Antibacterial activity of some plant extracts against Rathyibacter tritici

The ever expanding world population requires the production of huge quantities of food, but our efforts are hampered due to various diseases caused by phytopathogens. There is an urgent need for a quantum jump in productivity, particularly of the major crop wheat, that too with the present trend of declining per capita availability of arable land. In order to maintain the productivity, more and more chemicals are being added in the natural environment, which enter the food chain through water, soil and air as a result it seriously affect the human health1. According to the WHO survey, more than 50,000 people in developing countries are annually poisoned and 5,000 die as a result of the effects of toxic agents, used in agriculture. In India 35,000-40,000 tons of hazardous chemicals are sprayed on the crops every year, instead of helping the poor, these chemicals are causing cancer, sterility and death2. So there is an urgent need to develop sustainable methods for these horrible diseases. As plants and their product are known to possess various secondary metabolites, which showed inhibitory effect against the growth of pathogens, therefore, Biosciences, Biotechnology Research Asia Vol. 5(1), 283-288 (2008)


INTRODUCTION
The ever expanding world population requires the production of huge quantities of food, but our efforts are hampered due to various diseases caused by phytopathogens.There is an urgent need for a quantum jump in productivity, particularly of the major crop wheat, that too with the present trend of declining per capita availability of arable land.In order to maintain the productivity, more and more chemicals are being added in the natural environment, which enter the food chain through water, soil and air as a result it seriously affect the human health 1 .According to the WHO survey, more than 50,000 people in developing countries are annually poisoned and 5,000 die as a result of the effects of toxic agents, used in agriculture.In India 35,000-40,000 tons of hazardous chemicals are sprayed on the crops every year, instead of helping the poor, these chemicals are causing cancer, sterility and death 2 .So there is an urgent need to develop sustainable methods for these horrible diseases.As plants and their product are known to possess various secondary metabolites, which showed inhibitory effect against the growth of pathogens, therefore, the plants and their product should be utilized to combat the diseases causing pathogens.Keeping these problems in view, efforts are underway to search economic safe phytochemicals, which could be utilized for disease control.Thus in continuations of previous paper 3 , the object of present study was to exploit the potential of some more plants extracts against Rathyibacter tritici for selecting the most potent plant possessing antibacterial activities.

MATERIAL AND METHODS
The various parts of each plant were collected from different region of Haryana and its neighboring states on the basis of their traditional values (Table 1).The collected plant materials were thoroughly washed with tap water, followed with distilled water and then kept in dark under the filter papers at room temperature till completely dry.Each sample was individually grounded into powder form for preparation of extract.The bacteria Rathyibacter tritici used for the study was procured from cultures stocks of our Department.The culture was maintained at 4°C on Nutrient Agar medium with periodic sub-culturing.Western Peninsula Infusion of the plant is used to wash sores 20 .
and Baluchistan

Antibacterial tests
Fifteen percent plants parts extracts was prepared by brewing in boiling water for 15 minutes followed by centrifugation at 12000 rpm for 15 minutes.The supernatants were collected in screwcapped vials and sterilized by autoclaving for 15 minutes at 121°C and the pH was adjusted to 7.0 4 .
The assay for antibacterial activity of each plant part extract was tested by agar diffusion method 5 .Bacterial suspensions were cultured in peptone water for 6-8h and 0.2ml of this culture was spread on Mueller-Hinton agar in Petri dishes.Wells (8mm diam) were cut in agar plates and were filled 0.1ml of 15% plants extracts.The plates inoculated with Rathyibacter tritici were incubated at 37°C.The resulting zone of inhibition was measured after 24 h.Each combination of isolates and antimicrobial agent was repeated three times.The isolate which showed clear zone of inhibition more than 12mm including the 8mm well size were considered sensitive and those with less than 12mm as resistant.
Minimum Inhibitory Concentration (MIC) was determined by the agar dilution method 6 where plants samples concentration ranged from 0.25%-3.0%and defined as the lowest concentration that prevented visible growth of microorganisms after incubation for 40hours at 37°C.

