American-Eurasian J. Agric. & Environ. Sci., 18 (3): 105-114 2018 ISSN 1818-6769 © IDOSI Publications, 2018 DOI: 10.5829/idosi.aejaes.2018.105.114

Antifungal Activities of Oil Against Root Rot and Wilt Pathogens of Tomato Plants

Marian Thabet and Walaa Khalifa

Department of Plant Pathology, Faculty of Agriculture, Ain Shams University, Cairo, Egypt

Abstract: The antifungal activities of clove essential oil at various concentrations (0, 0.5, 1, 2, 4% v/v) were evaluated in vitro and in vivo against tomato root rot and wilt pathogens. Several fungal pathogens were isolated of infected tomato plants collected from different locations of Giza, Monufia and Qalyubia governorates, Egypt. The purified isolates were identified as Fusarium oxysporum, F. solani, F. semitectum and Rhizoctonia solani. All fungal isolates were tested for their pathogenic ability on tomato plants under greenhouse conditions. In vitro, clove oil exhibited an inhibitory effect against the mycelial growth of all pathogens. All tested concentrations significantly reduced mycelial linear growth of the tested fungi compared to respective controls. Complete growth inhibition was observed in Fusarium oxysporum and Rhizoctonia solani when clove oil was applied at concentration of 4%. Effect of clove oil on fungi morphological features was studied by light microscope. Observations showed the disruption of the fungal growth and conidial malformation as well. In case of Fusarium oxysporum, clove oil decreased the conidial numbers, but increased the production of chlamydospores. Additionally, the alleviative effects of clove oil application on disease incidence and severity were also studied. Clove oil at concentration of 4% resulted a highly significant decrease in disease both incidence and severity. Therefore, it could be suggested that application of clove oil as potent antifungal agent could be used for controlling tomato root rot and wilt diseases.

Key words: Antifungal Clove oil Fusarium oxysporum Rhizoctonia solani Fusarium solani Root rot Wilt

INTRODUCTION cause an annual loss of about 20% in the potential yield worldwide [4]. Tomato (Solanum lycopersicum Mill.) is one of the Control of root rot and wilt diseases depends most important vegetable crops in the world, widely mainly on fungicides [5]. Nowadays, the intensive cultivated in tropical and sub-tropical regions. It has a application of chemical fungicides has been reported to very high nutritive value with antioxidant and curative cause adverse effects on plants, soil environment and properties [1]. Tomato plants are infected by several human health [6]. Moreover, it leads to develop resistant soil-borne fungal pathogens such as Fusarium spp and strains of the pathogens against fungicides [7]. For these Rhizoctonia solani which cause serious diseases as root reasons, alternative methods with emphasis on the rots and wilt [2]. Fusarium wilt, caused by Fusarium resistance inducers for controlling the diseases have been oxysporum f.sp. lycopersici, is considered one of the studied by several researchers to minimize fungicide limiting factors in tomato production areas. It has become application and decrease cost of plant production. one of the most prevalent and damaging diseases are considered to be environmentally because, it can be soil-borne, air-borne or carried in plant safe as they are biodegradable and have little or nil residues and can be recovered from any part of the plant toxicity to non-target animals [8, 9]. Recently, it has been [3]. Tomato damping-off and root rot diseases caused by reported that some plant essential oils have become a Rhizoctonia solani and Fusarium spp. are the most potent antimicrobial agents against foliar and soil-borne destructive diseases in tomato production areas which pathogens [10].

Corresponding Author: Thabet Marian, Department of Plant Pathology, Faculty of Agriculture, Ain Shams University, Cairo, Egypt. 105 Am-Euras. J. Agric. & Environ. Sci., 18 (3): 105-114, 2018

