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Article Type: Original Article

Antifungal activity of various essential oils against Rhizoctonia solani and Macrophomina phaseolina as major bean pathogens

Abbreviated running heading: Efficacy of essential oils on fungi

Nima Khaledi, Parissa Taheri*, Saeed Tarighi

Department of Crop Protection, Faculty of Agriculture, Ferdowsi University of Mashhad, P.O.Box: 91775-1163, Mashhad, Iran

Corresponding author: P. Taheri E- mail: [email protected] Tel: 0098 51 8795612 Fax: 0098 51 8787430

Abstract Aims: The main objective of this study was to investigate the effect of various essential oils (EOs) to decrease activity of cell wall degrading enzymes (CWDEs) produced by fungal phytopathogens which are associated with disease progress. Also, effect of seed treatment and This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jam.12730 This article is protected by copyright. All rights reserved.

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foliar application of peppermint EO and its main constituent, menthol, on diseases caused by two necrotrophic pathogens on bean was investigated.

Methods and Results: Antifungal activity of EOs on Rhizoctonia solani and Macrophomina

phaseolina, as bean pathogens, was evaluated. The EOs of Mentha piperita, Bunium persicum

and Thymus vulgaris revealed highest antifungal activity against fungi. The EO of M. piperita had the lowest minimum inhibitory concentration (MIC) for R. solani among three EOs tested. This pathogen did not grow in presence of M. piperita, B. persicum and T. vulgaris EOs at 850, 1200 and 1100 ppm concentrations, respectively. The B. persicum EO had the lowest MIC for M. phaseolina as this fungus did not grow in presence of M. piperita, B. persicum and T. vulgaris EOs at concentrations of 975, 950 and 1150 ppm, respectively. Hyphae exposed to EOs showed structural changes. Activities of cellulase and pectinase, as main CWDEs of pathogens, decreased by EOs at low concentration without effect on fungal growth. Seed treatment and foliar application of peppermint EO and/or menthol significantly reduced the development of bean diseases caused by both fungi. Higher capability of menthol than peppermint EO in decreasing diseases on bean was observed.

Conclusions: Reducing CDWEs activity is a mechanism of EOs effect on fungi. Higher antifungal activity of menthol compared to peppermint EO was observed not only in vitro but also in vivo.

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Significance and Impact of the study: Effect of EOs on CWDEs involved in pathogenesis is described in this study for the first time. Menthol can be used as a botanical fungicide to control destructive fungal diseases on bean.

Keywords: Bunium persicum, Cellulose, Essential oil, Macrophomina phaseolina, Mentha

piperita, Menthol, Pectinase, Rhizoctonia solani, Thymus vulgaris.

Introduction Common bean (Phaseolus vulgaris L.) is one of the most abundant crops worldwide which is

known as a source of plant protein for human consumption. Also, it improves the soil fertility by fixing atmospheric nitrogen via the root nodules. Bean plants are attacked by different soilborne pathogens including Rhizoctonia solani (Kuhn) and Macrophomina phaseolina (Tassi) Goid, which are among the most important fungal pathogens of this crop plant (Nerey et al.

2010; Amusa et al. 2007). The damage caused not only leads to yield loss and reduced level of

nitrogen fixation, but also results in increasing pathogen density in soil (El-Gali et al. 2012).

R. solani is an important necrotrophic pathogen, with a broad host range and little effective resistance in crop plants (Nikraftar et al. 2013). To date, R. solani has been grouped into 13 anastomosis groups (AGs), which are different in pathogenicity, morphological characteristics and DNA sequences (Carling et al. 2002). Stem and root rot caused by R. solani is a major constraint leading to severe yield losses in bean production. Various AGs of R. solani have

been identified causing bean root rot, the most frequently reported being AG 2-2 and AG 4 (Balali and Kowsari 2004; Nerey et al. 2010). Macrophomina phaseolina (Tassi) Goid causes

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dry root rot, charcoal rot, seedling blight and ashy stem blight in several hosts, mainly bean plants (Su et al. 2001; Gachitua et al. 2009). It is one of the most destructive necrotrophic soil borne pathogens, infecting about 500 plant species (Mihail and Taylor 1995). Outbreaks of charcoal rot disease associated with M. phaseolina caused reduction of seed production and yield losses ranging from 46 to 74% in many bean-growing regions worldwide (Amusa et al. 2007; Mayek-Perez et al. 2003).

Several methods were suggested for controlling M. phaseolina and R. solani, i.e.

biological control (Huang et al. 2012; Sreedevi et al. 2011), chemical control (Mahmoud et al.

2006) and application of plant extracts and EOs (Javaid and Naqvi 2012; Seema and Devaki 2010). The most general method to control these pathogens is the use of fungicides. However, development of resistance in fungi to fungicides and increasing residual hazardous effects on human health and negative effects in the environment leads to focus on search for finding plant derivatives that can reduce fungal pathogencity (Aslam et al. 2010).

