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Management of Seedling Disease of Cucumber by Using Rhizosphere Antagonistic Bacteria

K M Alam*
Scientific Officer
Plant Pathology Division, BARI Gazipur

D Sarkar
Scientific Officer
Pulse Research Centre, BARI, Ishurdi Pabna

M A U Zaman
Scientific Officer
Rice Farming Systems Division, BRRI Gazipur

F Begum
Assistant Professor
Department of Plant Pathology, BSMRAU Gazipur

KA Bhuiyan
Professor
Department of Plant Pathology, BSMRAU Gazipur

Abstract/ Executive Summary:

A total of 47 bacteria were isolated from rhizosphere soil samples, collected from different locations of Bagerhat district in Bangladesh. Initially, all the isolates were tested for antagonism against virulent isolates of Rhizoctonia solani and Sclerotiumrolfsii in dual plate culture, where 4 isolates were found to be antagonistic against both of the target fungi. Following a series of biochemical assays, all the antagonistic isolates were identified as Bacillusspp. However, in dual plate culture test, the antagonistic Bacillus isolates demonstrated a higher level of antagonism against S. rolfsii compared to R. solani. Interestingly, the thermo stable components of all Bacillusisolates completely inhibited the colony growth of both S. rolfsii. On the other hand, the thermo stable components Bacillusisolate AB4 showed the highest potentiality to inhibit mycelial growth of R. solani. In the pot experiment, the isolate AB4 also showed highest disease reduction (69.56 and 71.46%) against R. solani and S. rolfsii, respectively. In cucumber growth promotion test, it was found that all the Bacillusisolates significantly influenced plant height, root length, leaf area, fresh shoot weight, dry shoot weight, fresh root weight and dry root weight.

Keywords: Rhizosphere, Antagonistic bacteria, Seedling disease, Cucumber

Location: In the laboratory and glasshouse of the Department of Plant Pathology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Salna, Gazipur 1706

Start Date: 00-00-2010

End Date:

Program Area: Pest Management

Commodity: Diseases, Cucumber

Objectives:

This aimed to isolate the rhizosphere bacteria and to assess their antagonism against seedling disease of cucumber caused by S.rolfsiiand R.solani

Methodology:

Study site The study was conducted in the laboratory and glasshouse of the Department of Plant Pathology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Salna, Gazipur 1706 during 2010. Isolation and Characterization of bacteria Six soil samples were collected from Bagerhat district, Bangladesh. Bacteria were isolated from individual sample following the soil dilution plate technique. Isolates were purified following single colony purification of the colonies with different appearance (color and colony shape). The purified isolates were preserved temporarily in 15% glycerol solution at 4^oC. Dual culture experiment was carried out to observe antifungal activity of bacterial isolates against the pathogenic isolates of S. rolfsii and R. solanion PDA plates. Those bacteria which developed inhibition zone on dual culture plate against the pathogenic fungus on were preserved in 10% LKB (20 g/l peptone, 1.5 g/l K2HPO4, 15 ml glycerol and 1.5 g/l MgSO4) media with 10 g skim milk at -80^oC. A series of biochemical test such as Gram staining, growth in CAG medium, growth at 7% NaCl, growth at pH 5.7 in PD broth, oxidase test, catalase test, gliding motility test and growth at 45^oC were conducted to characterize the antagonistic bacterial strains. The selected four strains of antagonistic bacteria genera were identified the following Bergey’s Manual of Determinative Bacteriology (Hortet al., 1994). Dual Culture Assay The study was conducted following CRD with three replicates. A 5mm disc from the margin of 3 days old fungal culture was placed at more or less centre of the PDA plate. Thereafter, aloopful of overnight grown antagonistic bacterial isolates (each) 10⁸CFU/ml suspension was sketch in straight line at 30 mm apart from the fungal disc. All plates were incubated in the dark for 25^oC until the mycelia of S. rolfsii and R. solani fully covered the control plates. The radius of the fungal colony towards and away from the bacterial colony was measured. Degree of antagonism was determined by measuring the radial growth of pathogen with bacterial culture and control. The percentage of inhibition was calculated using the equation suggested by Riunguet al., 2008. Thermo stable antifungal components of Bacillus sp. Bacillus spp. were incubated individually in 250 ml flask containing 200 ml YP (yeast peptone) broth for 7 days at 25^oC. Thereafter, supernatant was collected from the culture after 10 min.centrifuging at 15000 rpm. Required yeast, peptone and agar forpreparing 200 ml YPA medium were added to 200 mlsupernatant and then autoclaved at 121^oC under 1.1 kg/cm2pressures for 20 minutes. Five millimeter block of S. rolfsiiand R. solaniwas placed on the center of petri dish containing supernatant and incubated at 30^oC for 72 hours. Growth of S. rolfsiiand R. solanion YPA platesat 30^oC for 72 hours wasalso observed to determine the presence of thermo stable components in supernatant and their antifungal capacity. Diameter of mycelia growth was measured in mm using a ruler.Estimation of nitrogenase enzyme activity (acetylene reduction assay) One milliliter of the culture was transferred into an air-tight 30 ml bottle containing 10 ml of Nfb semi-solid medium (5 g malic acid, 0.5 g K2HPO4, 0.2 g MgSO4.2H2O, 0.1 g NaCl, 0.02 g CaCl2and 0.5 % bromothymol blue in 0.2 N KOH (2 ml), 1.64% Fe-EDTA solution (4 ml), 0.02 g yeast extract, 2 g agar). After the pellicle formation (72 h), the bottles to be injected with 5% (v/v) acetylene gas with simultaneous removal of the same volume of air. The bottles then incubated at 30°C for 24 h. 1 ml of gas to be withdrawn and transferred to 7 ml Vacutainer TW tubes. The ethylene gas produced to be assayed using G-300 gas chromatograph equipped with a FID and 1-m Porpak N column.

Source of Information: Eco-friendly Agril. J. 10(05):58-64, 2017 (May)

Article Link (Online Journal): www.efaj-international.com

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

From the collected six rhizosphere soil samples, 47 bacteria were isolated through soil dilution technique. Among them, four bacterial isolates showed antagonism against S. rolfsii and R. solani in in-vitro test on dual plate culture. These four antagonistic bacteria were identified as gram positive Bacillus through morphological and biochemical characterization. These four Bacillus isolates showed efficacy in controlling seedling disease of cucumber in pot experiment where S. rolfsiiand R. solani were inoculated in soil. In another pot test, we observed that Bacillus isolates had also some growth promotion effect on cucumber seedling. As this experiment was conducted in controlled conditions, so further experimentation are required in field conditions to evaluate the efficacy of those Bacillus isolates against seedling disease of cucumber.

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