Antibacterial activity of plant extracts on phytopathogenic Xanthomonas campestris pathovars
Dr K.A. Raveesha, Agricultural Microbiology Laboratory, Department of Studies in Botany, University of Mysore, Manasagangotri, Mysore—570 006, India.
Aqueous extracts from leaves of 30 higher plants, collected from different localities, were screened in vitro for antibacterial activity against different pathovars of the phytopathogenic bacterium, Xanthomonas campestris. Eight plant species showed antibacterial activity, based on the zone of inhibition in a diffusion assay. Significant antibacterial activity was observed in the aqueous extracts of Prosopis juliflora, Oxalis corniculata and Lawsonia inermis. The susceptibility of different pathovars of X. campestris to these plant extracts varied. The antibacterial activity of extracts of a few plants was comparable with that of the synthetic antibiotics, bacterimycin and streptocycline. The study indicates the potential of these plant extracts in the management of diseases caused by X. campestris in several important crop plants.
At present, quick and effective management of plant diseases and microbial contamination in several agricultural commodities is generally achieved by the use of synthetic pesticides ( Sbragia 1975 ; Agrios 1997). However, the incessant and indiscriminate application of these chemical pesticides has caused health hazards in animals and humans due to their residual toxicity ( Ambridge & Haines 1987 ; Anonymous 1998). In recent years, a large number of synthetic pesticides have been banned in the western world because of their undesirable attributes such as high and acute toxcity, long degradation periods, accumulation in the food chain and an extension of their power to destroy both useful and harmful pests ( Barnard et al. 1997 ). In developing countries such as India, they are still being used despite their harmful effects ( Anonymous 1996a ; Wodageneh & Wulp 1997). Many pathogenic micro-organisms and insect pests have developed resistance against chemical pesticides ( May 1985 ; Urech et al. 1997 ; Williams & Heymann 1998 ; Witte 1998). This seriously hinders the management of diseases of crops and agricultural products ( Dekker 1987). Considering the deleterious effects of synthetic pesticides on life supporting systems, there is an urgent need for alternative agents for the management of pathogenic micro-organisms ( Bolkan & Reinert 1994 ; Rice et al. 1998 ).
Green plants represent a reservoir of effective chemotherapeutants and can provide valuable sources of natural pesticides ( Balandrin et al. 1985 ; Hostettmann & Wolfender 1997). Reports are available on the use of active agents from higher plants, in place of chemical fungicides, that are non-phytotoxic, more systemic and easily biodegradable ( Fawcett & Spencer 1970). Reports are also available on the use of several plant by-products, which possess antimicrobial properties, on several pathogenic bacteria and fungi ( Deans & Svoboda 1990 ; Diker et al. 1991 ; Heisey & Gorham 1992 ; De Pooter et al. 1995 ; Lis-Balchin & Deans 1996 ; Hili et al. 1997 ). However, no attempts have been made to elucidate the mechanism of the antibacterial activity of higher plant extracts on phytopathogenic bacteria. Therefore, antibacterial activity was screened in some higher plants against an important phytopathogenic bacterium, Xanthomonas campestris, which is known to cause significant losses in many crop plants due to leaf spot, leaf blight etc.
Materials and methods
Fresh leaves of 30 different plant species free from disease were collected. The leaves were washed thoroughly 2–3 times with running tap water and once with sterile distilled water. Leaf material was then air-dried on a sterile blotter under shade.
Samples (50 g) of thoroughly washed and dried leaves of each plant species were macerated with 100 ml sterile distilled water in a Waring blender (Waring International, New Hartford, CT, USA) for 10 min. The macerate was first filtered through double-layered muslin cloth, then centrifuged at 4000 g for 30 min ; the supernatant fluid was filtered through Whatman no. 1 filter paper and heat-sterilized. The extract was preserved aseptically in a brown bottle at 5 °C until use ( Gupta et al.1996).
Authentic pure cultures of phytopathogenic Xanthomonas campestris pv. malvacearum (X.c.pv.m) from cotton (Gossypium herbaceum L.), Xanthomonas campestris pv. phaseoli (X.c.pv.p ) from French bean (Phaseolus vulgaris L.) and Xanthomonas campestris pv. vasicatoria (X.c.pv.v) from tomato (Lycopersicon esculentum Mill.) were obtained from DANIDA Lab, D.O.S. in Applied Botany and Biotechnology, University of Mysore, India.
