The isolate MML2501 was obtained from the culture collection of the Biocontrol and Microbial Metabolites Lab (MML), Centre for Advanced Studies in Botany, University of Madras, Chennai. The isolate was originally isolated from the groundnut rhizosphere soil.

Fungal pathogens were maintained on potato dextrose agar (PDA) and the isolate MML2501 was maintained on nutrient agar (NA). The bacterium was also preserved at -20°C as glycerol (15%) stocks.

The Bacillus sp. MML2501 was identified based on the 16S rDNA sequence homology

The isolate Bacillus licheniformis MML2501 was screened for antagonistic activity against the following phytopathogens: Fusarium oxysporum, Fusarium udum, Rhizoctonia solani, Macrophomina phaseolina, Pyricularia oryzae, Alternaria alternata and Curvularia lunata on PDA by dual culture technique. The mycelial discs measuring 9 mm were cut out from 4 days old culture of fungal phytopathogens pre-cultured on PDA plate and inoculated at the center of a fresh petriplate containing PDA. The isolate MML2501 was streaked in the periphery of each petriplate and incubated at room temperature. The growth of fungal phytopathogens was observed periodically and the growth inhibition was measured after 6 days of incubation.

The Mueller-Hinton agar (Hi Media) was prepared as per the manufacturer instructions, poured into petriplates and allowed to solidify. B. licheniformis MML2501 was inoculated evenly on the surface of the medium by swabbing the culture and kept for 2-3 min for the agar surface to dry. Then a disc containing 14 antibiotics (Hi-media) with specific concentration was placed on the surface of the culture with the help of a sterile forceps and slightly pressed so that the disc adhered to the surface of the agar. Plates with out antibiotic discs served as control. The plates were incubated at room temperature for 4 days and the zone of inhibition around each antibiotic disc was observed, measured to determine the resistance and sensitivity of B. licheniformis MML2501.

The tolerance of B. licheniformis MML2501 to various heavy metals was studied as follow. A loop full of overnight grown culture was streaked on Nutrient agar plate amended with various concentrations (2 – 10 mM) of heavy metals viz., cobalt, zinc, magnesium, iron, copper, mercury, silver, cadmium, arsenic, lead and selenium. The plates were incubated at room temperature for 24 h and observed for the growth of B. licheniformis MML2501. The MSM plates without heavy metals served as controls.

The tolerance of B. licheniformis MML2501 to various commercial fungicides was studied as follow. A loop full of (24hr) grown culture was streaked on Nutrient agar plate amended with various concentrations (100 – 1000 ppm) of commercial fungicides viz., propiconazole, tridemorph, carbendazim, mancozeb, hexaconazole and tricyclazole. The plates were incubated at room temperature for 48 h and observed for the growth of B. licheniformis MML2501. The Nutrient agar plates without fungicide served as controls.

The efficiency of B. licheniformis MML2501 to produce SA was tested in casamino acids medium with different concentrations of casamino acids (0.1 - 0.8 %) at different pH (3 - 10), temperature (20 - 55 ° C) and in static and shaken conditions. The broth cultures were incubated for 4 days and cells were separated by centrifugation at 8000 rpm for 10 min. All the harvesting procedures were carried out in dim light with samples maintained in covered ice baths.

The SA was extracted from the acidified culture supernatant. The filtrate was adjusted to pH 2 with 1M HCl and extracted twice with double volume of ethyl acetate. To 1 ml of the extraction, 2 ml of 2 M FeCl₃ and 1 ml of distilled water were added. The SA reacts with 2 M FeCl₃ to form a purple iron + SA complex in the aqueous phase with a maximum absorbance at 527 nm. The difference in absorbance was recorded at a Beckman DU40 spectrophotometer. The SA concentration in extraction was determined using a calibration curve of standard SA.

The extracted SA was evaporated to dryness in a flash evaporator, solubilized in a minimal volume of methanol and spotted on pre coated silica gel plates. Then the plates were developed in a solvent system consisting of chloroform:acetic acid:ethanol at the ratio of 95:5:2.5 (v/v). The plates were viewed in blue fluorescence emission under UV light (256 nm) immediately after removal from the developing chamber. The SA was detected by observing a UV reflected band with an R_f value corresponding to that of the standard SA.

