MATERIALS AND METHODS

Soil sample collection: Soil samples were collected from the rhizosphere of C. equisetifolia grown in agro-forestry plantation at Moolimagalam, Karur district, Tamil Nadu, South India lies at N 11°.08’ 00", E 77°.98’39". The study was conducted in November, 2013-January, 2014.The soil samples were collected up to 15 cm depth by random sampling method and brought to laboratory in sterile bags and stored in refrigerator at 4°C until further analysis. The methods described by Verma⁸ was followed to determine the soil physico-chemical analysis viz., pH, organic carbon, Electrical Conductivity (EC), Nitrogen, Phosphorous, Potassium, Calcium and Magnesium at Soil Testing Laboratory of the Institute of Forest Genetic and Tree breeding, India.

Enumeration of bacterial isolates: Serial dilution and plating techniques as described by Subba Rao⁹ with modification were adopted for enumerating bacterial population from soil samples. The Nutrient agar and Trypticase soy agar media with 5% NaCl concentration w as used for enumeration of bacterial isolates. After 24-48 h incubation at 37°C, the colonies appeared with different morphology were selected for further study. The Colony Forming Units (CFU) per gram soil was determined.

Effect of various concentrations of sodium chloride (NaCl) on the growth of isolates: The level of salt tolerance of the bacterial isolates was determined by Nagarajan and Natarajan¹⁰. The nutrient broth supplemented with various concentrations of NaCl (5, 10,15 and 20%) was inoculated with overnight grown bacterial isolates and incubated for 48-72 h at 37°C. Nutrient broth without extra addition of NaCl was maintained as control. Optical density at 600 nm was measured using UV-Vis spectrophotometer (HITACHI, U-2000 series).

In vitro evaluation of antagonistic potential of bacteria using dual culture method: Pathogen cultures of F. oxysporum, R. solani and T. vesiculosum isolated from diseased samples of C. equisetifolia were grown on Potato Dextrose Agar (PDA) at 28°C for 96-120 h, pure cultured and stored at 4°C for further study.

The selected bacterial isolates were assessed for their potential bio-control activity using dual culture technique¹¹. An agar disc of 4 mm was cut from an actively grown 96 h pathogen culture and placed on the surface of fresh PDA medium with different concentrations of NaCl (0, 5, 10, 15 and 20%) at the centre of the petri plates. A loopfull of actively growing bacterial isolate was streaked opposite to the fungal disc, approximately 3 cm from the centre. The petri plates inoculated with the plant pathogen and without bacterial isolates were kept as control. All the petri plates were incubated at room temperature for 7 days. Degree of antagonism was determined by measuring the radial growth of the plant pathogen with bacterial culture and control. The percentage of mycelial growth inhibition was calculated using the following formula described by Rahman et al.¹².

(1)

Where:

Effect of diffusible and volatile metabolites on the growth of fungal pathogen: To evaluate the production of diffusible antibiotic compounds from bacterial isolate, 1 mL of overnight grown bacterial culture was transferred into 50 mL nutrient broth and shaked at 37°C for 6 days in shaker orbital incubator. The bacterial culture was centrifuged and supernatant was mixed with melted PDA. A 5 mm disk of a pure culture of fungal pathogen was inoculated at the centre of the petri plate and incubated at 28°C for 5 days¹³.

To evaluate the production of volatile compounds, the antagonistic bacterial suspensions were streaked on petri plates containing nutrient agar and 2% glucose. After incubation at 28°C for 24 h, other petri plates containing PDA were inoculated in the centre with a 5 mm disc of a 4 day old fungal pathogenic culture. The two petri plates with no tops were placed face to face, sealed together and incubated at 28°C for 5 days¹³. The experiments for each selected antagonistic isolates were performed against all fungal pathogens with at least three replications. The percent of inhibition was determined by measuring the diameter of fungal mycelium and compared with the growth of control (without bacterial isolate).

Molecular characterization of the best isolate: The 16S rRNA sequences of selected salt tolerant antagonistic bacterial isolate was performed by following Edwards et al.¹⁴, Edgar¹⁵ and Cho et al.¹⁶ methods. The obtained sequence was blast using NCBI blast similarity search tool and the sequences were deposited in Gene Bank.

Preparation of inoculum and extraction of cured metabolites: As per the procedure followed by Battu and Reddy ¹⁷, the inoculum preparation and metabolite extraction from selected bacterial isolate was carried out. The selected bacterial isolate was grown in 100 mL nutrient broth under shaking condition (120 rpm) at 28°C for 120 h. The culture was centrifuged at 10,000 rpm for 15 min to get the cell free filtrate. These culture filtrates were used to study the efficacy against fungal pathogens. Crude metabolites were extracted from the effective growth medium by partitioning with organic solvents viz., chloroform, petroleum ether, ethyl acetate, hexane, acetone and methanol¹⁸.

