Identification and molecular phylogeny analysis using random amplification of polymorphic DNA ( RAPD ) and 16 SrRNA sequencing of N 2 fixing tea field soil bacteria from North Bengal tea gardens

Random amplification of polymorphic DNA (RAPD) amplification genomic DNA of 23 selected laboratory cultures of bacteria using RAPD revealed their polymorphism. Polymerase chain reaction (PCR) amplification of the bacterial 16SrDNA was performed using 704F GTAGCGGTGAAATGCGTAGA and 907R CCGTCAATTCCTTTGAGTTT primer, sequenced and accessed in NCBI (No. KY636356, KY631488, KY 860028, KX587470, KX665547, KY631489, KX608591, KY636360, KY671245, KY631490, KX587469, KY859856, KX665546, KX608590, KX587468, KY859798, KY636357, KY636361, KY 636359, KY631491, KY 859855, KY636358, KY636362) after submitting the contig. FASTA sequence in NCBI database was seen. All most all 23 bacterial strains (viz. TS-1-16, TS-4-23 DJ-1-22, DS-1-20, AS-1-4, DJ1-24 , DJ-1-10, DJ-1-46) showed strong homology with free living nitrogen fixing soil bacteria, also showing (98 to 100%) identity and E-value of 00 with Burkholderia spp, Strain-S-9-19, Str-S-9-15, SP2386, Stenotrophomonas maltophilla, strn-MM-3-3, Str-D-3,LP-05, Bacillus cereus Strn-FORC021and Azospirillum sp TSH51 gene, having good nitrogen fixing capacity. Phylogenetic tree analysis among the 23 isolates and between the different strains from GenBank showed close similarity. Most of the isolated bacterial strain identified as a member of the genus Burkholderia sp, Stenotrophomonas maltophila, Herbaspirillum sp, Acinetobacter johnsonii, Methylobacter, SP-T-20, and Bacillus cereus, Azospirillum sp would be consider to be the most suitable bioferiliser for organic and conventional tea gardens of North Bengal, India.


Introduction
Tea Camellia sinensis (L) O. "Kuntze" of family Theaceae is the most commonly used beverage in India and in the world.Tea is an evergreen shrub that mainly grows in tropical and subtropical areas.It is thought to have originated in East Asia somewhere between China and Burma.India is the world's second largest tea producer country next to China.In the financial year 2015 to 2016, India has recorded tea production of 1,233 million kg (mn kg) and exports crossing 230 mn kg after 35 years.The top five teas producing countries are China, India, Kenya, Sri Lanka and Turkey (http://www.gktoday.in/blog/keyfacts-about-tea-production-in-india/).India has around 563.98 thousand hectares of tea cultivated land (December 2013).Assam is the highest Indian agricultural soil that contains low nitrogen and cellulose, and therefore the self-sustaining free-living nitrogen-fixing micro flora would be of great advantage if their identity is known and their ability is properly exploited.The reduction of chemical fertilizers by the application of biological fertilizers is mainly based on the bacteria involved in nitrogen fixation as one of the suitable steps in sustainable agriculture (Vejan et al., 2016).
In recent times, PGPR got more attention and it has been used as potent biofertilizers (Richardson et al., 2009;Compant et al., 2010) as prolonged use of chemical fertilizers is perilous to soil, as well as, human health and also detiorate the crop quality (Islam et al., 2013).Alternative biotechnological approaches are adapted in different agriculture practices to not only increase the crop production and plant growth, but also to maintain soil health.It has been reported that inoculation of Azospirillum biofertiliser or liquid near the rhizosphere of tea significantly increased growth.Although, research about PGPR impact on the tea plants is still poorly organized, especially in the Northeast region of India including North Bengal tea growing region.The productivity of tea is decreased remarkably due to intensive application of chemical fertilizers for a prolonged period (Sharma et al., 2014).
Therefore, there is a growing demand to explore the indigenous micro flora associated with the tea rhizosphere soil not only to reduce the application of chemical fertilizer, but also for the benefit of plant, soil health and the environment.The 16S rRNA represents the right candidate to study bacterial evolution, ecology, phylogenetic relationships among taxa, bacterial diversity and quantification of the relative abundance of taxa of various ranks (Hugenholtz et al., 1998).Whereas, random amplified polymorphic DNA (RAPD) fingerprinting explores genetic polymorphisms (Teaumroong and Boonkerd, 1998) in bacteria.RAPD fingerprinting has been used for strain identification and to determine the genetic diversity within a field population of pinkpigmented facultative methylotrophs (Balachandar et al., 2008), Rhizobium isolates (Rajsundari et al., 2009), Photorabdus and Xenorabdus isolates (Moghaieb et al., 2017).
There was scanty report on molecular identification of free living N 2 fixing PGPR of North Bengal tea gardens of West Bengal.A preliminary investigation on isolation and characterization of free-living soil bacteria from tea gardens of Terai, Dooars and Darjeeling district West Bengal have been carried by the present research group.Morphological and biochemical evaluation of free living N 2 fixing tea rhizospheric and tea soil bacteria of North Bengal tea gardens has also been investigated (Bhaduri et al., 2018).
Keeping the background information, the present study has been undertaken for molecular identification and to understand the genetic diversity of free living N 2 fixing soil bacteria from tea garden soil of North Bengal to be used as biofertiliser.

