Ten soil samples were collected aseptically from crystallizer ponds of inland solar saltern located eastern side of the Sambhar salt lake (Latitude 26°58′N and Longitude 75°05′E), Jaipur, India (Figure 1). Among them, a soil sample was subjected to physiochemical analysis following standard methods to explore the physiochemical characteristics of the collection site. Soil pH, electrical conductivity (EC) and total nitrogen were determined according to Jackson (1973). Organic matter (organic carbon) was determined according to the method of Walkley and Black (1934). Total sodium, potassium and magnesium were estimated using atomic absorption spectrophotometer (AA-6200, Shimadzu, Japan).

Three composite samples were prepared from the collected soil samples. The composite samples were homogenized and air-dried in a laminar flow hood, ground lightly with a sterile pestle, heated for 2 h at 60–65°C and finally stamped onto the isolation agar media using sterile cotton plug as described earlier (Gontang et al., 2007; Jose and Jebakumar, 2012). In another method, 1 g of composite soil sample was suspended in 4 ml sterile water containing 1% NaCl, heated for 6 min at 55°C, vigorously shaken and further diluted (1:4) in sterile water, and 50 μl of each dilution was spread with a sterile glass rod onto agar-based isolation media. The isolation media (IM) contained following components: IMa, 10 g of starch, 4 g of yeast extract, 2 g of peptone, 10 g of NaCl, 18 g of agar, and 1 l of distilled water; IMb, 10 g of starch, 4 g of yeast extract, 20 g of NaCl, 2 g of NH₄SO₄, 1 g of MgSO₄.7H₂O, 1 g of K₂HPO₄, 22 g of agar, and 1 l of distilled water; and IMc, 10 g of starch, 0.3 g of casein, 2 g of KNO₃, 4.6 g of sodium chloride, 2 g of K₂HPO₄, 0.05 g of MgSO₄.7H₂O, 0.02 g of CaCO₃, 0.01 g of FeSO₄.7H₂O, 1 mg of ZnSO₄.7H₂O, 18 g of agar and 1 l of distilled water. The isolation media amended with filter-sterilized cycloheximide (100 μg/ml) and nalidixic acid (5 μg/ml) to suppress the growth of gram-negative bacteria and fungi to favor the selective isolation of actinomycetes. The isolation plates were incubated at 30°C for 2–12 weeks to acquire slow growing and rare actinomycetes. Actinomycete like leathery colonies appeared after 2 weeks of incubation were selected and repeatedly streaked on IMb to get the pure isolates. The pure actinomycete like isolates were maintained either on IMb or trypticase soy agar (Himedia, India) slants supplemented with 1% of NaCl (w/v); for long-term storage, cultures were maintained in 18% glycerol at −20°C.

Isolates were grown in trypticase soy broth supplemented with 1% NaCl for 10 days and the genomic DNA of each isolate was extracted following standard phenol-chloroform extraction procedure (Hopwood et al., 1985). The 16S rDNA was amplified from genomic DNA obtained from the actinomycete isolates by PCR with eubacterial universal primer pair 27F 5′-AGAGTT TGA TCC TGG CTC AG-3′ and 1492R 5′-GGT TAC CTT GTT ACG ACT T-3′ (Lane, 1991). The reaction mixture contained 25 ng of DNA as template, 1X reaction Buffer (10 mM Tris pH 8.3, 50 mM KCl, 1.5 mM MgCl₂), 200 μM of each dNTP, 10 pM of each primer and 0.05 U of Taq DNA polymerase (Sigma, USA). PCR conditions consisted of an initial denaturation at 94°C for 5 min; 31 cycles at 95°C for 30 s, 54°C for 90 s, and 72°C for 120 s; and a final extension at 72°C for 5 min. The amplification reactions were performed in Bio-Rad thermal cycler (MyCycler, Bio-Rad, USA) and the amplification products were examined by 1% agarose gel electrophoresis.

