003e (Moulin et al., 2001). Later, this symbiovar was found in different \u003cem\u003eParaburkholderia\u0026nbsp;\u003c/em\u003espp. nodulating plants such as \u003cem\u003eMimosa flocculosa\u003c/em\u003e, \u003cem\u003eMimosa candollei\u003c/em\u003e, \u003cem\u003eMimosa\u0026nbsp;\u003c/em\u003e\u003cem\u003ecaesalpiniifolia\u003c/em\u003e, \u003cem\u003eMimosa\u003c/em\u003e\u003cem\u003ediplotricha\u003c/em\u003e, \u003cem\u003eMimosa pudica\u003c/em\u003e,\u0026nbsp;and \u003cem\u003ePiptadenia gonoacantha\u003c/em\u003e (Paulitsch et al. 2020; Martinez-Romero\u0026nbsp;et al.\u0026nbsp;2024). Furthermore, the\u003cem\u003enifH\u003c/em\u003e gene of\u0026nbsp;strains\u0026nbsp;WGlv3\u003csup\u003eT\u003c/sup\u003e and WGtm5\u003csup\u003eT\u003c/sup\u003e was 100% identical to that of \u003cem\u003eCupriavidus taiwanensis\u0026nbsp;\u003c/em\u003estrains and \u003cem\u003e“Cupriavidus neocaledonicus”\u003c/em\u003e STM 6160 isolated from \u003cem\u003eMimosa pudica\u003c/em\u003e from different parts of world including French Guiana, New Caledonia, China, and Taiwan (Amadou et al. 2008;\u0026nbsp;Mishra et al. 2012;\u0026nbsp;Clerissi\u0026nbsp;et al. 2018; Liu et al. 2020). Notably, similar results were found when we used \u003cem\u003enodC\u003c/em\u003e gene sequences for the Blastn search (see Table S6), indicating that \u003cem\u003eParaburkholderia caribensis\u0026nbsp;\u003c/em\u003estrain SKND8 belong to sv. \u003cem\u003etropicalis\u003c/em\u003e. Similarly, strains belonging to the new species of \u003cem\u003eCupriavidus\u003c/em\u003e share the undescribed symbiovar found in case of \u003cem\u003eCupriavidus taiwanensis\u003c/em\u003e strains and \u003cem\u003e“Cupriavidus neocaledonicus”\u003c/em\u003e STM 6160 isolated from \u003cem\u003eMimosa pudica\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSymbiotic genes-based phylogeny resulted in the unique clustering of \u003cem\u003eM pudica\u003c/em\u003e rhizobia. Contrary to the 16S rRNA gene and core gene phylogenies, the \u003cem\u003enifH\u0026nbsp;\u003c/em\u003eand \u003cem\u003enodC\u0026nbsp;\u003c/em\u003egene phylogenetic tree placed the strain SKND8 close to \u003cem\u003eParaburkholderia diazotrophica\u003c/em\u003e JUA2-8\u003csup\u003eT\u003c/sup\u003e and \u003cem\u003eParaburkholderia\u0026nbsp;\u003c/em\u003esp\u003cem\u003e.\u0026nbsp;\u003c/em\u003eLEh15\u003csup\u003eT\u003c/sup\u003e, respectively. The \u003cem\u003enodC\u003c/em\u003e gene sequences of SKND8 strain were identical to the strains of \u003cem\u003eParaburkholderia\u003c/em\u003e isolated from the hosts like Brazil originating \u003cem\u003eM\u003c/em\u003e.\u003cem\u003e\u0026nbsp;pudica\u0026nbsp;\u003c/em\u003e(Dall’Agnol et al. 2017)\u003cem\u003e, M. claussenii\u0026nbsp;\u003c/em\u003e(de Pires et al. 2019), \u003cem\u003eM\u003c/em\u003e.\u003cem\u003ecaesalpiniifoli\u0026nbsp;\u003c/em\u003e(Martins et al. 2015;\u0026nbsp;de Oliveira et al. 2019) and other mimosoid legumes (Bournaud et al. 2013; Silva et al. 2018)\u003cem\u003e.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWhile\u0026nbsp;symbiotic genes\u0026nbsp;phylogeny placed the strains\u0026nbsp;WGtm5\u003csup\u003eT\u003c/sup\u003e and WGlv3\u003csup\u003eT\u003c/sup\u003e close to the members of \u003cem\u003eCupriavidus taiwanensis\u003c/em\u003e species complex, consisting of \u003cem\u003eCupriavidus taiwanensis\u003c/em\u003e SWF 66322,\u003cem\u003e\u0026nbsp;“Cupriavidus neocaledonicus”\u003c/em\u003e STM 6160,\u003cem\u003e\u0026nbsp;Cupriavidus taiwanensis\u003c/em\u003e STM 6041and \u003cem\u003eCupriavidus taiwanensis\u003c/em\u003e SWF66294 strains (Fig. S2 and S3)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDetermination of host range and Symbiotic efficiency of isolates\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInoculation of surface sterilized seeds of \u003cem\u003eVigna radiata, Pisum sativum, Vigna aconitifolia, Cicer arietinum, Vigna unguiculata, Arachis hypogaea, Trigonella foenum-graecum, Phaseolus vulgaris, Cyamopsis tetragonoloba\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Lens culinaris\u003c/em\u003e seeds with four test strains significantly influenced the shoot and root length, nodule number and plant dry weight compared to the control. The uninoculated controls did not show nodulation in none of the species, which confirmed the aseptic conditions of the experiment. The shoot length varied from 5.3 to 63.6 per plant, with strain WGtm5\u003csup\u003eT\u003c/sup\u003e exhibited the highest efficient shoot length for \u003cem\u003eVigna unguiculata\u003c/em\u003e test plant in comparison to uninoculated control, which was 57.6 cm. The difference between the test plant shoot length and control plant shoot length was 28.8 cm indicated 100% efficiency in term of shoot height enhancer of WGtm5 strain on cowpea plant. While WGlv3\u003csup\u003eT\u003c/sup\u003e strain found to be least potent shoot height inducer (4%) as recorded efficient shoot length was 13.86 cm for groundnut test plant (Fig. 4).