Plant essential oils (EO) are concentrated volatile cultural properties, morphological and microscopical hydrophobic liquids extracted from different parts of the characteristics according to the identification keys of aromatic plants [11]. They offer a variety of functions for Leslie and Summerell [21] and Sneh et al. [22]. the plants together with (i) protecting themselves of heat or cold, (ii) attracting or repelling insects and (iii) using Pathogenicity Test: Pathogenicity tests were carried out chemical ingredients in the oil as defense equipment. using the method of Haggag and El-Gamal [23] to As well, EO have shown promising results in vitro studies investigate the pathogenic potentiality of the isolated for their antifungal effects on mycelial growth and fungi as well as, to determine the most aggressive isolates spore germination against many phyto-pathogenic fungi that used in this study. Sterilized pots (10 cm diameter) such as Rhizoctonia solani, Fusarium moniliforme and were filled with disinfested soil. Inoculum of both F. oxysporum [12]. The extent of inhibition depends on Fusarium spp and R. solani isolates were prepared by the concentration of essential oils [13]. The pathway of growing each isolate in bottles containing sterilized activity of these complexes against fungi is unidentified sorghum grain media then incubated and kept for 15 days but may be related to their general capability to soften or at 25±2°C. Soil infestation was achieved by mixing the otherwise dislocate the reliability of cell membranes and inoculum of each isolate with the soil at the rate of 3% cell walls [14]. Clove oil has been known as a potential (w/w) potential inoculum. Four-weeks-old tomato organic pesticide derived from different parts of the clove seedlings were transplanted into the infested pots plant Eugenia aromatic [15]. It has been described to (2 seedlings per pot). The disease incidence was have useful antimicrobial effects on different types of determined by recording the percentage of dead plant pathogens such as fungi, bacteria and nematodes seedlings, 45 days after transplanting. [16, 17]. It has been shown to inhibit the growth of several soil-borne fungi [18]. In vitro Antifungal Activity Assay: The influence of The objectives of the present work were: (a) isolate antifungal activity of clove oil on the radial growth of and identify the causal agents of tomato root-rot and wilt Fusarium spp and Rhizoctonia solani was performed by disease from different Egyptian governorates and the agar medium assay according to Tatsadjieu et al. [24]. determination their pathogenicity. (b) to investigate the Potato Dextrose Agar (PDA) media with different inhibitory effects of different concentrations of clove oil concentrations of clove oil (0.5, 1, 2 and 4 % v/v) were on the mycelial growth and spore germination of all prepared by adding appropriate quantity of clove oil to isolated fungi. (c) to study the efficacy of clove oil as soil the melted media, followed by addition of Tween drench against the invasion of tomato plants by soil- 20 (100 µl to 100 ml of media) for the dispersion. About borne fungi under greenhouse conditions. 20 ml of the medium were poured into glass Petri-dishes (9 cm). Then inoculated at the center with a mycelial disc MATERIALS AND METHODS (0.5 cm diameter) taken from the margins of 4–6 days old Fusarium spp or Rhizoctonia solani cultures. Three Source of Plant Material and Clove Oil: Tomato replicates were conserved for each treatment. (Solanum lycopersicum L. cv. Super strain B) seeds were Additionally, positive control (without clove oil) was obtained from Unit of Vegetable Crops Seeds, Horticulture inoculated following the same procedure. Petri-dishes Research Institute, Agricultural Research Centre, Giza, were incubated at 25°C and the colony diameter was daily Egypt. Clove oil (containing 75 - 80% as the major measured until control petri dishes were fully covered ingredient) was purchased from Sigma Aldrich, Germany. with mycelia. The percentage of Mycelial Growth Inhibition (MGI) Isolation and Identification of the Causal Pathogens: was calculated according to the equation suggested by The fungal pathogens were isolated from naturally Djordjevic et al. [25] as follows: Mycelial Growth infected tomato plants showing typical root rot and wilt Inhibition (MGI) % = (Do – De) / Do x 100 diseases symptoms according to Davet and Rouxel [19]. Samples were collected from Giza, Monufia and Qalyubia where: Do = the diameter of the mycelia growth in the Governorates, Egypt. All emerged fungi were purified positive control – 0.5 cm using the hyphal tip technique according to Barnett De = the diameter of the mycelia growth in the oil and Hunter [20]. The identification was based on their supplemented plates – 0.5 cm

