Abstract: In an attempt to establish new control practices with low mammalian toxicity and low persistence in the environment against plant pathogens, crude extracts of seven plant species (Cassia senna, Caesalpinia gilliesii, Thespesia populnea var. acutiloba, Chrysanthemum frutescens, Euonymus japonicus, Bauhinia purpurea and Cassia fistula) were evaluated against Alternaria solani in tomato under laboratory and greenhouse conditions. Furthermore, GC-MS analysis was carried to identify the biologically active components of the most effective extract against A. solani. Moreover, the safety of the most effective extract was evaluated with respect to histological changes in treated rats’ organs. The results showed that, B. purpurea was most effective plant extract against early blight pathogen under laboratory and greenhouse conditions. The GC-MS analysis for the most effective plant extract showed the presence of different bioactive chemical components than known by its antifungal activity. The most effective plant extract showed low toxicity on rats relative to control. The results revealed that, the using of plant extracts can be regarded as effective and safe control of A. solani in tomato.
INTRODUCTION
More than 800 million people in developing countries do not have adequate food supplies and at least 10% of food is lost due to plant diseases (Strange and Scott, 2005). Plant diseases are caused by pathogens such as fungi, bacteria, nematodes and viruses. Compared to other plant parasites, fungi cause the greatest impact with regard to diseases and crop production losses either foliage or post harvest losses of fruits and vegetables through the decay resulted from fungal plant pathogens.
Tomato (Lycopersicon esculentum Mill.) is an important vegetable crop grown throughout the world. Tomato early blight disease caused by Alternaria solani become the most destructive in all over the world and yield losses up to 80% (Singh, 1985; Mathur and Shekhawat, 1986; Chandravanshi et al., 1994). The control of tomato early blight disease has been almost exclusively based on the application of chemical pesticides. Several effective pesticides have been recommended against this pathogen but they not considered a long-term solution, due to concerns of expense, exposure risks and the hazards of its residues. Moreover, the development of resistance of pathogenic fungi towards synthetic pesticides is a great problem that can affect significantly the efficacy of chemical fungicides. Thus to find safe, efficacious and environmentally friendly fungicides considered as source of major concern (Mdee et al., 2009).
Presently, the search for natural products with novel uses, particularly related to pest management is very important task. The use of plant extracts has been shown to be eco-friendly and effective against many plant pathogens (Khallil, 2001; Hawamdeh and Ahmad, 2001; Saadabi, 2006; Gachomo and Kotchoni, 2008; Thobunluepop, 2009; Latha et al., 2009; Moslem and El-Kholie, 2009; Duru and Onyedineke, 2010). Most of these substances were evaluated in order to find a safe alternative control methods to the human and the environment. Therefore, research should focus not only on the efficacy of botanical extracts against the target pests but also their safeties on human health in demand. Although, the assessment of enzymes activity in the blood is generally a sensitive measure of compounds toxicity than histopathological changes that can be assessed within a shorter time. The tissue alterations considered a confirmatory and supporting diagnostic role in the case of certain abnormalities in blood sampling (Cornelius et al., 1959). Most of the selected extracts in this study were known by their natural origin and safety to human health (Park et al., 2005; Panda and Kar, 1999). Moreover, no evidence of teratogenic or genotoxic activity has been detected by using of plant extracts to control pests (Mengs et al., 2004; Mitchell et al., 2006). This is besides the fact these plants are available in high amount in Egypt.
Therefore, the present work was designed to investigate the efficacy of certain plant extracts against the early blight pathogen, caused by A. solani, under laboratory and greenhouse conditions. This study has also to identify of the biologically active components of the most effective plant extract against the tested plant pathogen. Finally, it has been done to evaluate the toxicity of the most effective plant extract on rats with respect to histology changes in treated rats’ organ relative to control.