RESULTS
The activity of the plants extracts against the bacterial growth of Rathyibacter tritici is presented in Table 2

Minimum inhibitory concentrations (MIC)
The MIC of Clerodendron inerme was observed 3.0% while the MIC of four plants samples i.e.Brassicae juanca, Capparis decidua, Cassia fistula and Cuscuta reflexa were observed 2.0% for the test bacteria.Anthocephalus cadamba, Cannabis sativa and Casuarinae equisetifolia showed 1.0% Minimum Inhibitory Concentrations while Acacia arabicae and Azadirachta indica showed 0.5% Minimum Inhibitory Concentrations for the test bacteria Rathyibacter tritici as presented in Table 2.

DISCUSSION
Considering the need for an alternative eco-friendly approach to control the phyto pathogens, it was believed to be worthwhile to screen the antibacterial effects of locally available flora.The results obtained are indicating of the differential activities of the plant extracts against the bacterial growth of Rathyibacter tritici because many of these extracts have shown very strong inhibition against the bacterial growth of test bacteria (Table 2) and a definite potential for new effective bactericides.Among the plants whose extracts were found to be effective are Acacia arabicae, Casuarinae equisetifolia and Cuscuta reflexa.
The seed extracts of Acacia arabicae showed strong inhibitory effect against the bacterial growth of test bacteria (Fig. 1), which might be due to the presence of some antimicrobial secondary metabolites in the plant sample, some phytochemicals have also been reported in literature 7 and possess various medicinal properties 8,9 , hence, the spray of the extracts of Acacia arabicae could be used for protecting plants against pathogenic organisms instead of synthetic chemicals.
The seed extracts of Casuarinae equisetifolia also found strong effective against the bacterial growth of test bacteria, which might be due to the presence of some antimicrobial agents.Various reports were available about the medicinal, biological and economical properties 8,10,11 ; hence, the spray of the extracts of Casuarinae equisetifolia could be used for protecting plants against pathogenic organisms.
The antimicrobial activities of plants studied have also been found registered in various literature i.e.Azadirachta indica 12,13 , Cannabis sativa 14 , Capparis decidua 15 , Cassia fistula 16 .

Minimum inhibitory concentration
In general the MIC of various plants samples ranges from 0.5% -3.0% as presented in table 2. The test bacteria Rathyibacter tritici was observed sensitive at very low concentrations of the aqueous extracts of Acacia arabicae and Azadirachta indica.Minimum Inhibitory Concentrations was found slightly higher in case of.Brassicae juanca, Capparis decidua, Cassia fistula and Cuscuta reflexa against the test bacterium while Clerodendron inerme was observed to show inhibitory effect against the Rathyiabacter tritici at very higher concentrations as compared to others tested plants samples (Table 2).The variations in the Minimum Inhibitory Concentrations might be due to slight differences in phytochemicals composition.Earlier, various reports were found registered in literature about the variations in Minimum Inhibitory Concentrations of plants samples for phytopathogens 4,17 .
Since the extracts of Anthocephalus cadamba, Brassicae juanca, Clerodendron inerme and Cuscuta reflexa used in this study have not been tested before as inhibitor of phytopathogenic bacteria, therefore, they are the new addition to this field of study.The presence of various secondary metabolites such as alkaloids, quaternary alkaloids, coumarins, flavanoids, steroids/terpenoids, phenols etc. have been reported in the various plants extracts 10,15,18 which may be responsible for the antibacterial properties of the plant studied.

Table 2 : Antibacterial activity and minimum inhibitory concentrations (MIC) of plant extracts against Rathyibacter tritici
and Fig. 1.It is commonly observed that out of 20 plants parts extracts tested the seed extracts of Acacia arabicae (22.5mm) and seed extracts of Casuarinae equisetifolia (22.0mm) showed marvelous inhibitory effect against the bacterial growth of Rathyibacter tritici.The strong inhibitory effect was shown by whole plant extracts