MATERIALS AND METHODS
Pure cultures of previous study (Bhaduri et al., 2018) were used as experimental sample for this investigation, 23 strains for 16SrRNA analysis and 22 strains for RAPD analysis (El-Fiki, 2006).

Isolation of genomic DNA
Genomic DNA was isolated from selected pure bacterial isolates of three different region sample screened on the basis of salt tolerance, antibiotic resistance total N content, etc. following the cetyl trimethylammonium bromide (CTAB) method (Gomes et al., 2000).The isolated genomic DNA was treated with RNase and then subjected to Agarose Gel (0.8%) electrophoresis to check the purity of DNA.

RAPD
Random Amplification of Polymorphic DNA of selected strains to observe the genetic variability between them was carried out at Xcelris Lab.Ahmedabad, Gujrat using two RAPD primer P1v(5' to 3'): GTG TGT GTG TGT GTG TGT GT, (20) nts., El-Fiki 2006 and P2:OPQ1 (5' to 3'): GGGACGATGG (10) nts (Balachandar et al., 2008;Rajsundari et al., 2009;Moghaieb et al., 2017).PCR was carried out in a final reaction volume of 25 μl in ABI Veriti Thermal Cycler.Amplification reactions were performed in a 25 μL volume, containing: 20 mM Tris-HCl (pH 8.4), 50 mMKCl, 2.5 mM MgCl2, 200 μM each of dNTPs, 1 μM primer, 30 ng of genomic DNA and 1.5 U of Taq DNA polymerase.The reaction mixture was flooded with two drops of mineral oil, initial denaturation for 5 min at 95°C, the amplification, then continued for 35 cycles consisting of 30 s at 94°C, 30 s at 36°C and 60 s at 72°C followed by a 7 min final extension at 72°C.Amplification product was separated by gel electrophoresis on precast 1.2% agarose gel and visualized under ultra-violent (UV) illumination after staining with ethidium bromide and Gel Documented on Gel Documentation System (Lee et al., 2012).

PCR amplification of 16SrRNA gene
PCR amplification of 16SrRNA was performed using 8F AGAGTTTGATCCTGGCTCAG.

Sequencing of 16S rRNA
The PCR amplicon (1.4 kb approximately) was purified with ExoSap enzymatic purification as per the manufacturer , s instruction (ABI).After the purification, the products were subjected to Sanger sequencing using ABI, 3730XL DNA analyzer using BdT v3. 1 chemistry.Each forward and reverse reaction of PCR amplified products were sequenced separately.Forward and Reverse DNA sequencing reaction of PCR amplicons of respective samples was carried out using BDT v3. 1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer.

Construction of phylogenetic tree
The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura et al. ( 2004) model.Evolutionary genetics analysis uses maximum likelihood, evolutionary distance, and maximum parsimony methods were conducted in MEGA 5 software (Tamura et al., 2011).The RAPD profile derived phylogeny was performed by Xcelris Lab.

RESULTS AND DISCUSSION
Purified genomic DNA isolated from bacterial strains after resolving in 0.8% agarose gel reveals their good yield and large genome size (Figure not shown).

PCR amplification of 16SrRNA gene and sequencing
Twenty three isolated bacterial genomic DNA was Bhaduri et al. 657 amplified with forward and reverse sequencing primer.PCR amplified fragments of approximately 1.4 kb in size are sequenced.