MATERIALS AND METHODS
Plants and preparation of crude extracts: The leaves of seven medicinal plant species (Cassia senna, Caesalpinia gilliesii, Thespesia populnea var. acutiloba, Chrysanthemum frutescens, Euonymus japonicus, Bauhinia purpurea and Cassia fistula) were collected from a local nursery at Kafr El-Sheikh, Monofia, Gharbia and Alexandria Governorates, Egypt. C. senna (Alexandrian Senna) belonging to the family Fabaceae, is native to tropical Africa and cultivated in Egypt and Sudan. C. gilliesii (bird of paradise) belonging to the family Fabaceae, is native to tropical America, mainly Argentina and Uruguay. T. populnea var. acutiloba (Portia Tree) belonging to the family Malvaceae, is native to South Africa. C. frutescens (marguerite daisy) belonging to the family Asteraceae, is native to the Canary Islands. E. japonicus (Japanese Spindle) belonging to the family Celastraceae, is native to Japan, Korea and China. B. purpurea (Purple camel’s foot) belonging to the family Fabaceae, is native to South China. C. fistula (Cassias) belonging to the family Fabaceae, is native to southern Asia. The different leave samples were oven dried for 24 h at 70°C and then, finely ground into a powder using a blender. Each sample (25 g) was extracted twice with 300 mL of methanol at room temperature for 2 days. The extracts were filtered through Whatman filter paper. The combined filtrate was concentrated to dryness by rotary evaporation at 40°C.
Pathogen and plant cultivar source: The A. solani isolated from tomato plant was obtained as culture slant from the Department of Mycology and Plant Disease Survey, Plant Pathology Research Institute, Giza Egypt. Tomato, (Lycopersicon esculentum) plant, cultivar Super Strain, was obtained from Legumes Department, Gemmiza Agriculture Research Station. Cultures were grown on potato dextrose agar in 9 cm diameter Petri dishes for 14 days and incubated at 28±1°C. The resultant fungal growth was providing the amount of water used amounts of sterilized distilled water for 2 min. The suspension was filtered through two layers of sterilized cheesecloth. The spore suspension was counted using a hemocytometer under the microscope and was adjusted to 106 spores mL-1 according to the methods described by Shahin and Shepared (1979).
The tested fungicide: The tested fungicide used in this study was metalaxyl with a trade name of vicomil 50% WP used at field rate of five 370 g ha-1 and produced by Kafr-El-Zayat Company for chemicals and pesticides, Egypt. This fungicide recommended for control early blight disease in vegetables crops in Egypt.
Screening of plant extracts efficacy against A. solani under laboratory conditions: The seven extracts and metalaxyl were tested for their efficacy against A. solani in a completely randomized design. The efficacy of the tested plant extracts and fungicide was determined as percent of inhibition in the growth of selected fungus relative to the control treatment. Four concentrations for each plant extract (50, 100, 150 and 200 ppm) and four concentrations for the fungicide (1, 10, 25 and 50 ppm) were used. The required concentrations for plant extracts and fungicide were obtained by adding the appropriate amount of stock solution used to 60 mL portions of auto-calved PDA cooled to about 45°C. Four Petri dishes, 9 cm in diameter, were used as a replicate for each concentration of each treatment, including control. Control treatment was carried out without adding fungicide or plant extracts. Each dish was inoculated in the center with a disk (5 mm diameter) bearing the mycelium growth from A. solani culture (5 days old culture). The dishes were sealed with parafilm to avoid the evaporation of volatile compounds. The Dishes were incubated at 28°C until the full growth (mycelium reaching the edge of the plate) of the control treatment. The inhibition percentage of radial growth of A. solani was calculated using the formula suggested by Vincent (1947). Each the experiment (all concentrations for each treatment) was repeated three times. The inhibition percentage was calculated as shown in Eq. 1:
| (1) |
where, A is the radial growth of the tested fungus in control, B is the radial growth of the tested fungus in treatment and %I is percentage of radial growth inhibition.
Efficacy of the tested plant extracts against A. solani under greenhouse conditions: These experiments were carried under greenhouse conditions. Seeds of tomato (L. esculentum), cultivar Super Strain were planted in 17 cm diameter plastic pots filled with 2 kg/each of un-sterilized-loamy-clay soil. The plants were irrigated normally and the treatments were applied on the plants after 4 weeks from planting. The highest concentrations of tested plant extracts (150 and 200 ppm) were applied as foliar treatment on tomato early blight grown under greenhouse conditions. Metalaxyl was used as standard fungicide against A. solani at recommended dose level. Tomato plants in control were sprayed with water only at the same intervals used in plant extracts application. The tested plant extracts were applied twice, once at 15 days prior to inoculation with the pathogen and once 7 days before inoculation. The inoculation of tomato plants with A. solani spore suspension at concentration level of 106 spore mL-1 was carried out by sprayed it on tomato seedlings 10 days after transplanting using an atomizer. Inoculated seedlings were covered with black polyethylene bags for 48 h and kept in the green house at 30-32°C. Diseased plants were assessed weekly starting with the first symptom appearance till the end of the growing period. Twenty five leaves for every treatment were used to record the disease parameters according to the method of Awad (1980). Disease severity was assessed 8 days after inoculation according to the scale of Chirst (1991) and the efficacy of each treatment was calculated using Eq. 1:
| (2) |
Where:
| DSC | = | Disease severity under control |
| DST | = | Disease severity under treatment |
Chemical composition of the most effective plant extract: GC/MS analysis was carried to identify the components of the most effective plant extract (B. purpurea) according to the method described by Duarte-Almeida et al. (2004).
