Isolation, Characterization and Phylogenetic Analysis of Nodule-Associated Bacteria from Mimosa Pudica L.

The interaction between rhizobia and other nodule-associated bacteria assists to mitigate nutrient stress in leguminous plants by fixing atmospheric nitrogen and synthesizing plant growth regulators. The beneficial effects of microbial inoculants emphasize the need for further research and their use in modern agriculture. The present study describes the isolation, molecular identification, characterization, and phylogenetic analysis of nodule- associated bacteria from Mimosa pudica Linnaeus. Isolation and phenotypic characterization of nodule-associated bacteria were carried out according to standard procedures. Molecular characterization of the isolates was performed using 16S ribosomal RNA. Plant growth promoting the ability of selected isolates was analyzed by assessing indole acetic acid production, nitrogen- fixing ability and organic acid production. Evolutionary distance and relatedness were analyzed using the neighbor-joining method. Thirteen nodule-associated bacteria were isolated and identified using 16S rRNA gene sequencing. The selected isolates such as Rhizobium sp. CU8 and three other co-resident non-rhizobial nodule-associated bacteria (Bacillus cereus MY5, Ralstonia pickettii MY1 and Lactococcus lactis MY3) exhibited plant growth promotion and other potential microbial activities. Phylogenetic analysis revealed the genetic relatedness and evolutionary significance of all the thirteen isolates reside in the root nodule of M. pudica. The present study identified four isolates with plant growth promoting properties. L. lactis MY3 is the first report as a co-resident plant growth promoter from the root

The members of Leguminosae, are associated with endophytic, root nodule-associated bacteria (NAB) which ameliorates nutrient stress by fixing atmospheric nitrogen (N 2 ) and producing plant growth promoters. The genus Mimosa received considerable attention in recent years because of its potential to fix atmospheric nitrogen. Biological nitrogen fixation is ecologically important, contributing ~100-290 million tons of nitrogen annually to the natural ecosystem and enhancing the growth of agronomically important forage and crop plants. Biological nitrogen fixation (both symbiotic fixers and non-symbiotic fixers) reduces the use of synthetic nitrogen fertilizers.
Plant growth promoting bacteria (PGPB) enhance plant growth by fixing atmospheric nitrogen and its assimilation to the plant, production of siderophores to chelate iron and its absorption, solubilization of minerals such as phosphorus, synthesis of phytohormones, augmenting plant nutrient uptake 1, 2 and the production of substances like antibiotics 1 . In addition, the PGP microbes express abiotic stress tolerance like extreme temperature, drought, salinity, pH, heavy metal and pesticide pollution 3 .
PGPB has beneficial effects on legume growth and some strains enhance the nodulation and nitrogen fixation by effective interaction between plant and rhizobia 4 . Most of the nodulating bacteria are free-living rhizobacteria, however, some are intracellular or intercellular endophytes 5 and gain advantage of being protected from environmental stresses and microbial competition 6 . The endophytes and epiphytes are the two different types of plant growth promoting rhizobia associated with host tissue. There are many endophytic and epiphytic bacteria which are directly or indirectly involved in plant growth and development. Endophytic bacteria live in plant tissues without affecting the normal metabolism of the host or gaining any benefit other than a noncompetitive environment inside the host. It has been demonstrated that bacterial endophytes play a beneficial role in host plants, such as growth promotion and biological control of pathogens 7,8,9 . Legume root nodules may contain microbes other than rhizobia 10, 11 , however, the function of these co-residents in the nodules is yet to be fully elucidated, and their main role might be to assist rhizobia during the nodule infection process and to promote plant growth 12,13 .
Modern agriculture faces challenges, such as loss of soil fertility, fluctuating climatic factors, and increasing pathogen and pest attacks. The sustainability and environmental safety of agricultural production rely on ecofriendly approaches like the use of biofertilizers, biopesticides, and crop residue recycling. Increasing the food quality and quantity, without affecting sustainable plant productivity, and maintaining environmental quality is the principal aspect from the Agricultural and Ecological standpoint. The importance of nitrogen-fixing and plant growth promoting bacteria and their gene conservation can contribute better to sustain agriculture and 16S ribosomal RNA typing is used to identify microorganisms. The 16S rRNA based phylogenetic analysis revealed the relatedness of genus Rhizobium, Bacillus, Ralstonia, Burkholderia, cupriavidus and Lactococcus isolated from the root nodule of M. pudica. Our understanding of microbial interactions in the rhizosphere must be complemented by combining the basic and applied studies.
The beneficial effects of microbial inoculants, particularly nitrogen-fixing and plant growth promoters (PGP), from the root nodule of M. pudica accentuate the need for research and its application in modern agriculture. The present study is focused on the isolation, identification, characterization and comprehensive evaluation of phylogenetic relationship based on 16S rRNA gene of potential nitrogen fixers and plant growth promoters from root nodule of M. pudica.

