INTRODUCTION

    Due to plant diseases every year nearly 10-20% of the total world food production was decreased and leads toloss of billions of dollars. Chemical control is offernonspecific in its effect, may can cause killing beneficial organismsand it may have undesirable health and environmentalpollution risk. Intensifiedused of fungicides has resulted in accumulation oftoxic compound potentially hazardous to human andenvironment and also in the build up of resistance of thepathogens. In order to tackle these national and globalproblems, effective alternatives to chemical control arebeing employed. Biological control is nature friendlyapproach that uses specific micro-organisms, whichinterfere with plant protection. Biological control by anantagonism is a potential, no chemical and ecofriendlyapproach for managing plant diseases (Bailey et al., 2004).

     Fusarium oxysporum is a soil borne fungal pathogen that attacks plants through roots at all stages of plant growth, causes major economic losses by inducing necrosis and wilting symptoms in many crop plants (Cotxarrera, et al., 2002). The disease caused by this fungus is characterized by wilted plants, yellowed leaves and root rot minimal or absent crop yield (Nemec et al., 1996). Fusarium wilt of tomato caused by Fusarium oxysporum f. sp. Lycopersici (FOL) is the major limiting factor in the production of tomato. The disease causes great losses, especially on the susceptible varieties of tomato especially when soil and air temperature are rather high during the warm season (Agrios, 1997, Mandal et al., 2009). The interaction between F. oxysporum and F. solani causes a root-rot disease complex that severely damages this important crop (Klotz, 1973).

    Biological control of plant diseases is considered as one of the viable alternative methods to manage plant diseases (Barakat & Al-Masri, 2005; Pal & Gardener, 2006). The application of biological controls using antagonistic microorganisms has proved to be successful for controlling various plant diseases in many countries (Janisiewicz, 2000). Most of these studies were on the control of root and soil-borne plant pathogens and, to a lesser extent of foliar pathogens (Whipps & Lumsden, 2001).

     The antifungal activity of 36 plant aqueous extracts have been evaluated against F.oxysporuin, R.solani and B.cinerea in vitro testes and under greenhouse condition on watermelon and tomato plants (Elkaffash and Al-Menoufi,2003). They found that clove extract completely inhibited the mycelial growth of the three tested fungi whereas garlic extract suppressed the mycelial growth of F.oxyspornna and B.cinerea by 100 % and R.solani by 88 %. In the same year, Dawood et al., (2003) evaluated 11 aqueous plant extracts for their antifungal activity against Fusarium solani, Fusarium oxysporum, Rhizoctonia solani, Sclerotinia sativa, Alternaria alternata, Aspergillus flavus, Botrytis cinerea, Sclerotium rolfsii and S.batiticola. They stated that Spearmint (Mentha viridis) extract had no effect on mycelial growth of any of the tested fungi.

The objective of this study was to evaluate the potential of the  plant extracts of some wild plants in controlling the root rot fungi Rhizcoctonia solani and Fusarium solani which infected Cantaloupe plant under in vitro condition  .

1.              Materials & Methods

1.1.        PHYTOPATHOLOGICAL STUDIES

1.1.1.  Sampling

Naturally diseased Cantaloupe root showing wilting symptoms, Cantaloupe plants showing wilting and root rots  were collected from different districts of Egypt namely Sadat city, Noberia, el kataba, and Alex. Egypt road K. 76. Collected samples were transfered to laboratory in plastic bags to be used in pathogen isolation.

1.1.2.      Phytopathogenic Fungi 

 Isolation, identification and pathogenicity test were carried out for, Fusarium solani and and  Rhizoctonia solani. The isolates were grown on potato – dextrose  agar (PDA) medium (see-appendix) in petri dishes, then transferred to PDA slants and kept in refrigerator at 4°C as a stock cultures.

1.1.3.      Isolation, purification and identification of Fusarium solai

Diseased cantaloupe plants showing different degrees of wilt disease were collected from different regions of, (Egypt Sadat city, Noberia, El kataba, and Alex. Egypt road K. 76).

The affected roots were washed thoroughly with running tap water and cut into small portions before immersing in Sodium hypochlorite (5% chlorin) for one minute. The surface was washed with distilled water and finally dried between two sterilized filter papers. Then they were directly placed into Petri dishes containing potato dextrose agar medium (PDA) and incubated at 28^oC for 3-5 days. Examination was carried out when fungal growth appeared out from the incubated materials. All the isolated fungi were purified using single spore or the hyphal tip techniques suggested by Dhingra and Sinclair (1985). The purified fungi were identified according to their morphological features according to Booth (1985). Stock cultures were maintained on PDA slants and kept in a refrigerator at 5-10°C and were sub-cultured on fresh medium every 6-8 weeks.

