Molecular Biotyping, Phylogenetic Analysis of Indole Acetic Acid Producing Rhizobium Sp. CU8 and Co-inhabitant Non-rhizobial Nodule Associated Bacteria From Mimosa Pudica L.

The interaction between rhizobia and other nodule associated bacteria assist to mitigate nutrient stress in leguminous plants by xing atmospheric nitrogen and synthesizing plant growth regulators. Benecial effects of microbial inoculants emphasize the need for further research and their use in modern agriculture. The present work describes the isolation, identication, plant growth promoting properties and phylogenetic analysis of nodule associated bacteria from Mimosa pudica L. Isolation and characterization of nodule associated bacteria were done according to standard procedures. Molecular characterization of the isolates was performed using 16S ribosomal RNA. Plant growth promoting ability was analyzed by quantifying the levels of Indole acetic acid. Evolutionary distance and relatedness was analyzed using neighbor joining method. Rhizobium sp. CU8 and three other co-resident non-rhizobial nodule associated bacteria (Bacillus cereus MY5, Ralstonia pickettii MY1 and Lactococcus lactis MY3) exhibiting nitrogen xation, plant growth promotion and other potential microbial activities were characterized. Phylogenetic analysis revealed the genetic relatedness and evolutionary signicance of Rhizobium sp. CU8 and other co-inhabitant non-rhizobial nodule associated bacteria from M. pudica. Present study identied the four isolates as potential biofertilizers due to their nitrogen xing and growth promoting characteristics. L. lactis MY3 is the rst co-resident nitrogen xer and plant growth promoter reported from the root nodules of M. pudica.


Introduction
The members of Leguminosae, are associated with endophytic, root nodule associated bacteria (NAB) which ameliorates nutrient stress by xing atmospheric nitrogen (N 2 ) and producing plant growth promoters. The genus Mimosa received considerable attention in the recent years because of its potential to x atmospheric nitrogen. Biological nitrogen xation 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 xation (both symbiotic and nonsymbiotic) reduces the use of synthetic nitrogen fertilizers.
Plant growth promoting bacteria (PGPB) enhance plant growth by xing 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 (Glick 1995;Kloepper 1992) and the production of substances like antibiotics (Glick 1995). In addition, the PGP microbes express abiotic stress tolerance like extreme temperature, drought, salinity, pH, heavy metal and pesticide pollution (Gopalakrishnan et al. 2015).
PGPB has bene cial effects on legume growth and some strains enhance the nodulation and nitrogen xation by effective interaction between plant and rhizobia (Parmar and Dadarwal 1999). Most of the nodulating bacteria are free-living rhizobacteria, however, some are intracellular or intercellular endophytes (Sturz et al. 2000) and gain advantage of being protected from environmental stresses and microbial competition (Kobayashi and Palumbo 2000). 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 bene t other than a noncompetitive environment inside the host. It has been demonstrated that bacterial endophytes have bene cial effects on host plants, such as growth promotion and biological control of pathogens (Downing and Thomson 2000;Ryu et al. 2005;Sturz et al. 1999).
Legume root nodules may contain microbes other than rhizobia (Martínez-Hidalgo and Hirsch 2017; Zgadzaj et al. 2015), however, the function of these co-residents in the nodules is yet to be fully elucidated, their main role might be to assist rhizobia during the nodule infection process and to promote plant growth (Chibeba et al. 2020;Peix et al. 2015).
Modern agriculture faces challenges, such as loss of soil fertility, uctuating climatic factors and increasing pathogen and pest attacks. Sustainability and environmental safety of agricultural production relies on eco-friendly approaches like use of biofertilizers, biopesticides and crop residue recycling. From the agricultural and ecological viewpoints, "how can we increase food quality and quantity, and to improve the sustainable plant productivity, while maintaining environmental quality?" the importance of nitrogen xing and plant growth promoting bacteria, their gene conservation can contribute better in sustainable agriculture and 16S ribosomal RNA typing is used to identify microorganisms. The 16S rRNA based phylogenetic analysis revealed the distribution of genotypes in Rhizobium sp., B. cereus, R. pickettii and L. lactis. Our understanding of microbial interactions in the rhizosphere must be complemented by combining the basic and applied studies.
The bene cial effects of microbial inoculants, particularly nitrogen xing 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, identi cation, characterization, screening and comprehensive evaluation on phylogenetic relationship based on 16S rRNA gene of potential nitrogen xers and plant growth promoters from root nodule of M. pudica.