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Minimal Inhibitory Concentration (MIC) (defined as 3 50-75% root discoloration with one wilted leaf. the lowest concentration of clove essential oil in which no 4 = up to 75% root discoloration or more than one leaf growth occurred) was determined. wilted. 5 = dead seedlings. Microscopical Observations: The effects of different concentrations of clove oil on the structural and The percentage of disease severity index (DSI) morphological characters of the isolated fungi were was estimated using the following formula according to investigated using a light microscope (Leica – DM 2500). Song et al. [28]. Representative samples were taken from the periphery of 8-day-old fungal colony grown on PDA at 28°C DSI % = (d X number of plants in that grade) / (d max × containing the essential oil. The samples were fixed in n) ×100 lacto-phenol–trypan blue stain and then microscopically investigated [26]. where: d= is the disease rating of each plant d max = the maximum disease rating Effects of Clove Oil on Disease Incidence and Severity n = the total number of plants/samples examined in each under Greenhouse Conditions: The most effective replicate. concentration of clove oil (4%) that showed great inhibition on the mycelial growth of the tested fungi in Plant Growth Parameters of Tomato Plants: Six weeks vitro was selected to evaluate its activity against after transplanting, five replicates of each treatment were Fusarium spp and Rhioctonia solani under greenhouse destructively harvested and immediately separated into conditions. Clove oil was emulsified with 0.05% Tween roots, leaves and stems. The plant height, number of 20 at concentrations of 4% (v/v) before application [27]. branches/plant and fresh weight of the plant shoot were It was used as seedling root dipping for 12 h before recorded. The root segments were then washed transplanting. Tomato seedlings (four-weeks-old) were thoroughly with running water, blotted on tissue paper transplanted into plastic pots (30 cm diameter) containing and their fresh weighs were determined. Representative 3kg sandy loam soil (1:1 w/w). Pots were artificially samples of roots and shoots were then oven dried at 70 °C infested with fresh inoculum of the tested fungus, for 72 h for dry weights and water contents. Fusarium sp. and Rhizoctonia solani at the rate 3 % (w/w) of soil weight. There were 10 replicates per Statistical Analysis: All data sets were analyzed by treatment, each pot contains 2 tomato seedlings. Similar variance analysis (ANOVA) using SAS software. The numbers of pots only infected with pathogen were served separation of means was done by using the Least as a control. Pots were arranged in a completely Significant Difference (LSD) test at P 0.05 (30). randomized block design then kept at 22–28°C, 70–80% relative humidity and 12 h photoperiod and watered as RESULTS AND DISCUSSION needed and fertilized as usual. Percentage of disease incidence was calculated for Isolation, Identification and Pathogenicity Test: Four each individual treatment by dividing the number of fungi were isolated from symptomatic tomato plants that symptomatic plants over the total number of plants were collected from different governorates in Egypt according to Song et al. [28]. (Giza, Monufia and Qalyubia). All fungi were purified Disease severity of both root rot and wilt was and identified based on their cultural properties, estimated at 10 days interval for 45 day after transplanting morphological and microscopical characteristics. Obtained using a rating scale of (0 – 5) based on root discoloration results revealed that the most dominant fungi were or leaf yellowing grading, according to Abdou et al. [29] identified as Fusarium solani from Giza, Rhizoctonia as follow. solani from Qalyubia and Fusarium oxysporum and Fusarium semitectum from Monufia. Each purified fungus 0 = neither root discoloration nor leaf yellowing. was tested for its pathogenic ability on tomato plants 1 1-25% root discoloration or one leaf yellowing. under greenhouse conditions. The tested fungi were 2 25-50% root discoloration or more than one leaf obviously varied in their ability to cause root rot or wilt yellowing. infection of tomato plants. The most aggressive fungi