Toxicity assessments: The used adults Wistar male rats (Rattus norvegicus) with 8 weeks old and 80-100 g in weight were obtained from Faculty of Medicine, Tanta University. Wister rats were housed in wire cages under standard conditions with free access to drinking water and food. The rats were kept in temperature-controlled room with 14 h light and 10 h dark cycles. The rats were given a standard diet as described by Romestaing et al. (2007). Before treatment, rats were left two weeks during feeding for adaptation. The animals were randomly divided into two groups each comprising of three animals. The first group was for the treatment with the most effective plant extract (21 days) and the second group was for control. The most effective plant extract (B. purpurea) against the tested fungus were administered one time to rats orally at concentration level of 500 mg kg-1 body weight. Rats in control treatment were orally administrated with equal amount of almond oil. After 21 days of treatment, the rats were sacrificed under anesthesia. Then, specimens from kidney and lung were taken from each treatment and kept in neutral buffered formalin 10% for histopathological tests. The histopathology test was carried out at Histopathology Laboratory, Department of Histopathology, Faculty of Veterinary Medicine, Kafr El-Sheikh University according to the method described by Bancroft et al. (1996).
Statistical analysis: Data were subjected to the analysis of variance test and Newman-Keuls’s multiple range test using a computer program SAS (Version 6.12, SAS Institute Inc., Cary, USA). The level of significance was 0.05.
RESULTS
Effect of the tested plant extracts on radial growth of A. solani: Seven plant species, belonging to the various families were selected and evaluated for their antifungal activity against A. solani, the causal of early blight disease in tomato. All the tested plant extracts at different concentration levels inhibited the radial growth of A. solani, relative to control. The leaf extract of T. populnea var. acutiloba was the most effective one against A. solani with the inhibition percentage of 79.4%, followed by C. frutescens, C. gilliesii, C. senna, E. japonicus, B. purpurea and C. fistula with inhibition percentages of 73.3%, 71.1, 67, 59.4, 56.4 and 54.2%, respectively (Table 1). However, the fungicide metalaxyl as a recommended compound against A. solani was still the most effective treatment compared to all tested botanical extracts. The efficiency of the tested botanical extracts was concentration dependent since it’s toxicity against A. solani increased with the increasing of their concentrations level.
Efficacy of plant extracts on tomato early blight pathogen under greenhouse conditions: The protective action of the tested plant extracts relative to the recommended fungicide metalaxyl against A. solani in Tomato plant that evaluated under greenhouse conditions is shown in Table 2. The results showed that metalaxyl was the most effective treatment against A. solani followed by C. gilliesi, T. populnes var. acertiloba, C. frutesscens, C. fistula, C. senna, B. purpurea and E. japnicus, respectively either at concentration level of 150 or 200 ppm.
| Table 1: | Efficacy of the tested plant extracts and metalaxyl against early blight disease pathogen (A. solani) under laboratory conditions |
| Lower case letters in this column indicate separation of means according to the Student Newman Keuls multiple range test (p<0.05) | |
| Table 2: | Efficacy of the tested plant extracts and metalaxyl against early blight disease pathogen (A. solani) under greenhouse conditions |
| Lower case letters in this column indicate separation of means according to the Student Newman Keuls multiple range test (p<0.05) | |
The results also implied that the efficacy of the plant extracts was higher at a concentration level of 200 ppm than that of 150 ppm (efficacy was concentration dependent). There was no phytotoxicity of the tested plant extracts on tomato was observed Moreover, one of the most important notices that, there is no phytotoxicity of the tested plant extracts on tomato was recorded.