The 16S rDNA amplification products were purified by using PCR product purification spin kit (HiPurA, HiMedia, India) following the protocol suggested by the manufacturer. To identify the number of polymorphic groups and select the representative strains among the actinomycete isolates, aliquots of purified 16S rDNA amplicons were subjected to amplified ribosomal DNA restriction analysis with HaeIII and HinfI following the previously described procedure (Jose and Jebakumar, 2012). The digestion reactions were carried out in 20 μL reaction mixture containing 1X recommended buffer, 1 μL (10U) of restriction enzymes and 10 μL of 16S rDNA amplicons at 37°C for 3 h. The digested restriction fragments were electrophoresed in 2.5 % agarose gel using TE buffer. The gel was stained with ethidium bromide and visualized under UV transilluminator. Strong and clear bands were scored in a binary data form and used for similarity and clustering analysis in numerical taxonomy analysis program package, NTSYS-pc 2.02i (Rohlf, 1998). Similarities among the isolates were calculated by Jaccard's coefficient (Jaccard, 1912) and the dendrogram was constructed using UPGMA method (Nei and Li, 1979).

The PCR amplicons of 5 actinomycete isolates with representative ARDRA profiles were sequenced by Applied Biosystems 3730XL DNA Analyzer using same primer set as used in PCR amplification. All the sequences obtained from sequencing were analyzed and edited by using BioEdit software (Hall, 1999). Initially all the 16S rDNA sequences were compared to sequences in GenBank by use of the Basic Local Alignment Search Tool online service (Altschul et al., 1990) to determine approximate phylogenetic position. Sequences then were aligned using ClustalX (Thompson et al., 1997) with related 16S rDNA sequences retrieved from GenBank. Neighbor-joining phylogenetic tree was inferred from the 16S rDNA sequences of 5 representative isolates and selected members of related genus within the order Actinomycetales by using suitable programs of the PHYLIP (phylogeny inference package) version 3.68 (Felsenstein, 2008). The robustness of tree topology was assessed by bootstrap analysis of the neighbor-joining data sets on 1000 resamplings using the same PHYLIP package. Manipulation and tree editing were done by using TreeView.

All the representative isolates obtained from ARDRA analysis were subjected to morphological characterization on IMb. Inoculated plates were incubated at 29°C for 12 days and their morphology was visually observed.

Salt tolerance of the actinomycete isolates was determined on starch nitrate medium prepared with series of NaCl concentrations; 0, 50, 70, 100, 150, 180, 200, 250, and 300 g/L (~0–5 M) following Kushner (1978). Dry weights of mycelial pellets were quantified by drying at 60°C as a measure of growth on different salt concentrations.

Antimicrobial profile of all the actinomycte isolates was recorded against a range of bacterial and fungal strains using primary and secondary screening methods. Bacterial test strains viz., Bacillus subtilis MTCC 441, Klebsiella pneumoniae MTCC 109, Salmonella typhi MTCC 733, Proteus vulgaris MTCC 426 and Staphylococcus aureus MTCC 3160 were obtained from the IMTECH, Chandigarh, India. Fungal test strains viz., Fusarium oxysporum, Aspergillus nigre, and Alternaria alternata were obtained from Tamilnadu agricultural college and research centre, Madurai, India. The bacterial cultures were maintained either in Mueller Hinton broth (MH broth) or in nutrient broth. The fungal cultures were maintained in potato dextrose agar.

All the actinomycete isolates were primarily screened for antibacterial and antifungal activity against the test microorganisms using agar plug method (Eccleston et al., 2008) and dual culture method (Harveson and Kimbrough, 2000), respectively.

