Bobbrala V, Rao G. S, Maduri D. B, Naidu K. C. Biocide Potentialities of Different Plant Methanolic Extracts Against Crown Gall Bacteria viz Agrobacterium Tumefaciens. Biomed. Pharmacol. J.2009;2(1)

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Manuscript received on :February 10, 2009
Manuscript accepted on :March 10, 2009
Published online on: 12-11-2015

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Varaprasad Bobbrala¹, G. Srinivasa Rao¹, D. Bindu Maduri¹ and K. Chendrashekara Naidu²

¹For U Biosciences, A/4A, Park lane Residency, East point colony, Visakhapatnam - 530 017, India.

²Department of Botany, Andhra University, Visakhapatnam India.

Corresponding Author E-mail:varaprasadphd@rediffmail.com

Abstract

In this contemporary investigation fifty different plants used in traditional Indian medicine were examined against crown gall disease causing phytopathogen i.e. Agrobacterium tumefaciens using agar well diffusion method. The antimicrobial activities of fifty plant methanolic extracts demonstrated varyious inhibition effects on A. tumefaciens. Except the following six plants such as A. ilcifolius, A. officinalis, C. sativum, P. rubra, R. communis, and T. pumila were not exhibited the antimicrobial activity against A. tumefaciens. Among the selected fifty plants 88% of plants given remarkable bioactivity where as only the 12% of plants did not give antimicrobial activity against A. tumefaciens.

Keywords

Agrobacterium tumefaciens; Crown gall disease; Indian medicinal plants; Soxhlet extraction

Introduction

tumefaciens is the causal agent of Crown Gall disease (the formation of tumors) in over 140 species of dicots. It is a rod shaped Gram Negative soil bacterium. A. tumefaciens is an alfa proteobacterium of the family Rhizobiaceae, which includes the nitrogen fixing legume symbionts. Unlike the nitrogen fixing symbionts, tumor producing Agrobacterium are parasitic and do not benefit the plant. The wide variety of plants affected by Agrobacterium makes it of great concern to the agriculture industry (1). Economically, A. tumefaciens is a serious pathogen of grape vines, stone fruits, nut trees, sugar beets, horse radish and rhubarb.

The plants that possess therapeutic properties on the animal or plant body are generally designated as medicinal plants. A medicinal plant is any plant which, in one or more of its organ contains substance that can be used for therapeutic purpose or which is a precursor for synthesis of new drugs. The plants which ecologically synthesis and accumulate some secondary metabolites like alkaloids, glycosides, tannins, volatile oils, minerals and vitamins, they possess medicinal properties. Higher and aromatics plants have been used traditionally in folk medicine as well as to extend the shelf life of foods, showing inhibition against bacteria, fungi and yeasts (2). Biologically active compounds from natural sources have always been a great interest for scientists working on infectious diseases (3). The use of phytochemicals as natural antimicrobial agents commonly called “biocides” is gaining popularity (4). There is growing interest in correlating phytochemical constituents of plant with its pharmacological activity (5).  The main advantage of natural agents that they do not enhance the antibiotic resistance, a phenomenon commonly encountered with the long-term use of synthetic antibiotics. It has been reported that the higher plants have shown to be a potential source for the new antimicrobial agents (6). The antimicrobial compounds from plants may inhibit bacterial growth by different mechanisms than those presently used. Antimicrobials therefore, may have a significant clinical value in treatment of resistant microbial strains (7). In particular, the antimicrobial activities of plant oils and extracts have formed the basis of many applications including raw and processed food preservation, pharmaceuticals, alternative medicine, and natural therapies (8). According to the above information the authors selected and collected fifty different medicinal plants from coastal region of Andhra Pradesh. They were extracted by using soxhlet apparatus and have been evaluated antibacterial activity against A. tumefaciens.