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Table 1: Effect of clove oil on the radial growth of some isolated fungi of tomato plants Fusarium oxysporum Fusarium solani Fusarium semitectum Rhizoctonia solani ------Treatments MG (cm) MGI% MG (cm) MGI% MG (cm) MGI% MG (cm) MGI% Control 9a ±0.00 0e ±0.00 9a ±0.00 0e ±0.00 9a ±0.00 0e ±0.00 9a ±0.00 0e ±0.00 Clove oil 0.5% 7.5b ±1.00 16.6b ±11.00 7.6b ±0.52 15.5d ±5.88 7.9b ±0.28 12.9d ±3.18 7b ±1.00 22.2d ±11.10 Clove oil 1% 5.0c ±0.40 44.4e ±4.00 6.4c ±0.75 28.8c ±8.39 5.5c ±0.50 39.2c ±5.59 4.5c ±0.41 50c ±4.64 Clove oil 2% 1.66d ±0.76 81.3b ±8.00 3.7d ±0.50 58.8b ±5.59 3.4d ±0.92 62.2b ±10.34 1.0d ±0.20 84b ±2.29 Clove oil 4% *0.5e ±0.00 100a ±0.00 0.9e ±0.36 91.8a ±19.80 1.2e ±0.81 88.5a ±11.70 *0.5e ±0.00 100a ±0.00 * The diameter of the initial disk (0.5 cm) was recorded in the case of full inhibition of mycelium. MG (cm) = Mycelia Growth (cm), MGI% = Mycelia Growth Inhibition %. For each column, means followed by the same letter are not significantly different according to Least Significant Difference (LSD) test at (P 0.05).

Fig. 1: Mycelial growth of different isolated fungi after 8 days incubation with different concentrations of clove oil (0, 0.5, 1, 2, 4 %) were F. semitectum (M1) followed by F. solani (G2), as vivo. The obtained data in Table (1) and Fig. (1) show that they recorded 95% and 86%, respectively. Meanwhile, there was a significant inhibition in radial growth of all the F. oxysporum (M2) and R. solani (K1) had the least tested fungi due to clove oil application. The inhibition potentiality of infection to tomato plants (80% and 70%, was high at the lowest application rate (0.5%) and respectively). These results are in harmony with those improved by increasing the tested concentration to 4%. reported by Haggag and El-Gamal [23]. The inhibition in linear growth of F. semitectum and F. solani was lower than that observed for F. oxysporum Inhibition of Mycelial Growth of the Pathogenic Fungi and Rhizoctonia solani in all tested concentrations. in vitro: Clove oil has antimicrobial activity against a However, Rhizoctonia solani and F. oxysporum didn’t variety of soil-borne fungi, plant pathogens and show any mycelium growth at concentration 4% (Fig. 1). mycotoxigenic fungi [31- 33]. The main chemical Minimum inhibitory concentration (MIC) was tested for components of clove oil are eugenol, acetyl eugenol, each pathogen and the results showed that MIC of clove iso-eugenol and -caryophyllene [34]. These phenolic oil was 4%, which completely inhibited the growth of compounds are responsible for the antibacterial and F. oxysporum and Rhizoctonia solani. This concentration antifungal properties of essential oil [35]. The current led, however, to 88.5 and 91.8% reductions in the growth work aimed to evaluate the antifungal activities of clove of F. semitectum and F. solani respectively. Hence, it was essential oil as a resistance inducer against F. oxysporum, selected for application in pot experiments under F. solani, F. semitectum and R. solani in vitro and in greenhouse conditions.