The actinomycetes were initially streaked over three different Production Media (PM) solidified with 2% agar: PM1, 10 g of starch, 4 g of yeast extract, 5 g of NaCl, 2 g of NH₄SO₄, 1 g of MgSO₄.7H₂O, 1 g of K₂HPO₄ and 1 l of distilled water; PM2, 10 g of starch, 0.3 g of casein, 2 g of KNO₃, 4.6 g of NaCl, 2 g of K₂HPO₄, 1 0.05 g of MgSO₄.7H₂O, 0.02 g of CaCO₃, 0.01 g of FeSO₄.7H₂O, 1 mg of ZnSO₄.7H₂O, 18 g of agar and 1 litre of distilled water; and PM3, 10 g of starch, 4 g of yeast extract, 5 g of NaCl, 2 g of NH₄SO₄, 2 g of MgSO₄.7H₂O, 1 g of K₂HPO₄, 1 gm of CaCO₃, 0.010 g of FeSO₄.7H₂O, 0.001 g of ZnSO₄.7H₂O, 0.001 g of MnCl₂.4H₂O, 0.001 g of CuSO₄.5H₂O and 1 l of distilled water. The plates were incubated at 29°C for 10–20 days to attain enough growth over the production media.

For the initial antibacterial screening, agar plugs of 6 mm in diameter were cut from the 10 days old agar plates and plugged into the wells bored using sterile cock borer (diameter of 6 mm) in Mueller Hinton agar plates seeded with different bacteria. The agar plugged plates were incubated at 37°C for 24 h and observed for zone of inhibition around the inserted agar plugs.

For antifungal screening, the actinomycetes were point inoculated on production media at 30 mm distance from the center of plate. Fungal mycelial-disks (6 mm in diameter) prepared from growing margin of cultures of test fungal strains and placed in the center of plate. Antifungal activity was indicated as mycelia growth of fungal isolates was inhibited in the direction of active actinomycete isolates.

Those actinomycetes showed positive antimicrobial activity in the primary screening were subjected to antimicrobial compound production in submerged culture and their antimicrobial proficiency was confirmed in triplicates by disc diffusion method (Bauer et al., 1966).

Spore suspensions of active actinomycetes were prepared in distilled water from cultures grown on ISP-4 medium supplemented with 2% of NaCl and 0.4% yeast extract (w/v). The suspensions were added either to ISP-2 broth or modified ISP4 broth in 250 ml Erlenmeyer flasks at a rate of 10⁸ spores in 50 ml liquid medium incubated on a shaker at 120 rpm at 30°C for 10 to 15 days. From the seed cultures, 25 ml aliquots were transferred to 250 ml production media (PM1, PM2, and PM3) and the flasks were incubated for 10–20 days at 30°C while shaking at 120 rpm. The culture broths were centrifuged at 10,000 rpm for 10 min to separate the mycelial biomass. Ethyl acetate was added to the supernatants in 1:1 proportion and the mixtures were agitated for 45 min. The solvent layers were separated from broths and centrifuged at 5000 rpm for 15 min to remove traces of fermentation broth. The ethyl acetate fractions were evaporated and the resultant crude compounds were resuspended in 50 μl of methanol which were then assayed for antimicrobial activity.

For the evaluation of antimicrobial activity, Mueller Hinton agar plates were inoculated with indicator strains by spreading the microbial inoculums on the surface of the media. The extracts were loaded on 6 mm sterile discs, dried and placed on the surface of the medium inoculated with bacterial test strains and the plates were incubated at 37°C for 24 h. At the end of incubation period, inhibition zones formed around the disc were measured with transparent ruler in millimeter. In the case of antifungal assay the same procedure was practiced with fungal strains on potato dextrose agar instead of Mueller Hinton agar.

The 16S rRNA gene sequences of representative strains have been deposited in the GenBank database under the accession numbers KC012637- KC012641.

Physico-chemical characteristics of a saltern soil sample (Table 1) were comparable with that of other solar salterns previously reported elsewhere (Cai et al., 2009). The soil was alkaline in nature with pH 9.1. Electric conductivity was measured at 14.21 dSm⁻¹ with predominance of sodium and chloride. At the time of sample collection, the temperature of the site was measured at 29°C.

A total of 14 actinomycete isolates including slow growing and rare actinomycete were isolated from saltpan soil samples using two sample processing methods and three different isolation media with prolonged incubation period. Among the sample-processing methods, stamping facilitated the isolation of 9 (64%) actinomycete strains including members of some rare genera. Appearance of actinomycete like colonies on isolation media stamped with a ground soil sample shown in Figure 2. All the isolates were found to grow slowly on agar media.

