Abstract: In the present study, the antifungal activity of Cinnamomum zeylanicum Blume, Trigonella foenum-graecum L., Eucalyptus globulus Labill, Eruca sativa L. and Allium sativum L. extracts were investigated against soil borne pathogenic fungi. The results showed the plant extracts had inhibitory activities on the mycelial growth and spore germination of these fungi. The results exhibited C. zeylanicum and E. globulus at 1000 ppm gave the highest inhibition on the mycelial growth of the tested fungi except Fusarium verticillioides (Sacc.) that giving 48.5 and 54.4% inhibition, respectively. Whereas, A. sativum was the best extract effective against F. verticillioides (F. moniliforme) which produced 55.9% reduction in the mycelial growth. Extract of C. zeylanicum completely inhibited spore germination of the three Fusarium species tested. Also, E. globulus completely inhibited spore germination of F. oxysporum f. sp. melonis (Leach. and Currence) Snyd. and Hans. While, A. sativum gave 100% inhibition of the spore germination of F. solani at 1000 ppm concentration.
INTRODUCTION
Muskmelon (Cucumis melo L.) is affected by various diseases which in turn produce heavy loss to the crop. The diseases include wilt and root rot causing by soil borne fungi. These fungi are the important that causing great reduction in the field in Egypt (El-Sheshtawi et al., 2014). Fungicides are necessary to manage plant diseases and to maintain high crop yields. However, indiscriminate utilization of these fungicides has frequently resulted in adverse ecological effects, as disturbing the environmental stability of soils and making plants still more susceptible to diseases (Mancini et al., 2008). Increasing public concern on environmental issues requires alternative disease management systems which are less fungicides based on naturally occurring compounds (Cuthbertson and Murchie, 2005). Chemical pesticides utilization is a very popular practice to manage several plant pathogenic fungi as compare to natural one that are prepared from plants or plant parts. Nonetheless, consumer now demands less use of synthetic fungicides as a result of the residual toxicities, pollutive nature and non-biodegradability of agrochemicals (Avasthi et al., 2005). Researchers have revealed the plant extracts as source of natural fungicides that make good efforts for new fungicides development (Arokiyaraj et al., 2008; Brindha et al., 2009). Meanwhile, various spices and herbs have been utilized for centuries as preservatives for foods and medicinal purposes, some of them possess antifungal potential in combination and are considered as alternatives to conservative antifungal agents (Nwaopara et al., 2008). There are several studies reported that the phytochemicals of Melia azedarach, Euclyptus citriodora and Alstonia scholaris showed fungicidal activity against pathogenic fungi (Lloyd et al., 2005). The essential oil from Cymbopogon citratus showed inhibition of F. oxysporum f. sp. cubense (Guimaraes et al., 2011). Also, De Oliveira et al. (2008) showed that Lippia sidoides was effective in suppressing the mycelial growth of F. oxysporum f. sp. cubense with similar results imposed by carbendazin which demonstrates the possibility for utilizing these sources without losses in efficacy compared to current products. The present study was carried out to investigate the antifungal activity of plant extracts of some plant species against soil borne pathogenic fungi isolated from diseased rhizospher and soil of muskmelon.
MATERIALS AND METHODS
Soil samples and plant parts showing wilt and root rot characteristic were collected from the field of muskmelon. The samples were taken from Dakahliya governorate, Egypt. The plant parts were examined under microscope to confirm the presence of pathogens. An infected plant roots were cut into pieces (2-3 mm), then it is surface sterilized with 0.1% sodium hypochlorite (NaOCl) for 30 sec. The root samples were washed three times with sterilized distilled water and transferred aseptically on Potato Dextrose Agar (PDA). The plates were incubated at 25±2°C and observations were made daily for emergence of culture. After the development of the fungal colonies stock cultures were prepared using PDA in test tubes and stored at 4°C. All pathogens were isolated from infected muskmelon identified according to Barnett and Hunter (1998), Booth (1977) and Kora et al. (2005).
Preparation of plant extracts: Bark of C. zeylanicum, seeds of T. foenum-graecum, leaves of E. globulus, leaves of E. sativa and bulb of A. sativum were washed with distilled water. Leaves were surface sterilized with 1% sodium hypochlorite solution for 1 min followed by several washings with sterilized water. Fifty gram of plant material of each of the five test plant species was taken in 100 mL of sterilized distilled water and blended for 5 min at low speed and left for 1 h. The blended materials were then passed through muslin cloth and finally filtered through Whatman filter paper No.1 to obtain a 50% w/v stock solution.
Aqueous extract bioassays: To determine the inhibitory effects of plant extracts, selected concentrations of the plant extracts were incorporated into the PDA and poured into Petri dishes. Agar discs (0.5 cm in diameter) were placed in the center of Petri dishes containing PDA with the corresponding plant extract at different concentrations. Petri dishes were sealed with parafilm. The plates without the plant extract were used as control. The plates were incubated for 4 days and mycelium growth was measured.