isolation of nodule-associated bacteria
Bacterial isolates were obtained from the root nodules of M. pudica grown in different locations in the University of Calicut campus (11°08'01.0"N, 75°53'19.0"E; 11°08'00.4"N, 75°53'17.5"E). The nodule-associated bacteria (NAB), were isolated from healthy pink coloured root nodules, washed thoroughly using running tap water, surface sterilized using 70% (v/v) ethanol for 30s, 0.1% (w/v) HgCl 2 for 2 min and washed thrice with sterile double distilled water under aseptic condition for one min 14 . Nodules were crushed using a sterile glass rod and the extracts were plated onto Yeast Mannitol agar medium pH 6.8, supplemented with Congo red dye (0.025 gl -1 ). The cultures were incubated at 28±2ºC for 24-48hrs. Single colony-forming units were checked for purity by repeated transferring on to nutrient agar medium having pH-7 15 . Pure cultures were maintained on a nutrient agar medium with regular subculturing and used for analysis.

Phenotypic characterization
Phenotypic characterization based on the morphological and biochemical characters were done on bacterial isolates grown in nutrient agar medium using Bergey's manual of systematic bacteriology 16 . The morphological characters were listed using gram staining, motility test by hanging drop method and endospore staining by malachite green method using a phase contrast microscope. Biochemical analysis was performed using indole production, hydrolysis of urea, methyl red (MR) test, voges proskauer (VP), citrate utilization and nitrate reduction test. The intrinsic antibiotic resistance of the isolates was determined by the disc method with Ampicillin (Amp) (10 mcg/disc), Tetracycline (TE) (30 mcg/disc), and Penicillin G (PG) (10 IU/disc) 17 .

Molecular characterization dna extraction, 16s ribosomal rna typing and sequencing
Bacterial genomic DNA was extracted and purified using CTAB method 18 . The purified DNA was quantified using a Nanodrop 2000 spectrometer (UV scanning Thermo scientific). PCR amplification of the 16S rRNA gene was carried out using the universal primers 1-27F (AGAGTTTGATCCTGGCTCAG) and 1495R (CTACGGCTACCTGTTACGA) 19 . Amplification was performed in thermocycler with following PCR conditions: 30 cycles of 94 p C for 45 s, 50 p C for 1 min, and 72 p C for 1.30 min with initial denaturation at 94 p C for 3 min and final extension at 72 p C for 10 min. The band size was verified using agarose gel electrophoresis. The PCR products were cleaned and sequenced from Agrigenome Lab Pvt Ltd, Cochin, Kerala. Cloned 16S rRNA sequences were minimally edited and manually aligned using Bioedit software. Species identification and homology of the sequences were identified using BLAST (https://www.ncbi. nlm.nih.gov/BLAST/). The cloned 16S rRNA sequences were submitted to GenBank, NCBI and accession numbers were obtained.

Characterization of plant growth promoting potential of bacterial isolates
The plant growth enhancement potential of the four isolates was verified using their potential to produce indole acetic acid, organic acid and capacity to fix atmospheric nitrogen in plants.

