An Alternative Approaches for the Control of Sorghum Pathogens Using Selected Medicinal Plants Extracts

Sorghum (Sorghum vulgare L.) belongs to the tribe Andropogonae of the grass family Poaceae. The genus Sorghum is characterized by spikelet’s borne in pairs. Sorghum is treated as an annual, although it is a perennial grass and in the tropics it can be harvested many times. Sorghum crop production has considerably increased in several countries during the past few years. Sorghum is the fifth important cereals after wheat, rice and maize and are significant dietary food for one-third of the world population, these crops are the principal sources of energy, protein, vitamins and minerals for millions of the poorest people in these regions and sustain the lives of the poorest rural people and will continue to do so in the foreseeable future. India is the world's second largest producer of Sorghum. Like all crops, grain Sorghum is subject to infectious diseases which can sometimes limit production. Sorghum is susceptible to fungal and bacterial micro flora under certain environmental conditions. These mycoflora not only threaten plant growth but also affect food quality, causing huge economic losses. Every year, seed and seedling diseases of grain Sorghum are common in India. Grain Sorghum root rot can be a considerable problem in Sorghum production.


Introduction
Sorghum (Sorghum vulgare L.) belongs to the tribe Andropogonae of the grass family Poaceae. The genus Sorghum is characterized by spikelet's borne in pairs. Sorghum is treated as an annual, although it is a perennial grass and in the tropics it can be harvested many times. Sorghum crop production has considerably increased in several countries during the past few years. Sorghum is the fifth important cereals after wheat, rice and maize and are significant dietary food for one-third of the world population, these crops are the principal sources of energy, protein, vitamins and minerals for millions of the poorest people in these regions and sustain the lives of the poorest rural people and will continue to do so in the foreseeable future. India is the world's second largest producer of Sorghum. Like all crops, grain Sorghum is subject to infectious diseases which can sometimes limit production. Sorghum is susceptible to fungal and bacterial micro flora under certain environmental conditions. These mycoflora not only threaten plant growth but also affect food quality, causing huge economic losses. Every year, seed and seedling diseases of grain Sorghum are common in India. Grain Sorghum root rot can be a considerable problem in Sorghum production.
Synthetic pesticides are nowadays widely used for the control of plant diseases throughout the world because of their higher effectiveness in controlling disease causing organisms. However, excessive and unsystematic application of these chemicals has created several environmental and health hazards and also some phytopathogens have been developed resistance (Rhouma et al., 2009). Therefore, there is an urgent need to search for effective, safe and biodegradable alternative pesticides. Diseases of cultivated crops remain the major limitation to increased agricultural production. Therefore, protection of plants from pathogens remains a primary concern of agricultural scientists. Despite serious environmental implications associated with the increased use, chemical fungicides remain the first line of defense against bacterial and fungal pathogens.
Natural plant products and their analogues are an important source of new agricultural chemicals (Cardellina, 1988, Gulter, 1988. Medicinal plants as a group comprise approximately 8000 species and account for around 50% of all the higher flowering plant species of India. Over one and a half million practitioners of the Indian System of Medicine use medicinal plants in preventive, promotive and curative applications. In recent years, secondary plant metabolites (Phytochemicals), previously with unknown pharmacological activities, have been extensively investigated as a source of medicinal agents (Krisharaju et al, 2005). Plants have been formed the basis of natural pesticides, that make excellent leads for new pesticide development (Newman et al., 2000). The potential of higher plants as a source of new drugs is still largely unexplored. Hence, last decade witnessed an increase in the investigation on plants as a source of new biomolecules for human disease management (Grierson and Afolayan, 1999). Green plants are found to be an effective reservoir for the bioactive molecules and can provide valuable sources for the discovery of natural pesticides (Akhtar et al., 1997). Therefore in recent years medicinal plant extracts are intensively analyzed with an aim of isolating novel bioactive compounds.