1.1.4.      Isolation, purification and identification of Rhizoctonia solani

The collected diseased cantalope roots showing damping-off disease were collected from different regions of washed in running tap water followed by sterile water. Using sterilized scalpel, roots were cut into small pieces (1-2 cm²). The pieces were then transferred into 0.1% hypochlorite solution (disinfectant solution) for 3 minutes for surface sterilization. Surface sterilized pieces were then washed several times with sterilized water to wash out the remaining disinfectant solution. The pieces were then dried on sterilized filter papers. Using sterilized forceps, dried pieces were then transferred into Petri dishes containing potato dextrose agar medium (PDA) supplemented with antibacterial agent (L-chloramphenicol 5mg\L and streptomycin sulphate 5mg\L). The dishes were then incubated at 28C^o. and checked for fungal growth two days later. The resulting isolates were purified using mycelium fragment. (Shabana, 1987). Pure cultures of the isolated fungi were identified according to the cultural properties, morphological and microscopical characteristics described by Domsch et al., (1980)

1.2.  COLLECTION OF WILD PLANTS

Seven  wild plants (Table 1) were collected from canal banks, fields and markets of various localities in El menofoia  governorate and were taxonomically identified by Prof. Dr. Mohamed Fathy Azazy Surveys of Natural Resources Dept , Environmental Studies and Research Institute, University of Sadat City according to Tackholm (1974) and adopted according to Boulos (1999-2005).

Table (1): The collected wild plants

1.3.  PREPARATION OF PLANT EXTRACTS

Plant materials were dried in the shade at room temperature, ground using electrical mill into fine powder and extracted by soaking in methanol at the rate of 1:3 (w/v) for 48 hours.

The extracts were filtered through cheese cloth under a strong hand pressure and the solvent was dried under vaccum at 60-65C^o using a rotary evaporator. The extracted residue was dissolved in dimethyl sulfoxide (1mg /ml). The crude extracts were preserved under refrigeration until use (Dawood et al.,2003). 

1.4.  Screening of different plant extracts for antifungal activities against the selected phytopathogenic fungi

Seven plant extracts (Table 1) were examined for their inhibitory activities against some selected phytopathogenic fungi by using agar diffusion technique (Deans and Ritchie,1987).

 Dry potato dextrose agar plates were inoculated by spreading spore suspensions of constant fungal inoculum. In case of R.solani, discs were putted on the surface of plates because this fungus grow as mycelia or sclerotia. Wells were made in the inoculated potato dextrose agar plates for plant extracts and essential oils inoculation (200μ). All plates were incubated at 26±2^oC for 72 hours. The diameter of inhibition zones were determined for all tested fungi except R.solani, the radius of inhibition zones were measured.

1.5.  Phytochemical analysis by HPLC:

The chemical composition of Sargssum species extract were performed in central labs unit, Faculty of Agriculture, Cairo University, Gizza, Egypt for detected phenolic, flavonoids and other active componds.

1.6.  Ultrastructure studies (T.E.M):

The method described here is an example for fixation, contrastation and embedding only. For optimal results especially the fixation needs to be adapted to the investigated tissue.It is of major importance that the fixing agent is able to completely.Penetrate the specimen as soon as possible. For this reason only very small pieces of tissue (~ 1 mm³) should be fixed if no perfusion fixation (fixative is directly infused into a larger vessel of a deeply anaesthized animal) should be possible. The preparation should be quick enough, i.e. 5 minutes after tissue does no longer receive oxygen, and it began to show first signs of degeneration of ultra-structures. Perfusion or immersion fixation of the tissue using a modified Karnovsky (1965) solution:2.5% buffered glutaraldehyde + 2 % paraformaldehyde in 0.1 M sodium phosphate buffer pH 7.4, leave tissue overnight at 4° C,wash 3 x 15 minutes (min.) in 0.1 M sodium phosphate buffer + 0.1 M Sucrose, postfix 90 min.in 2 % sodium phosphate buffered osmium tetroxidepH7.4, wash 3 x 15 min in 0.1 M sodium p, after drying for ~15 min sections may be investigated in a transmission electron microscope . Ultrathin sections were observed at 160 kV using a JEOL JEM -2100 at Electron MicroscopyUnit, Mansoura University, Egypt.

1.7.  Statistical analysis:

All values represent the mean of triplicate determinations. Data were statistically analyzed by using one way analysis of variance (ANOVA) according to SPSS (1999). The least significant difference is abbreviated as LSD and measured at P≤0.05.

2.      RESULT

2.1.  Psychopathological Studies

2.1.1.    Isolation, purification and identification of the causative organism of wilt disease (Fusarium solani)

The causative organism of wilt disease was isolated from diseased cantaloupe roots collected from different regions of Egypt namely Sadat city, Noberia, el kataba, and Alex. Egypt road K. 76. These isolates were identified as Fusarium solani (Fig.1) according to morphological and microscopic features (mycelial development and spore formation) according to Booth (1985). The identification was confirmed by Mycology Center, Assuit University as(Fusarium solani 9704 AUMC).

