INTRODUCTION

Medicinal plants have been used for centuries to cure human diseases^[1,2]. It has been proved that many of these plants exhibit general antifungal and antibacterial activities.

Plant diseases caused by plant pathogenic fungi are among the most important factors that limit vegetable production in Jordan. To reduce yield losses, farmers apply large quantities of fungicides every year. The continuous application of chemicals will lead to destroy the ecosystem and result in outbreaks of new strains of fungi that are difficult to control. To minimize the side effects of chemical application many efforts have been done to utilize the antimicrobial activity of plant extracts. Several studies showed the importance of natural chemical as a possible source of non-phytotoxic, systemic and easily biodegradable alternative^[3-6].

Studies on the antifungal activity of medicinal plants against plant pathogens are rare. Therefore, the aim of this study was to investigate the antifungal activity of various medicinal plants (milfoil, chamomile, field southern wood, small flowered bind wood white sage and large flowered sage) collected from different location in Jordan against the following plant pathogenic fungi (Phytophthora infestans, Fusarium oxysporum, Rhizoctonia solani, Stemphlium solani and Mucor sp.).

MATERIALS AND METHODS

Plant material: Six plants collected from different areas in Jordan (between March and May of 2003) were used in this study (Table 1).

Fungal strains: The plant pathogenic fungi used in this study were collected from various location in Jordan: phytophthora sp., Fusarium oxysporum, Rhizoctonia solani and Mucor sp. (Table 2). All fungal isolates were identified at species or genus level and deposited in fungal collection bank at Department of Biotechnology of Al-Balq’a Applied University, Al-Salt, Jordan.

Fungal isolates were maintained on Potato Dextrose Agar (PDA, Difco Laboratories, Detroit, MI USA) and the culture were stored at room temperature and subculturing once a month. The isolates were allowed to grow for 7-10 days before they were used in the microbial studies.

Preparation of extracts: Plant material was dried in shade at room temperature and then ground by using a blander. A 250 g of powdered plant material were soaked in 1.25-1.5 L of 95% ethanol for 5 days at room temperature.

Table 1:	Medicinal plants collected from Jordanian environment and used from extraction preparation

Table 2:	Plant pathogenic fungi collected from various locations in Jordan

The mixture was stirred daily by shaker for regular infusion. After a five-day period, the extract was filtered by using Whatman filter paper No. 1 (ALBET^R). The filtrate was dried using a rotary evaporator at 60°C. The final dried extract was stored in labeled sterile glass bottles at -20°C until used^[7].

Antifungal activity: Extracts from the six test plants were diluted in ( Dimmethyl Sulfoxide ) DMSO (10 mL as final volume). The 10 mL of DMSO including the plant extract was added to 240 mL of PDA to give a final concentration of 100, 250 and 500 ppm for each extract and then the resulting medium was poured in plates. Control plates received only DMSO in PDA without plant extract. Inoculum plugs from the actively growing margin of petri plate cultures of each fungal isolate was placed face down in the center of each pertri plate using a 10 cm long spring loaded plunger of 5 mm diameter. Each isolate was inoculated on 3 plates for each extract and incubated for 7-10 days at 28°C. Control plates were run along each fungal isolate and crude extract, following the same procedure as above.

Starting two days after inoculation, radial growth was recorded daily for 7 days or until the plates were overgrown. The percentage of fungal growth inhibition = [(growth in control - growth in sample)/(growth in control) X 100] where growth was measured in mililiter m as colony diameter^[8]. The values reported for minimum inhibitory concentration were average of three readings.

RESULTS AND DISCUSSION

Extracts from 6 medicinal plants from Jordan were tested against 5 phytopathogenic fungi to determine their antifungal. Antifungal activity, was measured by the MIC (Minimum Inhibition). Different concentrations of each extract were tested: 100, 250 and 500 ppm. All the fungi tested in the growth inhibition assay showed various degrees of sensitivity to the 6 plant extracts obtained (Table 3).

For S. solani 66.2% of fungal growth was inhibited with E1 at 100 ppm, followed by E6 (44.4%) at 500 ppm, E₂ (31.4%) at 500 ppm, E₃, E₄ and E₅ had a weak antifungal effect against S. solani (Table 3).

All extracts had a weak effect on Mucor sp. Except extract (E1) (60.4% inhibition at 250 ppm).

R. solani showed 51.6% of fungal growth inhibition by E₁ at 500 ppm followed by E₅ (29.8%) at 500 ppm, E₆, E₃, E₂ and E₄ had very weak antifungal effect against R. Solani (Table 3).

Fusarium oxysporum showed 47.9% of fungal growth inhibition by E₁ at 500 ppm, followed by E₅ (14.1%) at 500 ppm, E₂, E₃, E₆ and E₄ had very weak antifungal effect against F. oxyporum.

For Phytophthora sp. 28.81% of fungal growth was inhibited with E₁ at 500 ppm, followed by E₄ (20.3%) at 100 ppm. E₅, E₆, E₃ and E₂ had very weak antifungal effect against phytophthora sp.

The highest growth inhibition of all fungi was observed with S. indica (E₁), which gave (66.2%) of inhibition for stemphylim solani, followed by Mucor (60.46%), Rhizoctonia solani (51.6%), Fusarium (47.9%) and phytophthora (28.8%) (Table 3).

Table 3:	Antifungal activity of plant extracts from six medicinal plants pathogenic fungi isolated in Jordan

All extracts showed high antifungal activity against stemphylim solani E₁ (66.28%), E₆ (44.4%), E₂ (32.8%) E₃ (36.9%), E₅ (31.9%) and E₄ (29.7%) (Table 3).

The significance of indigenous products for plant disease control has been investigated in other studies and encouraging results are reported^[9-11].

This is the first report of antifungal activity (against plant pathogenic fungi) of extracts from medicinal plants collected from the Jordan environment. The preliminary results presented in this study shed the light on the ability of extracts from medicinal plants to be used against different fungi that cause plant diseases, but further studies are need to investigate the inhibitory activities of extracts from other medicinal plants on various fungi and plant pathogenic bacteria that cause severe yield losses to different crops in Jordan. In addition, efforts should be done to identify the active compounds that cause growth inhibition and try to integrate these compounds in the control programs used to reduce yield losses caused by different plant pathogens.

ACKNOWLEDGMENTS

The authors would like to thank The Higher Council For Science and Technology for their financial support. Thanks also to rawan Abu-Taleb and Wesam Shahrour for their technical assistance.