\u003c/p\u003e\n\u003cp\u003eThe plant dry weight of all test plants was measured on a\u0026nbsp;digital weighing balance and compared with the uninoculated control of each species. The mean values of test plant dry weight ranged from 13.76±0.30 to 30.76±0.85 (mean ± SD) mg per plant\u003cem\u003e. Vigna unguiculata\u0026nbsp;\u003c/em\u003especies inoculated with strain WGtm5\u003csup\u003eT\u003c/sup\u003e recorded the highest efficient increased dry weight which was increased by 8.7 mg when compared with control (Fig. 5). In contrast, Mat bean inoculated with the strain SKND8 recorded the lowest plant dry weight\u0026nbsp;which was decreased by 0.04 mg; however, no significance difference was found in this treatment. Except few treatments such as SKND8 with \u003cem\u003eVigna aconitifolia\u003c/em\u003e and WGlv3\u003csup\u003eT\u003c/sup\u003e with \u003cem\u003eArachis hypogaea\u003c/em\u003e, all the representative isolates found to be effective on all the test plants.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, there was a significant variation in the number of nodules that formed on the roots of the test plants, including the host plants (Fig. 6). Three out of four strains effectively nodulated \u003cem\u003eM. pudica\u003c/em\u003e, while strain WGmk3 failed to nodulate. Additionally, strains SKND8, WGtm5\u003csup\u003eT\u003c/sup\u003e and\u0026nbsp;WGlv3\u003csup\u003eT\u003c/sup\u003e were able to form nodules in the roots of \u003cem\u003eVigna radiata\u003c/em\u003e, \u003cem\u003eVigna unguiculata\u003c/em\u003e, and \u003cem\u003ePisum sativum\u003c/em\u003e. The mean nodule number per plant varied from 2.33±0.57 for treatment WGtm5\u003csup\u003eT\u003c/sup\u003e with \u003cem\u003eVigna radiata\u0026nbsp;\u003c/em\u003eto 9.33±0.57 for treatment SKND8 with \u003cem\u003ePisum sativum.\u0026nbsp;\u003c/em\u003eThe strain SKND8 induced 4.66±0.57,8.33±0.57 and 9.33±0.57 nodules in \u003cem\u003eVigna radiata\u003c/em\u003e, \u003cem\u003eVigna unguiculata\u003c/em\u003e and \u003cem\u003eP. sativum\u003c/em\u003e, respectively. Both the strains WGtm5\u003csup\u003eT\u003c/sup\u003e and WGlv3\u003csup\u003eT\u003c/sup\u003e were able to nodulate \u003cem\u003eVigna radiata\u003c/em\u003e and \u003cem\u003eVigna unguiculata\u003c/em\u003e. However, these nodules might likely not be effective or may have some other bacteria, as in this study we did not perform the re-isolation of root-nodulating bacteria from these nodules. Noteworthy, \u003cem\u003eCupriavidus\u003c/em\u003e sp. isolated from \u003cem\u003eMimosa pudica\u003c/em\u003e exhibited nodulation in \u003cem\u003eV. unguiculata\u003c/em\u003e (Silva et al. 2012). Furthermore, beta-rhizobia including \u003cem\u003eParaburkholderia\u003c/em\u003e have been reported to nodulate\u003cem\u003e\u0026nbsp;V. unguiculata\u0026nbsp;\u003c/em\u003e(Castro et al. 2017; Ramrez et al. 2020; Muindi et al. 2021).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMorphology, Physiology, and chemotaxonomy of new taxa\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe colonies of strains WGtm5\u003csup\u003eT\u003c/sup\u003e and WGlv3\u003csup\u003eT\u003c/sup\u003e were circular, translucent, gummy, cream-white, smooth and round with entire margin on YEMA-Cr media. Excessive amount of extracellular polysaccharides were observed around the colonies of both strains when grown on YMA plates. Microscopic study inferenced that the cells of strains WGtm5\u003csup\u003eT\u003c/sup\u003e and WGlv3\u003csup\u003eT\u003c/sup\u003e were short rod-shaped, motile and Gram-stain negative. Both strains can grow at temperatures ranging from 20 ℃ to 40 ℃ but not at 10 or 50 ℃. The optimum temperature for their growth was 30 ℃.\u0026nbsp;In terms of pH, strains WGtm5\u003csup\u003eT\u003c/sup\u003e and WGlv3\u003csup\u003eT\u003c/sup\u003e displayed growth across a range of 5–8, with the best growth observed at pH 7.0.\u0026nbsp;Both the strains were able to tolerate 1% of NaCl (w/v), but fail to grow in 2% NaCl (w/v).\u0026nbsp;In enzyme detection, strains WGtm5\u003csup\u003eT\u003c/sup\u003e and WGlv3\u003csup\u003eT\u003c/sup\u003e tested positive for nitrate reductase enzyme and catalase enzyme test. Furthermore, a comparison of the physiological and biochemical traits of the newly discovered strains in this investigation with the closely related members \u003cem\u003eC. taiwanensis\u003c/em\u003e species complex was carried out (Table 1). The growth ranges for temperature and pH in the cases of \u003cem\u003eCupriavidus taiwanensis\u003c/em\u003e LMG 19424\u003csup\u003eT\u003c/sup\u003e, \u003cem\u003eCupriavidus necator\u003c/em\u003e LMG 8453\u003csup\u003eT\u003c/sup\u003e and \u003cem\u003eCupriavidus nantongensis\u003c/em\u003e X1\u003csup\u003eT\u003c/sup\u003e were consistent with the ranges reported earlier (Makkar et al. 1987; Estrada-de los Santos et al. 2012; Sun et al. 2016). It is worth noting that, both strains WGtm5\u003csup\u003eT\u003c/sup\u003e and WGlv3\u003csup\u003eT\u003c/sup\u003e could grow at 1% (w/v) NaCl, while fail to grow at 2% (w/v) NaCl, which is contrary to the type strains of other species of \u003cem\u003eCupriavidus\u003c/em\u003e (Sun et al. 2016).