GenBank accession followed by homology searching
Twenty three (23) GenBank Accession were obtained after submitting the contig FASTA sequence.Most of the 23 isolated bacterial strain demonstrated strong homology with known nitrogen fixing bacteria.1).

Evolutionary genetics analysis and phylogenetic tree
Phylogenetic tree analysis between the 23 isolated strains showed close similarity among the strains.The tree reveals that there are 8 main groups consisting of two closely related strains.The group 1 consists of strains of TS-4-23 and DS-1-16 which resembles the Burkholderia sp functioning as a free living N 2 fixer and belonging to PGPR activity (Hayat et al., 2010).
Grou p 2 contained strains of TS-4-12 and DJ-1-46 which resembles the Burkholderia cepacia and Azospirillum sp TSH51 gene, which solely functions as a free living N 2 fixer and displaying PGPR activity.Group 3 contained strains of DJ-1-3 and DJ-1-22 which resembles the Burkholderia sp different strain which solely functions as a free living N 2 fixer and PGPR activity.Group 4 contained strains of AS-4 and DJ-1-24 which resembles the S. maltophilla different strain which solely functions as a free living N 2 fixer and PGPR activity (Fouzia et al., 2015).Group 5 contained strains of TS-3-15 and DS-1-20 which resembles the S. maltophilla different strain which solely functions as a free living N 2 fixer and PGPR activity.Group 6 contained strains of TS-3-27 and DJ-1-10 which resembles the Bacterium str-CH-2 and Bacillus cereus strn-FORC021which solely functions as a free living N 2 fixer and PGPR activity.Group 7 contained strains of TS-4-16 and DS-2-10 which resembles Herbaspirillum sp different strain which solely functions as free living as well as endophytic (certain strain) N 2 fixer and PGPR activity in tea plant (Zhan et al., 2016).
Group 8 contained strains of DS-2-8 and DS-2-9 which resembles Methylobacter, SP-T-20 and uncultured Ralstonia sp., Clone-3P-3-2 which solely functions as a free living N 2 fixer and PGPR activity.The other seven strains are distantly related to these clusters having nitrogen fixing and plant growth promoting activity (Figure 1) (Hayat et al., 2010).

