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Plant Extract Regulating Pectate Lyase Production in Soft Rotting Bacterial Isolates

Nazmun Nahar
Department of Biotechnology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh

Nazmun Nahar
Department of Biotechnology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh

Md. Manjurul Haque
Department of Environmental Science, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh

Md. Abu Ashraf Khan
Department of Plant Pathology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh

Md. Ashraful Haque
Department of Biotechnology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh

Md. Manjurul Haque
Department of Environmental Science, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh

Md. Abu Ashraf Khan
Department of Plant Pathology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh

Md. Ashraful Haque
Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh

Abstract/ Executive Summary:

Soft-rotting bacteria caused diseases by secreting the plant cell-wall-degrading enzymes mainly pectate lyase (Pel). In this study, a total of 20 bacterial strains were isolated from soft rotted fruits and vegetables. Among them, 11 isolates produced soft rot symptom on potato tubers. All these isolates were partially characterized. When these isolates were tested for their Pel production in different media, the isolate P1 produced significantly higher amount of Pel than other isolates in M63 glycerol minimal medium. Interestingly, the isolate P1 grown on M63 glycerol minimal medium supplementing with 0.4% of polygalacturonic acid (PGA) and M63 glycerol minimal medium plus 0.4% of PGA and 1% of various plant extracts, Pel production was induced in M63 glycerol minimal medium containing 0.4% of PGA and hyper-induced in M63 glycerol minimal medium plus 0.4% of PGA and 1% of various plant extracts. However, Pel synthesis was not hyper-induced in high concentrations of potato extract. Thus, not only plant extracts but also concentrations of plant extract may be important for hyper-induction of Pel.

Keywords: Soft rotting bacterial isolates, Pectate lyase, Hyper-induction, Plant extracts

Location: Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh

Start Date: 00-06-2014

End Date: 00-05-2015

Program Area: Pest Management

Commodity: Diseases

Objectives:

To isolate and characterize soft rotting bacteria from diverse plant species affected by disease, and
To investigate the effect of plant extracts on the induction of Pel production in soft rotting bacteria.

Methodology:

The experiment was conducted in the laboratory of Biotechnology and Plant Pathology, Faculty of Agriculture, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur during June 2014 to May 2015. In order to isolate soft rotting bacteria, diseased potato tubers and plants (Solanum tuberosum L.), mango fruits (Mangifera indica L.), onion (Allum cepa L.), melongena L.) and apple (Malus domestica) were collected from local markets, experimental and farmer’s fields. A portion of each sample was sliced with water, then a loop of sample was streaked on yeast extract peptone (YP) agar plates and incubated at 28°C for 48 h. Maceration tests were done using potato tubers. The isolate caused soft rot symptom on potato tubers was selected. KOH test was done as described previously with a few modifications. In brief, two drops of 3% KOH solution were placed on a clean glass slide, then a loop full of 24-h old bacterial culture grown on YP agar plate was added and mixed properly. The viscosity of bacterial suspension was tested within 10 seconds. A loop was pushed in suspension and pulled out gently. The gram negative bacterial suspension produces a fine thread of slime while the gram positive bacteria do not produce such thread but are watery. On YP agar plate) was placed on a clean glass slide then a drop of 3% hydrogen peroxide (H₂O₂) solution was added and mixed with the culture. Production of gas bubbles indicated positive reaction. The test was performed as described by Hugh and Leifson (1953). The broth contained 2.0 g of peptone dissolved in 1L distilled water and the pH was adjusted to 7.0. The 5 mL of broth was dispensed into the test tubes, plugged and autoclaved. Test tubes were cooled to 45-50⁰C, then 10% filter sterilized glucose solution was added aseptically. The medium was stab inoculated with a small loop containing 24-h old bacterial cells (two test tubes were inoculated). One test tube was covered with sterile liquid paraffin to a depth of 8-12 mm. The test tubes were then incubated at 28°C for 48 h and were observed for production of acid manifested by yellow color. Fermentative bacteria produced acid in both test tubes while oxidative bacteria produced acid only in uncovered medium. To check the growth and Pel production, each isolate was grown in various growth media such as M63 glycerol minimal medium (per liter, 2.5 g of NaCl, 3 g of KH₂ HPO₄, 2 g of (NH₄)₂SO₄, 0.5 mg of FeSO₄, 2 g of thiamine hydrochloride, and 0.2% (wt/vol) of glycerol), Luria-Bertani (LB) medium (1% of tryptone, 0.5% of yeast extract, 0.5% of NaCl, pH 7.0) and yeast extractpeptone (YP) medium (1% of peptone, 0.5% yeast extract, pH 6.8). For the growth of the bacterial isolates, 50 µL of each bacterial isolate was inoculated in glass test tube containing 5 mL of M63 glycerol minimal medium then incubated at the indicated temperatures. The optical density (OD) was measured after 24 h incubation with spectrophotometer at 660. Bar indicate error bar with standard error for data of three independent experiments. However, Pel specific activity was determined as described previously. In brief, isolates were grown in the medium until OD₆₆₀ reached at 1.0 then 1 ml culture was centrifuged at 15000 rpm for 5 min to remove the cell debris. The supernatant was used for assaying Pel activity. A 10 µl of sample solution was added to 990 µl of the reaction buffer (0.05% PGA), 0.1 M Tris -HCl pH 8.5, 0.1 mM CaCl₂, pre-warmed to 30⁰C. After a proper mixing of the solution, the increase in optical density at 230 nm was measured every minute. One unit of Pel activity was defined as the amount of the enzyme that produced a change in absorbance of 0.001 at 230 nm in 1.0 min. The mean of Pel activity from five independent experiments was expressed as the specific activity (U/OD₆₆₀).

Source of Information: The Agriculturists 13(2): 81-88 (2015); ISSN 2304-7321

Article Link (Online Journal): http://banglajol.info/index.php/AGRIC/article/download/26594/17844

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Different soft rotting bacterial isolates were isolated from different diseased fruits and vegetable samples. All the isolates were only partially characterized. Thus, 16S rRNA gene sequence is needed to confirm the genus and species of the bacteria. Among the isolates, P1 produced higher Pel than the other isolates. Media composition and concentration of plant extracts played an important role in hyper induction of Pel synthesis but many crucial questions remains unanswered. The study will help us to know how bacterial soft rot pathogens cause disease and respond to the plant components.

Knowledge Management: Journal