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA total of 17 different cellular fatty acids, in different quantities, were detected in strains WGtm5\u003csup\u003eT\u003c/sup\u003e and WGlv3\u003csup\u003eT\u0026nbsp;\u003c/sup\u003e(Table 2). It was noted that the fatty acid compositions of the strains WGtm5\u003csup\u003eT\u003c/sup\u003e and WGlv3\u003csup\u003eT\u003c/sup\u003e were identical to the type strains of \u003cem\u003eCupriavidus taiwanensis\u003c/em\u003e, \u003cem\u003eCupriavidus necator\u003c/em\u003e, \u003cem\u003eCupriavidus nantongensis\u003c/em\u003e and \u003cem\u003eCupriavidus alkaliphilus\u003c/em\u003e with differences in their proportions (Makkar et al. 1987; Chen, et al. 2001; Estrada-de los Santos et al. 2012; Sun et al. 2016). The major fatty acids detected were C\u003csub\u003e14:0\u003c/sub\u003e, C\u003csub\u003e14:0\u003c/sub\u003e 2OH, C\u003csub\u003e15:1\u003c/sub\u003e ꙍ6c, C\u003csub\u003e16:0\u003c/sub\u003e, C\u003csub\u003e16:1\u003c/sub\u003e ꙍ5c, C\u003csub\u003e16:0\u003c/sub\u003e 2OH, C\u003csub\u003e16:1\u003c/sub\u003e 2OH, C\u003csub\u003e17:0\u003c/sub\u003e, C\u003csub\u003e17:0\u003c/sub\u003e cyclo, C\u003csub\u003e18:0\u003c/sub\u003e and C\u003csub\u003e18:1\u003c/sub\u003e 2-OH for strains WGtm5\u003csup\u003eT\u003c/sup\u003e and WGlv3\u003csup\u003eT\u003c/sup\u003e. Noticeably, C\u003csub\u003e19:0\u0026nbsp;\u003c/sub\u003efatty acid was present in strain WGlv3\u003csup\u003eT\u003c/sup\u003e in comparison to WGtm5\u003csup\u003eT\u003c/sup\u003e and other reference type strains.\u003c/p\u003e"},{"header":" Conclusions","content":"\u003cp\u003eThe microsymbionts of \u003cem\u003eMimosa pudica\u003c/em\u003e in India exhibit\u0026nbsp;a distinct geographic distribution, with \u003cem\u003eParaburkholderia caribensis\u003c/em\u003e being prevalent in the Eastern Himalayan region and two new species of \u003cem\u003eCupriavidus\u003c/em\u003e identified in the Western Ghats. Moreover, \u003cem\u003eParaburkholderia caribensis\u003c/em\u003e strain SKND8 was placed within the sv. \u003cem\u003etropicalis\u003c/em\u003e on the basis of \u003cem\u003enifH\u003c/em\u003e and \u003cem\u003enodC\u003c/em\u003e genes sequence analyses.\u0026nbsp;Additionally, a non-rhizobial strain belonging to \u003cem\u003eHerbaspirillum huttiense\u003c/em\u003e was isolated from the root nodules of \u003cem\u003eMimosa pudica\u0026nbsp;\u003c/em\u003ein the Western Ghat. Based on the genomic features such as phylogenetic grouping, ANI and dDDH, and phenotypic characteristics, two Western Ghat strain represent two new species within the genus \u003cem\u003eCupriavidus\u003c/em\u003e. We propose\u0026nbsp;the names\u0026nbsp;\u003cem\u003eCupriavidus mimosa\u003c/em\u003e sp. nov. for the type strain WGtm5\u003csup\u003eT\u003c/sup\u003e and \u003cem\u003eCupriavidus gehlotii\u003c/em\u003e sp. nov for the type strain WGlv3\u003csup\u003eT\u003c/sup\u003e.\u0026nbsp;Our analyses indicate that these new species are part of the\u0026nbsp;\u003cem\u003eCupriavidus taiwanensis\u003c/em\u003e species complex,\u0026nbsp;which includes 12 genospecies.\u0026nbsp;Of these, four are already named, while the others await taxonomic descriptions. Notably, seven genomes, including that of the strain\u0026nbsp;WGtm5\u003csup\u003eT\u003c/sup\u003e,\u0026nbsp;were placed with in the newly proposed\u0026nbsp;\u003cem\u003eCupriavidus mimosa\u003c/em\u003e, while \u003cem\u003eCupriavidus gehlotii\u003c/em\u003e is currently represented only by the strain WGlv3\u003csup\u003eT\u003c/sup\u003e. The cross-nodulation tests demonstrated that the test strains could elicit nodules on the roots of\u003cem\u003e\u0026nbsp;Vigna radiata\u003c/em\u003e, \u003cem\u003eVigna unguiculata\u003c/em\u003e, and \u003cem\u003ePisum sativum\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDescription of \u003cem\u003eCupriavidus mimosae\u003c/em\u003e sp. nov.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCupriavidus mimosa\u0026nbsp;\u003c/em\u003e(mi.mo'sae. N.L. gen. n. \u003cem\u003emimosae\u003c/em\u003e. of the plant genus \u003cem\u003eMimosa,\u0026nbsp;\u003c/em\u003efrom which the strain was isolated).\u003c/p\u003e\n\u003cp\u003eCells are Gram-negative, aerobic, short rod-shaped and motile. Colonies are round, smooth, raised, translucent, gummy, small and cream-white and grow 0.1 to 0.2 cm in diameter after 48 hours of growth on YMA medium at 30℃. Optimum growth was observed at a pH of 7.0, a temperature of 30℃ and a NaCl concentration of 0.25% (w/v). Strains do not grow at temperatures 10℃ and below 10℃ or at 50℃ and above 50℃, at pH values of below 5.0 and above 8.0, or at NaCl concentrations above 1.0% (w/v). The major fatty acids detected were C\u003csub\u003e14:0\u003c/sub\u003e,\u0026nbsp;C\u003csub\u003e14:0\u0026nbsp;\u003c/sub\u003e2OH, C\u003csub\u003e15:1\u0026nbsp;\u003c/sub\u003eꙍ6c, C\u003csub\u003e16:1\u0026nbsp;\u003c/sub\u003eꙍ5c, C\u003csub\u003e16:0\u003c/sub\u003e 2OH, C\u003csub\u003e16:1\u003c/sub\u003e 2OH, C\u003csub\u003e17:0\u003c/sub\u003e, C\u003csub\u003e17:0\u003c/sub\u003e cyclo, C\u003csub\u003e18:0\u003c/sub\u003e, C\u003csub\u003e18:1\u003c/sub\u003e 2-OH, with a high amount of C\u003csub\u003e16:0\u0026nbsp;\u003c/sub\u003efatty acid Positive for catalase, oxidase and nitrate reductase.