Partial purification of bioactive compound using Thin Layer Chromatography: Silica gel 60 F_25-4 alumina backed plates (Merck) (10×10 cm) was used for separation and identification of inhibitory fractions from solvent extracted bacterial broth culture¹⁹. The solvent system used to separate the compounds was Chloroform: Methanol in 9:1 ratio. The aliquots (10 μL) of each extract were spotted and the solvent front was allowed to run for 1/4th of the Thin Layer Chromatography (TLC) plate. The running lane was then air dried thoroughly and elution of compound detected at 254 nm. The RF value was calculated for all bands and obtained bands were scraped into different micro-centrifuge tubes and extracted with appropriate solvent used for extraction. Centrifugation was carried out to remove the silica residue, the supernatant was collected and stored for further use.

Antifungal property of TLC fraction: The individual fractions were tested for antifungal activity by well diffusion method described by Valgas et al.²⁰. The PDA plates were swabbed with fungal pathogen (F. oxysporum) and 100 μL extract was filled in the well, incubated for 28°C for 48 h. The zone of clearances was measured and the fraction showing maximum zone of inhibition stored for further study.

GC-MS analysis of effective TLC fraction: Most effective TLC fraction was analyzed using GC-MS to predict the presence of inhibitory compounds in TLC purified fraction obtained from the bacterial isolate KS-21. The GC-MS (Thermo Trace GC Ultra VER: 5.0) equipped with a DB35-MS fused silica capillary column and GC interfaced to a Mass Selective Detector (MS-DSQ-II) with XCALIBUR software. For GC-MS detection, an electron ionization system with ionization energy of -70 eV was used. Helium gas was used as carrier at a constant flow rate of 1 mL min^–1 and 2 μL sample injected; injector temperature was 250°C and ion source temperature of 200°C maintained. The oven temperature was programmed from 70-200°C at the rate of 6°C min^–1, held isothermal for 1 min and finally raised to 260°C at 6°C min^–1. Interface temperature was kept at 250°C. Total GC run time was 40.51 min. The relative percentage of the extract constituents was expressed as percentage with peak area normalization. Interpretation on mass spectrum of GC-MS was done using the database of National Institute of Standard and Technology (NIST) having more than 62,000 patterns. The mass spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST library.

Statistical analysis: All data were analysed using one way analysis of variance (ANOVA) and the significant difference among the means were compared by Duncan’s Multiple Range Test (DMRT) a t p = 0.05 level using SPSS PC+ Student ware (Version, 1990), statistical software PSS Inc. SAS²¹.

RESULTS

Soil characterization: The soil texture of the study area was found to be sandy loam and pH was neutral (7.3) range. The Electrical Conductivity (EC) of the soil was found to be 8.0 dS m^–1, it indicated the salinity of the soil. Organic carbon was very low in the range of 0.95%. It is the major factor indicated the health of the soil. Available nitrogen was found to be low (53.6 kg ha^–1), phosphorus and potassium were in normal range 24.9 and 995.6 kg ha^–1, respectively. The presence of calcium and magnesium was in the range of 66.8-100 g and 14.0 mEq/100 g.

Enumeration of bacterial population: The Colony Forming Units (CFU) per gram of soil in nutrient agar was 50.1×10⁶ and in Trypticase soy agar it was 40.5×10⁶. A total of 10 colonies with different morphology from nutrient agar and 6 colonies from Trypticase soy agar plates were selected, pure cultured and stored for further analysis.

Screening for salt tolerance: The salt tolerant study with various concentrations of NaCl showed different levels of tolerances in bacterial isolates. All the bacterial isolates showed maximum growth in nutrient agar and difference was observed in salt incorporated medium. Among sixteen isolates, KS-21, KS-23, KS-24, KS-25, KS-28 and KS-33 exhibited significant salt tolerances up to 20%, the different salt tolerance capacity of all the isolates was represented in Fig. 1.

In vitro dual culture assay: There was only slight difference in antagonistic property of bacterial isolates under normal and in saline condition. Among six isolates tested, the bacterial isolate KS-21 exhibited maximum percent of inhibition against all the pathogens. Range of inhibition was found to be high against F. Oxysporum followed by R. solani.

Fig. 1:	Effect of different concentration of NaCl on growth performances of C. equisetifolia rhizosphere isolates

All the isolates showed minimum inhibition against T. vesiculosum in the range of (6-25%) The isolate KS-28 showed no inhibition against T. vesiculosum in salt amended medium. The presences of different concentrations of NaCl showed only minor variation in antagonistic property (Fig. 2-4). It proved the salinity tolerance of selected bacterial isolates.