 Materials and Methods

Plant material and preparation of the extracts:

The plant materials of fifty plant species (Table. 1) were collected from different places at Visakhapatnam. The collected plants were identified and authenticated by Professor M. Venkaiah, Department of Botany, Andhra University, Visakhapatnam, Andhrapradesh. Voucher specimens have been deposited at the Herbarium of our department. The selected parts of different medicinal plants were cut into small pieces and shade dried at room temperature for fifteen days, finely powdered plant materials were successively extracted with methanol using soxhlet apparatus. The different extracts obtained were subsequently concentrated under reduced pressure to get their corresponding residues. The extracts were screened for antimicrobial activity using the method described under the section.

Table 1: list of investigated medicinal plants.

Microorganism employed

The plant extracts were assayed for antimicrobial activity against one reference bacterial strain Agrobacterium tumefaciens (MTCC 2250) obtained from Microbial Type Culture Collection & Gene Bank (MTCC), Chandigarh. And a strain isolate from soil and identified with biochemical examination was used in this present research. The bacteria was grown in the nutrient broth at 37⁰C and maintained on nutrient agar slants at 4⁰C until used.

Antimicrobial analysis

The Methanolic extracts of fifty different plant extracts were screened for antimicrobial activity by agar well diffusion method (9) with cork borer of size 6.0mm.  The overnight cultures grown in nutrient broth was used for inoculation of nutrient agar plates. An aliquot (0.02ml) of inoculums was introduced to molten nutrient agar and poured on petri dish by pour plate technique. After solidification the appropriate wells were made on agar plate by using cork borer. In agar well diffusion method 0.05ml of methanolic extracts of fifty different plant extracts were introduced serially after successful completion of one plant analysis. Incubation period of 24-48hours at 37⁰C was maintained for observation of antimicrobial activity of plant extracts.  The antimicrobial activity was evaluated by qualifying zones of inhibition of bacterial growth surrounding the plant extracts. The complete antimicrobial analysis was carried out under strict aseptic conditions and results were represented in Table: 2.

Table: 2 Minimum inhibitory Concentration (MIC) analysis of fifty plants methanolic extracts on A. tumefaciens.   Click here to View table

Results and Discussion

The antimicrobial screening of fifty different methanolic plant extracts were reported in table 2. The antimicrobial activities of different medicinal plants (Table.1) methanolic extracts were studied in this investigation. Among the selected fifty plants forty four plants given remarkable bioactivity where as only the six plants A. ilcifolius, A. officinalis, C. sativum, P. rubrum, R. communis and T. pumila were not exhibited antimicrobial activity against A. tumefaciens. The antimicrobial activity was observed by forty four plants with variable concentrations, not with the same concentrations. Based on this criterion plants can be divided as A, B and C and called them as highest activity plants, moderate activity, and least activity plants respectively. The plants given activity up to 50mg/ml, 55-100mg/ml, and 110-300mg/ml concentrations were treated as group A, B and C respectively. The plants B. montana, P. pterophorus, S. dulcis, T. chebula and W. somnifera, were given bioactivity up to 50mg/ml concentration. Among them the highest activity was exhibited by S. dulcis at 15mg/ml concentration against A. tumefaciens and Terminalia chebula also given very nice activity with 25mg/ml conc. Most of the tested plants have reported moderate activity with 55-100mg/ml conc. Among them A. farnaciana, C. forskohlii, G. arborea, H. antidysenterica, H. populipolia, H. sueolences, L. camara, M. azedarach and P. pinnata were shown good moderate activity with 75mg/ml concentration and finally least bioactivity was found with only one plant H. bengalenses at 300mg/ml conc. Therefore, this result may suggest that methanolic extracts of screened plants would be helpful in treating crown gall disease in plants. In particular, the authors may recommend that the methanolic extract of S. dulcis can be used as potent biocide to treat the crown gall disease caused by A. tumefaciens. The observed findings suggested the further work on all the selected plants to evaluate their potential for use as antibacterials to treat human and plant related diseases.

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