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Invitro Evaluation of Botanicals and Biocontrol Agents against Pomegranate Bacterial Blight Pathogen

Seema J Patel1, Shalini D B2, Sripriya P A2 , Priyanka S2, Sowmya B M2
  1. Assistant Professor, Department of Biotechnology, GM Institute of Technology, Davangere, Karnataka, India
  2. UG Student, Department of Biotechnology, GM Institute of Technology, Davangere, Karnataka, India
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In India, pomegranate is regarded as a “vital cash crop”. Successful cultivation of pomegranate in recent years has met with different traumas such as pest and diseases. Among diseases bacterial blight caused by Xanthomonasaxonopodispv. Punicae is a major threat. In the present study the bacteria were isolated from infected pomegranate plant; fruit, leaves and stem and subjected to bio chemical analysis to identify Xanthomonas. Ethanol, methanol and aqueous extracts of four botanicals and three bio control agents were evaluated for their antibacterial activity against the harmful pathogen which was isolated from the infected pomegranate plant. Among these, the ethanol extracts of Azadirachtaindica (Neem) and bio control agent T.viride showed significant inhibitory effect. These were applied to the infected plant and were tested.


Botanicals, pomegranate, biocontrol, bacterial blight.


Pomegranate (Punicagranatum L.) is an ancient fruit, belonging to the smallest botanical family Punicaceae. Pomegranate is a good source of carbohydrates and minerals such as calcium, iron and sulphur. It is rich in vitamin-C and citric acid is the most predominant organic acid in pomegranate [1]. The bark of the stem, root and rind of the fruit is used for slimming, control of dysentery, diarrhea and killing tapeworms [2].
In Karnataka, this crop has spread across different districts viz., Bijapur, Bellary, Bagalkot, Koppal, Chitradurga, Belgaum, Davangere, Tumkur, Bangalore and Gulbarga. Pomegranate, the commercial crop of the farmer turned as a bane after the outbreak of bacterial blight. Since 2002, the disease has reached the alarming stage and hampering the Indian economy vis-à-vis export of quality fruits. The disease accounted up to 70 – 100 per cent during 2006 in Karnataka and Maharashtra resulting in wipe out of pomegranate during the year 2007, the total output of pomegranate production in India was down by 60 per cent [3]. During 2008 – 09 the disease has reached its alarming stage bringing substantial damage to the crop and heavy loss to the farmers. However, the magnitude of the disease severity has raised several questions to researchers, farm managers, administrators, private firms and above all the farmers to safeguard the cultivation of this dollar earning crop.
Disease is characterized by the appearance of small, irregular water soaked, dark colored spots on leaves resulting in premature defoliation. Pathogen also infects stem and branches causing girdling and cracking symptoms. Spots on fruit are dark brown, irregular slightly raised with oily appearance, which split opens with „L‟/„Y‟ shaped cracks at final stages. Under severe condition it destroys the entire orchard and causes heavy economic losses. There are large number of chemicals are available in the market as bactericides and their bioefficacy and suitability needs to be verified by in vitro and field studies, so as to incorporate the effective ones in the management package [4].


Isolation and identification of bacteria from infected stem and fruit:
Pomegranate fruits and stem showing typical symptoms of bacterial blight were collected from the field (farm in Chitradurga district) and the bacterium was isolated by dilution plate technique. Isolation of the pathogen is done within 48 hours after collection of samples on nutrient agar medium. Pure culture is established by repeated single colony transfer method. A reference culture was obtained from UAS, Dharwad.
The morphological characteristics such as cell shape and gram staining characters of the isolates were studied. The physiological and biochemical characters of the isolates were studied for hydrogen sulphide production, urease production and acid from different sugars viz., glucose, arabinose, lactose etc.
Koch‟s postulates were followed to prove pathogenic nature of Xanthomonas isolates. For pathogenicity test, inoculation has to be made by spraying bacterial suspension of 48 hours old culture onto the injured and uninjured stem and fruits of healthy plants. The characteristics symptoms were observed on pomegranate leaves after four days of inoculation as small water soaked lesions. After six days of inoculation it turned brown to black coloured lesions, which later developed into angular to irregular shaped spots along the margins, veins and veinlets of the leaf lamina. Reisolations were carried out from these lesions for each isolate and comparisons were made with original culture to confirm the identity of the pathogen. The reisolated culture resembled the original mother culture and thus pathogenicity test was confirmed [5].
Evaluation of efficacy of plant extracts against bacterial pathogen
Fresh leaves and bulb of different medicinal plants (Neem, Tulsi, Ginger, Garlic) are collected and washed, shade dried and then powdered using the blender and stored in air tight bottles[6]. 10 g of powder is added to 100 ml of solvent (methanol, ethanol, water) in a conical flask and plugged with cotton wool. After 42 hours the supernatant is collected and the solvent evaporated to make the crude extract and stored at 4o C [7].
Antibacterial activity testing
Antibacterial activity of aqueous extract, solvent extracts was determined by cup diffusion / agar well diffusion method on nutrient agar medium. Cups/ wells are made in nutrient agar plate using cork borer (5 mm) and inoculum of bacteria is spread on the solid plates with a sterile swab moistened with the bacterial suspension. Then aqueous extract, solvents extracts are placed in the wells made in inoculated plates. Similarly each plate carried a blank with solvent only in the center to serve as a control [7].
Determination of the efficacy of biocontrol agents against the bacterial pathogen
Cultures of Pseudomonas fluorescens (NCIM 2174), Bacillus subtilis(NCIM 2548) and Trichodermaviride(NCIM 1060) are obtained from NCIM, Pune and used as biocontrol agents. Effect of bio control agents was studied by well diffusion method [8] and dual culture plate method [6].


Pomegranate fruits showing typical symptoms of bacterial blight are collected from the field and the bacterium was isolated by dilution plate technique. By gram staining technique the bacteria was identified to be gram negative rod shaped bacteria. Then the Hi media standard test kit for the identification of gram negative bacteria was used for further identification of bacteria.
Field application:Two plants in the field were used for the field application study for a month.Farmer sprayed the botanical extract of neem on the diseased plant once in week. It avoided spreading of disease. The spraying of the biocontrol agent Trichodermaviride with sterile water once in a week helped in fruit development.


In the present market, demand of Indian pomegranate fruit is decreasing because of using more chemical pesticides to control the disease, so with the present work we are stressing on using botanicals and biocontrol agents for the disease management and for yielding more crop.


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