RAPD
RAPD analysis of isolated bacterial genomic DNA reveals a little polymorphism pattern (Figure 2).Among the two primers tested only primer P1 was proper for amplification.The bacterial isolates DS-2-10; DS-2-8; DS-1-25; TS-4-24 gave no response  at RAPD amplification and rest of the 18 isolates showed amplification.The DNA amplified fragment varied in size ranges from 100 bp to 1.5 kb.The dendrogram result of polymorphic band showed similarity between the organisms as exhibited by 18 strains (Figure 3).The strain DS-1-20 is closely related to the cluster of DS-1-18, TS-4-12, DS-1-16 and DS-1-26.The strain AS-1-4 shows similarity to cluster containing TS-1-6 and TS-4-23, the former is closely related to the cluster formed by TS-3-27, DJ-1-46 and DS-2-9.The strains DJ-1-10 and TS-3-15 are closely related to each other and distantly related to a cluster formed by AS-1-2 and DJ-1-24, the latter two are related to a cluster formed by DJ-1-22 and DJ-1-3.AS-1-1 strain could not produce polymorphism and hence cannot relate to the cluster (Figure 3).
The genus Stenotrophomonas comprises of about eight species.Strains of the most common species, Stenotrophomonas maltophilia, have a function that includes beneficial effects of plant growth (Ryan et al., 2009).S. maltophilia is an ubiquitous, aerobic, nonfermentative and Gram-negative bacillus that is closely related to the Pseudomonas species (Calza et al., 2003).The genus Stenotrophomonas has pathogenic effect and they are resistant to certain antibiotics and susceptible to Chlorampenicol which we have already investigated in our previous study.Nahi et al. (2016) studied the effect of herbicide on nitrogenase and N 2 fixing capacity of Stenotrophomonas maltophilia (Sb 16).Bacteria of the genus Azospirillum (α-subclass of Proteobacteria) are known as plant growth promoting rhizobacteria (Okon, 1994.They were isolated from the rhizosphere of many grasses and cereals all over the world, in tropical as well as in temperate climates (Patriquin et al., 1983).Due to cell shape, growth behavior and habitat within grass roots, genus Herbaspirillum were previously thought to be a new Azospirillum species.However, RNA-RNA hybridization experiments reveal no relationship with Azospirillum spp or Aquaspirillum itersonii (Falk et al., 1986).Herbaspirillum seropedicae, Herbaspirillum frisingense and Herbaspirillum lusitanumable are reported to fix nitrogen (Baldani et al., 1986;Kirchhof et al., 2001;Valverde et al., 2003).The endophytic Herbaspirillum sp WT00C isolated from the tea plant, seems to have a potential ability to promote tea-plant rooting and budding due to its capability of producing indole-3-acetic acid (IAA), ammonia and siderophores (Zhan et al., 2016).Bacterial species of the genus Acinetobacter are ubiquitous in nature (Bergogne-Berezin and Towner, 1996).
In recent years, members of the genus Acinetobacter have been isolated from the rhizo-sphere of different plants ( Kuklinsky-Sobral et al., 2004;Roberts et al., 2005;Nakayama et al., 2007;Li et al., 2008).In India, A. indicus was described for the first time in soil samples collected from hexachlorocyclohexane dump sites (Malhotra et al., 2012).Strains belonging to the genus Acinetobacter, and their plant growth-promoting properties have been reported in the literature (Sachdeva et al., 2010).The presence of different species of Acinetobacter was worked in the rhizosphere of three agricultural wheat fields of Pune, India.The genetic diversity of Acinetobacter species using metagenomics study in the wheat rhizosphere was assessed by denaturing gradient gel electrophoresis (DGGE) of 16 SrRNA genes PCR products.Plant growth-promoting traits such as nitrogen fixation, siderophore production and mineral solubilization were reported in in vitro culture of Acinetobacter isolates (Sachdeva et al., 2010).From the perusal of literature it has been revealed that, in India no work has been done with Acinetobacter sp in tea field soil for their study related to biofertiliser or PGPR.Auman et al. (2001) reported nitrogenous and utilize N 2 as a nitrogen source by some methane-oxidizing bacteria (methanotrophs).There are two types of methanotrophstype I and type II.Type II methanotrophs and members of the type I genus Methylococcushave been shown to be capable of nitrogen fixation, while type I methanotrophs are not (Dedysh et al., 2000;Murrell and Dalton, 1983;Oakley and Murell, 1988).The genus Ralstonia established in 1995 by Yabuuchi et al. (1995) accommodate species previously known as Alcaligeneseutrophus, Pseudomonas solanacearum and Pseudomonas pickettii.Ralstonia eutrophaisolated from sludge, soil and R. basilensis from waste-water (Steinle et al., 1998).Chen et al. (2001) isolated several strains of Ralstonia from Mimosa as a symbiont nitrogen fixer, the most promising one is Ralstonia taiwanensis, cells are Gram negative, non spore forming rod shaped and mean cell size which ranges from 0.5 to 0.7 m width and 0.8 to 2.0 m in length (Chen et al., 2001).Gulati et al. (2011)  fixing as well as PGPR activities as evident from homology searching can be used as potent bioferiliser for organic and conventional tea gardens especially in North Bengal.The present investigation will suggest an insight to the tea growers of North Bengal and researchers as readily established "Bio accelerant" or" Bio fertiliser".

Conclusion
Since tea is a non-leguminous plant, the search for free living N 2 fixing soil bacteria in tea growing areas is gaining momentum day by day.Few good strains have been identified to be used as a potential N 2 fixer in tea field.The isolated bacterial strain identified as a member of the genus Burkholderia sp, Stenotrophomonas maltophila, Herbaspirillum sp, Acinetobacter johnsonii, Methylobacter, SP-T-20, and Bacillus cereus, Azospirillum sp can be the Bhaduri et al. 661 right candidates as potent bioferiliser for organic and conventional tea gardens of North Bengal, India.Hence, it is evident from the homology searching that our isolated strain would be ascribed as a member of the respective genus until and unless DNA-DNA hybridization and other biochemical parameter have been tested.The study reveals a thorough investigation regarding the molecular identification of free-living N 2 fixing bacteria; however, their field application is still needed in further study.

Figure 1 .
Figure 1.Phylogenetic tree between the 23 isolated bacterial strain showing homology (interrelationship) between them and highest matching with the GenBank Accession in National Center for Biotechnology Information.

Figure 3 .
Figure 3.Total homology analysis of 22 Bacterial Genomic DNA derived from RAPD profile showing their genetic relatedness.

Table 1 .
Homology and annotation of 23 GenBank accession of isolated N2 fixing Bacterial strain.