Plant materials
Fifty medicinal plants (Table-1) were selected in this study based on the information collected from literature (Warrier et al., 1994-1996and Pullaiah, 2002. All the plant materials were collected in and around Visakhapatnam over the course of the respective growth season during February to April in the year 2005 because of the extracts were generally rich in antibacterial agents after the flowering (sexual) stage and plants from stressful environments (Mitscher et al., 1972). Plant materials were identified with the help of Gamble, "Flora of the Presidency of Madras" and later verified by comparison with the authentic specimens available in the herbariums of NBRI, Lucknow and the Department of Botany, Andhra University, Visakhapatnam. Voucher specimens were deposited in the herbarium of the Botany Department, Andhra University, Visakhapatnam.

Solvents and chemicals used
All chemicals were purchased from Qualigens fine Chemicals, Mumbai and SD fine chemicals, Mumbai. All culture media components and antibiotics used in this study were procured from Hi Media, Mumbai, India.

Tested organisms
Based on the disease index of Sorghum (Horne and Frederiksen., 1993) crops in which five phytopathogenic microorganisms were selected to screen the antimicrobial inhibition of the selected plant extracts listed in Table-2. The organisms used were procured from Microbial Type Culture Collection & Gene Bank (MTCC), Chandigarh. The lyophilized form of pure strain is reconstituted in sterile water and produced a suspension of the microbial cells. Inoculation was done with sterile inoculating loop to liquid broth medium. Liquid cultures are then incubated to allow cell replication and adequate growth of the culture, for use in bioassays. Following incubation, liquid cultures are refrigerated to store for further use. Typically, 24 hours will provide sufficient growth to allow visibly thick spread of the microbes as required for bioassay. The bacterial strains are maintained and tested on Nutrient Agar (NA) and Potato Dextrose Agar (PDA) for fungi.

Preparation of plant extracts
The collected plant materials were chopped into small pieces shade dried and coarsely powdered in Willy mill. The coarsely powdered material weighed and extracted with hexane, chloroform, methanol and water in sequential order of polarity using a soxhlet extractor for five to six hours at temperature not exceeding the boiling point of the solvent. For each gram of dry material 2ml of solvent was used. The extracted solvents were filtered through Whatman no-1 filter paper and subsequently concentrated under reduced pressure (in vacuo at 40°c) using a rotary evaporator. The residue obtained were designated as crude extracts and stored in a freezer at -20°c until assayed.
The dried plant extract residues obtained were redissolved in 0.1% Dimethyl Sulfoxide (DMSO) to get different concentrations (100mg/ml, 300mg/ml and 500mg/ml) of crude extracts and filtration through a 0.45μm membrane filter and stored in sterile brown bottles in a freezer at 20°c until bioassay.
The prepared hexane, chloroform, methanol and water extracts samples were tested for antimicrobial activity against the test organism's the plant pathogens using the agar cup plate method. Streptomycin (5µg) was placed as a positive control in all plates and inoculated with bacteria and for the bacterial cultures used that was incubated at 37°C for 18-24 hours. Bavistin (5µg) was placed as a positive control in all plates inoculated with fungi and for the fungal cultures that were incubated at 26°C for 36-48 h. The microbes were plated in triplicates and average zone diameter was noted.

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.

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 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.

Procedure
Peeled potatoes (20grams) were cut into small pieces and boiled with 100ml of water for 30 minutes. The pieces are crushed during boiling and the pulp was removed after cooling by filtration through muslin cloth. Dextrose (2grams) and agar (1.5grams) were added and the volume is made up to 100ml. the medium is then distributed in 20ml quantities in two 250ml conical flasks and were sterilized in an autoclave at 121ºC (15lbs/sq. in.) for 30min. the medium was inoculated using 4 days cultures of the test organisms in aseptic condition and transferred to sterile 6 inch diameter petri dishes and allowed to set at room temperature for about 10 minutes. Four cups of 6mm diameter bore in medium at equal distance were made in each agar plate by using sterile borer.
Hexane, chloroform, methanol and water extracts in different concentrations (100mg/ml, 300mg/ml, and 500mg/ml) to get the final drug concentration 5mg/well, 15mg/well, and 25mg/well respectively, control (DMSO) and standard (Bavistin 5μg/ml), were transferred 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 even against some antibiotic-resistant strains (Kone et al., 2004).

