Phytochemical Analysis and Antifungal Activity of Fruit Leaves Extracts on the Mycelial Growth of Fungal Plant Pathogens

The damage to crops caused by fungal plant pathogens has required the use of range of antifungal control agents. Among pesticides used to protect crops, fungicides were perceived until recently as relatively safe. However, the 1986 National Academy of Sciences (NAS) report on pesticide residues on food indicated that fungicides pose more of a carcinogenic risk than insecticides and herbicides together [1]. Furthermore, the use in crop protection of many synthetic fungicides that have various degrees of persistence has now been cautioned due to their carcinogenicity, teratogenicity and other residual toxicities. Several of the synthetic fungicides are reported to cause adverse effects on treated soil ecosystems because of their non-biodegradable nature [2,3]. Synthetic fungicide residues are suspected to present a significant health risk to consumers, and demand is increasing to find safe alternatives. Additionally, continued use of fungicides leads to an increase in resistance by plant pathogens, creating a need for finding biological alternatives with these pesticides. Present activities to find both natural and synthetic fungicides focus on finding compounds that are safe to humans, environment and delicate ecosystems [4,5].


Introduction
The damage to crops caused by fungal plant pathogens has required the use of range of antifungal control agents. Among pesticides used to protect crops, fungicides were perceived until recently as relatively safe. However, the 1986 National Academy of Sciences (NAS) report on pesticide residues on food indicated that fungicides pose more of a carcinogenic risk than insecticides and herbicides together [1]. Furthermore, the use in crop protection of many synthetic fungicides that have various degrees of persistence has now been cautioned due to their carcinogenicity, teratogenicity and other residual toxicities. Several of the synthetic fungicides are reported to cause adverse effects on treated soil ecosystems because of their non-biodegradable nature [2,3]. Synthetic fungicide residues are suspected to present a significant health risk to consumers, and demand is increasing to find safe alternatives. Additionally, continued use of fungicides leads to an increase in resistance by plant pathogens, creating a need for finding biological alternatives with these pesticides. Present activities to find both natural and synthetic fungicides focus on finding compounds that are safe to humans, environment and delicate ecosystems [4,5].

Plant materials
Leaves of thompson seedless grape (Vitis vinifera cv. Sultana), flame seedless grape (Vitis vinifera cv. Roumy Ahmer), zizyphus (Zizyphus spina-christi cv. Willd), pomegranate (Punica granatum cv. Baladi) and fig (Ficus carica cv. Sultani), belong to families Vitaceae, Rhamnaceae, Punicaceae and Moraceae, respectively, were used in this study. Samples were collected, cutting the leaves of plants, from the Experimental Station of the Faculty of Agriculture within the campus of the University of Mansoura, which located in the north of Egypt. Leaves were cleaned, dried in the shade, and then grounded to a fine powder using a grinding mill. Leaves powder (3 Kg) of each plant was extracted by soaking at room temperature for six times with methanol (30 L). Extracts were concentrated to nearly dryness under reduced pressure by using rotary evaporator at 45°C to achieve the crude methanolic extracts.

Plant pathogens
Five plant pathogenic fungi, Alternaria solani, Botrytis cinerea, Botrytis fabae, Fusarium oxysporum and Fusarium solani, were isolated from diseased potato plants, diseased fruits of tomato, diseased broad bean plants, naturally infected tomato and artichoke, respectively. All the diseased plants and the diseased fruits were collected from Mansoura, Egypt. All fungal strains were grown on potato dextrose agar (PDA) or czapek agar (CZA) media and purified using single spore or hyphal tip techniques. Identification of the pure cultures was accomplished according to Barnett and Hunter [19]; Leslie and Summerell [20]; Elad et al. [21] by cultural properties, morphological and microscopical characteristics. Stock cultures of each strain were maintained on PDA at 5°C. For use in antifungal activity assay, the fungi were subcultured onto PDA in Petri dishes (9 cm diameter), and incubated at 25°C for 7-10 d.