Inhibitory effect of plant extracts on spore germination: The antifungal effect of plant extracts on conidial germination of the three Fusarium species used were tested using different concentrations of aqueous plant extracts by spore germination method using cavity slides (Maji et al., 2005). Spore suspension of the pathogens was prepared aseptically from 7 days old pure culture. Fifty microliter of spore suspension and 50 μL of different concentrations of aqueous leaf extracts were taken on separate cavity slides. One cavity was maintained as control without adding any extract. All treatments were maintained in triplicates. And the cavity slides were incubated at ambient temperature (25±2°C) in moist chambers for 24 h. After the incubation period, observations were made under microscope to calculate the Percentage Inhibition (PI) by counting the number of spore germinated and the total number of spores in different microscopic view.
RESULTS
Inhibitory effect of plant extracts against F. oxysporum f. sp. melonis (Leach. and Currence) Snyd. and Hans: The extract of C. zeylanicum was found highly effective in suppressing the growth of F. oxysporum f. sp. melonis. All the concentrations of the extract significantly reduced the mycelial growth of the fungus in vitro. There was 46.1-78.6% reduction in fungal mycelial growth due to different concentrations of the extract (Table 1). This was followed by E. globulus extract, E. sativa and T. foenum-graecum extract giving 69.3, 66.1 and 62.8% reduction in the mycelial growth of F. oxysporum f. sp. melonis, respectively. In case of spore germination, the maximum inhibition of spore germination was recorded in C. zeylanicum and E. globulus extract which completely inhibited spore germination at 1000 ppm concentration (Table 2). This was followed by E. sativa and A. sativum with 95.7 and 93.0% inhibithion in spore germination, respectively. Significant results were also observed in T. foenum-graecum extract which showed average results with 54.2% inhibition in spore germination.
| Table 1: | Effect of plant extracts on radial growth of F. oxysporum f. sp. melonis |
| R.G: Radial growth, Inh %: Inhibition (%) | |
| Table 2: | Effect of plant extracts on spore germination of F. oxysporum f. sp. melonis |
| S.G: Spore germination, Inh %: Inhibition (%) | |
| Table 3: | Effect of plant extracts on radial growth of Rhizoctonia solani |
| R.G: Radial growth Inh.%: Inhibition (%) | |
| Table 4: | Effect of plant extracts on radial growth of F. verticillioides |
| R.G: Radial growth, Inh %: Inhibition (%) | |
| Table 5: | Effect of plant extracts on spore germination of F. verticillioides |
| S.G: Spore germination, Inh %: Inhibition (%) | |
Inhibitory effects of plant extracts against Rhizoctonia solani kühn: Data in Table 3 showed that all plant extracts had inhibitory activity against R. solani. The C. zeylanicum extract induced growth inhibition zone (80.2%) at a concentration of 1000 ppm. On the other hand, another plant extract used showed moderate reduction ranging from 47.1% (E. sativa) to 52.6% (A. sativum).
Inhibitory effects of plant extracts against F. verticillioides (Sacc.): In vitro antifungal activities of plant extracts showed that all studied plant extracts had inhibitory effect on F. verticillioides. The extracts obtained from the bulb of A. sativum showed the highest antifungal activity against the pathogen that produced 55.9% reduction in mycelial growth (Table 4). This was followed by E. globulus extract by 54.4% suppersion in mycelial growth. Conversely, E. sativa extract gave the lowest effect against F. verticillioides giving 40.4% reduction in mycelial growth. On the other hand, the effect of different extracts of selected plants was observed on spore germination of F. verticillioides. The extract of C. zeylanicum at 1000 ppm was found to be most effective in reducing the spore germination with 100%, followed by E. globulus extract with 96.5% reduction (Table 5).
Inhibitory effect of plant extracts against Sclerotinia sclerotiorum (Lib.) de bary: The activity of the plant extracts against the mycelial growth of S. sclerotiorum is presented in Table 6. It was noticed that out of five plant extracts tested, leaf extract of E. globulus (62.0%) showed maximum inhibitory effect against the mycelial growth of S. sclerotiorum followed by C. zeylanicum extract (55.7%). On the other hand, T. foenum-graecum produced the lowest effect against S. sclerotiorum with 30.0% reduction in mycelial growth.
| Table 6: | Effect of plant extracts on radial growth of Sclerotinia sclerotiorum |
| R.G: Radial growth, Inh %: Inhibition (%) | |
| Table 7: | Effect of plant extracts on radial growth of F. solani |
| R.G: Radial growth, Inh %: Inhibition (%) | |
| Table 8: | Effect of plant extracts on spore germination of F. solani |
| S.G: Spore germination, Inh %: Inhibition (%) | |
Inhibitory effect of plant extracts against Fusarium solani (Mart.) Sacc: It is evident from Table 7 that C. zeylanicum extract showed antifungal activity against F. solani with 71.0% reduction in mycelial growth followed by E. globulus extract that produced 66.1% reduction in mycelial growth of the pathogen. Oppositely, the extract of E. sativa exhibited moderate activity against F. solani with 42.3% reduction. On the other hand, the extracts of C. zeylanicum and A. sativum at 1000 ppm completely suppressed spore germination. Also, the extract of E. globulus and E. sativa highly effective on spore germination with 98.2 and 91.7% inhibition in spore germination of F. solani, respectively (Table 8). Conversely, the T. foenum-graecum extract produced the least reduction in spore germination with 29.7%.