Antibacterial activity of aqueous plant extracts was determined by the cup diffusion method on nutrient agar medium ( Anonymous 1996b). The sterile medium (15 ml) in Petri dishes was uniformly smeared with cultures of X. campestris pathovars. Wells (5 mm diameter) were made in the centre of each Petri-dish, to which 50 μl of the different plant extracts were added. The treatments also included 50 μl bacterimycin (3 μg ml−1) and streptocycline (1 μg ml−1) for comparison. For each treatment, 24 replicates were maintained.
The plates were incubated at 26 °C for 24 h and the size of the resulting zone of inhibition, if any, was determined. The data were subjected to analysis of variance ( anova).
Results and discussion
Of the 30 plant extracts screened, eight showed significant antibacterial activity as evidenced by a zone of inhibition. These plant species were Acacia arabica, Achras zapota, Enterolobium saman, Lawsonia inermis, Oxalis corniculata, Prosopis juliflora, Punica granatum and Viscum orientale. The other 22 plant species, which did not show any inhibition against all the tested bacteria, were Aegle marmelos Corr., Aloe vera L., Azadirachta indica A., Juss. Catharanthus roseus (L.), G. Don., Clerodendron inerme Gaertn., Coleus aromaticus Benth., Cuscuta chinensis Lam., Delonix regia Raf., Embelica officinalis Gaertn., Euphorbia pulcherrima Willd., Hibiscus vitifolius L., Lantana camara L., Macrosolen parasiticus (L.) Danser., Mimosa pudica L., Moringa oleifera Lam., Origanum majorana L., Plumbago zeylanica L., Polyalthia longifolia KH.f. & T., Pongamia glabra Vent., Salvia officinalis L., Sapindus laurifolius Vahl. and Tabebuia argentea Britt.
Aqueous leaf extracts of the eight plant species, when tested on X. campestris pathovars with the two antibiotics, bacterimycin and streptocycline, and the zone of inhibition of these plant extracts, were significant. The antibacterial activities measured as the zone of inhibition of aqueous leaf extracts of eight plant species and the antibiotics on the pathovars of X. campestris are shown in Table 1.
Table 1 A. ntibacterial activity measured as a zone of inhibition (mm) of aqueous leaf extracts of eight plant species and two antibiotics on three pathovars of Xanthomonas campestris
|Acacia arabica Willd.||17·79±0·37||19·00±0·53||16·25±0·53|
|Achras zapota L.||12·96±0·72||18·54±0·18||10·50±0·65|
|Enterolobium saman Prain.||18·21±0·12||13·75±0·67||12·33±0·46|
|Lawsonia inermis L.||16·58±0·73||25·38±0·58||23·42±0·91|
|Oxalis corniculata L.||16·58±0·42||28·17±0·46||16·33±1·15|
|Prosopis juliflora Swartz.||18·71±0·52||23·25±0·51||23·29±1·79|
|Punica granatum L.||12·67±0·17||13·50±0·10||16·58±0·53|
|Viscum orientale Willd.||17·25±0·45||23·71±0·15||16·50±0·65|
|F-value (df ; 9, 230)||99·331 *||234·336 *||69·472 *|
Xanthomonas campestris is a rod-shaped, Gram-negative bacterium which causes bacterial blight, common blight, fuscous blight, cankers and leaf spots in French bean, cotton, paddy, tomato etc. Although losses due to the pathogen are difficult to estimate, it is known to cause significant yield loss ( Hirano & Upper 1983). Many antibiotics are used to control these diseases. The increased awareness of the environmental problems associated with these antibiotics has led to the search for non-conventional chemicals of biological origin for the management of these diseases. Bactericides of plant origin can be one approach to disease management because of their eco-friendly nature ( Bolkan & Reinert 1994). The products of plant origin are of greater advantage to the user, the public, and the radical environmentalists.
Laboratory screening of plant extracts has given encouraging results, indicating their potential use in the management of diseases caused by Xanthomonas species. As the bacterium is known to be transmitted through seeds ( Richardson 1990), one important application of the plant extracts is as a seed protectant. Prosopis juliflora, O. corniculata and L. inermis appear to be promising in this respect. Further work is underway in this laboratory to screen for their efficacy in managing seed-borne bacterial diseases in different crops.
The authors wish to thank Prof. H.S. Shetty, D.O.S. in Applied Botany and Biotechnology, for providing cultures. The authors are grateful to D.O.S. in Botany for providing facilities and the University of Mysore for financial assistance.