Preliminary phytochemical tests of crude plant extracts
Crude extracts were analyzed to detect the presence of terpenes, tannins, flavonoids, saponins, alkaloids, carbohydrate and/or glucosides, phenolic glucosides and resins according to Harborne [22].

Determination of total polyphenols and total flavonoids content
Total phenolics content of air dried leaves were determined by Folin-Ciocalteau method according to Lin and Tang [23]. Gallic acid was chosen as standard of total phenolics for making the standard curve (200-1600 mg/l). Concentration of total phenolics content expressed as milligram gallic acid equivalents GAE/g. Total flavonoids content of air dried leaves were determined using aluminum chloride colorimetric method, which described by Chang et al. [24]. Quercetin was chosen for making standard curve of flavonoids (0-50 mg/l). Concentration of total flavonoids content was expressed as milligram quercetin equivalents QE/g.

Identification of polyphenols by HPLC
Phenolic compounds of plant samples were extracted according to Ben-Hammouda et al. [25]. Identification of individual phenolic compounds of plant samples was performed on a Hewlett-Packard HPLC (Model 1100), using a hypersil C 18 reversed phase column (250×4.6 mm) with 5µm particle size. Injection by means of Rheodyne injection valve (Model 7125) with 50 µm fixed loop. Phenolic compounds of each sample were identified by comparing their relative retention times with those of the standards mixture chromatogram. The concentration of an individual compounds was calculated on the basis of peak area measurements.

Antifungal activity of plant extracts on the mycelial growth of test fungi
Extracts were dissolved in dimethyl sulphoxide (DMSO) and added to PDA medium immediately before it was poured into Petri dishes (9 cm diameter) at 40-45°C to obtain a series of concentrations (1, 2 and 4 mg/ml). Control plates were treated with DMSO alone, and three replicates per treatment were used. Plates were incubated at 25 ± 2°C. Colony growth diameter was measured after the fungal growth in the control treatment had completely covered Petri dishes. Percentage of mycelial growth inhibition was calculated from the formula: Mycelial growth inhibition=[(diameter of control-diameter of sample)/ diameter of control]×100

Statistical analysis
Statistical analyses of all experimental data were done using the statistical software package CoStat [26]. All comparisons were first subjected to one way analysis of variance (ANOVA) and significant differences between treatment means were determined using Duncan's multiple range test at P<0.05 as the level of the significance [27].

Total polyphenols and total flavonoids content
Total polyphenols include several classes of phenolic compounds that are secondary plant metabolites and integral part of human and animal diets. Flavonoids are a large group of the phenolic compounds consisting mainly of flavonols, flavanols and anthocyanins. Phenolic compounds can play an important role in preventing body cells and organs from injuries by hydrogen peroxide, damage by lipid peroxides and scavenging or neutralizing free radicals [39]. It has been reported that free radical scavenging and antioxidant activity of many medicinal plants are responsible for their therapeutic effect against cancer, diabetes, tissue inflammatory and cardiovascular diseases [40]. Also, it was found that high total phenols content increase antioxidant activity and there is a linear correlation between phenolic content and antioxidant activity in fig leaves extract [41].
Zizyphus leaves had the highest concentration of total polyphenols (147.47 mgGAE/g). Also, pomegranate and fig leaves had high values, which were 122.52 and 123.51 mgGAE/g, respectively. For total flavonoids, zizyphus leaves contained the highest amount (16.35 mgQE/g) followed by flame seedless leaves by 15.93 mgQE/g. Whereas, thompson seedless and pomegranate leaves contained 13.25 and 10.95 mgQE/g, respectively ( Table 2).
These results are in agreement with Monagas et al. [13]. They found that the total polyphenols was 112 ± 18 mgGAE/g of methanolic extract of Vitis vinifera leaves. Additionally, Orhan et al. [42] suggested that the total phenolic contents of V. vinifera leaves were 205.79 ± 8.89, 57.17 ± 6.5 and 37.97 ± 0.90 mgGAE/g for ethyl acetate, butanol and remaining aqueous fractions, respectively. Also, Orha et al. [43] suggested that the total phenolic contents of V. vinifera leaves were 89.4, 216.0, 91.2 and 68.6 mgGAE/g for chloroform, ethyl acetate, butanol and remaining aqueous fractions, respectively. Although, chloroform and ethyl acetate fractions contain 59 and 206 mgQE/g of total flavonoids, respectively, total flavonoids were not detected in butanol fraction and remaining aqueous fraction had a traces of total flavonoids. While, Aseri et al. [44] represented that the total phenols content was 1.81 mg/g in control Punica granatum leaves on fresh weight basis. Pari and Suresh [45] showed that the ethanolic extract of grape leave contain phenolic compounds in a concentration of 98.84 ± 9.29 mgGAE/g. Allam et al. [46] found that 20, 40 and 60 mg of fig leaves aqueous extract contain 3.05, 6.10 and 9.14 mg/g extract of total phenols, respectively. While, the previous amounts of the extracts contain 0.22, 0.45 and 0.67 mg/g extract of total flavonoids, respectively.

