Isolation of halophilic bacteria
In this study, fifty halophilic bacterial isolates were isolated from the saline soils. They have salt tolerance range of 1- 17 % NaCl, and classified them as halotolerant (1-6 % NaCl w/v) and halophilic (6-15 % NaCl w/v) and extremophiles (15-30 % NaCl w/v) based on growth in nutrient broth supplemented with different NaCl concentrations. Out of these fifty bacterial isolates, forty-one isolates classified as halotolerant and nine as halophilic based on in vitro assay.
Physiological properties of halophilic bacterial isolates
In this study, six morphological types of bacteria were observed under compound microscope (Olympus) at 1000 x magnification viz., rods, small rods, long rods, coccus, streptococcus, and staphylococcus. In gram staining, twenty eight isolates were gram positive and twenty two were gram negative. The pigmentation in halophilic bacteria varied from creamy white to milky white, red and yellow (Figure 1). Based on growth pattern in the broth, the isolates were classified as aerobic, facultative anaerobe and obligate anaerobes for pellicle formatting, throughout and bottom growing isolates. In biochemical tests, the halophilic bacterial isolates showed diversified results and displayed in Table 1. Thirty-six isolates showed positive for nitrate reduction test, fourteen isolates were positive for MR (Methylene Red) test, ten isolates positive for indole production, eleven isolates positive for citrate utilization test, thirty-seven isolates positive for starch hydrolysis test, ten isolates positive for H2S production test, all the isolates positive for the cytochrome c oxidase and catalase tests. In carbohydrate utilization test, forty-one isolates positive for lactose test, thirty-three isolates positive for mannitol, ten isolates positive for rhamnose, thirty isolates positive for sucrose, twenty-seven isolates positive for sorbitol, thirty-nine isolates positive for arabinose, sixteen isolates positive for adonitol and glucose utilizing bacteria from saline soils (Table 2).
Production of extracellular enzymes
The chitinase activity was limited to few natural isolates; in the present investigation, a total of fourteen halophilic bacteria found with chitinase activity. Based on colloidal chitin degradation, Bacillus sp. (HB-25) was recognized as best solubilizer with halo zone of 12.1 mm in plate, followed by Staphylococcus sp. with 9.33 mm and Pseudomonas stutzeri (HB-30) with 3 mm solubilization zone. In gelatin liquefaction test, forty-four isolates liquefied the gelatin, thirty seven isolates positive for amylase production.
Selection of antagonistic halophilic bacterial isolates
The antifungal activity of halophilic bacteria was examined on potato dextrose agar using dual culture method. Out of the fifty halophilic bacterial isolates screened for their antifungal activity against Sclerotium oryzae ten isolates inhibited the mycelial growth (Figure 2), forming a clear halo zone. The isolate, Pseudomonas stutzeri (HB-10) inhibited the S. oryzae up to 34.4 %, and Staphylococcus xylosus (HB-39) up to 33.33 %. Similarly, the radial growth of Rhizoctonia solani, inhibited by eight halophilic bacterial isolates under in vitro conditions. The halophilic bacterial isolate, Bacillus cereus HB-48 inhibited the radial growth of R. solani up to 38.9 % (Figure 3) and Lysinibacillus sphaericus (HB-50) up to 33.3 %. In this study, radial growth of R. solani inhibited more than S. oryzae. Using this approach, eight isolates were selected for further study based on criteria such as biocontrol activity and extracellular enzyme activity.
Plant growth promoting attributes
The ability of halophilic bacteria to solubilize and release phosphorous, zinc and potassium at different concentrations of NaCl (3, 6 and 10 % w/v) was studied in greater detail based on plate assay. In potassium releasing test, out of eight halophilic bacterial isolates selected, two isolates namely, P. stutzeri (HB-10) and Bacillus cereus (HB-48) showed halo zones at 6 and 10 % NaCl (w/v). Clear halo zones gradually increased over a period in all the isolates. Insoluble tri-calcium phosphate solubilized by five isolates, viz.,P. stutzeri (HB-10), B. albus (HB-17), B. safensis (HB-28), P. stutzeri (HB-30), L. sphaericus (HB-50) at 3, 6, and 10 % salt concentration. The isolates showing solubilization zones > 5 mm were classified as halophilic phosphate solubilizing bacteria. P. stutzeri (HB-30) demonstrated the highest solubilization at 3 % salt (11.20 mm), while three halophilic bacteria had no sign of solubilization. In the presence of 6 and 10 % NaCl (w/v), the phosphate solubilization was much lower.
The zinc solubilization ability of halophilic bacterial isolates was evaluated at different salt concentrations (3, 6 and 10 % w/v) based on the diameter of halo zone. Zinc solubilization varied with the isolate and salt concentration. Among all, four isolates showed positive for zinc carbonate solubilization and six isolates positive for zinc oxide solubilization. Maximum zone of 10.03 and 13.20 mm was observed for Pseudomonas stutzeri (HB-30) in ZnCO3 and ZnO amended medium respectively (Table 3). The zone of solubilization was competitively high in ZnO amended medium as compared to ZnCO3 at 3 and 6 % NaCl (w/v) concentrations. The zone of solubilization increased up to 6 % NaCl thereafter decreased. The halophilic bacterial isolates which sowed positive for zinc, phosphorous and potassium solubilization were subjected to inoculation of bromothymol blue near solubilization zones for evaluation of their effect on pH. Decreased in pH was evaluated based on the changes in color from blue to yellow near halo zones, all the positive isolates showed yellow color formation.