The HPLC system used was having C18 column (Gemini) with a mobile phase, 2.3 g monobasic ammonium phosphate in 850 ml of water, 150 ml acetonitrile, 1 g sodium pentane sulphonate salt with pH adjustment 2.5 using phosphoric acid. The configuration of system is Shimadzu prominence. The flow rate is 1 µl and the injecting volume is 20 µl. For each sample, the peak heights corresponding to the retention times of SA were measured on chromatographs from detector, and the F/FC ratio was also calculated. The ratios from each of three replications of each culture filtrate extraction were compared to an SA standard.

According to the 16S rDNA sequencing analysis the Bacillus sp. MML2501 was identified as Bacillus licheniformis and designated as MML2501. The 16S rDNA sequence of B. licheniformis MML2501 generated with 1549 bases was deposited in the GenBank with the accession number of EU344793 and the culture was deposited in IMTECH and the deposition number is 8525.

Screening of MML2501 against a wide range of plant pathogens revealed that it effectively inhibited the mycelial growth of Macrophomina phaseolina, Fusarium oxysporum, Fusarium udum, Bipolaris oryzae, Pyricularia oryzae, Alternaria alternata and Curvularia lunata. The maximum zone of inhibition (ZOI) was observed against Bipolaris oryzae (2.2 cm) followed by Macrophomina. phaseolina (2.0 cm). However In other pathogens, the ZOI ranged from 1.0 cm to 1.9 cm (Table 1).

Figure 1

Table 1: Antagonistic activity of MML2501 against plant pathogens

Values are mean of three replicates. In a column, values followed by the same letter are not significantly different at P = 0.05.

B. licheniformis MML2501 exhibited resistance to ampicillin, streptomycin, bacitracin, cephalotitin, erythromycin and oxytetracycline. It showed sensitivity to remaining 14 antibiotics (Table 2).

Figure 2

Table 2: Antibiotic sensitivity/resistance pattern of MML2501

B. licheniformis MML2501 exhibited good tolerance to zinc, cobalt and selenium and moderate tolerance to manganese, cadmium and silver. It showed slight tolerance to lead and sensitivity to iron, mercury and arsenic (Table 3).

Figure 3

Table 3: Heavy metal tolerance spectrum of MML2501

B. licheniformis MML2501 exhibited good tolerance to propiconazole and tridemorph. It exhibited moderate tolerance to tricyclazole and slight tolerance to carbendazim, hexaconazole and mancozeb (Table 4).

Figure 4

Table 4: Fungicide tolerance spectrum of MML2501

The optimal conditions for the SA production by B. licheniformis MML2501 were standardized.

Among the static and shaken conditions, culturing B. licheniformis MML2501 at 100 rpm was found to be optimum for the production of SA (14 µg/ml) on the 2 ^nd day of incubation (Fig. 1).

Figure 5

Among different pH tested, the pH 7.0 favoured the maximum SA production of 16 g/ml as against 7-12 g/ml in rests of the pH (Fig. 2).

Figure 6

Among different temperature tested, B. licheniformis MML2501 produced maximum SA of 17 g/ml at 30 ° C. The SA production in rest of the temperature ranged between 5 g/ml and 15 g/ml. It has been observed that in both the lower and higher temperature (20, 25, 45 ° C and 50 ° C), there was no SA production (Fig. 3).

Figure 7

Different concentrations of casamino acids as a substrate ranging from 0.1-0.8% was tested for the production of SA, in which 0.4% was found to be optimum for the production of SA with 18 µg/ml on the 2 ^nd day of incubation (Fig. 4).

Figure 8

The SA produced by the B. licheniformis MML2501 was confirmed by the blue bands that appeared on pre-coated silica gel viewed under UV illumination. The R_f value of SA (0.61) produced by B. licheniformis MML2501 was matched with the R_f value (0.61) of the authentic SA (Fig. 5).

Figure 9

The extracted SA from the culture filtrate of B. licheniformis MML2501 showed similar peak to that of the authentic SA peak in HPLC analysis. The retention time of the extracted SA is 5.24 min (Fig. 6) as that of the authentic SA (Fig. 7).

Figure 10