Sites and isolation of endophytic bacteria

Endophytic bacteria were isolated from surface-sterilized sodicity tolerant polyembyonic mango root stock ML-2 and GPL-1 (the plants can grow normally at the level of pH-9.53 and electrical conductivity EC- < 4.0). The rhizosphere soil total Na⁺ and K⁺ concentrations of GPL-1 and ML-2 were 21.20 meq / L; and 0.126 meq / L respectively. Two tolerant polyembryonic mango plants were collected from the experimental farm of Central Soil Salinity Research Institute Regional Research Station, Lucknow, India, located at 260 47?45?? to 260 48?13?? N latitude and 800 46?7?? to 800 46?32?? E longitude, lying in the Central part of Indo-Gangetic plain, in June 2012. Roots, stems and leaves from each plant were separated and washed extensively first in several changes of 0.01 M EDTA and then in distilled water to remove any non-specifically bound Na⁺ / K⁺ and dried to constant weight. Dried plant tissues were ground into a fine power, and digested with HNO₃:H₂SO₄:HClO₄ (10:1:4). Digested material was diluted to 10 ml and the sodium (Na⁺) / potassium (K⁺) content was analyzed with the help of a flame photometer (Furman, 1975). Plant samples were washed with tap water followed by three rinses with deionized water and then separated into roots, stems and leaves. Healthy plant tissues were sterilized by sequential immersion in 75% (v/v) ethanol for 2 min, and 1% mercuric chloride for 1 min and washed three times with sterile water to remove surface sterilization agents. To confirm the surface disinfection process was successful, water from the final rinse was plated on Nutrient agar. Plant tissue materials (0.2 g) were ground by a mortar and pestle in the presence of 5 ml of sterile distilled water. Serial dilutions were spread on plates containing 0.1 to 3.0 M range NaCl₃ supplement of nutrient agar medium with or without NaCl₃. The medium was found to be most suitable for the isolation of sodium tolerant bacteria in preliminary experiments. NaCl₃ resistant colonies were picked randomly and purified by streaking three to four times on the same media. Sixteen endophytic bacterial isolates growing well on sub culturing were finally selected and stored on slants for further study.

Analysis of extracellular enzyme activity

Endophytic bacterial isolates were analysed for production of four enzymes under (0.1, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 M) NaCl stress, that is, protease, amylase, cellulase and lipase by plate methods. Proteolytic activities of the cultures were studied in a medium containing skimmed milk (HiMedia, Mumbai). Zones of precipitation of paracasein around the colonies in the next 48 h were taken as evidence of proteolytic activity. The presence of amylolytic activity was determined using starch agar medium (HiMedia, Mumbai). After inoculation of endophytic bacteria and incubation at 28^°C for 5 days, the plates were flooded with 0.3% I^­2 and 0.6% KI solution and a clear zone around the growth indicated hydrolysis of starch. For cellulase activity, mineral-salt agar plate 5 containing 0.4% (NH₄)₂SO₄, 0.6% NaCl, 0.1% K₂HPO₄, 0.01% MgSO₄, 0.01% CaCl₂ with 0.5% carboxymethylcellulose and 2% agar (HiMedia, Mumbai) were surface inoculated. Iodine solution was used to detect cellulase activity as described.

Growth pattern of effective isolates at different NaCl concentrations

Growth of salt tolerant bacteria was measured at different NaCl concentration (0.1, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 M). L-Broth having different NaCl concentrations was prepared and autoclaved. For preparation of inoculum, bacterial cells harvested from overnight culture were washed and resuspended in sterile distilled water to adjust the OD at 106 cells per ml at 600 nm. 100 μl of inoculum was given in all media having different NaCl concentrations and incubated at 37°C on shaker with 150 rpm shaking for 24 h. Bacterial growth was monitored at 600 nm (Spectrophotometer S200, R & M Marketing, England). Three replicates per strain were taken for each salt concentration.

Vigour index studies

Plant growth promoting (PGP) activities of the current strains were assessed using seedling vigour index by pot culture studies in saline sodic soils of pH 9.40, electrical conductivity (ECe) 6.72 dSm^-2; sodium (Na⁺) 21.20 meq / L. The vigor index was calculated by using the formula as described by Abdul-Baki and Anderson (1973).