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Our findings are in agreement with those obtained by proteins. They may change the permeability and fluidity Sharma, et al. [36] and Furdos [37], which revealed that of cell membrane and disintegrate fungal hyphae [43]. clove essential oil, have the ability to inhibit radial growth, The microscopical observations are in agreement with spore germination and to reduce the dry weight of the study conducted by each of LaTorre et al. [44] and Fusarium oxysporum f. sp. lycopersici. Similar type of Sharma et al. [36]. They found that clove oil exhibited study was conducted by Juniawan, et al. [38] who considerable alterations in hyphal morphology and reported the sensitivity of Fusarium spp. against all the inhibited conidial germination of F. oxysporum f. sp. tested essential oils, but clove oil was the most effective lycopersici in comparison to those of control. They among the tested oils. On the other hand, Abdulaziz and confirmed that clove oil provided a complete inhibition Younes [39] investigated the antifungal activity of four after 24 and 48 h thus, probably killing the conidia. On the medicinal plants, (cinnamon, anise, black seed and clove) other hand, the microscopic observations of Soylu et al. against pea root-rot fungus (Rhizoctonia solani). The [45] for Sclerotinia sclerotiorum showed changes in the highest antifungal activity was recorded for clove extract hyphal morphology due to clove oil application. Shriveled (4%) which cause complete growth inhibition for hyphal aggregates reduced hyphal diameters and lyses of Rhizoctonia solani. hyphal wall were commonly observed in oregano- or fennel oil-treated mycelium, compared with controls. Effect of Clove Oil on Fungal Structures: There are several possibilities for antifungal mechanism of essential Effect of Clove Oil on Disease Incidence and Severity oils (EOS) described by researchers. For bioactivity, the under Greenhouse Conditions: Many in vivo studies EOS pass through the cell wall and cytoplasmic membrane have reported the efficacy of essential oils against [40]. Many authors emphasized that the antimicrobial Fusarium spp and Rhizoctonia solani [46, 47]. The effect of essential oil elements has been dependent on present study was planned to investigate the effect of their hydrophobicity and partition in the microbial clove oil (4%) on reduction the incidence and severity of plasmatic membrane [41, 42]. wilt and root rot diseases on tomato plants under The effects of different dosages of clove oil on the greenhouse conditions at 6 weeks after transplanting in morphological characterization of different pathogens soil infested with each of Fusarium spp and R. solani. were investigated. Microscopical investigations revealed Data illustrated in Figure (4) clearly indicate that clove oil that all concentrations of clove oil caused cytotoxicity to (4%) reduced significantly (P 0.05) the percentages of the tested fungi resulting in abnormal growth of mycelia, wilt and root rot disease incidence and severity on swollen of hypha and conidial malformation. Clove oil tomato plants compared with the untreated control treatment decreased significantly the conidial numbers, plants. Actually, it decreased F. oxysporum and R. solani but increased the production of chlamydospores relative incidence by 66 and 75%, while F. solani and to the control (untreated) and also mycelium that showed F. semitectum were reduced by 50 and 40%, respectively. regular cell structures with homogenous cytoplasm However, clove oil treatments decreased the disease (Figs. 2 & 3). Particularly, F. oxysporum and Rhizoctonia severity of F. oxysporum and R. solani by 72 and solani treated with clove oil at concentrations of 1% and 81%, respectively, whereas, those of F. solani and 2% exhibited irregular branching of the terminal hyphae, F. semitectum were reduced by 54 and 46%, respectively. severely collapsed mycelium and malformation in the These results are in harmony with those conducted by morphology of the hyphae. In some cases, the hyphal LaTorre et al. [44]: Estrada et al. [18] and Sharma et al. cytoplasm was autolysis. While the production of [36] which revealed that increasing concentration of the Fusarium oxysporum conidia was severely impaired due clove oil can reduce the incidence and severity of to all clove oil concentrations. The presence of Fusarium wilt of tomato under greenhouse conditions chlamydospores was increased in proportion to the compared to control. Furthermore, Hamad et al. [48] increase of essential oil concentration (Figs. 2 &3). mentioned that clove oil applied on guava seedlings roots Such modifications may be related to the effect of the completely inhibited F. oxysporum and R. solani. essential oil as enzymatic reactions regulating wall synthesis. Therefore, volatile phenolic compounds Effect of Clove Oil on Plant Growth Parameters: (, eugenol, ) may interfere with cell wall Fungal infection with each of F. oxysporum, F. solani, enzymes (enzyme inhibition), possibly through reaction F. semitectum and R. solani negatively affected the with sulfhydryl groups or through interactions with overall plant growth, the effect was less severe in plants

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Fig. 2: Light micrographs showing the abnormality of Fusarium oxysporum mycelia due to clove oil application. (A) Hyphae growing on control medium. (B & C) effects of clove oil at concentration of (1%). Note: deformations of conidia and severely collapsed mycelium (large black arrows), chlamydospores formation (small black arrows). (D & E) effects of clove oil at concentration of (2%). Note: irregular branching of the terminal hyphae (large black arrows), autolysis of the hyphal cytoplasm and necrosis. Bar = 40 µm.

Fig. 3: Light micrographs showing the abnormality of Rhizoctonia solani mycelia due to clove oil application. (A) Hyphae growing on control medium. (B & C) effects of clove oil at concentration (1%). (D & E) effects of clove oil at concentration of (2%). Note: deformations of hyphal shape (large black arrows), cytoplasmic coagulation, hyphal shrinkage and necrosis (small black arrows). Bar = 40 µm.