Leaves extract
Total polyphenols (mgGAE/g)  There were no available authentic samples to identify these unknown compounds which may be some polyphenolic derivatives.

Antifungal activity of plant extracts on the mycelial growth of test fungi
All methanolic leaves extracts showed an extensive antifungal activity against the different fungal pathogens tested (Table 4). However, fungal sensitivity varied according to the species. Extract of zizyphus significantly reduced the mycelial growth of all tested fungi from 64.44 (for F. oxysporum) to 95.56% (for B. fabae) at the concentration of 4 mg/ml (Figure 1). These were followed by pomegranate extract, which caused a significant decrease on the fungal growth ranged from 57.78 (F. oxysporum) to 94.44% (B. fabae) at the same concentration ( Figure  1). There was no significant difference between inhibitory effects of zizyphus and pomegranate on the growth of B. fabae at 4 mg/ml. Also, extracts of zizyphus followed by pomegranate were the most effective among the plant species tested against the growth of the fungi at the concentrations of 1 and 2 mg/ml (Figures 2 and 3). Extract of flame seedless had more effectual antifungal than thompson seedless at all concentrations. F. oxysporum and F. solani showed high resistance for all methanolic extracts (Table 4). In general, There were positive relationships between the concentration of the methanol extracts and the inhibition rate on mycelia growth of all tested fungi.
According with the obtained results for HPLC analysis, ten polyphenols compounds have been identified in the methanolic extract of zizyphus leaves, besides; the main component found in this extract was phenol. The high antifungal activity of zizyphus extract is possibly due to phenolic compounds. Phenolic compounds are one of the major families of secondary metabolites in plants, and they are of nearly 10,000 individual compounds [49]. Phenolic or polyphenol can be defined chemically as a substance which possesses a benzene ring with one or more hydroxyl groups, with evidence that increased hydroxylation results in increased toxicity [50]. These polyphenols are very important for plants to contribute to resistance to microorganisms, herbivores and insects [51]. Generally, the active antifungal compounds of most plant extracts are phenolic compounds. This may be due to that their antifungal mode of action might be related to that of other compounds.
These results were agreed with Brown and Morra [52] and Nita-Lazar et al. [53]. They suggested that the activation of the phenolic pathway is known in the plant physiology to be part of the defense response against phytopathogenic microorganisms. Also, Sisti et al. [54] showed that the phenolic agents are active against pathogenic microorganisms for humans and animals.
Many potential modes of action by which phenolics counteract envelopment of pathogenic agents have been suggested, from the impairment of enzymatic processes involved in energy production and structural component synthesis by weakening or destroying the    permeability barrier of the cell membrane by altering the physiological status of the cells or affecting nucleic acids synthesis [55]. In the same way, Galvan et al. [8] mentioned that plant secondary metabolites have great potential as a source of effective antifungal agents, for example, plant-derived compounds such as hydroquinones, naphthoquinones, alkaloids and flavonoids, have shown diverse antimicrobial activities including antifungal activities. Batovska et al. [14] found that the grapevine leaves possess a resistance towards several fungi, Plasmopara viticola, Oidium tukeri and Botrytis cinerea as biomarkers for the fungal resistance.