Vigor index= Percent germination x Seedling length (shoot length + root length)

Molecular profile of endophytic bacterial

Bacterial DNA from the elite strains showing higher extra cellular activities were extracted according to the method given by Araujo et al. (2002), with amplification of 16S rDNA being performed in a 25 μl reaction mixture, which consisted of 25 ng genomic DNA, 10× reaction buffer with 15 mM MgCl₂, 2.5 mM each of dNTPs, 0.2 mM bacterial universal primer (27F-AGAGTTTGATCMTGGCTCAG) (1492R-GGYTACCTTGTTACGACTT), and 1 unit of Taq DNA polymerase (Bangalore Genei Pvt Ltd, India). The reaction was carried out in a thermal cycler (PTC-200, MJ Research Inc, USA). The DNA amplification program was set to a 5 min initial denaturation at 94°C followed by 30 cycles of 1 min at 94°C for denaturation, 1 min at 54°C for annealing, 2 min at 72°C for extension, and ended with a final 10 min extension at 72°C. The reaction products were separated by running 5 μl of the polymerase chain reaction mixture in 1.2% (w/v) agarose gel and staining the bands with ethidium bromide (Sambrook et al., 1989). The Restriction Fragment Length Polymorphism (RFLP) assay involved restriction enzyme digests and the 16S rRNA gene PCR products using five different enzymes. Reactions were performed in a 30 μl reaction mixture, which consisted of 10 μl of each PCR product, 2 μl of 10× Fast digest green buffer, 17 μl nuclease free water and 1 μl (1FDU) for restriction enzyme cleavage by either AluI, Hae III, EcoRI, MspI or BamHI (Fermentas, USA). Digestions were performed for 5 min at 37°C to ensure that complete fragmentation was achieved. Digested DNA fragments were analysed in a 1.5% (w/v) agarose gel with 10 mg / ml of ethidium bromide and photographed on a gel documentation system. For cluster analysis, the data were converted to a binary matrix, where the digit 1 or 0 represented the presence of or absence of DNA band in the gel. NTSYSpc 2.20 (Exeter software, New York) was used to analyze the fingerprinting. The data were analysed using the neighbor-joining tree construction method to generate the Dice similarity coefficient. Similarity coefficients were used to construct the dendrogram using the Dice coefficient in clustering (Damodaran et al., 2012). Statistical stability of the branches in the cluster was estimated by bootstrap analysis using Winboot software program (Yap and Nelson, 1996). The principle co-ordinate analysis was performed with NTSYS software with the Dice (1945) coefficient.

Sequencing and phylogenetic analysis

Purified PCR products from the 16S rRNA genes of pure culture isolates representing all major RFLP patterns were sequenced on an ABI3730 automated sequencer (Applied Biosystems, USA) with primers 27 f and 1492 r. All reference sequences were obtained from the National Center for Biotechnology Information (NCBI) and the 16S rDNA similarity sequences searches were performed using the BLASTN tool in the NCBI website. These sequences were aligned using the multiple sequence alignment program, CLUSTAL X (Thompson, 1996). The method of Jukes and Cantor (1969) was used to calculate evolutionary distances. A phylogenetic dendogram was constructed by the neighbor-joining method and tree topologies were evaluated by performing bootstrap analysis of 1,000 data sets using MEGA 3.1 (Molecular Evolutionary Genetic Analysis).

Nucleotide sequence accession numbers

The sequence obtained in this study was deposited in the GeneBank nucleotide sequence database under the accession numbers (KC433665 and KC433667).

Principle coordinate analysis

The positioning of 16 bacterial endophytes based on principle coordinate 1 and 2 is given in Figure 3. The scatter plot of Dice similarity showed three major groups of bacterial endophytes belonging to salt tolerant endophytes (group I) and those below 1.0 M NaCl concentration and negative for hydrolysis enzyme activity (group II, group III). Three bacterial endophytes (CSR-M-03, CSR-M-07, CSR-M-12) separated from the three groups. The principle coordinates 1 and 2 encompassed 59.42 and 38.96% of total variation, respectively.

Confirmation of enzyme producing salt tolerant endophytes

Identification of salt tolerant enzyme producing strains clustered in group I were verified by sequencing the 16S rRNA genes and validated with BLAST (ncbi.nlm.gov.blast/Blast.cgi) database. This method was carried out as follows: DNA was extracted from the isolates by a standard kit (Xcelgen-Germany). Then amplification of 16S rRNA gene (27F-AGAGTTTGATCCTGGCTCAG; 1492R-ACGGCTACCTTGTTACGACTT) was performed by PCR method and eventually the products were sequenced on an ABI 3730xl Genetic analyzer (Applied Biosystems, USA). The results (Figure 4) show that all of the potential strains belonged to the genus Bacillus, with the strain CSR-M-06 identified as Bacillus sp., CSR-M-08 as Bacillus clausii, CSR-M-09 as Bacillus pumilus and CSR-M-16 as B. licheniformis.

