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Biological Control of Bacterial Leaf Spot Disease of Papaya (carica Papaya) Through Antagonistic Approaches Using Medicinal Plants Extracts and Soil Bacteria

Md. Firose Hossain
Department of Genetic Engineering and Biotechnology, Faculty of Life and Earth Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh

SM Zia Hasan
Department of Genetic Engineering and Biotechnology, Faculty of Life and Earth Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh

Zannati Ferdous Zaoti
Department of Genetic Engineering and Biotechnology, Faculty of Life and Earth Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh

Habiba
Department of Genetic Engineering and Biotechnology, Faculty of Life and Earth Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh

Md. Faruk Hasan
Department of Genetic Engineering and Biotechnology, Faculty of Life and Earth Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh

Md. Asadul Islam
Department of Genetic Engineering and Biotechnology, Faculty of Life and Earth Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh

Biswanath Sikdar*
Professor Joarder DNA and Chromosome Research Lab., Department of Genetic Engineering and Biotechnology, Faculty of Life and Earth Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh

Abstract/ Executive Summary:

The present investigation was carried out to isolate the causal agent of bacterial leaf spot disease of papaya and evaluation of its biological control through different extracts from selected medicinal plants and antagonistic microorganisms. The causal organism was isolated on nutrient agar medium at 370C for incubation period of 16 hours. Molecular detection of the isolated bacteria confirmed by PCR using specific primers 27F (5′-AGAGTTTGATCCTGGCTC3′) and 1391R (5′-GACGGCGGTGTGTRCA-3′) which amplified approximately 1600 bp DNA fragment of the isolated bacteria. Four different types of solvents Methanol, Ethanol, Acetone, Petroleum ether were used to prepare the extract from Lantana camara, Pongamia pinnata, Rauwolfia serpentina, Cymbopogon citrates, Tagetes erecta. Among them the ethanol extract of Lantana camara showed highest 13.5 mm zone of inhibition at 60µg/ml concentration and methanol extract showed 17mm zone of inhibition at 100µg/ml concentrations respectively against isolated bacteria. Antagonistic soil bacteria were isolated from different soil samples from Rajshahi University premises. Isolate 1 and 2 of soil bacteria showed highest 12±0.2 mm inhibition zone at 40µl/disc concentration in disc diffusion method and isolate 3 of soil bacteria showed 16±0.5 mm inhibition zone against bacterial leaf spot disease causing pathogen at 50 µl/well concentrations in well diffusion method. This study will be helpful for effective biological control of bacterial leaf spot disease of papaya causing less harm to the environment compare to the traditionally available chemical control.

Keywords: Papaya, Bacterial leaf spot, Biological control, Antagonistic, Pseudomonas carica papaya.

Location: Department of Genetic Engineering and Biotechnology, Faculty of Life and Earth Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh

Start Date:

End Date:

Program Area: Pest Management

Commodity: Extract (plant, seed), Papaya, Diseases

Objectives:

The present study was designed to screen the antagonistic activity of soil bacteria and extracts from selected medicinal plants to biological control of bacterial leaf spot disease of papaya.

Methodology:

