**2.5 Antibacterial activity**

The antimicrobial activity of the chloroform, methanol and water extracts of each sample was evaluated by using well diffusion method or cup plate method of Murray *et al.,* (1995) modified by Olurinola, (1996). Which is the most widely used type for identifying the antimicrobial activity, which exploit diffusion of antimicrobial compounds through agar media to demonstrate inhibition of bacteria and fungi.

### **2.5.1 Composition of nutrient agar medium**


#### **2.5.2 Procedure**

This assay performed by two methods agar disc diffusion and agar well diffusion. In these two methods the agar well diffusion essay was used to screen for antimicrobial activity of the hexane, chloroform, methanol and water extracts of different plant species. In agar well

An Alternative Approaches for the Control of

against all other microorganisms.

*Rhizoctonia solani* at less than 50mg/ml concentration.

all the pathogens tested in this study.

**3. Results** 

even against some antibiotic-resistant strains (Kone *et al.,* 2004).

**2.7 Minimum inhibitory concentration (MIC) assays** 

Sorghum Pathogens Using Selected Medicinal Plants Extracts 133

to the cups of each agar plate, incubated at room temperature (28ºC) and examined for inhibition zones of 36 hours of incubation. The results of different studies provide evidence that some medicinal plants might indeed be potential sources of new antibacterial agents

Based on the preliminary reports all the medicinal plants were identified to have potent antimicrobial activity and Minimum Inhibitory Concentrations (MIC) of the extracts was determined according to Elizabeth, (2001). A final concentration of 0.5% (v/v) Tween-20 (Sigma) was used to enhance crude extract solubility. A series of two fold dilution of each extract, ranging from 0.2 to 100 mg/ml, was prepared. After sterilization, the medium was inoculated with 3μl aliquots of culture containing approximately 105 CFU/ml of each organism of 24 hours slant culture in aseptic condition and transferred into sterile 6 inch diameter petridish and allowed to set at room temperature for about 10 minutes and then kept in a refrigerator for 30 minutes. After the media solidified a number 3-cup borer (6mm) diameter was properly sterilized by flaming and used to make three to five uniform cups/wells in each petridish. A drop of molten nutrient agar was used to seal the base of each cup. Different plant crude extracts ranging from 0.2 to 100mg/ml were added to the cups/wells of each petridish and the control plates without plant extract. Inhibition of organism growth in the plates containing test crude extracts was judged by comparison with growth in blank control plates. The MICs were determined as the lowest concentration of extracts inhibiting visible growth of each organism on the agar plate. Similarly the MICs of methanol extracts were determined

Among the 50 plant methanol extracts screened thirteen plant extracts showed antibacterial and antifungal activity by zone of inhibition. These results indicated that the plant extracts showed antibacterial as well as antifungal activity. Hexane, chloroform and aqueous extracts were showed very less activity against all the phytopathogens hence only the methanol extracts reports was analyzed. The methanol extracts activities were increased with increasing concentrations. However, the activity produced by the extract was low when compared with that of the standard. The methanol extracts of fifty medicinal plants (Table-1) showed broad spectrum of antimicrobial activity against the test organisms (Table-2) using agar cup plate method. The plant species were *Adenocalymna allicia*, *Acacia farnaciana*, *Avicenia officinales*, *Bridilia Montana*, *Coleus forskohlii*, *Phyllanthus niruri*, *Grewia arborea*, *Melia azadirach, Ocimum sanctum*, *Peltophorum pterocarpum*, *Scoparia dulcis, Terminalia chebula* and *Withania somnifera*, showed a significant activity against *Macrophomina phaseolina*,

Of all *Terminalia chebula* and *Melia azadirach* showed remarkable largest zones of inhibition against all the phytopathogens tested. Antimicrobial activities are different medicinal plants were represented in Table 3 and 4. Fruit extract of *Terminalia chebula* showed less than 2mg/ml and *Melia azadirach* below 15mg/ml concentrations showed significant activity on

diffusion method peptone (0.5 grams), meat extract (1.0 grams), sodium chloride (0.5 grams) and agar (1.5 grams) were dissolved in small quantity of distilled water with the aid of heat on water bath and the volume was made up to 100 ml with purified water. The pH of the nutrient broth was adjusted to 7.2 using 5M sodium hydroxide, and then sterilized in an autoclave maintained at 121ºC (15lbs/sq. in.) for 20 minutes.

After sterilization, the medium was inoculated with 3μl aliquots of culture containing approximately 105 CFU/ml of each organism of 24hours slant culture in aseptic condition and transferred into sterile 6 inch diameter petridishes and allowed to set at room temperature for about 10 minutes and then kept in a refrigerator for 30 minutes. After setting a number 3 cup borer (6mm) diameter was properly sterilized by flaming and used to make three to five uniform cups/wells in each petridish. A drop of molten nutrient agar was used to seal the base of each cup. The cups/wells were filled with 50µl of the different extracts of 100mg/ml, 300mg/ml, and 500mg/ml so final drug concentration will be 5mg/well, 15mg/well, and 25mg/well respectively and allow diffusing of plant extract into the medium for about 45 minutes.

Standard drugs Streptomycin (5μg/ml), control (0.1% DMSO) were transferred to the cups of each agar plate by means of sterile pipettes under a laminar flow unit. The solvents used for reconstituting the extracts were similarly analyzed. The plates thus prepared were left for 2 hours in a refrigerator for diffusion and then kept in an incubator at 37ºC. After 24 hours, the agar plates were examined for inhibition zones, and the zones were measured in millimeters. The zones of inhibition were measured with antibiotic zone scale in mm and the experiment was carried out in triplicates.