Medicinal plants are potential sources of antimicrobial compounds, which could be

used in the management of plant diseases (Balbi-Peña et al. 2006). Research on plant extracts and EOs which may substitute the use of agrochemicals or contribute to the development of new compounds seems to be extremely important. EOs are promising alternative compounds which have inhibitory effect on fungal growth.

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Various EOs could be useful alternative substances to be replaced with synthetic fungicides in the plant disease management (Gwinn et al. 2010; Nguefack et al. 2013). They

contain a variety of volatile molecules such as terpenes and terpenoids, phenol-derived aromatic and aliphatic components (Bakkali et al. 2008). EOs and their components are

gaining high level of interest due to their relatively safe status, wide acceptance by consumers, and exploitation for potential multi-purpose functional uses (Ormancey et al. 2001). General antifungal activity of various EOs is well documented (Chang et al. 2008; Pitarokili et al. 1999; Meepagala et al. 2002; Nguefack et al. 2012).

Seema and Devaki (2010) studied antifungal activity of various EOs against R. solani.

The EO of cinnamon was found to be the most effective, as it caused complete inhibition of the pathogen at 500 ppm concentration. Also, leaf EO and its constituents from Calocedrus macrolepis var. formosana Florin were strongly inhibitory to the growth of R. solani (Chang

et al. 2008). The EOs of Thymus vulgaris (Zambonelli et al. 2004), Salvia fruticosa (Pitarokili

et al. 2003), Mentha piperita (Zambonelli et al. 2004), and Monarda spp. (Gwinn et al. 2010) also reduced the mycelial growth of R. solani.

In the case of M. phaseolina, fungitoxicity effects of leaves and seeds of 15

angiospermic taxa against this fungus were investigated by Dwivedi and Singh (1998). They reported the best fungitoxic capability among their tested plants for the EO obtained from seeds of Trachyspermum ammi L. (Sprauge). Actinidine which was isolated from the EO of Nepeta clarkei was effective in vitro against M. phaseolina (Saxena and Mathela 1996).

Kazmi et al. (1995) and Alice et al. (1996) reported that neem oil was more or equally

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effective compared to synthetic fungicides such as benomyl and carbendazim against M. phaseolina.

Although previous studies have identified reduction in the growth of R. solani and M.

phaseolina via application of various EOs, there is no report on their effects on pathogenesis mechanisms of these destructive fungal pathogens so far. Therefore, the objectives of present study were (i) to screen and select effective EOs of various medical plants against the mycelial growth of R. solani and M. phaseolina in vitro, and (ii) to determine the potential of very low concentrations of selected EOs, without any effect on the fungal growth, in reducing activity of CWDEs such as pectinase and cellulase which are involved in the infection process of theses fungi on the host plant. In addition, the effect of seed treatment and foliar application of M. piperita EO and menthol, as its main constituent, on decreasing progress of the diseases caused by both pathogens on bean plants was investigated in greenhouse conditions.

Materials and methods Plant pathogenic fungi Two phytopathogenic fungi used in this study were obtained from Phytopathology

Laboratory in Ferdowsi University of Mashhad, Iran. R. solani anastomosis group (AG) 4 HG-I is the causal agent of stem and root rot in several plants. M. phaseolina causes ashy stem blight disease on various plant species. Both fungal isolates were highly virulent on bean plants. These phytopathogenic fungi were maintained on potato dextrose agar (PDA) medium at 4˚C, and sub-cultured at monthly intervals.

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Plant materials All plant species were collected from Khorassan-Razavi and Tehran provinces in Iran (Table 1). The species were identified, and a voucher sample was kept in the laboratory of Botany (Ferdowsi University of Mashhad, Iran).

Extraction of EOs All plants were washed with distilled water and dried at room temperature in the shade away from direct sunlight. Then, the plant tissues were crashed and passed through a 10 mesh sieve. For isolation of the EOs, 100 g of dried plant materials were subjected to hydro-distillation for about 3 h, using a Clevenger apparatus (Tripathi et al. 2008). The oil was dried over anhydrous Na2SO4 and preserved in sealed glass bottles. It was protected from light by

wrapping in aluminium foil and stored at 4°C until used.

Screening for antifungal activity and fungal growth inhibition in vitro The tests were performed using the agar medium assay described by Tatsadjieu et al. (2009). For preliminary assessment and screening the efficacy of EOs obtained from various plants, the effect of each EO at 2000 ppm concentration on growth of the pathogens was investigated. PDA media treated with the EOs were prepared by adding appropriate quantity of each EO to melted medium, followed by addition of Tween 20 (0.01 %) to disperse the EO in the medium. Each Petri-dish was inoculated at the center with a mycelial disc (10 mm diameter) taken at the periphery of R. solani or M. phaseolina colony grown on PDA at 28°C for 72 h.