\u0026nbsp;Assimilates D-fructose, dextrose, sodium acetate and sodium citrate, but does not assimilate the sucrose, lactose, D-mannitol, sodium succinate, sodium propionate and sodium pyruvate.\u003c/p\u003e\n\u003cp\u003eThe genome sequence has a DNA G + C content of 67.0%, spanning 6,703,278 bp. \u0026nbsp;The NCBI accession numbers for the 16S rRNA (OM281132), \u003cem\u003eatpD\u003c/em\u003e (OM574816), \u003cem\u003erecA\u0026nbsp;\u003c/em\u003e(OM574813), \u003cem\u003egyrB\u003c/em\u003e (OM574814), \u003cem\u003ednaK\u003c/em\u003e (OM574815), \u003cem\u003enodC\u003c/em\u003e (OM574818) and \u003cem\u003enifH\u0026nbsp;\u003c/em\u003e(OM574817) genes, and genome sequence\u0026nbsp;GCA_023952485.1 (JALHRY000000000)\u0026nbsp;of strain WGtm5\u003csup\u003eT\u003c/sup\u003e. The type strain is WGtm5\u003csup\u003eT\u003c/sup\u003e (MCC4888\u003csup\u003eT\u003c/sup\u003e = KACC 22828\u003csup\u003eT\u003c/sup\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDescription of \u003cem\u003eCupriavidus gehlotii\u0026nbsp;\u003c/em\u003esp. nov.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCupriavidus gehlotii\u0026nbsp;\u003c/em\u003e(geh.lo'ti.i. N.L. gen. n.\u0026nbsp;gehlotii, of Gehlot, in the name of Professor Hukum Singh Gehlot (Dept. of\u0026nbsp;Botany, JNV University, Jodhpur, India), to honour his contribution to the research of\u0026nbsp;indigenous nitrogen-fixing bacteria from different region of India).\u003c/p\u003e\n\u003cp\u003eCells are Gram-negative, aerobic, short rod-shaped and motile. Colonies are round, smooth, raised, translucent, gummy, small and cream-white and grow 0.1 to 0.2 cm in diameter after 48 hours of growth on YMA medium at 30℃. Optimum growth was\u0026nbsp;observed at a pH of 7.0, a temperature of 30℃ and a NaCl concentration of 0.25% (w/v). Strains do not grow at temperatures 10℃ and below 10℃ or at 50℃ and above 50℃, at pH values of 4.0 or above 8.0, or at NaCl concentrations above 1.0% (w/v). The major fatty acids detected were C\u003csub\u003e14 : 0\u003c/sub\u003e,\u0026nbsp;C\u003csub\u003e14 : 0\u0026nbsp;\u003c/sub\u003e2OH, C\u003csub\u003e16 : 1\u0026nbsp;\u003c/sub\u003eꙍ5c, C\u003csub\u003e17 : 0\u003c/sub\u003e, C\u003csub\u003e17 : 0\u003c/sub\u003e cyclo, C\u003csub\u003e18 : 0\u003c/sub\u003e, C\u003csub\u003e18 : 1\u003c/sub\u003e 2-OH, with a high amount of C\u003csub\u003e16 : 0\u0026nbsp;\u003c/sub\u003efatty acid. Positive for catalase, oxidase and nitrate reductase. Assimilates D-fructose, dextrose, sodium acetate, D-mannitol (weakly) and sodium citrate, but does not assimilate the sucrose, lactose, sodium succinate, sodium propionate and sodium pyruvate.\u003c/p\u003e\n\u003cp\u003eThe genome sequence has a DNA G + C content of 67.10%, spanning\u0026nbsp;6,755,320\u0026nbsp;bp. \u0026nbsp;The NCBI accession numbers for the 16S rRNA (OM281131), \u003cem\u003eatpD\u003c/em\u003e (OM574810), \u003cem\u003erecA\u003c/em\u003e (OM574807), \u003cem\u003egyrB\u003c/em\u003e (OM574808), \u003cem\u003ednaK\u003c/em\u003e (OM574809), \u003cem\u003enodC\u003c/em\u003e (OM574812) and nifH (OM574811) genes, and genome sequence\u0026nbsp;GCA_023952475.1 (JALHRX000000000)\u0026nbsp;of strain\u0026nbsp;WGlv3\u003csup\u003eT\u003c/sup\u003e. The type strain is WGlv3\u003csup\u003eT\u003c/sup\u003e (MCC4890\u003csup\u003eT\u003c/sup\u003e = KACC 22827\u003csup\u003eT\u003c/sup\u003e).\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Prof. Arnab Sen, Department of Botany, University of North Bengal, Siliguri, for providing access to his laboratory for the isolation of \u003cem\u003eMimosa pudica\u0026nbsp;\u003c/em\u003eroot-nodulating bacteria from the Eastern Himalayan region. We thank the National Centre for Microbial Resource, Pune for various research facilities. This work used the computational and storage services (TARS cluster) provided by the IT department at Institut Pasteur, Paris. The authors would like to thank Prof. H. S. Gehlot of JNV University, Jodhpur, India, for granting us permission to propose a new species named in his honour.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.S. conceived the study, designed and performed the experiments, analysed the results and wrote the manuscript. M.K. and A.P. helped with the experiments. K.M.M. assisted with genome assembly, and G.K.G. supervised the study. P.R. performed genomic analyses, analysed the results and wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study do not involve any work on or with animals.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAltschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215 (3), 403\u0026ndash;410, http://dx.doi.org/10.1016/ S0022-2836(05)80360-2.\u003c/li\u003e\n\u003cli\u003eAmadou, C., Pascal, G., Mangenot, S., Glew, M., Bontemps, C., Capela, D., ... \u0026amp; Masson-Boivin, C. (2008). 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Twenty years of paradigm-breaking studies of taxonomy and symbiotic nitrogen fixation by beta-rhizobia, and indication of Brazil as a hotspot of \u003cem\u003eParaburkholderia\u003c/em\u003e diversity. \u003cem\u003eArchives of Microbiology\u003c/em\u003e, \u003cem\u003e203\u003c/em\u003e(8), 4785-4803.\u003c/li\u003e\n\u003cli\u003ePoehlein, A., Kusian, B., Friedrich, B., Daniel, R., \u0026amp; Bowien, B. (2011). Complete genome sequence of the type strain Cupriavidus necator N-1.