Diffusible and volatile metabolites production: Diffusible metabolites from all the bacterial isolates showed significantly different (p = 0.05) antifungal activity against three fungal pathogens screened. There was no variation in antagonism of dual culture assay, diffusible and volatile metabolites productions were carried out without amendment of NaCl and it was observed that the level of mycelial growth inhibition was found to be high when compared with dual culture method. The filtered broth culture of KS-21 isolate showed significantly higher (p = 0.05) mycelium growth reduction against all fungal pathogens, followed by other bacterial isolates of KS-23, KS-24 and KS2-5. But minimum inhibitory effect was found against T. vesiculosum.

Fig. 2:	Percent of inhibition of F. oxysporum against C. equisetifolia rhizosphere isolates under salt stress

Fig. 3:	Percent of inhibition of R. solani against C. equisetifolia rhizosphere isolates under salt stress
	The mean of five replicates and bars indicates standard error

Fig. 4:	Percent of inhibition of T. vesiculosum against C. equisetifolia rhizosphere isolates under salt stress
	The mean of five replicates and bars indicates standard error

Fig. 5:	Effect of Diffusible Compounds (DC) and Volatile Compounds (VC) on the growth inhibition of F. oxysporum
	The mean of five replicates and bars indicates standard error

The isolates KS-28 and KS-33 did not have any impact on T. vesiculosum but growth of remaining two fungal pathogens was found to be controlled effectively. All the bacterial isolates exhibited maximum retardation of mycelium growth of F. oxysporum followed by R. solani. The results of the study revealed that the diffusible culture filtrates have an active compound which effectively inhibits the mycelia growth of fungal pathogens.

The volatile compound produced by six bacterial isolates also showed different antifungal activity. The bacterial isolate KS-21 showed effective inhibition of all the tested fungal pathogens, except T. vesiculosum where the growth was found to decrease when it was treated with volatile metabolites, the bacterial isolates KS-28 and KS-33 have no effect on T. vesiculosum and details are given in Fig. 5, 6 and 7.

Molecular characterization of salt tolerant antagonistic isolate (KS-21): Based on BLAST similarity search tool, the sequence shows closest relativity with Bacillus pumilus. The nucleotide sequence determined in this study has been deposited in GeneBank under accession number KX023322.

Assessment of antifungal activity: Ethyl acetate extract of B. pumilus showed band with R_F value 0.82 obtained from TLC. Activity of band exhibited maximum zone of inhibition (31 mm) against F. oxysporum, followed by Hexane (22 mm) and Methanol extract TLC band (15 mm), no inhibition was observed in petroleum ether TLC band.

Identification of bioactive compounds: Ethyl acetate extract of B. pumilus revealed 19 biologically active compounds. The GC-MS peak area of compounds were represented in Fig. 8 and its molecular formula, molecular weight, retention time are given in Table 1.

Fig. 6:	Effect of Diffusible Compounds (DC) and Volatile Compounds (VC) on the growth inhibition of R. solani
	The mean of five replicates and bars indicates standard error

Fig. 7:	Effect of Diffusible Compounds (DC) and Volatile Compounds (VC) on the growth inhibition of T. vesiculosum
	The mean of five replicates and bars indicates standard error

Table 1:	Bioactive compounds of isolate KS-21 identified by using GC-MS

The compounds identified and occurred in higher probability are 1,3-Bis (4-chlorophenyl)-1,4-dihydrobenzo[f]quinazoline (89.72), Syn-2-prop-1-3-diol (42.01), 3-Methyl-5-lanosta-4,8-dien-3-one (38.60), 9-Octadecenamide (33.80%), Cis-3-chloroall alcohol (32.93%).

Fig. 8:	GC-MS analysis of active compounds from KS-21 ethyl acetate extract
	The mean of five replicates and bars indicates standard error

DISCUSSION

The higher EC and deficient nitrogen level observed in soil used for bacterial isolation indicated higher salinity level. Mousivand et al.²² reported that the different soil physico-chemical conditions can influence the bacterial cell number. Similar approach was carried out by Upadhyay et al.²³ and they isolated 24 saline tolerant rhizobacterial isolates from saline infested zone of wheat rhizosphere. They found that the isolates could withstand upto 8% NaCl concentration and screened its PGP activity. But in the present study, 16 bacterial isolates obtained from the rhizosphere of C. equsetifolia showed growth tolerance upto 12% NaCl.