Positive control (without EO) plates were inoculated following the same procedure. Plates were incubated at 28°C for 4 days and the colony diameter was recorded each day. The

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growth inhibition percentage was calculated according to the formula described by Abd-ElKhair and El-Gamal Nadia (2011) which is mentioned below:

Growth inhibition (%) = (C – P / C) × 100

Where, C is the diameter of mycelial growth in control plates, and P is the diameter of mycelial growth in treated plates.

Three replicates were used per treatment and the experiment was repeated three times.

The EOs with the highest levels of inhibition against the pathogens were selected for further experiments.

Nature of toxicity of EOs and determining minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC) and inhibitory concentration 50 (IC50) The nature of toxicity (fungistatic and/or fungicidal effect) of the EOs against fungi was determined as described by Thompson (1989) to determine if each EO had only fungistatic activity on the pathogens or also it could have fungicidal effect. The inhibited fungal mycelial plugs of the EO treated PDA plates were re-inoculated into fresh medium and revival of their growth was investigated to determine which concentration of each EO and menthol had fungicidal effect on the tested fungi.

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The MIC and MFC of the most effective EOs against both fungi were determined as described by Plodpai et al. (2013). The PDA plates were amended with various concentrations of EOs (0 to 2000 ppm). For enhancing the EO solubility, Tween-20, 0.01% (v/v) was added. Each plate was inoculated with a mycelial plug of R. solani or M. phaseolina. All plates were incubated in triplicate for each concentration at 28°C for 72 h. Plates with Tween-20, without any EO were used as control. The MIC values were determined as the lowest concentration of each EO that completely prevented the visible fungal growth.

To determine MFC, the mycelial plugs were obtained from each Petri dish treated with

the oil concentrations higher than MIC, cultured on PDA and incubated at 28°C for 72 h. MFC was defined as the lowest concentration at which no colony growth was observed after subculturing into fresh PDA medium. IC50 (concentration that produces a 50% inhibitory effect) values were graphically calculated from the dose-response curves based on

measurement at different concentrations as described by Chang et al. (2008). Inhibitory effect of the selected EOs at concentrations of 0.01 × IC50, and 0.1 × IC50 compared to the IC50 and control treatment for each EO were investigated in vitro.

Antifungal activity of main constituent of the EO of M. piperita Based on the results of a recent GC-MS analysis (Moghaddam et al. 2013) on exactly the same M. piperita plant sample used in this study, a survey was carried out on the antifungal effect of menthol, as a major constituent in the EO of M. piperita and its IC50, MFC, and MIC were determined as described before.

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Efficacy of the EOs was compared with some common fungicides, such as Carboxin (Vitavax) and Thiabendazole (Tecto 60) by the agar medium assay described before.

Determination of EO effects on hyphal morphology Hyphal morphology of R. solani and M. phaseolina grown in PDA plates containing IC50 concentration of M. piperita, B. persicum, and T. vulgaris EOs was investigated 3 days after culturing the fungi. Morphological changes resulting from EOs on hyphal growth were examined under light microscope (Olympus BX41, Japan).

Effect of EOs on the activity of pectinase and cellulase The efficacy of three selected EOs at 0.01 × IC50 concentration, which in previous experiments proved to have no effect on the fungal growth, in reducing the activity of pectinase and cellulase were determined using the methods described by Khairy et al. (1964) and Abdel-Razik (1970). Production of pectinase was carried out using a medium containing 4.6 g citrus pectin , 5.0 g yeast extract, 5.0 g peptone, and 5.0 g K2HPO4 in 1 liter of distilled

water and pH 7.2±0.2 as described by MacMillan and Voughin (1964). The same medium supplemented with 4.6 g carboxymethyl cellulose instead of citrus pectin was used for cellulose production. Each essential oil was added to the sterilized medium in each flask to obtain 0.01 × IC50 concentration. Then, the flask was inoculated with a 1 cm diameter mycelial plug of each fungus. Pectinase and cellulase activities were determined after 10 days of incubation at 28°C. The supernatants were obtained by filtration and centrifugation at 4695

g for 15 min at 4°C. Then, the supernatants were used for crude enzyme preparation. Three

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flasks were used as replicates for each treatment as well as the control and the experiment was repeated three times.