Production of indole acetic acid (iaa)
IAA production capacity of the isolates was identified using bacterial cultures grown in nutrient broth supplemented with 0.1% L-Tryptophan (w/v) incubated at 30p C for 48hrs. Indole acetic acid (IAA) production was analysed using the colorimetric method of Gordon and Weber 20 . IAA in the culture was quantified using a standard calibration curve prepared using gradient concentrations of IAA.

Production of organic acid
Assessed by growing bacterial culture in calcium carbonate agar [CaCO 3 5 gl -1 , glucose 50 gl -1 , yeast extract 5 gl -1 , agar 15 gl -1 ] medium and the clear zone around the colony confirmed the production of organic acid.

16s rrna-based phylogenetic analysis
Phylogenetic analysis based on the16S rRNA sequence was performed using MEGA 7.0 program 22 based on neighbor-joining statistical method 23 and the branching support of 1000 bootstrap 24 . The phylogenetic tree construction based on 16S rRNA sequences from noduleassociated bacteria isolated from M. pudica was aligned using ClustalW. The model selection was performed using MEGA 7 22 based on the lowest Bayesian Information Criterion (BIC) value 25 .

statistical analysis
Using the SPSS software (27.0V, SPSS, Chicago, USA), one-way ANOVA was performed to analyze the concentration of IAA in the isolates after 48hrs. Statistical analysis was carried out according to Tukey's test (Pd"0.05). The data were an average of 4 separate experimental observations with three independent replicates (n=3).

results and disCussion isolation of root nodule associated bacteria
A total of 13 root nodule-associated bacteria were isolated from M. pudica. All the isolates were purified and subcultured on nutrient agar medium (pH-7). The isolated pure bacterial strains were characterized using morphological, biochemical and molecular techniques.
The nodule surface sterilization was aimed to allow the obtention of nodule-associated bacteria 26 resulting in the isolation of thirteen nodule-associated bacteria from the root nodule of M. pudica. Out of the 13 NAB obtained, nine were non-rhizobial nodule associated GenBank accession numbers provided for the 16S rRNA gene sequence of thirteen nodule-associated bacteria are given in Table 1.
Rhizobia are a functional class of soil bacteria having a nitrogen-fixing symbiosis with legumes, also termed legume nodulating bacteria (or LNB). The ability to nodulate legumes is spread among the alpha and beta-subclasses of Proteobacteria. Beta-rhizobia was originally described in 2001 in two parallel studies: the first study identified Burkholderia tuberum and B. phymatum from Aspalathus carnosa and Machaerium lunatum plant respectively which were belongs to the family Papilionoideae and the second study isolated R. taiwanensis from two Mimosa species which was later named as Cupriavidus taiwanensis 31 . Verma 32 has demonstrated the widespread occurrence of beta rhizobia as symbionts in Indian Mimosa species.
It has previously been documented that many non-rhizobial endophytes are often associated with root nodules of a variety of legumes 30, 15, 10 and the genetic diversity of these endophytes is often high 13,10 . Among these, Bacillus and Pseudomonas are particularly common 13, 10 and these genera are well-recognized for their roles in plant growth promotion and biocontrol over soilborne pathogens 34 . These two genera are also prominent among rhizoplane bacteria of a variety of plants. Thus, the high diversity of root nodule-associated bacteria in Mimosa and the predominance of Bacillus and Pseudomonas was not unexpected inside the M. pudica nodule, as seen in previous studies 4 .