DISCUSSION
The present study demonstrated that the plant extract such as C. zeylanicum, T. foenum-graecum, E. globulus, E. sativa and A. sativum had considerable effect on the growth rate and spore germination of soil borne pathogenic fungi. The extract of C. zeylanicum indicated considerable antifungal activity against mycelial and spore germination of soil borne fungal pathogens. Our results agree with those obtained by Monteiro et al. (2013) who demonstrated that C. zeylanicum was effective at 500 ppm against the mycelial growth and germination of conidia of Botrytis cinerea and Alternaria alternata. Hadi and Kashefi (2013) stated that C. zeylanicum extract was the most effective against F. oxysporum followed by Mentha piperita, Allium hirtifolium and A. sativum. Al-Taisan et al. (2014) found that the cinnamon oil at 10 ppm compelety inhibited the mycelial growth of S. sclerotorum and the minimum inhibition concentration was 2 ppm. This high activity of C. zeylanicum could be attributed to the presence of Cinnamic aldehyde (57.73%) and 2-propenal, 3-phenyl (16.20) (Hadi and Kashefi, 2013). Boniface et al. (2012) studied the antimicrobial activity of cinnamon oil. The results proved the essential oil had fungicidal activity against Penicilium digitatum and F. oxysporum.
Eucalyptus globulus extract was very potent against all the selected pathogenic fungi. Tabanca et al. (2001) found that the E. globulus oil exhibited a very strong activity against the fungus Candida albicans in all concentrations.
Results of the present study indicates that the tested extracts showed fungicidal activity against the tested pathogens and can be exploited as natural fungitoxicant to manage the growth of pathogenic fungi and thus reduce the dependence on the fungicides. This high antifungal activity of E. globulus extract may be attributed to the presence of some compounds. The major component was 1, 8-cineole (85.8%), β-pinene (7.2%) and β-myrcene (1.5%). Other compounds identified in the extract and oil of E. globulus obtained were β-pinene, limonene, α-phellandrene, λ-terpinene, linalool, pinocarveol, terpinen-4-ol and α-terpineol. The E. globulus oil consisted mostly of oxygenated monoterpenes and monoterpene hydrocarbons (Damjanovic-Vratnica et al., 2011).
In the present study, it was noticed that the extract of A. sativum revealed considerable antifungal activity against the tested pathogens. Okigbo et al. (2009) evaluated the fungicidal effects of A. sativum against some phytopathogenic fungi; Penicillium oxalicum, F. solani, M. phaseolina Botryodiplodia theobromae, F. oxysporum and Aspergillus niger. Syzygium aromaticum and A. sativum showed 100% inhibition of the mycelial growth of A. niger at 20% concentration (Avasthi et al., 2005). The obtained results revealed A. sativum had effective inhibition the mycelial growth of all tested pathogenic fungi and spore germination of the three Fusarium species. Allium sativum bulbs extract had antifungal activity against the mycelial growth of Fusarium pallidoroseum (Jocob and Sivaprakasam, 1994; Appleton and Tansey, 1975). Bowers and Locke (2000) showed that the antifungal activity of A. satvium extract against the mycelial growth and spore germination of Fusarium solani f. sp. melongenae. This fungicidal activity of A. sativum possibly related to organosulphur compound including allicin (Hovana et al., 2011). These compounds showed better antifungal activity than both antibiotics streptomycin and ampicillin (Ilic et al., 2012). Also, Durairaj et al. (2009) stated that allicin exhibits its antimicrobial activity mainly by immediate and total inhibition of RNA synthesis. Furthermore, garlic extract is known to inhibit cell wall synthesis because it inhibits transpeptidation enzymes involved in the cross-linking (Durairaj et al., 2009).
CONCLUSIONS
Because the extracts of C. zeylanicum (cinnamon), T. foenum-graecum (fenugreek), E. globulus (eucalyptus), E. sativa (rocket) and A. sativum (garlic) are found effective against the growth and spore germination of the test organisms. Therefore, this study suggests that the extracts of these spices would be helpful in treating diseases in plants caused by soil borne pathogens. In conclusion, the findings of this study confirmed that plant extracts can be used as natural fungicides against the growth of pathogenic fungi and thus reduce the dependence on the synthetic fungicides.
ACKNOWLEDGMENT
This project was supported by King Saud University, Deanship of Scientific Research, College of Science Research Center. I would like to express my sincere gratitude and deep gratefulness to all our colleagues of the Department of Botany and Microbiology, King Saud University for their valuable criticism and advice.