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Fig. 4: Effect of clove essential oil (4 %) on root rot and wilt disease incidence and severity on tomato plants caused by Fusarium spp and Rhizoctonia solani under greenhouse conditions.

Table 2: Effect of clove oil on the growth parameters of tomato plants Control F. oxysporum F. solani F. semitectum R. solani ------Trait H. N. T. H. T. I. N. T. I. T. I. N. T. I. T. I. N. T. I. T. I. N. T. I. T. NB/ per plant 8.00a±1.00 8.30a±0.57 4.60b±0.57 8.30a±0.57 4.00b±1.00 7.30a±1.52 3.30c±1.52 6.00b±1.00 5.00b±1.00 8.60a±0.57 Ph [cm] 34.60a±1.52 35.00a±4.00 19.60b±3.21 36.30a±5.50 17.60b±2.51 27.60a±6.42 15.00c±2.51 24.00b±1.00 23.30b±2.88 38.00a±4.00 RFW [g] 5.18a±0.84 5.26a±0.30 1.39b±0.35 4.34a±0.48 1.15c±0.04 3.21b±0.07 0.94c±0.03 2.66b±0.48 1.75b±0.02 5.20a±0.70 SFW [g] 8.96a±0.56 9.00a±0.50 4.15b±0.17 8.33a±0.51 4.50c±0.36 7.28b±0.19 4.45c±0.27 6.18b±0.07 4.56b±0.63 9.11a±0.44 RDW (%FW) 10.09a±3.702 9.91a±1.12 26.18c±4.12 15.80b±1.14 28.58b±1.24 13.39a±1.01 19.08b±2.82 12.81a±1.72 18.65a±2.39 14.69a±1.74 SDW (%FW) 13.70a±2.32 14.43a±1.97 14.22a±3.67 10.57a±1.24 11.33ab±1.60 10.39b±0.34 8.28c±0.85 11.16b±0.85 15.12a±2.47 17.17a±2.94 For each column, means followed by the same letter are not significantly different according to Least Significant Difference (LSD) test at (P 0.05). NP=number of branch /plant, Ph= plant height (cm), RFW= root fresh weight (g), SFW = shoot fresh weight, RDW= root dry weight (g), SDW= shoot dry weight (g), H.N.T= healthy plant un-treated, H.T= healthy treated, I.N.T= infected un-treated, I.T. = Infected treated infected with R. solani. The number of branches per their fresh weights were reduced by 53, 50, 50 and plant was steadily decreased due to infection with 50% in plants infected with F. oxysporum, F. solani, F. oxysporum, F. solani, F. semitectum and R. solani by F. semitectum and R. solani, respectively (Table 2). 42, 50, 58 and 37%, respectively, compared to the controls Fungal infections remarkably altered the dry weight (Table 2). Heights of infected plants were also declined by (% fresh weight), the effect was varied between 43, 50, 56 and 32%, respectively, compared to the under- and aboveground plant parts. Root dry weights respective controls. As a general trend, plant biomass was were increased by 159, 183, 89 and 85% in plants significantly suppressed upon fungal infection, with more infected with F. oxysporum, F. solani, F. semitectum and severe effects on the root parts (Table 2). The infected R. solani, respectively. However, the shoot dry weights plants exhibited about 73, 77, 80 and 66% reductions in were slightly increased only in plants infected with their root fresh weight upon infection with F. oxysporum, F. oxysporum and R. solani, but decreased in those F. solani, F. semitectum and R. solani, respectively. infected with F. solani and F. semitectum (Table, 2). Shoot parts were less sensitive to fungal infection, as Whereas clove oil treatment (4%) did not significantly