\u003c/li\u003e\n\u003cli\u003eRahi, P., Giram, P., Chaudhari, D., Kiran, S., Khullar, A., Chandel, M., ... \u0026amp; Mahajan, B. (2020). Rhizobium indicum sp. nov., isolated from root nodules of pea (Pisum sativum) cultivated in the Indian trans-Himalayas. Systematic and Applied Microbiology, 43(5), 126127.\u003c/li\u003e\n\u003cli\u003eRahi, P., Prakash, O., \u0026amp; Shouche, Y. S. (2016). Matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF MS) based microbial identifications: challenges and scopes for microbial ecologists. Frontiers in Microbiology, 7, 1359.\u003c/li\u003e\n\u003cli\u003eRam\u0026iacute;rez, M. D. A., Espa\u0026ntilde;a, M., Lewandowska, S., Yuan, K., Okazaki, S., Ohkama-Ohtsu, N., \u0026amp; Yokoyama, T. (2020). Phylogenetic analysis of symbiotic bacteria associated with two Vigna species under different agro-ecological conditions in Venezuela. \u003cem\u003eMicrobes and Environments\u003c/em\u003e, \u003cem\u003e35\u003c/em\u003e(1), ME19120.\u003c/li\u003e\n\u003cli\u003eReiner, K. (2012). Carbohydrate fermentation protocol. \u003cem\u003eenergy\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e, 12.\u003c/li\u003e\n\u003cli\u003eRosconi, F., Davyt, D., Mart\u0026iacute;nez, V., Mart\u0026iacute;nez, M., Abin‐Carriquiry, J. A., Zane, H., ... \u0026amp; Fabiano, E. (2013). Identification and structural characterization of serobactins, a suite of lipopeptide siderophores produced by the grass endophyte \u003cem\u003eHerbaspirillum seropedicae\u003c/em\u003e. \u003cem\u003eEnvironmental Microbiology\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e(3), 916-927.\u003c/li\u003e\n\u003cli\u003eSasser, M. (2001). Tracking\u0026rdquo; a strain using the sherlock microbial identification system (MIS). \u003cem\u003eTechnical Note\u003c/em\u003e, \u003cem\u003e102\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eSheu, S. Y., Chou, J. H., Bontemps, C., Elliott, G. N., Gross, E., James, E. K., ... \u0026amp; Chen, W. M. (2012). \u003cem\u003eBurkholderia symbiotica\u003c/em\u003e sp. nov., isolated from root nodules of Mimosa spp. native to north-east Brazil. \u003cem\u003eInternational Journal of Systematic and Evolutionary Microbiology\u003c/em\u003e, \u003cem\u003e62\u003c/em\u003e(Pt_9), 2272-2278.\u003c/li\u003e\n\u003cli\u003eSheu, S. Y., Chou, J. H., Bontemps, C., Elliott, G. N., Gross, E., dos Reis Junior, F. B., ... \u0026amp; Chen, W. M. (2013). \u003cem\u003eBurkholderia diazotrophica\u003c/em\u003e sp. nov., isolated from root nodules of \u003cem\u003eMimosa\u003c/em\u003e spp. \u003cem\u003eInternational Journal of Systematic and Evolutionary Microbiology\u003c/em\u003e, \u003cem\u003e63\u003c/em\u003e(Pt_2), 435-441.\u003c/li\u003e\n\u003cli\u003eSilva, F. V., Sim\u0026otilde;es-Ara\u0026uacute;jo, J. L., Silva J\u0026uacute;nior, J. P., Xavier, G. R., \u0026amp; Rumjanek, N. G. (2012). Genetic diversity of Rhizobia isolates from Amazon soils using cowpea (\u003cem\u003eVigna unguiculata\u003c/em\u003e) as trap plant. \u003cem\u003eBrazilian Journal of Microbiology\u003c/em\u003e, \u003cem\u003e43\u003c/em\u003e, 682-691.\u003c/li\u003e\n\u003cli\u003eSilva, V.C., Alves, P.A.C., Rhem, M.F.K., dos Santos, J.M.F., James, E.K., Gross, E. (2018) Brazilian species of Calliandra Benth. (tribe Ingeae) are nodulated by diverse strains of \u003cem\u003eParaburkholderia\u003c/em\u003e. \u003cem\u003eSyst. Appl. Microbiol\u003c/em\u003e. 41 (3), 241\u0026ndash;250. https://doi.org/10.1016/j.syapm.2017.12.003.\u003c/li\u003e\n\u003cli\u003eVerma, S. C., Chowdhury, S. P. \u0026amp; Tripathi, A. K. (2004). Phylogeny based on 16S rDNA and \u003cem\u003enifH\u003c/em\u003e sequences of \u003cem\u003eRalstonia taiwanensis\u003c/em\u003e strains isolated from nitrogen-fixing nodules of \u003cem\u003eMimosa pudica\u003c/em\u003e, in India. \u003cem\u003eCanadian Journal of Microbiology\u003c/em\u003e. 50(5), 313-322. https://doi.org/10.1139/w04-020\u003c/li\u003e\n\u003cli\u003eSun, L. N., Wang, D. S., Yang, E. D., Fang, L. C., Chen, Y. F., Tang, X. Y., \u0026amp; Hua, R. M. (2016). \u003cem\u003eCupriavidus nantongensis\u003c/em\u003e sp. nov., a novel chlorpyrifos-degrading bacterium isolated from sludge. \u003cem\u003eInternational Journal of Systematic and Evolutionary Microbiology\u003c/em\u003e, \u003cem\u003e66\u003c/em\u003e(6), 2335-2341.\u003c/li\u003e\n\u003cli\u003eTamura, K., Stecher, G., \u0026amp; Kumar, S. (2021). MEGA11: molecular evolutionary genetics analysis version 11. \u003cem\u003eMolecular Biology and Evolution\u003c/em\u003e, \u003cem\u003e38\u003c/em\u003e(7), 3022-3027.\u003c/li\u003e\n\u003cli\u003eVincent, J. M. (1970). A manual for the practical study of the root-nodule bacteria. \u003cem\u003eA manual for the practical study of the root-nodule bacteria.\u003c/em\u003e\u003c/li\u003e\n\u003cli\u003eWeisburg, W. G., Barns, S. M., Pelletier, D. A., \u0026amp; Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. \u003cem\u003eJournal of Bacteriology\u003c/em\u003e, \u003cem\u003e173\u003c/em\u003e(2), 697-703 \u003c/li\u003e\n\u003cli\u003eXia, M., Zhang, S., Shen, L., Yu, R., Liu, Y., Li, J., ... \u0026amp; Zeng, W. (2021). Optimization and characterization of an antioxidant exopolysaccharide produced by \u003cem\u003eCupriavidus pauculus\u003c/em\u003e 1490. \u003cem\u003eJournal of Polymers and the Environment\u003c/em\u003e, 1-10.