Pectinase activity was determined based on the amount of reducing sugar (D-galacturonic acid) released in the culture supernatant. The amount of D-galacturonic acid was determined by dinitrosalicylic acid colorimetric method of Colowich (1995). Briefly, 0.5 ml of cell free supernatant was incubated with 0.5 ml of 1% pectin in 0.1 mol l-1 acetate buffer with pH 6.0

and the reaction mixture was incubated at 40°C for 10 min in static condition. After adding 1 ml of 3, 5-dinitrosalicylic acid (DNS) reagent, the mixture was boiled for 5 min at 90°C. The reaction was stopped by adding 1 ml of 1% Potassium sodium tartrate. Then, the mixture was diluted by adding 2 ml of de-ionized water and absorbance was measured at 540 nm. One unit of pectinase activity was defined as the amount of enzyme that released 1 μmol of galacturonic acid per minute according to the standard curve. The standard curve was drawn based on the absorbance in different concentrations (μg ml-1) of D-galacturonic acid.

Cellulase activity was investigated using the method of Wood and Bhat (1988). Carboxymethyl cellulose was used as substrate for cellulase assay as reported in the literature (Han et al. 1995; Deshpande 1984; Ding et al. 2001). Briefly, 0.5 ml of cell free supernatant was mixed with 1 ml of 0.7% carboxymethyl cellulose in 0.05 mol l-1 acetate buffer with pH 4.8 and the reaction mixture was incubated at 50°C for 60 min in static condition. After adding 2 ml of DNS reagent, the mixture was boiled for 10 min at 100°C. The reaction was stopped by adding 1 ml of 40% Potassium sodium tartrate. The absorbance was measured at 550 nm and the amount of reducing sugar released was calculated from the standard curve of

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glucose. One unit of cellulase activity was defined as the amount of enzyme that catalyzed 1.0 μmol of glucose per minute during the hydrolysis reaction.

Effect of peppermint EO and menthol on progress of diseases caused by R. solani and M. phaseolina on bean Greenhouse experiments were done using seeds of Phaseolus vulgaris cv. Naz (obtained from bean research institute, Khomein, Iran). The seeds were surface sterilized in 1% sodium hypochlorite solution for 5 min, rinsed twice in sterile distilled water, and placed in petri dishes on sterile wet filter paper. For seed treatment with peppermint EO or menthol, different concentrations of the EO or menthol (IC50, 0.1 × IC50, and 0.01 × IC50) were obtained by suspending in distilled water containing 0.01% Tween 20. In control, the seeds were soaked in sterile distilled water containing 0.01% Tween 20. The seeds were soaked in each treatment for 5 min before sowing in soil. After 3 days at room temperature, germinated seeds were sown in 15 cm-diameter plastic pots containing a combination of clay, sand and leaf compost with the ratio of 1:1:1 (v/v) which had been autoclaved at 121°C for 30 minutes on 2 successive days. The plants were grown in greenhouse (28±2°C; 16/8 h light/dark photoperiod), and irrigation process was carried out when needed.

Wheat grains colonized by R. solani or M. phaseolina were prepared as inoculum (D'aes et al. 2011). In the greenhouse experiments, pathogenicity tests on 2-week-old bean plants were carried out on the stem near the soil line as previously described (Nikraftar et al. 2013). Colonized wheat grains were filled 1 cm above the soil surface, which contained 3 g of wheat grain inoculum per pot. Sterilized wheat grains which were placed on PDA without the fungus

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The plant cell wall is composed of polysaccharides and proteins. The wall polysaccharides are often classified into cellulose, hemicelluloses, and pectin. Cellulose is the main load-bearing structure and is composed of β-1, 4 linked glucan chains (Harholt et al. 2010). Pectic substances are structural polysaccharides, present in the middle lamella and primary cell wall of plants. Pectins are high molecular weight acid polysaccharides, made up of α-1, 4 linked D-galacturonic acid residues (Ridley et al. 2001). For cell wall degradation, exocellular cellulolytic and pectolytic enzymes are produced by phytopathogens which are capable of attacking each of these major polymeric components (Abd-El-Khair and El-Gamal Nadia 2011). Cellulolytic enzymes serve as invasive agents that enable the pathogen to penetrate host tissue (Olutiola and Cole 1976). Bateman (1964) reported that R. solani produces cellulase which may assist penetration of the fungus into host cells. Pectinase activity was regarded to be the most important predictor for virulence of R. solani (Mondal et al. 2013; Weinhold and Bowman 1974). Degradation of plant cell walls by M. phaseolina is due to pectinase and cellulase secretion. The development of intercellular mycelium and its close association with cell wall in cortex are correlated with pectinase concentration (Ijaz et al. 2012).

Current study revealed that three selected EOs were capable of decreasing cellulase

and pectinase activity of both fungi investigated. In overall, the EO of M. piperita was more

effective in reducing pectinase activity of R. solani and M. phaseolina at most of the time points investigated. Also, the EOs of B. persicum and M. piperita had higher effect in

reducing cellulase activity of both pathogens compared to the thyme oil. These results suggested that the EO of tested plants differed in their capability for reducing the activity of

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Javaid, A. and Naqvi, S.F. (2012) Evaluation of antifungal potential of Cenchrus pennisetiformis for the management of Macrophomina phaseolina. World Acad Sci Engineer Technol 69, 32-835.