Although, special attention has been paid to endophytic bacterial isolated from sodicity tolerant polyembryonic mango root stock (GPL-1 and ML-2) in special environments such as sodium and heavy metal contaminated soils (Idris et al., 2004; Barzanti et al., 2007), no information is available about the composition of endophytic bacterial  communities  present  in  the  two dominant sodium tolerant and accumulating plants naturally growing on salt affected wasteland. The surface sterilization protocol is a critical prerequisite for isolation and identification of the endophytic bacterial that express plant growth promotion (PGP) traits at high salt concentrations (Kannan et al., 2014). The present study showed that there was a reduction in the bacterial diversity with increased sodium concentration in plant tissue parts. It has been reported earlier that Na⁺ concentration in tissue parts plays a prominent role in the microbial adaption process as environmental stress leads to reduced bacterial diversity (Borneman et al., 1996). In our study, we have cultured 40 isolates from different tissue portions of polyembryonic mango accessions grown in sodic soils and identified 16 pure cultures for further investigations. The majority of endophytes are identified as Bacillus sp. based on 16S rDNA sequencing and validated with BLAST (ncbi.nlm.gov.blast/Blast.cgi) database. A similar process was adopted in many previous characterization studies (Sturz et al., 2000; Kuklinsky-Sobral et al., 2004; Tank and Saraf, 2010).

In this study, 16 bacterial endophytic strains were isolated from both sodicity tolerant polyembryonic mango root stock GPL-1 and ML-2. Out of the 16 endophytes, four (CSR-M-06, CSR-M-08, CSR-M-09 and CSR-M-16) showed tolerance to high (3.0 M) NaCl concentration (Figure 1). It has also been reported previously that bacterial isolated from saline soil are more likely to withstand saline conditions (Upadhyay et al., 2009). Among the isolates, four strains possessed amylase, cellulase, protease and lipase enzyme activity. According to the 16S rRNA sequences the strains belonged to genus Bacillus with CSR-M-06 identified as Bacillus sp, CSR-M-08 as B. clausii, CSR-M-09 as B. pumilus and CSR-M-16 as B. licheniformis which were able to produce above four enzymes under high NaCl concentrations. The characterized strains showed optimum growth between 2.5 to 3.0 M of NaCl. Therefore, according to the salt tolerance (up to 3.0 M) these strains could be described as salt tolerant microorganisms (Fahimeh et al., 2013; Dave et al., 2006). In general, the hydrolysis enzymes of endophytes appear to be important for the colonization of plant roots with external stress condition (Quadt-Hallmann et al., 1997; Reinhold-Hurek and Hurek, 1998; Sakiyama et al., 2001). This hypothesis is supported by the endophytic bacterial possessing higher activity of amylase, cellulase, protease and lipase enzymes from young radish roots (Weon Taek Seo et al., 2010). Similarly, the presence of cellulolytic and pectinolytic enzymes produced by numerous endophytic bacterial such as Rhizobium sp. has been described (Al-mallah et al., 1987). In addition, bacterial enter the interior of the root by hydrolyzing wall-bound cellulose, auxin-induced tumors, water flow and wounds, or where the lateral roots branch (Al-Mallah et al., 1987).

Molecular characterization based on amplification of 16s rDNA revealed the presence of B. clausii, B. pumilus, B. licheniformis and Bacillus sp. Variation of 16S rDNA allow the inference of phylogenetic relationship among taxonomically related  as  well  as  distinct organisms (Espinosa-Victoria et al., 2009). Based on Amplified Ribosomal Restriction Analysis (ARDRA), different groups were obtained in the dendrogram and these results find support from the observations made by Chowdhury et al. (2007) who divided the bacterial into various groups based on ARDRA pattern and one representative from each of the 4 groups was selected for sequencing of a 16S rRNA gene fragment so as to compare sequence similarity. Kannan et al. (2014) revealed that apart from normally encountered endophytic microflora, Bacillus coagulans, Bacillus megatarium, B. pumilus and B. subtilis were encountered. Molecular characterized phyto-ameliorant grasses grown on the patches of undisturbed sodic soil with prominent rhizosphere encountered microflora B. pumilus and B. subtilis, respectively (Damodaran et al., 2014). In the present study, the bacterial endophyte identified in higher Na⁺/K⁺ in the root portion of sodicity tolerant polyembryonic mango root stock belonged to the same group of firmicutes reported previously (Bharathkumar et al., 2008; Islam et al., 2010).

Therefore, this salt tolerant stability and adaptation of salt tolerant strains with capability to produce amylase, cellulase, protease and lipase enzymes (higher than the other strains of other researcher isolates) suggest that these strains could be a good choice for commer-cialisation for cultivation of crops in normal and salinesodic soil applications. In conclusion, elucidation of the origin of the potential endophytes clarifies influence of these endophytes in reclamation of salt affected soils.

The author(s) have not declared any conflict of interests.