Phenotypic characterization
Phenotypic characteristics such as shape, gram's reaction, motility and spore formation and biochemical characterization like indole production, hydrolysis of urea, MR-VP, citrate utilization, nitrate reduction and antibiotic sensitivity are presented in supplementary Except B. cereus CUMY2 were negative to citrate utilization. Of the thirteen bacterial isolates tested for antibiotic such as tetracycline (30 µg/disc), penicillin-G (10 IU/disc), ampicillin(10 mcg/disc) and erythromycin (15 µg/disc) showed atleast sensitive to one antibiotic.
There were many reports on the diversity of microorganisms in the rhizosphere, the present study revealed nodule bacterial diversity exists even among the organisms associated with the nodules. According to Rajendran 14 probably all the organisms whose presence has a beneficial relation might get associated with the root nodules. The isolated NAB showed 80% similarity in the biochemical features examined. The morphological and microscopic features of the isolates were in congruence with the earlier reports of the species. In agriculture, the use of PGPB as inoculants is widely applied but only limited studies addressed their antibiotic resistance. Thus, the best practice is to do that systematically, to limit antibiotic resistance gene (ARG) distribution into the environment 35 and also the use of high quality, effective rhizobia on agriculture have contributed significantly to the economy of farming systems through the biological nitrogen fixation in the rhizosphere. However, the rhizosphere comprises large populations of antibiotic-producing microorganisms, which affect susceptible rhizobia 36 . Thus, antibiotic resistance is an extremely valuable and positive selection marker to select symbiotically effective bacteria. Our findings show that all the isolates were sensitive to at least one standard antibiotics and can be used as a safe biofertilizer candidate. Characterization of plant growth promoting activities iaa production potential of the isolates IAA production during the 48hr of growth was quantified in Rhizobium sp. CU8, B. cereus MY5, R. pickettii MY1 and L. lactis MY3 using Salkowski reagent. R. pickettii MY1 and Rhizobium sp. CU8 developed colour immediately after the addition of reagents indicating the formation of IAA and better IAA production was observed when the cultures were incubated for 25 min in dark. The highest quantity of IAA was produced in R. pickettii MY1 (49.8630±0.1779 µg/ml) followed by B. cereus MY5 (13.5159±0.2416 µg/ml), Rhizobium sp. CU8 (11.6895±0.1837 µg/ml) and L. lactis MY3 (4.9315 ±0.0790 µg/ml) (Fig. 1) after 48hrs of incubation, which was significant at P<0.05.
A diverse group of microbes, including free-living, epiphytic and tissue colonizing bacteria synthesizes IAA 37 . The four strains produced a considerable quantity of IAA, which is comparable with earlier studies on various bacteria including Rhizobium sp., B. cereus, R. pickettii and L. lactis 38,39,40,41 . According to Datta and Basu 42 , most of the studies reported that IAA-producing organisms are gram-negative, however, few Bacillus are known to produce IAA which is gram-positive strains 43 . The present study showed that B. cereus MY5 is IAA-producing gram-positive bacteria.

Production of organic acid
Among the four isolates, L. lactis MY3 showed a clear zone after 24hrs of incubation due to the degradation of calcium carbonate leading to the production of organic acid. The other three isolates don't show any clear zone around the colony.
L. lactis is a rare observation from the root nodule of M. pudica and can be used as an agent for plant growth promotion 44 . L. lactis develop organic acid indicating the interactions between PGPR and plants can enhance the secretion of organic acids, which play an important role in the process of the activation and absorption of insoluble nutrients by plants 45 .

Nitrogen fixing potential of the isolates
The four isolates, Rhizobium sp. CU8, B. cereus MY5, R. pickettii MY1 and L. lactis MY3 exhibited N 2 fixing ability grown in nitrogen-free malate medium containing bromothymol blue as an indicator. The Rhizobium sp. CU8, B. cereus MY5 and R. pickettii MY1 showed a significant colour change from pale green to pale blue indicating N 2 fixing ability within 24hrs. However, L. lactis MY3 developed the colour change only after 48hrs.
The interaction between rhizobia and other nodule-associated bacteria is of high relevance due to the N 2 fixation and other plant growth promotion capacities in leguminous plants 46,26 Zhao 47 reported endophytic non-rhizobial Bacillus cereus and Ralstonia spp. are potent N 2 fixers. The genus Rhizobium is the first bacteria participating in nitrogen fixation in legumes 48 . According to Higdon 49 , Lactococcal bacteria exist as a diazotroph in maize without nifHDKENB homologs and hypothesized that L. lactis isolates from the mucilage microbiota of Sierra Mixe maize possess genes enabling BNF activity and elucidated that all the important genes for the BNF trait in L. lactis underpinning the ability to fix atmospheric nitrogen present in the mucilagederived Lactococci, which supports the hypothesis that Lactococci can exist as diazotrophs.

