
Indigenous strains of
Twenty one isolates each of
Twenty random rhizosphere soil samples were collected from different tea gardens of Assam, north eastern region of India and stored in sterile plastic bags. The soil samples were air dried and isolation was done by the serial dilution technique (Krassilnikov, 1950). Morphologically distinct colonies were picked on the basis of their morphology (Kubicek and Harman, 2002) and purified on PDA.
All the isolates of
Where r1 was the radial growth of pathogen without
Initial the identification of
To prepare the cell-free culture filtrate, the antagonist was cultured in potato dextrose broth (Himedia Laboratories Pvt. Ltd., India) and incubated at 28°C on shaker incubator (150 rpm). Incubation period ranging from 8 to 24 days was assessed to determine the effect of incubation period of active metabolite production by SDRLIN1. Cell-free supernatants were collected at 2-days’ intervals by aseptic filtration through Whatman filter paper no. 44 followed by re-filteration through 0.20 μm cellulose acetate syringe filter. The growth inhibitory effects of the extracellular metabolites from culture filtrates were estimated by using the radial growth inhibition assay on PDA containing 50% (v/v) cell free filtrate. Fungal growth inhibition was expressed as the percentage of radial growth inhibition relative to the control (Behzad et al., 2008).
The strain SDRLIN1 was grown in potato dextrose broth with continuous shaking (150 rpm) at 28°C for 24 days. Cell-free supernatants were collected from 8 days incubation period, at 2-day intervals as described previously. Proteases activity was determined by Lowry et al. (1951) method using 1% casein in 50 mM phosphate buffer (pH 7.0). The absorbance of liberated tyrosine in the filtrate was measured at 750 nm. Cellulase activity was measured following the method of Miller (1959) with 0.5% pure cellulose (Sigma Co. USA) suspended in 50 mM phosphate buffer (pH 5.0). The absorbance was measured at 540 nm and the amount of reducing sugar released was calculated from standard curve of glucose. The β-1,3-glucanase activity was assayed according to the method of Singh et al. (1999) with slight modifications, using laminarin from
All the test phytopathogenic fungi namely
To assess the stability of the extracellular metabolites, 20 days old culture filtrates of strain SDRLIN1 were either treated with 0.1 mg/ml proteinase K (Thermo Scientific, Lithuania) at 37°C for 60 min or boiled for 45 min. The inhibitory effect of treated culture filtrates on the radial growth of
Mycoparasitism of the SDRLIN1 against test fungal phytopathogens was studied using slide culture method. Inocula from the antagonist and test fungus were placed 1 cm apart from each other on the surface of microscopic slides covered with thin (5.0-mm-thick) layers of PDA. The inoculated slides were incubated at 28°C for 3?5 days. At the end of the incubation period, regions where the hyphae of
The means and standard deviations of the inhibition levels, radial growth and activities of proteases, cellulase, β-1,3-glucanase, pectinase, chitinase and amylase were calculated. Data were analysed by one-way analysis of variance (ANOVA) using GraphPad Prism 5. Significant differences (
Out of the total 49 isolates of
The strain SDRLIN1 was identified as
20 days old cell free culture filtrate of the antagonist SDRLIN1 showed maximum inhibition activity against all the test fungal phytopathogens at 50% (v/v) concentration. The antagonistic activity was increasing in the exponential phase and was noted highest in the stationary phase (Fig. 4 and Fig. 5). The reduction in the radial growth of isolates of
The activity of extracellular cellulase, pectinase, β-1,3-glucanase, amylase, proteases and chitinase was determined at different growth phases from 8 days to 24 days at 2 days of interval. There was no cellulose, pectin, casein, laminarin, chitin or related substrate containing in the PD broth. The highest activity of extracellular enzymes namely cellulase (170.82 IU/L), pectinase (168.43 IU/L), β-1,3-glucanase (90.63 IU/L) and amylase (16.51 IU/L) was noted at 8 days of incubation period. The activity of the respective extracellular enzymes decreases gradually in the subsequent incubation periods. Contrary to this, the activity of proteases (20.61 IU/L) and chitinase (6.91 IU/L) was noted maximum at 20 and 14 days of incubation period respectively followed by subsequent decrease in the later phase of growth (Fig. 6).
Light microscope investigation revealed that extracellular metabolites in the culture filtrate of the antagonist produced cellular alterations in hyphal morphology including hyphal distortion, swelling and cytoplasm aggregation of the test phytopathogenic fungus (Fig. 7).
The antifungal activity of proteinase K treated culture filtrate of the antagonist against
SDRLIN1 showed parasitic behavior against all the test fungal phytopathogens namely
Antagonist potential of
Further screening of the strain SDRLIN1 against different isolates of
Cell free culture filtrate from different growth phases was used in the present study to demonstrate the possible best optimal stage of the growth period exhibiting optimum concentration of extracellular antifungal metabolite (s) corresponding to highest antagonistic activity of the strain
The antagonist showed statistically significant differences (
The higher antagonistic activity of the strain SDRLIN1 recorded against majority of the
The production of extracellular hydrolytic enzymes, apart from other secretory antifungal metabolites, by
The effects caused by extracellular metabolites of the strain SDRLIN1 on the hyphal morphology of the pathogen were also studied through light microscopy. A greater degree of alteration of hyphal morphology in the form of distortion, swelling and even aggregation of the cytoplasm was noted on PDA amended with 50% (v/v) stationary culture filtrate (Fig. 7). It has been reported that lytic enzymes such as β-1,3-glucanases, chitinases and proteases produced by antagonistic strains are responsible for such changes in the hyphal morphology (Cortes et al., 1998). The observations of this study further highlighted the importance of these lytic enzymes in the antagonistic activity of
On the basis of findings from
Microscopic study of the interaction region between test fungal pathogens with