Kagale, S., Marimuthu, T., Kagale, J., Thayumanavan, B. and Samiyappan, R. (2011) Induction of systemic resistance in rice by leaf extracts of Zizyphus jujuba and Ipomoea carnea against Rhizoctonia solani. Plant Signal Behav 6, 919-923.

Kamatou, G.P.P., Vermaak, I., Viljoen, A.M. and Lawrence, B.M. (2013) Menthol: a simple monoterpene with remarkable biological properties. Phytochemistry 96, 15-25.

Kazmi, S., Saleem, S., Ishrat, N., Shahzad, S. and Niaz, I. (1995) Effect of neem oil and benomyl on the growth of the root infecting fungi. Pak J Bot 27, 217–220.

Khairy, E.M., Sammour, H.M., Ragheb, A., Ghandour, M.F. and Aziz, K. (1964) A Laboratory Manual of Practical Chemistry. Cairo, Egypt: Dar El-Nahda El-Arabia. 1– 142

Lee, S.O., Choi, G.J., Jang, K.S. and Kim, J.C. (2007) Antifungal activity of five plant essential oils as fumigant against postharvest and soilborne plant pathogenic fungi. Plant Pathol J 23, 97-102.

MacMillan, J.D. and Voughin, R.H. (1964) Purification and properties of a polyglacturonic acid- transeliminase produced by Clastridium multiformentans. Biochemistry 3, 564572.

Mahmoud, Y.A.G., Aly, A.A., Omar, M.R. and Ismail, A.W.A. (2006) Variation in sensitivity among some isolates of Macrophomina phaseolina isolated from cotton roots to flutolanil fungicide. Mycobiology 34, 99-103.

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E

F

G

H

Fig. 4. A

Pectinase activity (μg/ml)

4500 4000 3500 3000 2500

Control

2000

Thymus vulgaris

1500

Bunium persicum

1000

Mentha piperita

500 0 0

24

48

72

96

120

144

Time (h)

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168

192

216

240

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Cytoplasmic coagulation or fragmentation in the hyphae was recorded (Figs. 3B, 3C, 3D, and 3H). In some cases, cell wall disruption and consequent hyphal lysis or necrosis were observed (Figs. 3F, 3G, and 3H).

Effect of the EOs on enzymatic activity of R. solani and M. phaseolina

Investigating the effect of three selected EOs on the activity of pectinase and cellulase secreted by R. solani and M. phaseolina revealed that 0.01 × IC50 concentration of these EOs, which did not have any effect on fungal growth, reduced activity of both enzymes in vitro. R. solani showed maximum pectinase and cellulase activity after 192 and 216 hours postculturing on liquid medium (hpc), respectively, and decreased afterward (Fig. 4). At most of the time points investigated, especially after 120 hpc, M. piperita and B. persicum oils had higher effects on reducing pectinase activity compared to the Thyme oil (Fig. 4A). In general, the highest reduction in the activity of cellulase in the case of R. solani was observed using the

EO of B. persicum (Fig. 4B).

Activity of pectinase and cellulase secreted by M. phaseolina reached to the maximum

level at 216 and 192 hpc, respectively (Fig. 5). In overall, highest reduction in pectinase activity of M. phaseolina was observed using the EO of M. piperita, followed by B. persicum and T. vulgaris (Fig. 5A). The EOs of B. persicum and M. piperita had higher effects compared to the thyme oil on reducing cellulse activity of M. phaseolina (Fig. 5B).

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Efficiency of peppermint EO and menthol on disease severity of each pathogen The data presented in Table 3 indicated that both seed treatment and foliar application of peppermint EO and/or menthol markedly reduced the development of been stem and root rot and charcoal rot caused by R. solani and M. phaseolina, respectively. Severity of the disease caused by each pathogen on bean significantly decreased by seed treatment using peppermint EO or menthol at IC50 concentration followed by 0.1 and 0.01 × IC50 concentrations. Similar results for both diseases were obtained in the experiments using foliar spray. Any phytotoxicity on the plant leaves at the low concentrations of peppermint EO and menthol was not observed in this study. In overall, higher levels of suppression were obtained for menthol compared to M. piperita (Table 3).

Discussion In this study, antifungal capability of the EOs obtained from various plants against R. solani and M. phaseolina was investigated using in vitro and in vivo assays. The effect of the most efficient EOs on the activity of CWDEs such as cellulase and pectinase, as virulence factors of these fungi, were demonstrated for the first time. Also, this is the first report on the effect of seed treatment and/or foliar spray of peppermint EO and its main constituent, menthol, on severity of the disease caused by each pathogen on bean.