Plant Materials In the present study bacterial leaf spot infected papaya plants were collected from Rajshahi University premises and were identified by Bangladesh Council of Science and Industrial Research (BCSIR), Binodpur, Rajshahi. Five medicinal plants Lantana camara (choitra), Pongamia pinnata, Rauwolfia serpentina, Cymbopogon citrates (lemon grass), Tagetes erecta were collected from the Botanical garden of Rajshahi University for the Preparation of plant extracts. Isolation of causal organism Disease infected leaves of papaya were first washed using distilled water and disinfested using a dilute sodium hypochlorite solution (10%) and rinsed thoroughly. We cut the infected area and placed on LB liquid media and incubated for 12 to 16 hours at 37oC for allow to growing bacteria. After the bacteria have grown into LB liquid medium, used a sterile loop to streak the bacteria onto a solid nutrient agar media plates and incubated for 12 hours at 37^oC. One of creamy white colony was picked by wire loop and streaked on another media plate for pure culture. Molecular identification of causal organism (Isolated bacteria) For genomic DNA isolation and purification, the isolated bacterial single colony was cultured in LB broth medium at 37ºC for overnight. The culture was centrifuged and the liquid was discarded. The total genomic DNA were isolated from bacterial mass by heat lyses and selective precipitation of cell debris and polysaccharides with CTAB (cetyltrimethylammonium-bromide), and the procedure was maintained as similar to Ausbel10 with slight modification in the incubation period and amount of chemicals which was used. The DNA was resuspended in TE buffer and quantified using a spectrophotometer then electrophoresed on 1% agar gel by comparison with DNA samples of known concentration. The amplification of 16S rRNA gene from the isolated DNA was done by PCR reaction in a thermo cycler (Nyx, Technic, Inc., USA), using the primers 27F (5′-AGAGTTTGATCCTGGCTC-3′) and 1391R (5′-GACGGCGGTGTGTRCA-3′). PCR was performed in volumes of 25µl, containing nuclease free ddH2O 15µl, dNTP mix 1.0µl, forward primer 1.0µl, reverse primer 1.0µl, DNA template 1.5µl, MgCl2 2.5µl, Taq buffer B 2.5µL and Taq polymerase (Takara, Japan) 0.5µl. The procedure was as following: initial denaturation at 95ºC for 5min; 35 cycles of denaturation for 40s at 95ºC, annealing for 1min at 65ºC, and extension for 2min at 72ºC; the final extension at 72ºC for 10 min, followed by cooling to 4ºC until the sample was recovered. Gel electrophoresis was used to visualize the PCR products lengths. 0.5x TBE buffer was used in agar gel and visualized under a UV transilluminator. Isolation of antagonistic microorganism (Soil bacteria) Soil samples were collected in the month of January 2017 from rhizopheric region of leguminous plants from Rajshahi University region and brought to Lab. Samples were collected to a depth of 5-7 inches from rhizopheric region. After collection, soil samples were immediately dried at room temperature for two to five days. 200gm of soil samples was taken for microbiological analysis and the remainder was discarded. Soil sample was collected from four different regions. Analysis of each soil samples was done separately. Bacterial-flora from the soil samples were isolated by serial dilution, plating and streaking on suitable growth medium11. Colony was found after incubation at 37⁰C for 16 hours. Screening of antagonistic activity of soil bacteria Screening of antagonistic activity was done by disc diffusion method and well diffusion method. Agar disc-diffusion testing developed in 1940 is the official method used in many clinical microbiology laboratories for routine antimicrobial susceptibility testing12. Disc diffusion method was done according to Hasan and Sikdar13. In disc diffusion method we inoculated causal pathogen on to the agar plate. Then we placed the filter paper discs (about 6 mm in diameter), containing the soil bacteria at 10µl, 20µl, 40µl and 50µl per disc concentration, on the centre of agar surface. The Petri dishes were incubated at 37⁰C for 16 hours. Then diameter of zones of inhibition was measured by millimeter (mm) scale.

Source of Information: Int. J. Pure App. Biosci. 6 (1): 1-11 (2018) ISSN: 2320 – 7051

Article Link (Online Journal): doi: http://dx.doi.org/10.18782/2320-7051.6137

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Bacterial leaf spot of papaya is very demolishing disease in Bangladesh. It can cause serious damage to papaya production and greatly hampers the economy. Pseudomonas caricapapayae caused this disease. Different types of chemical control have used to control this disease but it cause serious danger to animal diversity. In our study we used different types of medicinal plant and soil bacteria to inhibit the growth of causal pathogen of this disease in vitro as biological control. We found significant result for methanol and ethanol extract of Lantana camara as well as for soil bacteria in disc diffusion method regarding the growth inhibition of the causal pathogen. So findings of our present study would be helpful to biologically control the bacterial leaf spot disease of papaya.

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