111 Am-Euras. J. Agric. & Environ. Sci., 18 (3): 105-114, 2018 influence the morphological traits of non-infected tomato 4. Muriungi, S., E. Mutitu, J.W. Muthomi and plants, it led distinctly to alleviate some of the detrimental J. Muriungi, 2014. Efficacy of cultural methods in the effects of fungal infection (Table 2). Clove oil treatment control of Rhizoctonia solani strains causing significantly (P 0.05) enhanced the number of branches tomato damping off in Kenya. African Journal of per plant, plant height, root and shoot fresh weights of Food, Agriculture, Nutrition and Development, infected plants compared to non-treated infected ones 14(2): 8776-8790. (Table 2). These increases in growth may be attributed to 5. Devay, J.E., R.H. Garber and R.J. Wakeman, 1988. elicitor’s effect on physiological processes in plant such Field management of cotton seedling diseases in as ion uptake, cell elongation, cell division, enzymatic California using chemical and biological seed activation and protein synthesis [49]. These results are in treatments. In: Proceedings of Beltwiae Cotton agreement with those obtained by El-Mohamedy et al. Conference, National Cotton Council of Americana, [50] and Hamad et al. [48]. Memphis, TN, USA, pp: 29-35. 6. Huang, I.B., J. Keisler and I. Linkov, 2011. CONCLUSION Multi-criteria decision analysis in environmental sciences: ten years of applications and trends. Fusarium oxysporum, F. solani, F. semitectum and Science of the Total Environment, 409: 3578-3594. Rhizoctonia solani were isolated from infected tomato 7. Deising, H., S. Reimann and S. Pascholati, 2008. plants collected from different locations of Giza, Monufia Mechanisms and significance of fungicide resistance. and Qalyubia governorates, Egypt. In vitro, all Brazilian Journal of Microbiology, 39(2): 286-295. concentrations of clove oil reduced mycelial growth of all 8. Linde, J.H., S. Combrinck, T.J. Regnier and tested fungi. Microscopical observations showed the S. Virijevic, 2010. Chemical composition and disruption of the fungal growth and conidial malformation. antifungal activity of the essential oils of Lippia Moreover, Clove oil at concentration 4% highly decreased rehmannii from South Africa. South African Journal disease incidence and disease severity. Therefore, it could of Botany, 76: 37-42. be suggested that application of clove oil as a strong 9. Kumar, V., C.S. Mathela, A.K. Tewari and K.S. Bisht, antifungal agent could be used for controlling tomato root 2014. In vitro inhibition activity of essential oils from rot and wilt diseases. some Lamiaceae species against phytopathogenic fungi. Pesticide Biochemistry and Physiology, ACKNOWLEDGEMENTS 114: 67-71. 10. Hamini-Kadar, N., F. Hamdane, R. Boutoutaou, This research was financially supported by M. Kihal and J.E. Henni, 2014. Antifungal Department of Plant Pathology, Faculty of Agriculture, activity of clove (Syzygium aromaticum L.) essential Ain Shams University, Cairo, Egypt. oil against phytopathogenic fungi of tomato (Solanum lycopersicum L) in Algeria. Journal of REFERENCES Experimental Biology and Agricultural Sciences, 2(5): 448-454. 1. Olaniyi, J.O., W.B. Akanbi, T.A. Adejumo and 11. Pawar, V.C. and V.S. Thaker, 2007. Evaluation of the O.G. Akande, 2010. Growth, fruit yield and nutritional anti-Fusarium oxysporum f. sp. cicer and anti- quality of tomato varieties. African Journal of Food Alternaria porri effects of some essential oils. Science, 4: 398-402. World Journal of Microbiology & Biotechnology, 2. Singh, A.K. and S. Kamal, 2012. Chemical control of 23: 1099-1106. wilt in tomato (Lycopersicon esculentum L.). 12. Sameza, M.L., L.C. Mabou, S.N. Tchameni, International Journal of Horticulture, 2(2): 5-6. M.A. Bedine, F. Tchoumbougnang, P.M. Dongmo 3. Suarez-Estrella, F., C. Vargas-Garcia, M.J. Lopez, and F.B. Fekam, 2016. Evaluation of clove essential C. Capel and J. Moreno, 2007. Antagonistic activity oil as a mycobiocide against Rhizopus stolonifer and of bacteria and fungi from horticultural compost Fusarium solani, Tuber Rot Causing Fungi in Yam against Fusarium oxysporum f. sp melonis. Crop (Dioscorea rotundata Poir.). Journal of Protection., 26: 46-53. Phytopathology, 164: 433-440.

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