Phylogeny based on 16s rrna gene
The cloned 16S rRNA sequences were used to construct the phylogenetic tree using neighbor-joining (NJ) method with 1000 bootstraps. Models with the lowest BIC scores were considered to describe the best nucleotide substitution pattern. Bayesian Information Criterion (BIC) and Akaike Information Criterion (AIC) are the best-fit nucleotide-substitution models determined using MEGA 7.0. Models with the lowest BIC scores (Bayesian Information Criterion) are depicted as the best substitution pattern with 16S rRNA sequence of the 13 nodule-associated bacteria isolated from M. pudica provided TN93+I (Tamura 3-parameter model), with the lowest BIC score (11977.858), and lowest AIC score (11755.020).
The  (Fig. 2.). The optimal tree with the sum of branch length = 0.3866 is shown in Fig. 2. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The evolutionary distances were computed using the Tamura-Nei method and are in the units of the number of transitional substitutions per site. The analysis involved 13 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair. There were a total of 1482 positions in the final dataset. In NJ tree, the isolates in the first groups includes in the phylum Firmicutes and the Group II isolates belongs to phylum Proteobacteria. The branching where started from phylum to genus level.
The evolutionary history was derived using the neighbor-joining method and maximum Likelihood method based on the Tamura-Nei model 50 . The bootstrap consensus tree developed from 1000 replicates represented the evolutionary history of the taxa analyzed 24 . Branches corresponding to partitions reproduced in less than 50% of bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches 24 . The development of the bacterial taxonomy can be traced through earlier reviews by Jordan 51 , Graham 52 , Young 53 , Elkan 54 , and Martinez-Romero 55 , and the 13 nodule-associated bacterial isolates showed evolutionary relatedness and grouping in congruence with the bacterial taxonomic classification.

ConClusions
This study reports the isolation, molecular identification, characterization and phylogenetic relationship of the thirteen root nodule-associated bacteria of M. pudica. The biochemical analysis confirms the nitrogen-fixing potential, plant growth promotion and other potential microbial activities of the Rhizobium sp. CU8, B. cereus MY5, R. pickettii MY1 and L. lactis MY3. The bacteria with N 2 fixing capacity act as plant growth promoters and hence can be used as biofertilizers. L. lactis strain MY3 is a new report from the root nodule of M. pudica with plant growth promotion and N 2 fixation capacity. Phylogenetic analysis using neighbor-joining method showed the relatedness and evolutionary position of the isolates. The analysis showed that non-rhizobial bacteria, B. cereus MY5, B. cereus CUMY2, B. cereus MYB1, Bacillus sp. MYB5, Bacillus sp. MY2, Bacillus sp. CU2, Bacillus sp. CU3, B. thuringensis CUMY1, and L. lactis MY3 may co-exist with Rhizobium sp.CU8, Cupriavidus sp. MNMY3, Burkholderia sp. MY6 and R. pickettii MY1 in the root nodule of M. pudica. However, it requires further studies to assess the role of these isolates in N 2 fixation and plant growth promotion under pot culture as well as in field condition and these can be used as a potential biofertilizer.

aCKnowledgeMent
The author acknowledge the facilities provided by the Director, Interuniversity Centre for Plant Biotechnology, University of Calicut, for providing facilities

Conflicts of interest
The authors declare that they have no conflict of interest.

Funding source
This work was supported by the University of Calicut, Government of Kerala for the research grants (Grant numbers 11347/2016/Admn).

statement of informed consent
Authors declares that they have consented to participate in the manuscript and publish it.

ethical statement
This article does not contain any studies with human participants and/or animals performed by any authors. Formal consent is not required in this study.