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Research Detail

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D Afrin*
Deparment of Animal and Fish Biotechnology, Sylhet Agricultural University, Sylhet-3100, Bangladesh

M F Hossain
Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi-6205, Bangladesh

S M Z Hasan
Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi-6205, Bangladesh

M Khalekuzzaman
2Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi-6205, Banglad
Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi-6205, Bangladesh

B Sikdar
Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi-6205, Bangladesh

Citrus species are among the most important fruit crops in the world. Citrus bacterial spot disease is caused by the bacteria Xanthomona axonopodis pv. citrumelo which may cause huge damage of citrus production. The pathogen was isolated and cultured on LB liquid media at pH 7.5 and bacterial growth developed within 48 to 72 hours. Colonies on the nutrient agar media were straw yellow and creamy in color. Isolated bacteria identified by Gram staining, Endospore staining, KOH and MacConey agar test and the result showed that isolated bacteria were gram negative. It also showed positive result in case of Catalase, Simmons citrate agar, TSI, Oxidase and KIA test. In case of carbohydrate test, highest OD (0.26) was observed in Maltose containing medium of the isolate. The isolate was tested against ten plant extracts and fifteen antibiotics. The isolate was highly sensitive against cefotaxime, neomycin, streptomycin, gentamicin, chloramphenicol, azithromycin antibiotics and the susceptibility zones were 28±0.5, 26±0.5, 23±0.5, 17±0.5, 20±0.5, 17±0.5 mm respectively. Antimicrobial activity of ten plant extracts showed that the isolateis susceptible to Momordica charantia extract, revealed a wide antibacterial spectrum against the bacterial strain.

  Citrus bacterial spot, Infectious disease, Xanthomonas axonopodis, Gram negative bacteria, Antimicrobial activity.
  Department of Genetic Engineering and Biotechnology, University of Rajshahi
  00-11-2015
  
  Pest Management
  Bacteria

The present research work has been undertaken to characterize the bacteria of citrus bacterial spot disease through biochemical approaches and to analyze its control measures by different antibiotics and plant extracts. The goal of this present study was to provide useful data to citrus breeders and to characterize the isolated bacteria using some biochemical tests and to study the effect of some antibiotics and plant extracts against isolated bacteria.

Collection and processing of infected leaves The present investigation was conducted during the period of November 2015 at Professor Joarder DNA and Chromosome Research Laboratory in the Department of Genetic Engineering and Biotechnology, University of Rajshahi. Leaves from different sections of the same citrus plant were collected from Rajshahi University area and tested for the presence of X. axonopodis pv. citrumeloas described below by different morphological and biochemical tests. Infected plant leaves were surface disinfested using a dilute sodium hypochlorite solution (10%) and rinsed thoroughly. Surface-disinfested tissue was then placed in a LB liquid medium and allowed to grow bacteria into LB liquid medium by overnight. After that, a sterile loop was used to streak the bacteria onto a solid agar medium. The bacteria were allowed to grow for at least 48 hours at room temperature and examined periodically for colony growth. Both solidified and liquid media were used for the present study. Plating is essential to obtain single colony from bacterial culture. In order to identify bacteria, it is necessary to obtain a pure culture. This is done by using the streak-plate method. Isolation, plating, streaking and subculture were done manually. At each time of plating and streaking, precaution was taken to minimize cross contamination of samples. Biochemical characterization of the isolated bacteria Isolated bacteria were characterized by some morphological and biochemical tests. Colony morphology of the isolated bacteria on the agar plate was recorded after 24 h of growth on LB agar plate at 37°C. It was yellow and creamy white in color. Gram negative test was done on the basis of their physiological and chemical properties of cell wall of the test pathogen. In order to characterize bacteria a series of biochemical tests were conducted as described by Bergey’s Manual of Systematic Bacteriology (Bergey et al., 1994). Gram (+/-) staining test, Endospore (+/-) staining test, SIM test, Methyl red test (MR), Simmons citrate utilization test, MacConkey test, Catalase test, Triple Sugar Iron test (TSI), KOH test, Klinger’s Iron Agar test (KIA test), Oxidase test, Urease test, King’s medium B test and Carbohydrate utilization test were carried out using isolated bacterial colonies or broth cultures. In MacConkey test, MacConkey agar was used for the isolation of gram-negative enteric bacteria and the differentiation of lactose fermenting from lactose non-fermenting Gram-negative bacteria (Macconkey, 1905). In case of KOH test, bacteria were aseptically removed from the agar medium with a toothpick placed on a glass slide into a drop of 3 % KOH, and stirred for 10 seconds using a quick circular motion (Suslow et al.,1982). Antimicrobial and antibacterial activity screening test using different plant extracts and antibiotic against isolated bacteria Antibiotic susceptibility test and antimicrobial activity test were usually carried out to determine which antibiotic or plant extract will be most successful to treat this test pathogen. Antimicrobial activity test was done by using ten different plant extracts (Phyllanthus emblica, Azadirachta indica, Terminalia arjuna, Justicia adhatoda, Momordica charantia, Ocimum tenuiflorum, Aloe vera etc. plant leaf extracts and Allium sativum, Zingiber officinale, Allium cepa etc. plant raw materials). Agar disk diffusion method was used for the test (Alzoreky and Nakahar, 2003). Materials of ten plant species were harvested in November 2016 from different parts of Rajshahi University campus. The isolated bacterial strains were grown overnight in nutrient broths that were placed in the shaker at 37°C temperature and 150 rpm for the antimicrobial activity test. The antimicrobial activity of the plant extracts was evaluated and the resulting inhibition zones were measured in millimeter (mm) scale. Antibacterial activity test of fifteen antibiotics (Gentamicin, Erythromycin, Doxycycline, Tetracycline, Penicillin, Amoxycillin, Chloramphenicol, Clarithromycin, Cefotaxime, Neomycin, Kanamycin, Azithromyin, Ampicillin, Stretomycin and Carbenicillin) against isolated bacteria was also done carefully by serial dilution technique. Antibiotic disks were placed centrally on the respective plates and incubate overnight at 37°C. After overnight incubation zones were observed on the plate and measured with the help of millimeter (mm) scale. The entire test was performed manually and enough care was taken for plating, streaking and handling of the test pathogen.

  J. Sylhet Agril. Univ. 3(2):315-323, 2016 ISSN: 2308-1597
  www.jsau.com.bd
Funding Source:
1.   Budget:  
  

Biochemical and physiological test results of the isolated bacteria were observed. In the present research, it was found that the citrus bacterial spot bacteria were gram negative and endospore negative from the morphological and biochemical tests. Citrus bacterial spot bacteria was highly sensitive against streptomycin, gentamicin, chloramphenicol, neomycin, cefotaxime, azithromycin antibiotics and the susceptibility zones were 23±0.5, 17±0.5, 20±0.5, 26±0.5, 28±0.5, 17±0.5 mm, respectively. Antimicrobial activity of ten plant extracts showed that the isolate was susceptible to M. charantia extract, revealed a wide antibacterial spectrum against bacterial strains. This study showed that antibiotics and some plant extracts have some activity against the pathogen and may have application in protecting plants from developing CBS. Thus, this study confirms the efficacy of some plant extracts as natural antimicrobials and suggests the possibility of employing them in drugs for treatment of infectious diseases caused by the test pathogens which may contribute in our agricultural sector.

  Journal
  


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