Our data revealed that all EOs used in this study had considerable inhibitory effect on

mycelial growth of R. solani in vitro. Except the EOs of Ocimum basilicum and Juniperus polycarpus, all other EOs tested had considerable antifungal capability on M. phaseolina. Zingiber officinale and Foeniculum vulgare had moderate antifungal effect on M. phaseolina

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and high inhibition for R. solani. Antifungal activity of the EOs of these plants observed in our research is parallel with other reports (Bansod and Rai 2008; Sharma and Sharma 2011). Compared to other reports on high levels of antifungal activity of O. basilicum and J. polycarpus EOs (Al-Maskri et al. 2011; Tavares et al. 2012), there are some differences

between our results (in the case of M. phaseolina) and these reports. It could be caused by differences in the examined fungal species. Also, it might be due to the variation of chemical components present in each plant variety which the oil drived from it. Plant growth and the components of its EO may change with the region of collection, altitude, climatic conditions of each season, and also interaction of various microorganisms with plant tissues. So, we should be careful to recommend any EO without sufficient sampling and repetition.

The EOs of M. piperita, B. persicum and T. vulgaris showed maximum inhibition on

growth of both pathogens among all EOs tested. So, they were selected to be used in the rest of experiments. Antifungal activity of the peppermint EO against M. phaseolina (Moghaddam

et al. 2013) and R. solani (Abdel-Kader et al. 2011) were previously reported, which are in accordance with our results. No antifungal activity was previously reported for the volatile compounds of the M. piperita oil against R. solani (Lee et al. 2007). However, we observed

anti-R. solani activity for the EO of this plant species by mixing it with PDA medium. So, it might be possible that antifungal activity of the M. piperita EO was due to its non-volatile compounds. On the other hand, the chemical composition of plant EOs depends on various factors such as the plant variety, environmemtal conditions, and also plant-microbe interactions (Mucciarelli et al. 2007; Gwinn et al. 2010).

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Antifungal activity of thyme EO had been previously reported on several fungi, including R. solani (Lee et al. 2007; Abdel-Kader et al. 2011) and M. phaseolina (AbdelKader et al. 2011) which are in accordance to our data. However, there is not any report on the effect of thyme oil (at low concentration without any effect on fungal growth) on reducing the activity of CWDEs produced by fungi so far.

In our investigations, the EO of B. persicum was one of EOs with best inhibitory effect

on growth of both fungi in vitro. This is in accordance with the results obtained by Sekine et al. (2007), which demonstrated the best antifungal effect of B. persicum among 52 plant species tested against Fusarium oxysporum.

Antifungal activity of each EO increased with increasing its concentration. Minimum

concentration of the oil required to inhibit mycelial growth of fungi was different. Inhibitory effect of each EO on growth of fungi varied among different fungal species. This is in accordance with previous reports on different effects of an EO on various fungi or different isolates of a fungal species (Hashem et al. 2010; Chang et al. 2008).

The lowest levels of IC50, MFC and MIC were obtained for the M. piperita oil against

both fungal pathogens among three EOs tested. The IC50, MIC, and MFC values obtained for all three EOs were considerably lower than the values obtained for synthetic fungicides tested. These data suggest higher efficacy of the EOs in controlling both fungi compared to fungicides as previously reported for EOs obtained from other plants (Plodpai et al. 2013).

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Investigating fungistatic and/or fungicidal effects of the EOs showed that the T. vulgaris EO had fungicidal activity against both pathogens. The EOs of M. piperita and B. persicum revealed fungistatic effect on M. phaseolina and fungicidal activity against R. solani, which were similar to the effect of menthol on these fungi. These findings are in agreement with other reports indicating that the fungistatic or fungicidal effect of EOs could be different on various microorganisms (de Lira Mota et al. 2012; Soković et al. 2009).

Antifungal activity of M. piperita EO is supposed to be associated with high level of

menthol, as a cyclic monoterpene alcohol (Bupesh et al. 2007). Considerably lower IC50 and

MIC values obtained using menthol compared to M. piperita EO against both fungi suggested high antifungal potential of menthol, which was previously demonstrated against other microorganisms (reviewed by Kamatou et al. 2013). These results are of great importance, because of facilitating the utilization of individual components in an EO, instead of a mixture,

giving more predictability and probably less collateral effects.

In this study, microscopic observations of R. solani and M. phaseolina hyphae exposed

to EOs revealed alterations in hyphal morphology. Appearance of shriveled aggregates, reduced diameters and degradation of hyphal cell walls were commonly observed in the mycelia treated with M. piperita, B. persicum EOs, compared with thick, elongated, normal

mycelial growth in controls. Cytoplasmic coagulation was observed in M. phaseolina hyphal cells treated with T. vulgaris oil. The data obtained by microscopic observations are in accordance with previous studies in which EOs of aromatic plants caused morphological alterations on the fungal hyphae (Romagnoli et al. 2005; Soylu et al. 2010).