\u003c/li\u003e\n\u003cli\u003eYan, X., Wang, Z., Mei, Y., Wang, L., Wang, X., Xu, Q., ... \u0026amp; Wei, C. (2018). Isolation, diversity, and growth-promoting activities of endophytic bacteria from tea cultivars of Zijuan and Yunkang-10. \u003cem\u003eFrontiers in Microbiology\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e, 1848.\u003c/li\u003e\n\u003cli\u003eYoon, S. H., Ha, S. M., Kwon, S., Lim, J., Kim, Y., Seo, H., \u0026amp; Chun, J. (2017). Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. \u003cem\u003eInternational Journal of Systematic and Evolutionary Microbiology\u003c/em\u003e, \u003cem\u003e67\u003c/em\u003e(5), 1613.\u003c/li\u003e\n\u003cli\u003eYoung, J. P. W., Moeskj\u0026aelig;r, S., Afonin, A., Rahi, P., Maluk, M., James, E. K., ... \u0026amp; Tian, C. F. (2021). Defining the \u003cem\u003eRhizobium leguminosarum\u003c/em\u003e species complex. \u003cem\u003eGenes\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e(1), 111.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1: Differentiating characteristics of strain 1, WGtm5\u003csup\u003eT\u003c/sup\u003e; 2, WGlv3\u003csup\u003eT\u003c/sup\u003e; 3, \u003cem\u003eCupriavidus taiwanensis\u003c/em\u003e LMG 19424\u003csup\u003eT\u003c/sup\u003e; 4, \u003cem\u003eCupriavidus necator\u003c/em\u003e LMG 8453\u003csup\u003eT\u003c/sup\u003e; 5, \u003cem\u003eCupriavidus nantongensis\u003c/em\u003e X1\u003csup\u003eT\u003c/sup\u003e; 6, \u003cem\u003eCupriavidus alkaliphilus\u003c/em\u003e LMG 26294\u003csup\u003eT\u003c/sup\u003e in comparison to its closest phylogenetic neighbours. *+, positive; -, negative; W, weakly positive; ND, Not defined\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"937\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.5342%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCharacteristics\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.2479%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0726%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.5342%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSource of isolation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003eRoot nodules of\u003cem\u003e\u0026nbsp;Mimosa pudica\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003eRoot nodules of\u003cem\u003e\u0026nbsp;Mimosa pudica\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003eRoot nodules of\u003cem\u003e\u0026nbsp;Mimosa pudica\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003eRoot nodules of\u003cem\u003e\u0026nbsp;Mimosa pudica\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.2479%;\"\u003e\n \u003cp\u003eSludge sample\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0726%;\"\u003e\n \u003cp\u003eRhizosphere\u0026nbsp;in alkaline soils\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.5342%;\"\u003e\n \u003cp\u003e\u003cstrong\u003epH range for growth (optimum)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e5-8 (7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e5-8 (7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e6-9 (7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e6-9 (7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.2479%;\"\u003e\n \u003cp\u003e6-9 (7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0726%;\"\u003e\n \u003cp\u003e6-10 (7.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.5342%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTemperature range for growth (◦C) (optimum)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e20-40 (30)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e20-40 (30)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e15-42 (30)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e15-40 (30)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.2479%;\"\u003e\n \u003cp\u003e20-42 (30)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0726%;\"\u003e\n \u003cp\u003e15-42 (30)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.5342%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1% NaCl (w/v)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.2479%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0726%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.5342%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2% NaCl (w/v)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.2479%;\"\u003e\n \u003cp\u003ew\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0726%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.5342%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMotility\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.2479%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0726%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.5342%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCatalase\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.2479%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0726%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.5342%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNitrate Reductase\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.2479%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0726%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.5342%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ed-fructose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.2479%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0726%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.5342%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ed-glucose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.2479%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0726%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.5342%;\"\u003e\n \u003cp\u003e\u003cstrong\u003esucrose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.2479%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0726%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.5342%;\"\u003e\n \u003cp\u003e\u003cstrong\u003edextrose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.2479%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0726%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.5342%;\"\u003e\n \u003cp\u003e\u003cstrong\u003elactose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.2479%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0726%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.5342%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ed-mannitol\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.2479%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0726%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 25.5342%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ecitrate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003ew\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.2863%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.2479%;\"\u003e\n \u003cp\u003ew\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.0726%;\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Data for strain 3 from Chen, et al. (2001); 4 from Makkar et al. (1987); 5 from Sun et al. (2016) and for 6 from Estrada-de los Santos et al. (2012).\u003c/p\u003e\n\u003cp\u003eTable 2: Cellular fatty acid compositions (%) of strains WGtm5\u003csup\u003eT\u003c/sup\u003e and WGlv3\u003csup\u003eT\u003c/sup\u003e and related species of the genus \u003cem\u003eCupriavidus\u003c/em\u003e Strains: 1, WGtm5\u003csup\u003eT\u003c/sup\u003e; 2, WGlv3\u003csup\u003eT\u003c/sup\u003e; 3, \u003cem\u003eCupriavidus taiwanensis\u003c/em\u003e LMG 19424\u003csup\u003eT\u003c/sup\u003e; 4, \u003cem\u003eCupriavidus necator\u003c/em\u003e LMG 8453\u003csup\u003eT\u003c/sup\u003e; 5, \u003cem\u003eCupriavidus nantongensis\u003c/em\u003e X1\u003csup\u003eT\u003c/sup\u003e; 6, \u003cem\u003eCupriavidus alkaliphilus\u003c/em\u003e LMG 26294\u003csup\u003eT\u003c/sup\u003e.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"601\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFatty acids\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eC\u003csub\u003e14 : 0\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.21\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.29\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eC\u003csub\u003e14 : 0\u0026nbsp;\u003c/sub\u003e2OH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.04\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.81\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eC\u003csub\u003e15 : 1\u0026nbsp;\u003c/sub\u003eꙍ6c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eC\u003csub\u003e16 : 0\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e23.43\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e26.28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e28.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e27.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e28.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e31.6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eC\u003csub\u003e16 : 1\u0026nbsp;\u003c/sub\u003eꙍ5c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eC\u003csub\u003e16 : 0\u003c/sub\u003e 2OH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eC\u003csub\u003e16 : 1\u003c/sub\u003e 2OH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eC\u003csub\u003e17 : 0\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eC\u003csub\u003e17 : 0\u003c/sub\u003e cyclo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.20\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.56\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e11.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e12.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eC\u003csub\u003e18 : 0\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eC\u003csub\u003e18 : 1\u003c/sub\u003e 2-OH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eC\u003csub\u003e19:0\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eC\u003csub\u003e19 : 0\u003c/sub\u003e cyclo\u0026nbsp;ꙍ8c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" valign=\"top\" style=\"width: 601px;\"\u003e\n \u003cp\u003eSummed features*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e9.91\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e9.36\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e33.39\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e33.14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e32.