Materials And Methods
Isolation of nodule associated bacteria Bacteriae were isolated from the root nodules of M. pudica grown in different locations in the University of Calicut campus (geographic coordinates: 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 (Rajendran et al. 2012). Nodules were crushed using a sterile glass rod and the extracts were plated on to 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 transfer on to nutrient agar medium pH-7 (Vincent 1970). Pure cultures were maintained on nutrient agar medium with regular subculturing and used for analysis.

CHNS analysis
Soil samples collected from the rhizosphere of M. pudica was used for CHNS elemental analysis using a Euro Vector CHNS Analyser Model No: Ea 3000. Instrument parameters were carrier pressure 100kPa, purge ow 80 mL/min, oxygen ow 20 mL and run for 7 min. Temperature of front furnace was 980ºC and GC oven temperature was 105ºC. Sample (8.5-9.5 mg) was used for the analysis.

Phenotypic characterization
Bacterial isolates grown in nutrient agar medium were subjected to phenotype characterization based on the morphological and biochemical characters examined using Bergey's manual of systematic bacteriology (Bergeys 2001). The morphological characters were analysed 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 disc method with Ampicillin (Amp) (10 mcg/disc), Tetracycline (TE) (30 mcg/disc), and Penicillin G (PG) (10 IU/disc) (Cappuccino and Sherman 1983).
Molecular characterization DNA extraction, 16S ribosomal RNA typing and sequencing Genomic DNA of the three species was extracted and puri ed using CTAB method (Ausubel et al. 1995). The puri ed DNA was quanti ed using a Nanodrop 2000 spectrometer (UV scanning Thermo scienti c).
PCR ampli cation of the 16S rRNA gene fragment was carried out using the universal primers 1-27F (AGAGTTTGATCCTGGCTCAG) and 1495R (CTACGGCTACCTGTTACGA) (Lane 1991) at an annealing temperature of 50 C. The band size was veri ed 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 identi cation and homology between the sequences were identi ed using BLAST (https://www.ncbi.nlm.nih.gov/BLAST/). The cloned sequences were submitted to GenBank, NCBI.
Characterization of plant growth promoting potential of bacterial isolates Plant growth enhancement potential of the four isolates was veri ed by the production of indole acetic acid, organic acid and capacity to x atmospheric nitrogen in plants.

(a). Production of indole acetic acid (IAA)
The isolates with IAA production capacity was identi ed using bacterial culture grown in nutrient broth supplemented with 0.1% L-Tryptophan(w/v) incubated at 30 C for 48hrs. Indole acetic acid (IAA) production was analysed using colorimetric method of Gordon and Weber (1951). IAA in the culture was quanti ed using standard calibration curve prepared using gradient concentrations of IAA.  (Felsenstein 1981) and the branching support of 1000 bootstrap (Felsenstein 1985). The phylogenetic tree construction based on, 16S rRNA was initially performed using the cloned sequence along with sequences retrieved from GenBank and aligned with ClustalW. The model selection was performed using MEGA 7 (Kumar et al. 2016) based on the lowest Bayesian Information Criterion (BIC) value (Schwarz 1978). To compare the similarity or diversity, the nucleotide sequences of 40 selected Rhizobium, Bacillus, Ralstonia, Lactococcus species from different geographical regions were retrieved from NCBI, GenBank (http:/www.ncbi.nlm.nih.gov/ ). The list of sequences retrieved from NCBI with strain name, accession number and locations are given in online resource 1.

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 Turkey's test (P≤0.05). The data were an average of 4 separate experiments with three replicates (n=3).

Isolation and CHNS elemental analysis
Four nodules associated bacteria were isolated from the healthy nodules of M. pudica. The isolates were puri ed and subcultured on nutrient agar medium (pH-7). CHNS analysis of the rhizosphere soil showed nitrogen (0.274%), carbon (1.534%), hydrogen (2.094%) and sulphur (0%). The soil was acidic to slightly neutral with a mean pH value of 6.49 ± 0.1.

Phenotypic characterization
Phenotypic characteristics of the four isolates are presented in Table 1.