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The plant cell wall is composed of polysaccharides and proteins. The wall polysaccharides are often classified into cellulose, hemicelluloses, and pectin. Cellulose is the main load-bearing structure and is composed of β-1, 4 linked glucan chains (Harholt et al. 2010). Pectic substances are structural polysaccharides, present in the middle lamella and primary cell wall of plants. Pectins are high molecular weight acid polysaccharides, made up of α-1, 4 linked D-galacturonic acid residues (Ridley et al. 2001). For cell wall degradation, exocellular cellulolytic and pectolytic enzymes are produced by phytopathogens which are capable of attacking each of these major polymeric components (Abd-El-Khair and El-Gamal Nadia 2011). Cellulolytic enzymes serve as invasive agents that enable the pathogen to penetrate host tissue (Olutiola and Cole 1976). Bateman (1964) reported that R. solani produces cellulase which may assist penetration of the fungus into host cells. Pectinase activity was regarded to be the most important predictor for virulence of R. solani (Mondal et al. 2013; Weinhold and Bowman 1974). Degradation of plant cell walls by M. phaseolina is due to pectinase and cellulase secretion. The development of intercellular mycelium and its close association with cell wall in cortex are correlated with pectinase concentration (Ijaz et al. 2012).

Current study revealed that three selected EOs were capable of decreasing cellulase

and pectinase activity of both fungi investigated. In overall, the EO of M. piperita was more

effective in reducing pectinase activity of R. solani and M. phaseolina at most of the time points investigated. Also, the EOs of B. persicum and M. piperita had higher effect in

reducing cellulase activity of both pathogens compared to the thyme oil. These results suggested that the EO of tested plants differed in their capability for reducing the activity of

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CWDEs secreted by R. solani and M. phaseolina, which may be related to their chemical composition. Similarly, a previous report indicated that aqueous extracts of various plants reduced mycelial growth of R. solani and Fusarium solani, together with significant inhibition of polygalactronase and cellulase activites of these fungi (Abd-El-Khair and El-Gamal Nadia 2011). The present data revealed considerable decrease in pectinase and cellulase activities of M. phaseolina and R. solani, in all treatments having the EOs at 0.01 × IC50 concentration which did not suppress mycelial growth of the fungi in vitro. So, decrease in the activity of CWDEs may be a part of mechanism involved in reducing virulence of these fungi.

As is previously reported by other authors, foliar application of plant extracts or

essential oils could be capable of significantly reducing severity of plant diseases caused by soil-borne fungal pathogens, such as R. solani (Plodpai et al. 2013).

Investigations on

mechanisms of disease suppression by plant extracts and EOs have suggested that the active constituents of them may either act on the pathogen directly (Abdel-Monaim et al. 2011), or activate defense responses in host plants leading to reduction of disease progress (Kagale et al. 2011). In foliar application of EOs on plants to protect them against soil-borne fungi, it seems that activation of plant defense mechanisms might be involved in disease suppression, which could be an interesting subject for future researches. In vivo effects of various EOs and plant extracts against several soil-borne pathogens were examined not only by foliar spray (Plodpai et al. 2013), but also by soil or seed treatment (Abd-El-Khair and El-Gamal Nadia 2011; Hashem et al. 2010), so far. In the present research, first we used soil (data not shown) and seed treatment (Table 3) to evaluate the efficacy of peppermint EO and menthol in disease control. In some cases, soil treatment was effective similar to seed treatment. But in most of

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the cases, soil treatment was less effective in disease suppression than seed treatment (data not shown). One of reasons for this result might be the possibility of degrading the EO and menthol in soil. On the other hand, it seems that seed treatment leads to higher induction of plant defense mechanisms as previously demonstrated by Abd-El-Khair and El-Gamal Nadia (2011). So, based on these observations, we decided to present only the results of seed treatment and then, we carried out another experiment using foliar spray of different concentrations of peppermint EO and menthol. Finally, we compared the results of seed treatment with foliar spray to determine their capability in disease control. Greenhouse experiments indicated that, using peppermint EO and menthol as seed treatment or foliar spray (at 2 dpi) were effective in reducing bean diseases caused by both fungi in a dose dependent manner. Recently, Plodpai et al. (2013) used only foliar spray of plant extracts to control R. solani, causing rice sheath blight. They reported that foliar application of Desmos chinensis extracts is significantly effective in reducing the disease caused by R. solani on rice in a dose

dependent manner (Plodpai et al. 2013) which is confirming our data. Generally, menthol was more effective in decreasing the DI of each pathogen on bean compared to the M. piperita EO. Our study is the first in demonstrating the efficacy of plant EOs against M. phaseolina using seed treatment or foliar spray in vivo.