In this study, isolate KS-21 showed effective antagonistic activity against all the tested plant pathogens when compared with other bacterial isolates. Williams and Asher²⁴ reported that different antagonistic activities of bacterial isolates possess various mode of actions and the antifungal metabolites produced by the isolates may differ and the isolates are taxonomically different from each other. Edwards et al.²⁵ found that the genera belong to Bacillus and Pseudomonas was effective against fungal plant diseases. In the present study, they have suggested that production of antifungal compounds by the bacteria may be the cause for the constraint of fungal hyphal growth even there was no direct contact between bacterial colonies and pathogenic fungal mycelium, so that the fungal growth restriction was triggered by diffusion of substances into the agar medium. These results are in agreement with the findings made in the present study. Recently, Shrivastava and Kumar²⁶ reported that microorganisms with stress tolerance capacity and plant growth promotion attributes are very helpful in growth and development of plants under stressed environments.

Mousivand et al.²² observed that bacteria produce different antifungal metabolites with numerous antagonistic mechanisms and confirmed that the genetic diversity of different patho-types affect the bacterial antagonistic features. Various studies demonstrated that bacterial volatiles provide resistance to salt and droughtstress²⁷. The present study was in conformity with the above findings.

In the present study, the potent salinity tolerant bacterial isolates with antagonistic property was sequenced through 16S rRNA and identified with online BLAST in NCBI and it was identified as Bacillus pumilus. A similar finding was made by Upadhyay et al.²³ and they have reported the occurrences of Bacillus and Bacillus derived genera were found to be leading in root zone of wheat in saline soil. Spore-forming bacteria, typically Bacillus species are one of the major groups of soil bacteria. The production of a multilayered cell wall structure, formation of stress resistant endospores and secretion of peptide antibiotics, peptide signal molecules and extracellular enzymes are the common physiological properties to survive in stressed condition²⁸. Quantitative and qualitative variations in these traits allow for these bacteria to inhabit varied niches in agro ecosystems. Bacillus pumilus isolated in this study has salt tolerant capacity as well as antagonistic ability against different plant pathogens.

Investigation on microbial metabolites is gaining greater momentum in the agrochemical industry as a source for the development of new pesticide and fungicidal products. Several such products have been developed and used as bactericide, fungicide and insecticide in agriculture. A similar study was conducted by De Melo et al.²⁹ with different fungal pathogens and in their work they have used ethyl acetate and dichlromethene extract of B. pumilus for antifungal activity which showed maximum inhibition against Rhizoctonia solani, Pythium aphanidermatum and Sclerotium rolfsii.

In the present study, 19 active compounds were identified from B. pumilus through GC-MS analysis. Among them, Cis- 3-chloroall alcohol was one of the compounds with high probabilities (38.48). Belser and Castro³⁰ reported the biocidal property of this compound. Pyrrolo [1,2-a] pyrazine-1,4-dione (61.16) have anticancer activity. This compound was also identified in ethyl acetate extract of Micrococcus luteus by Sheela and Uthayakumari³¹. Similar compound was identified from B. pumilus isolated from mangrove sediment and its inhibition in bio-film formation was noticed by Mithun and Rao³². In the present study 9-Octadecenamide (33.80) was isolated from the sponge associated actinomycetes, Nocardio psisdassonvillei MAD08 which had the broad range of antimicrobial activity including anticandid activity reported by Selvin et al.³³.

In the current scenario the exploration for different microbes and their new bioactive compounds has gained momentum and has great potential in agricultural field for crop protection and enhancing yields. Thus, the B. pumilus strain identified in this study could be used in eco-friendly disease management of C. equsetifolia raised in salt stressed condition.

CONCLUSION

A good salt tolerant bio-control agent should be able to thrive and persist in salt affected environments. In the present study, the salt tolerant bio-control bacterial isolate from C. equisetifolia rhizosphere was found to be effective in controlling major fungal pathogens. However this in-vitro result suggested the use of B. pumilus and its secondary metabolites as best eco-friendly alternative against hazards chemical amendments for the control of fungal diseases in salt affected agroforestry systems.

SIGNIFICANCE STATEMENTS

This study discovers the secondary metabolites production of Bacillus pumilus isolated from C. equisetifoila rhizosphere soil that can be beneficial for formulation of bio-fungicides against major fungal diseases. This study will help the researcher to uncover the critical areas of salt tolerant antagonistic rhizosphere microbes isolated from saline agroforestry system that many researchers were not able to explore. Thus a new theory on use of this bacterial culture or its secondary metabolites as bio-fungicide can benefit farmers to grow tree plantations in salt affected areas.

ACKNOWLEDGMENTS

Authors are highly thankful to the Institute of Forest Genetics and Tree Breeding (IFGTB), Coimbatore, Tamil Nadu, India for providing all the facilities and encouragement to carry out the study.