PCR amplification of 16S rDNA yielded a single amplicon of ~1500 bp for all the isolates. Restriction digestion of these amplicons with HaeIII and HinfI yielded different profiles characterized by 2–4 fragments ranging from 100 to 800 bp in size for the different isolates. The actinomycete isolates were clustered into four groups in UPGMA dendrogram (Figure 3) inferred from ARDRA patterns obtained with HaeIII and HinfI. Group II has maximum of 9 isolates, group I and III have 2 isolates each and groups IV has single isolate.

16S rDNA of selected five isolates belonging to different clusters established by ARDRA was sequenced and used to determine the diversity among the isolates. The phylogenetic tree (Figure 4) inferred by 16S rDNA sequence indicated that they should be classified in following 3 genera belong to 2 families; Streptomycetaceae and Pseudonocardiaceae.

Morphology of representative actinomycete isolates was visually observed and their aerial and substrate mycelium are shown in Figure 5. All the isolates grew well on IMb with characteristic musty odour, dimorphic mycelium, spore formation and non-motile leathery colonies. Actinoalloteichus sp. JAJ71 grew well as white puffy colonies with dark bluish black diffusible pigment, white aerial mycelium and black substrate mycelium. Streptomyces sp. JAJ83 produced snow white aerial mycelium and gray substrate mycelium. Whereas, Streptomyces sp. JAJ73 produced white aerial mycelium and wheat color substrate mycelium. Pseudonocardia sp. JAJ77 produced white aerial mycelium and orange substrate mycelium without diffusible pigment. Pseudonocardia sp. JAJ82 grew with brown diffusible pigment, gray aerial mycelium and brown substrate mycelium.

The saltern based actinomycetes showed different levels of salt tolerance (Figure 6). Four actinomycete strains (JAJ74, JAJ75, JAJ76, and JAJ77) grew well in the absence of NaCl as well as up to 0.85 M NaCl while grew mildly beyond 0.85 M NaCl up to 1.2 M NaCl in the culture medium. Eight actinomycete strains (JAJ70, JAJ71, JAJ78, JAJ79, JAJ80, JAJ81, JAJ82, and JAJ83) grew optimally in the presence of 0.5–1.2 M and mildly in absence and 1.7 M NaCl. Strains JAJ72 and JAJ73 grew well in the presence of 0.5–2.0 M NaCl and tolerated maximum of 2.5 M NaCl in the culture medium.

In the primary screening, four actinomycetes (JAJ70, JAJ73, JAJ77, and JAJ82) were found to be active against at least one of the tested bacteria and fungi. Among the four antagonistic actinomycetes, JAJ73 and JAJ82 showed antifungal activity, JAJ77 showed antibacterial activity and JAJ70 showed both the antifungal and antibacterial activity. The actinomycetes showed antimicrobial activity in primary screening were further recognized to produce antimicrobial compound in the batch of submerged fermentation process with three different production media (Tables 2, 3). All the three production media, PM1, PM2, and PM3 supported antimicrobial compound production in all the antagonistic actinomycetes, however the measure of antimicrobial compound varied from one medium to another (Tables 2, 3). PM3 favored higher antimicrobial compound production in the all the actinomycetes. Actinoalloteichus sp. JAJ70 exhibited highest antifungal activity (up to 44 ± 1.2 mm) followed by Streptomyces sp. JAJ73 (14 ± 1.20 mm) and Pseudonocardia sp. JAJ82 (16 ± 1.52 mm) against the fungal strains. Furthermore, Actinoalloteichus sp. JAJ70 exhibited considerable antibacterial activity (up to 35 ± 0.88 mm) followed by Pseudonocardia sp. JAJ77 with up to 21 ± 1.15 mm diameter of zone of inhibition against the bacterial strains. Figure 7 shows antifungal activity by disc diffusion method for Actinoalloteichus sp. JAJ70 against three fungal strains.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.