On the basis of these results, the EOs of M. piperita, B. persicum, and T. vulgaris had

fungicidal and/or fungistatic activity against R. solani and M. phaseolina. Very low concentration (0.01× IC50) of these EOs was capable of decreasing the activity of CWDEs, as the main virulence factors of these fungi. The EO of M. piperita showed highest antifungal efficacy against both fungi, which could be associated with menthol as its main constituent.

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Peppermint EO decreased the DI of both pathogens on bean. So, peppermint and menthol may represent new alternative disease management strategies. However, there is a need for more studies aimed at correlating their potent antifungal activity in vitro and practical use in field to

prove their safety for environmental application.

Acknowledgments We thank Ferdowsi University of Mashhad, Iran, for financial support of this research.

Conflict of interest No conflict of interest declared

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Table 1. List of plant species, common names, family and part used in the preliminary screening for antifungal properties.

No. Plant species Family Common names Plant part used 1 Mentha piperita Labiatae Peppermint leaves 2 Ocimum basilicum Lamiaceae Basil leaves 3 Mentha aquatic Lamiaceae Water Mint leaves 4 Zingiber officinale Zingiberaceae Ginger rhizome 5 Foeniculum vulgare Apiaceae Fennel seeds 6 Juniperus polycarpus Cupressaceae Persian juniper leaves 7 Bunium persicum Apiaceae Black Caraway seeds 8 Thymus vulgaris Lamiaceae Thyme leaves

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Table 2. In vitro antifungal activity of the EOs and their main constituents compared to synthetic fungicides against mycelial growth of Rhizoctonia solani and Macrophomina phaseolina. Fungi Treatments

IC50a

R. solani MFCb

MICc

M. phaseolina IC50a MFCb MICc

Mentha piperita Bunium persicum Thymus vulgaris

400 450 600

1000 1500 1500

850 1200 1100

450 500 700

IN IN 1500

950 975 1150

menthol

250

1000

700

300

IN

500

2000 1000

4000 2000

4000 2000

2000 1000

IN 2000

4000 2000

EOs

Fungicides Carboxin Thiabendazole a

Inhibitory concentration with 50% inhibitory effect on the fungal growth (ppm) Minimum fungicidal concentration (ppm) c Minimum inhibitory concentration (ppm) IN: Ineffective b

Table 3. Efficiency of seed treatment and/or foliar spray using EO of Mentha piperita or its main constituent, menthol, to control bean diseases caused by Rhizoctonia solani and Macrophomina phaseolina under greenhouse conditions. R. solani M. phaseolina Application Treatment type DI* SE** DI* SE** Untreated control P. oil (IC50) P. oil (0.1 IC50) P. oil (0.01 IC50) M. (IC50) M. (0.1 IC50) M. (0.01 IC50)

Seed treatment Foliar spray Seed treatment Foliar spray Seed treatment Foliar spray Seed treatment Foliar spray Seed treatment Foliar spray Seed treatment Foliar spray Seed treatment Foliar spray

88.7±7.2a 80.1±5.4a 52.5±5.9bc 40.2±7.3c 76.2±4.3a 48.7±3.7c 81.2±4.7a 67.5±4.3b 49.1±4.2c 35.1±5.4d 69.7±2.4b 53.7±2.4bc 75.7±2.6b 63.7±2.4b

40.5±5.3b 68.3±2.3a 13.1±5.7d 49.2±3.8b 0.1±0.1e 16.9±2.1d 45.1±1.9b 74.6±1.9a 21.5±4.7c 36.5±4.7c 16.2±5.6d 23.8±5.6c

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95.3±4.7a 91.1±5.4a 90.6±7.4a 7.5±3.2e 90.6±5.9a 17.5±3.2e 93.7±6.7a 28.7±5.5d 57.7±4.3c 8.7±4.3e 80.1±3.5b 20.1±3.5d 82.7±2.4b 28.7±2.4d

5.4±3.6e 72.6±9.4b 5.1±3.1e 40.3±5.7c 1.9±1.9e 22.7±10.8d 38.9±10.5c 90.8±10.5a 15.8±11.1d 78.1±11.1ab 13.9±3.7d 68.1±3.7b

Accepted Article

* DI: Disease index of stem and root rot caused by R. solani and charcoal rot caused by M. phaseolina. ** SE: Suppression efficacy (%), P. oil: peppermint oil, M : menthol. The results are means ±standard errors of four replications. Means within a column indicated by the same letter were not significantly different according to Duncan’s multiple range test at the level P

Antifungal activity of various essential oils against Rhizoctonia solani and Macrophomina phaseolina as major bean pathogens.

The main objective of this study was to investigate the effect of various essential oils (EOs) to decrease the activity of cell wall degrading enzymes...
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