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Isolation, Identification, in Vitro Antibiotic Resistance and Plant Extract Sensitivity of Fire Blight Causing Erwinia Amylovora

Mohammed Amirul Islam
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science & Technology, Sylhet, Bangladesh.

Md Jahangir Alam
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science & Technology, Sylhet, Bangladesh.

Samsed Ahmed Urmee
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science & Technology, Sylhet, Bangladesh.

Muhammed Hamidur Rahaman
Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi-6205, Bangladesh.

Mamudul Hasan Razu
Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi-6205, Bangladesh.

Reaz Mohammad Mazumdar
BCSIR Laboratories Chittagong, Bangladesh Council of Scientific and Industrial Research, Chittagong-4202, Bangladesh

Abstract/ Executive Summary:

Erwinia amylovora is the causal organism of fire blight. The fire blight is widely spread in bacterial disease of plants from both epidemiological and economic points of view. Furthermore, the situation is worsening by the advent of increased antibiotic resistance in these bacteria. The study was aimed to determine the in vitro antibiotic and herbal sensitivity of E. amylovora isolated from plants available in Sylhet, Bangladesh. In this study, bacterial isolates taken from five fire blight infected plants like apple, pear, lemon, orange and olive plants were identified based on morphological, cultural and biochemical characteristics. All the isolates were tested for antibiotic sensitivity against five commonly used antibiotics and herbal sensitivity against five plants extract. Morphological, physiological and biochemical study of pure culture of suspected organism revealed E. amylovora bacteria which was found 100% resistant to Cefotaxime and 81.89% to Bacitracin. Chloramphenicol was found most effective as all the isolates were sensitive to it. Besides that, most of the isolates were susceptible to plant extracts and found maximum sensitive to Allium sativum and Syzygium cumini whereas resistant to V. amurensis. It can be concluded that the investigation of herbal treatment can be implicated for fire blight disease in contrast of antibiotic test in future.

Keywords: Fire blight; Erwinia amylovora; Antibiotic sensitivity, Plant extract

Location: Sylhet, Bangladesh.

Start Date:

End Date:

Program Area: Pest Management

Commodity: Diseases

Objectives:

To identify the bacteria on the basis of morphological, physiological, biochemical test and make a comparative study in vitro between antibiotic and plant extract sensitivity of the organism.

Methodology:

Total number of 21 diseased plant samples was collected from different nurseries of Sylhet city according to standard pathological procedure. Then, 1 ml of fruits rinsed water and fruit juices sample was taken to a test-tube containing 9 ml of sterile water and thoroughly mixed to get a 10^-1 dilution of the water sample. Again, 1 ml of 10^-1 dilution was transferred again to another 9 ml of sterile water in another test-tube and thoroughly mixed to get a 10^-2 dilution. In such way serial dilution of water samples were made up to 10^-4. Isolation of E. amylovora was done on Nutrient Agar (NA) or Leavan media which was prepared by dissolving 1g yeast extract, 2.5 g peptone, 2.5g NaCl, 25 g sucrose into 500 ml of distilled water, pH adjusted to 7.0-7.2 and sterilized by autoclaving at 121°C, 15 psi for 15 minutes. Then, transferred the suspected single colony from NA plate by sterile loop and inoculated on the King’s medium agar B (KB) which was prepared by peptone 20g, glycerol 10 mL, K₂HPO₄ 1.5g, MgSO₄·7 H₂O 1.5 g, agar 15 g, distilled water 1000 mL, pH adjusted to 7.0-7.2 and sterilized by autoclaving at 121° for 20 minutes. The plates were then incubated at 27°C for 2-3 days and observed daily for bacterial growth. Suspected colonies of E. amylovora (white, circular, mucoid, and curved) were selected and further purified again on KB agar at 27°C. This operation was repeated three to four times to be sure that pure cultures were obtained for identification tests and preserved it for next investigation. For identification of E. amylovora, colony morphology was studied when it appeared in KB and NA media. Moreover, Gram’s staining was performed as described by and growth of pure cultured isolates was measured at 39ºC and 4% NaCl containing NA media. Besides that, fluorescent test was done and under UV light at 366 nm after 48 h of incubated plate. Biochemical tests like oxidative-fermentative test, nitrate reduction test, citrate utilization test, urease test, sucrose fermentation test, TSI (Triple Sugar Iodine) test, mannitol fermentation test, gelatine hydrolysis were done. In antibiotic susceptibility test 250 µl of microbial inoculums of E. amylovora strain from cultured nutrient broth was spread on the surface of Mueller-Hinton agar (CM337-OXOID) by using a sterile L-shaped glass rod. Then in vitro five different commercially available antimicrobial discs Streptomycin (10 µg), Gentamycin (10 µg), Chloramphenicol (30 µg), Cefotaxime (5 µg), Bacitracin (10 µg) were applied on the inoculated plates according to the Kirby-Bauer. During susceptibility test same bacterial density was maintained by using Spectrophotometer at OD₆₀₀. After incubation, the plates were examined and the diameters of the zone of complete inhibition were measured in mm. The antimicrobial activity of five plants extracts were used in the herbal sensitivity experiments. Name and parts of the plants are given. After cleaning the selected parts of plant by sterilized distilled water, extracts were collected in a falcon tube and centrifuged at 4000 rpm for few minutes. Besides that wells of 5 mm diameter were punched into the agar plates with the help of sterilized cork borer and 50 µl of the plant extracts were added by using a micropipette to the wells made in the agar plate along with well diffusion 0.1 ml of diluted inoculum of the freshly cultured experimental strains. Inhibitory response of the herbal extracts was recorded according to the normal growth response of the bacteria after incubation at 28°C for 24 hour and zone of inhibition was measured by mm.

Source of Information: J Plant Pathol Microb 2014, 5(3), ISSN: 2157-7471

Article Link (Online Journal): http://dx.doi.org/10.4172/2157-7471.1000233

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

It can be concluded that, present study showed all the isolates of E. amylovora were resistant to at least one or more of commonly used antibiotics. So, emphasis must be placed on the development of effective bactericides and their proper use with knowledge of the appropriate dosage. On the other hand, herbal sensitivity test has paved the way the viable introduction of plants for the treatment of disease causing microorganism in cheaper cost and eco-friendly way. Therefore, it will be more beneficial to put emphasis on biological control of E. amylovora through plant extracts instead of antibiotics. Moreover, further researches are necessary to find more plant species and purify the antimicrobial substances present in the crude plant extracts effective against E. amylovora.

Knowledge Management: Journal