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e33.6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e27.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e27.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e22.51\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e20.74\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e20.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e21.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e18.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e15.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Summed features are combinations of fatty acids that cannot be separated by the MIDI system. Summed feature 2 = C\u003csub\u003e12 : 0\u0026nbsp;\u003c/sub\u003ealdehyde and/ or unknown ECL 10.9525; summed feature 3 = C\u003csub\u003e16:1\u0026nbsp;\u003c/sub\u003eꙍ7c and/or C\u003csub\u003e16:1\u003c/sub\u003e ꙍ6c; summed feature 5= C\u003csub\u003e18:0\u0026nbsp;\u003c/sub\u003eante and/or C\u003csub\u003e18:2\u0026nbsp;\u003c/sub\u003eꙍ6,9c; summed feature 8 = C\u003csub\u003e18 : 1\u003c/sub\u003eꙍ7c and/or C\u003csub\u003e18 : 1\u0026nbsp;\u003c/sub\u003eꙍ6c.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea\u0026nbsp;\u003c/sup\u003eData for strain 3 from Chen, et al. (2001); 4 from Makkar et al (1987); 5 from Sun et al. (2016) and 6 from Estrada-de los Santos et al. (2012).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"MALDI-TOF MS, core-gene phylogeny, rhizobia, Western Ghats, Eastern Himalayas","lastPublishedDoi":"10.21203/rs.3.rs-5391658/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5391658/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe invasive weed \u003cem\u003eMimosa pudica\u003c/em\u003e (wild) is widespread in India’s tropical regions and nodulated by beta-rhizobia, including \u003cem\u003eCupriavidus\u003c/em\u003e and \u003cem\u003eParaburkholderia\u003c/em\u003e. This study investigates the genetic diversity of rhizobia associated with \u003cem\u003eM. pudica\u003c/em\u003e in the Eastern Himalayan (EH) and Western Ghat (WG) regions of India. MALDI-TOF-MS and 16S rRNA gene phylogeny identified EH rhizobia as \u003cem\u003eParaburkholderia\u003c/em\u003e, while WG rhizobia belonged to \u003cem\u003eCupriavidus\u003c/em\u003e.\u003cem\u003e \u003c/em\u003eCore-gene phylogeny (\u003cem\u003ebac120\u003c/em\u003e genes) and average nucleotide identity (ANI) values confirmed EH strain SKND8 as \u003cem\u003eParaburkholderia caribensis\u003c/em\u003e. In contrast, WG strains WGtm5\u003csup\u003eT\u003c/sup\u003e and WGlv3\u003csup\u003eT\u003c/sup\u003e clustered within the \u003cem\u003eCupriavidus taiwanensis\u003c/em\u003e species complex but had ANI values below 95.1% with validly published species, suggesting they are novel species. Distinct clusters in \u003cem\u003ebac120\u003c/em\u003e gene phylogeny and ANI values below 95.8% further indicate that both strains represent different species. Major fatty acids identified in WGtm5\u003csup\u003eT\u003c/sup\u003e and WGlv3\u003csup\u003eT\u003c/sup\u003e included\u0026nbsp;C\u003csub\u003e16: 0\u003c/sub\u003e, C\u003csub\u003e16:1 \u003c/sub\u003eꙍ7c and/or C\u003csub\u003e16:1 \u003c/sub\u003eꙍ 6c and C\u003csub\u003e18: 1\u003c/sub\u003eꙍ 7c and/or C \u003csub\u003e18: 1\u003c/sub\u003e ꙍ 6c. Based on ANI values, core-gene phylogeny, and phenotypic data, we proposed two novel species, \u003cem\u003eCupriavidus mimosae \u003c/em\u003esp. nov for WGtm5\u003csup\u003eT\u003c/sup\u003e (MCC4888\u003csup\u003eT\u003c/sup\u003e = KACC 22828\u003csup\u003eT\u003c/sup\u003e) and\u003cem\u003e Cupriavidus gehlotii\u003c/em\u003e sp. nov for WGlv3\u003csup\u003eT\u003c/sup\u003e (MCC4890\u003csup\u003eT\u003c/sup\u003e = KACC 22827\u003csup\u003eT\u003c/sup\u003e). The presence of nodulation and nitrogen-fixation genes and nodule formation in \u003cem\u003eVigna radiata\u003c/em\u003e and \u003cem\u003eVigna unguiculata\u003c/em\u003e by strain SKND8, WGtm5\u003csup\u003eT\u003c/sup\u003e and WGlv3\u003csup\u003eT\u003c/sup\u003e highlighted their nitrogen fixation potential. Additionally, WG strain WGmk3, identified as \u003cem\u003eHerbaspirillum huttiense\u003c/em\u003e, lacks nodulation genes and may be an endophyte of \u003cem\u003eM\u003c/em\u003e. \u003cem\u003epudica\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Genome sequence-based identification of bacteria nodulating Mimosa pudica growing in Eastern Himalayas and Western Ghats of India and description of Cupriavidus mimosae sp. nov. and Cupriavidus gehlotii sp. nov","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-19 10:01:45","doi":"10.21203/rs.3.rs-5391658/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0a0655d5-7d59-491a-9c4f-29a4b9c68048","owner":[],"postedDate":"November 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-15T01:53:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-19 10:01:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5391658","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5391658","identity":"rs-5391658","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}