Siderophores production was evaluated on CAS agar medium and the type of siderophores were identified based on Arnow’s and Csaky’s tests. Strain HB-17 had a positive result for both Arnow’s and Csaky’s test and showed highest siderophore producing capacity with a siderophore unit of 49.34 %. The three isolates (HB-28, HB-30 and HB-39) studied had a strong reaction to Csaky’s test, which suggest that they produce hydroximate type of siderophores. Isolates showed varied yellow halo zone around the colony (Figure 4). Hydrogen cyanide (HCN) was produced by Pseudomonas stutzeri (HB-10), Bacillus safensis (HB-28) and Bacillus cereus (HB-48). Isolates were also checked with respective to their IAA production qualitatively and quantitatively. Among eight isolates, six isolates positive for IAA in quantities ranging from 27.56-58.76 μg ml-1. Strains HB-30 and HB-49 had the highest IAA production ability, yielding 58.76 and 46.83 μg ml-1 respectively.
The 16S rRNA sequence analysis was performed to eight isolates for elucidating the taxonomic position of isolated prospective halophilic bacterial strains. The strain HB-10 and HB-30 highly homologous (99.9 %) to P. stutzeri, two isolates were closely related to Bacillus cereus (HB-48 and HB-49), one isolate similar to Bacillus albus (HB-17), one isolate displayed a highest similarity (99.9 %) with Bacillus safensis (HB-28), one isolate had a close similarity with Staphylococcus xylosus (HB-39), and one to Lysinibacillus sphaericus (HB-50). The resulted sequences were also deposited in Gen Bank with the accession numbers MN098847.1, MN098870.1, MN122435.1, MN098871.1, and MN121550.1 and remaining three isolates are under process of getting the accession numbers.
Screening of halophilic bacteria for their antibiotic susceptibility
In vitro evaluation of antibiotic susceptibility using different antibiotics viz., Cephalothin (CEP-30 mcg), Clindamycin (CD-2 mcg), Cotrimoxazole (COT-25 mcg), Erythromycin (E-15 mcg), Gentamicin (Gen-10 mcg), Ofloxacin (OF-1 mcg), Penicillin-G (P-10 units), and Vancomycin (VA-30 mcg) showed varied responses. Isolates responded very differently to the Cephalothin (30 mcg), which is a β-lactam group antibiotic active against gram-positive and some gram-negative bacteria. The response of the isolates was recorded as viz.,Pseudomonas sp. (Gram negative), Pseudomonas sp. HB-11 (Gram negative), Pseudomonas sp.HB-12 (Gram negative), and Bacillus sp. HB-20 (Gram positive) were completely inhibited whereas some isolates namely, Bacillus sp. HB-8 (Gram positive), Pseudomonas sp. HB-13 (Gram negative), Staphylococcus sp. HB-14 (Gram positive), Pseudomonas sp. HB-35 (Gram negative), Bacillus sp. HB-36 (Gram positive), and Lysinibacillus sphaericus HB-50 (Gram positive) were completely resistant.
Clindamycin (2 mcg) belong to lincosamide group interferes with protein synthesis by binding with 50S part of ribosome. It is mostly active against anaerobes, gram positive cocci and less effective against enterobacteriaceae family members. Clindamycin showed complete inhibition of Bacillus sp. HB-6 (Gram positive, rod), Pseudomonas sp. (Gram negative, rod), Pseudomonas sp. HB-12 (Gram negative, cocci), and Bacillus sp. HB-20 (Gram positive, rod) isolates and some isolates showed complete resistance viz.,Pseudomonas sp. HB-13 (Gram negative, rod), Bacillus sp. HB-41 (Gram negative, rod), and Neisseria sp. HB-43 (Gram negative, coccus). Rest of the isolates showed intermediate to low susceptibility for clindamycin. Cotrimoxazole belongs to sulfonamide group, contains two components sulfamethoxazole and trimethoprim. In combination it acts as a bactericidal, sulfamethoxazole inhibits the formation of dihydrofolic acid and trimethoprim inhibits the dihydrofolate reductase. In the present investigation, cotrimoxazole showed complete inhibition of some isolates viz.,Pseudomonas stutzeri (HB-10), Bacillus sp. (HB-20), and Bacillus sp. (HB-23), rest of all isolates have low to moderate susceptibility. Isolates, Pseudomonas stutzeri (HB-10), Bacillus sp. (HB-20) and Bacillus cereus (HB-48) were completely susceptible to the Erythromycin, whereas remaining isolates showed moderate susceptibility. Few isolates viz.,Pseudomonas stutzeri (HB-10), Bacillus sp. (HB-20), and Pseudomonas sp. (HB-26) showed complete susceptibility to the Gentamicin, a broad spectrum antibiotic. Conversely, a maximum number of isolates showed susceptibility to the Ofloxacin viz., HB-6, Pseudomonas stutzeri (HB-10), Staphylococcus sp. (HB-15), Bacillus sp. (HB-20), Bacillus sp. (HB-21), Pseudomonas sp. (HB-26), Moraxella sp. (HB-27), Pseudomonas sp. (HB-35), Bacillus sp. (HB-37), Staphylococcus sp. (HB-38), and Staphylococcus xylosus (HB-39). Almost all the isolates were moderate to highly susceptible to oflaxacin. In contrary to the above results most of the isolates were resistant to Penicillin except few isolates namely, Pseudomonas sp. (HB-7), Pseudomonas stutzeri (HB-10), Bacillus sp. (HB-20), Clostridium sp. (HB-34), and Lysinibacillus sphaericus (HB-50). Vancomycin has less effect on bacteria which is evident from the appraised data. Isolates, Pseudomonas stutzeri (HB-10), Bacillus sp. (HB-20), and Clostridium sp. (HB-34) showed maximum susceptibility, rest were poorly inhibited.
Reproducibility of results
All the experiments were repeated, and performed independently at least three times. Data is presented as mean values.