Fig (1): Conidia of Fusarium solani (400x) light microscope

2.1.2.    Isolation, purification and identification of the causative organism of damping-off disease on bean (Rhizoctonia solani)

The causative organism of damping off disease was isolated from diseased cantalouperoots which collected from different regions of Egypt namely Sadat city, Noberia, el kataba, and Alex. Egypt road K. 76. The isolates obtained were purified using mycelium fragment and these isolates were identified as Rhizoctonia solani (Fig.4) according to morphological and microscopical characteristics by Domsch et al., (1980). The identification was confirmed by Mycology Center, Assuit University as (Rhizoctonia solani 6590 AUMC).

Fig (2):  Mycelium of R.solani (400 x) light microscope

2.2.  Screening of plant extracts for antifungal activities against Fusarium solani

      Seven plant extracts were screened for their antifungal activities against Fusarium solani. They are arranged in Table (2) and Fig. (3) according their antifungal actitives. Calotropis procera was the most active plant extract against Fusarium solani followed by Azadicachta indica Adrjuss, Cymbopogon proximus (hochst) staps, Cyperus rotundus L., Eucalyptus rostrata Schlecht,Nerium oleander L. and Pluchea dioscoridis L.

Table (2): Screening of plant extracts for antifungal activities against Fusarium solani

LSD = 2.1

●The recorded valued are the mean values of 3 replicates

* Significant

Non significant  

Fig (3): Screening of plant extracts for antifungal activities against Fusarium solani 

2.3.  Screening of plant extracts for antifungal activities against Rhizoctonia solani 

     Data in Table (3) revealed that seven plant extracts were screened for their inhibitory activities against Rhizoctonia solani.  They are arranged in Table 3) and Fig.(4) according to their inhibtorty activities. The plant extract of Calotropis procera was the most active against R.solani followed by Eucalyptus rostrata Schlecht, Azadicachta indica Adrjuss, Cyperus rotundus L., Nerium oleander L., Cymbopogon proximus (hochst) staps. and Pluchea dioscoridis L. respectively.

Table (3): Screening of plant extracts for antifungal activities against Rhizoctonia solani

LSD = 0.1

●The recorded valued are the mean values of 3 replicates

* Significant

Non significant  

Fig (4): Screening of plant extracts for antifungal activities against Rhizoctonia solani  

2.4.   Transmission electron micrographs showing the effect of plant extract of calotropis prcera on Fusarium solani.

     A: fusarium solani hypha grown on medium lacking plant extract of calotropis prcera. A cell of fusarium solani surrounded by a thin wall (W) and the cytoplasm (Cy) contains numerous organelles such as mitochondria (M). Note an electron-dense plasmalemma (arrow). Scale bar = 0.5 µ m (cited from Baka et al., 2002).

B and C: Fusarium solani hypha grown on medium containing plant extract of calotropis prcera at concentration of 0.3%. Fusarium solani cell show a thickening wall (w), disintegration of cytoplasm (Cy). Note large lipid bodies (L), vacuoles (v) and not recognized plasma membrane. Scale bar=0.5μm.

Fig. (5) Transmission electron micrographs showing the effect of plant extract of calotropis prcera on Fusarium solani.

2.5.  Phytochemical analysis by HPLC:

The results in Table (4) showed Phytochemical analysis of calotropis prcera extract showed presence of phenolic compound and flavonoids which responsible for antifungal activites.

Table (4): Phytochemical analysis by HPLC:

Discu Thus, our study was planned to replace the undesirable and unsafe chemical control by another effective, inexpensive and safe options for control of some root rot diseases  in Cantaloupe Several strategies were used for biological control of plant pathogens by using plant extracts (Assadi and Behroozine,1987 Natural plant extracts may provide an alternative to chemicals for controlling plant disease (Farag et al., 1989).

  The screening of antifungal activity of seven_plant extracts against phytopathogenic fungi;, F solani , R.solani, showed that plant extracts have antifungal activities against the tested phytopahogenic fungi. Among all plant extracts tested the plant extract of calotropis procaera proved to be the highest most effective growth inhibitor for all the tested phytopathogenic fungi. Also the results revealed that the other plant extracts have high antifungal activities against the tested phytopathogenic fungi. These results were in agreement with that of Wilson et al.,(1997) who tested extracts from 345 plants for their antifungal activities against Botrytis cinerea. They found that among the 345 plant extracts tested, 13 showed high levels of antifungal activities, with species of Allium predominating.

Also, Carcia and Lawas (1990) studied the antifungal activity of the crude water extract of 127 plant species against R.solani and they found that garlic extract (Allium sativum was the most effstive one   Mahmoud et al.,(2004), reported that, the plant extracts of Ipomoea carnea, Eucalyptus citriodora, Cuminum cyminum, Allium sativum  and Hyocyampus muticus have antifungal activities against Botrytis fabae and showed that the plant extracts of  Eucalyptus citriodora and Ipomoea carnea have high inhibition zones against B.fabae also have high ability  to decrease percentage of spore germination of B.fabae .

The antifungal activities of plant extracts may be attributed to the plant contents of secondary metabolites (e.g. phenolic, alkaloids, flavonoids, and terpenoids) that could adversely influence pathogen growth and development (Cowan,1999). Some plants impact on the growth and or development of others by releasing various chemical compounds called allelopathy (Jadhav et al.,1997).