Minimum inhibitory concentration (MIC) assays
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 10 5 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 against all other microorganisms.

Results
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 (

Discussion
Natural products isolated from higher plants have been providing novel, antimicrobial drugs. Historically, many plant oils and extracts, such as tea tree, clove, Etc. have been used as topical antiseptics, or have been reported to have antimicrobial properties (Hoffman 1987 andLawless 1995). It is important to investigate scientifically those plants which have been used in traditional medicines as potential sources of novel antimicrobial compounds (Mitscher et al., 1987). Also, the resurgence of interest in natural therapies and increasing consumer demand for effective, safe, natural products means that quantitative data on plant oils and extracts are required.
Majority of studies conducted the search of compounds with antimicrobial properties have targeted plants with a history of ethno botanical uses (Janovska et al., 2003), most of the medicinal plant species screened in this study were previously been surveyed for antimicrobial activities on human pathogens. And very few citations were reported on phytopathogens (Kaushik and Arora, 2003;Jaspal singh and Tripathi, 1993;Krishna kishore and Suresh pande, 2005;Meena and Goplakrishnan, 2005). The observed antimicrobial activity of these plant extracts, and isolated compounds were of highly remarkable.
The present study was designed to obtain information on the antimicrobial effect of 50 Indian medicinal plants on certain plant pathogenic microorganisms. The well diffusion/cup plate method was used in this study since it was found to be better than the disc diffusion method. All the medicinal plant extracts and isolated compounds showed antimicrobial activity against selected pathogens of Sorghum.
Hexane extracts never showed antimicrobial activity. The chloroform and water extracts showed very less antimicrobial activity compared with methanol extracts. This may be due to little diffusion properties of these extracts in the agar or because fresh plants contain active substances which may be affected or disappeared by the steps of extraction methods.
The methanol extracts of all the medicinal plant screened (Table-1) exhibited grater antimicrobial activity. According to Darout et al.,(2000) the antimicrobial action of methanol extracts is due to the compounds such as thiocynate, nitrate, chloride and sulphates beside other high polarity soluble compounds which are naturally occurring in most plant materials.
Methanolic extracts of T. chebula, B. Montana, M. azadirach, W. somnifera, O santum and P. pterocarpum showed greater antimicrobial activity. Terminalia chebula possessed 32-40% of tannin content and the antibacterial activity may be indicative of the presence of some metabolic toxins or broad-spectrum antibiotic compounds (Fundter et al., 1992). M. azadirach was exhibited good antimicrobial activity against most of the tested pathogens in this study. According to Jacobson, (1995) this activity is due to Nimbidin, extracted from M. azadirach demonstrated several biological activities. From this crude principle some tetranortriterpenes, including nimbin, nimbinin, nimbidinin, nimbolide and nimbidic acid have also been showing antimicrobial activities.
The observations reveal that tested medicinal plant methanol extracts activity against all phytopathogenic species. As evidenced, the fungal strains that were sensitive are M. phaseolina, R. solani species, C. graminicola and F. moniliforme found to be resistant strains. Among the tested medicinal plants methanol extracts against the phytopathogenic species, Terminalia chebula extracts showed greater antimicrobial activity on all plant pathogens.
In view of the changing agricultural policies throughout the world complete disease control is no longer a target of plant pathologist's reducing the threshold level using cost-effective and eco-friendly management option is the focus of the day. In this context identification of aqueous leaf extract of T. chebula and M. azadirach methanol extracts as bactericides and fungicides against the pathogens tested are highly significant recommendable. The result of these studies maybe helpful in developing/synthesizing the plant based natural fungicides and insecticides that may be for preventing and curing the common destructive diseases of Sorghum crop and other cereal crops. In this context the studied plant extracts is more appropriate and helpful in synthesizing the plant based biofungicides to reduce the pathogen population to lower economic threshold level using cost effective and eco friendly management. This will also offer a great help in facing the emergence spread of antimicrobial resistance.