Composition of the most effective botanical extract against A. solani: The identified compounds from the most effective botanical extract (B. purpurea) against A. solani are given in Table 3.
| Table 3: | The main constituents of B. purpurea extract identified by GC-MS analysis |
| Fig. 1(a-b): | Sections in kidney of rats treated with B. purpurea (b) at dose level of 500 mg kg-1 after 21 days of treatment relative to control (a) |
Eighteen compounds were identified from B. purpurea plant extract as shown in Table 3. The identified compounds are belonging to aldehydes, esters, alcohols and fatty acids.
Toxicity evaluation
The histopathological changes in the kidney: The normal structure of kidney
tissue was shown in Fig. 1a.
| Fig. 2(a-b): | Sections in lung of rats treated with B. purpurea (b) at dose level of 500 mg kg-1 after 21 days of treatment relative to control (a) |
For the rats treated with B. purpurea extract at dose level of 500 mg kg-1, the kidney tissue was the lung tissue is normal as control except for some collecting tubules (Fig. 1b).
The histopathological changes in the lung: The normal structure of lung tissue was shown in Fig. 2a. For the rats treated with B. purpurea extract at dose level of 500 mg kg-1, the tissue was normal as control with some lymphocytes infiltration and the blood vessels were observed to be engorged with blood (Fig. 2b). However, these changes still not significant relative to the normal tissue.
DISCUSSION
In the present study, the tested plant extracts showed antifungal activity against early blight pathogen in tomato. The efficacy of different plant extracts belonging to different species other than the tested botanical extracts against the A. solani either under laboratory or greenhouse conditions have been reported (Bergaoui et al., 2007; Tegegne et al., 2008; Chutia et al., 2009; Latha et al., 2009; Zaker and Mosallanejad, 2010). However, for the tested plant extracts especially the effective ones against early blight pathogen A. solani, this is considered the first report.
It was observed that, among the identified compounds from B. purpurea extract, some compounds such as, Alpha humulene , eugenol, tetradecanoic acid, oleic acid, Phytol, 9, 12, 15 octadecanoic acid and n-hexadecanoic acid were detected with high percentages relative to other detected compounds. The antifungal activity of B. purpurea extract against A. solani pathogen may be due to the presence of the previous fatty acids and its derivatives (Hammer et al., 2003; Bergaoui et al., 2007; Chutia et al., 2009; Ahmadi et al., 2010).
Although, the antifungal activity of tested plant extracts is mainly attributed to its major compounds but the synergistic or antagonistic effect of one compound in minor percentage in the mixture has to be considered (Ragas et al., 2005). Therefore, each component of the plant extract has its own contribution on biological activity of the extract.
The essential oils in the tested plant extracts as antimicrobial agents considered at low risk for resistance development by pathogenic microorganisms. It is believed that it is difficult for the pathogens to develop resistance to such a mixture of components with, apparently, different mechanisms of antimicrobial activity (Liu et al., 2008).
The botanical extracts as pest control agents present two main characters: the first is their safety to the people and the environment and the second is the less resistance development against it by the tested pathogen. Regarding to the safety, the toxicity evaluation of the most effective plant extract revealed that, there were some slight variations occurred sporadically in treated rats relative to control with the respect to enzymes markers and histopathology of treated organs. Moreover, the observed changes in the tissues were mostly uncorrelated with the dosages which mean the safety of the tested plant extract on human health. With the referring to resistance development, it is believed that, it is difficult for the insect to develop resistance to such a mixture of bioactive components with, apparently, different mechanisms of fungicidal activity (Liu et al., 2008).
CONCLUSIONS
The fungicidal activity of the extracts against A. solani indicates the potential of some plant species as a natural source of fungicidal material. Antifungal activity was confirmed in all the tested plant species, although the results showed that different plant extracts varied in their effectiveness in inhibiting the mycelia growth of different pathogens tested. In vivo results under greenhouse confirmed and could be a viable option for controlling A. solani because leaves are available in all seasons and it is a world wide invasive species. The ability of using botanical products as alternative of chemical control of plant pathogens is possible. This approach can contribute in reducing the amount applied of fungicides and subsequently minimize its hazards to the environment and human health. Work in this regards should continuing on other invasive species on isolating antifungal compounds and on field trials with promising extracts or compounds. Also, further research is needed in order to obtain information regarding the practical effectiveness of essential oils to protect the plants or the plant products without toxic effects.