Phylogeny based on 16S rRNA gene
The cloned sequence and the sequences retrieved from the GenBank 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. The TN93 + G (Tamura Nei Model) displayed the lowest BIC scores (10125.617-online resource 2) to construct consensus NJ tree from the aligned sequences (Fig. 2). The multiple sequence alignment based phylogram using MEGA 7.0 and TN93 + G model based on bootstrap analysis of 1000 replicates was performed to estimate the con dence of the tree topologies. The phylogenetic position of the Rhizobium sp. CU8, B. cereus MY5, R. pickettii MY1 and L. lactis MY3 in relation to other species of this genus is illustrated in Fig. 2; the numbers adjacent to nodes are the statistical frequency of the indicated species.
The genus Rhizobium and Ralstonia fall under the same phylum proteobacteria classi ed as subclass α and β proteobacteria. Based on 16S rRNA homology, both genera are placed in separate group originated from a single node. L. lactis and B. cereus were grouped as a separate clade.
Phylogenetic tree revealed that Rhizobium sp. CU8 and B. cereus MY5, showed highest relatedness with other members of the genus. Rhizobium sp. CU8 shows closest relatedness with Indian Rhizobium sp. S19. B. cereus MY5 shows highest similarity to B. subtilis IMG04 from India among other members of this genus. In the case of R. pickettii MY1 and L. lactis MY3, the maximum similarity of these two native strains were shown to R. pickettii CP12 from China and L. lactis KUMS-T18 from Iran.
Group I Rhizobium sp. CU8 and Group II Ralstonia pickettii MY1 showed 100% bootstrap support within the genus level. L. lactis MY3 in Group III is tightly clustered with L. lactis KUMS-T18 with bootstrap support 98%. In Group IV, B. cereus MY5 clustered with B. subtilis IMG04 with bootstrap support (> 50%). However, none of the isolates that clustered in different phylogenetic trees based on 16S rRNA analysis.

Discussion
The interaction between rhizobia and other nodule associated bacteria are of relevance in the improvement of N 2 xation and plant growth promotion in leguminous plants ( Barea  The morphological and microscopic features of the isolates were in congruence with the earlier reports. All the isolates were sensitive to standard antibiotics suggesting their prevention mechanisms. The isolates, R. pickettii MY1, L. lactis MY3, B. cereus MY5 and Rhizobium sp. CU8 exhibited important agrobiotechnological properties, such as nitrate reduction, production of organic acid, indole acetic acid and ability to x N 2 . Interestingly, urease activity was also observed in the isolates, indicating the importance of consortia with N 2 xing bacteria as a requirement for survival (Chibeba et al. 2020).
The present result con rms the nitrogen xation ability of R. pickettii MY1, B. cereus MY5, Rhizobium sp. L. lactis MY3 is a rare observation from the root nodule of M. pudica and can be used as an agent for plant growth promotion (Lamont et al. 2017). According to , Lactococcal bacteria exists 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 x atmospheric nitrogen present in the mucilage-derived lactococci, which supports the hypothesis that lactococci can exist as diazotrophs. L. lactis MY3 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 (Pii et al. 2015).
A diverse group of microbes, including free living, epiphytic and tissue colonizing bacteria synthesize IAA (Patten and Glick 1996 Datta and Basu (2000), most of the studies reported that IAA producing organisms are gram negative, however, few Bacillus known to produce IAA which is gram positive strains (Wahyudi et al. 2011). Present study showed that B.cereus MY5 is IAA producing gram positive bacteria.
The evolutionary history was derived using the neighbor joining method based on the Tamura-Nei model (Tamura and Nei 1993). The bootstrap consensus tree developed from 1000 replicates represented the evolutionary history of the taxa analyzed (Felsenstein 1985). Branches corresponding to partitions reproduced in less than 50% 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 (Felsenstein 1985). Rhizobium sp. CU8 and B. cereus MY5 showed highest similarity with native strains.

Conclusions
This study reports the isolation, molecular identi cation, characterization and phylogenetic relationship of the Rhizobium sp. CU8, R. pickettii MY1, L. lactis MY3, and B. cereus MY5 from the root nodule of M. pudica. The biochemical analysis con rms the nitrogen xing potential, plant growth promotion and other potential microbial activities of the obtained isolates. The bacteria with N 2 xing capacity act as plant growth promoters 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 xation capacity. Phylogenetic analysis using neighbor joining method showed the relatedness and evolutionary position of the isolates with native strains as well as other geographical locations retrieved from NCBI. The analysis showed that non-rhizobial bacteria, L. lactis MY3 and B. cereus MY5 may co-exist with Rhizobium sp. CU8 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 xation and plant growth promotion under pot culture as well as in eld condition and these can be used as a potential biofertilizer.