Funneliformis mosseae root colonization affects Anethum graveolens essential oil composition and its efficacy against Colletotrichum nymphaeae

Industrial Crops and Products 90 (2016) 126–134

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Funneliformis mosseae root colonization affects Anethum graveolens essential oil composition and its efficacy against Colletotrichum nymphaeae Kaivan Karimi a,c , Asadollah Babai Ahari a , Weria Weisany b,c , Ilaria Pertot c , Urska Vrhovsek c , Mahdi Arzanlou a,∗ a

Department of Plant Protection, University of Tabriz, Tabriz, Iran Department of Plant Agronomy, University of Tabriz, Tabriz, Iran c Research and Innovation Centre, Fondazione Edmund Mach (FEM), San Michele all’Adige, Italy b

a r t i c l e

i n f o

Article history: Received 2 February 2016 Received in revised form 19 June 2016 Accepted 21 June 2016 Keywords: Volatile organic compounds Dill Essential oil Anthracnose Pathogen

a b s t r a c t Essential oils have been widely used against a range of plant pathogens due to their antimicrobial effects. Mycorrhization of aromatic plants can enhance the concentration of essential oil components obtained from these plants. This study evaluated the effect of essential oils obtained from dill (Anethum graveolens), inoculated with Funneliformis mosseae, against Colletotrichum nymphaeae, the causal agent of strawberry anthracnose. Dill plants were inoculated with F. mosseae under field conditions, essential oils were extracted from the seeds of inoculated and non-inoculated dill and chemical analysis of their composition was carried out. The efficacy of non-inoculated and inoculated essential oil against C. nymphaeae was evaluated through in vitro experiments. Gas chromatography-mass spectrometry analysis showed that constituents were the same in essential oils extracted from seeds of inoculated and non-inoculated dill plants, however several components increased significantly with inoculated as compared with noninoculated treatments particularly known antimicrobial compounds including limonene and carvone. Essential oil from inoculated plants inhibited mycelium growth (contact and volatile phases) and conidia germination of C. nymphaeae more efficiently than oil from non-inoculated plants. Essential oil from both inoculated and non-inoculated dill plants affected the morphology in a similar manner and deterioration in the morphology was more evident when higher concentrations (500 and 1000 mg/L) were applied. The increase in antimicrobial efficacy due to F. mosseae may have a positive impact on the further development of dill essential oils for plant protection against diseases. This is the first evidence of a positive effect of F. mosseae colonization of dill on the antimicrobial activity of its essential oil against C. nymphaeae. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Essential oils (EOs) are volatile compounds extracted by distillation, or less frequently by solvents or steam, from aroma-yielding plants. Their composition is relatively complex and they may contain up to 20–60 compounds at various concentrations, which mainly include terpenoids, plus some other non-terpene components such as phenylpropanoids (Sangwan et al., 2001; Edris, 2007; Bakkali et al., 2008). Intensive application of synthetic chemical pesticides can negatively impact human health and the environment and may lead to an increased risk of develop-

∗ Corresponding author. E-mail address: [email protected] (M. Arzanlou). http://dx.doi.org/10.1016/j.indcrop.2016.06.024 0926-6690/© 2016 Elsevier B.V. All rights reserved.

ing pathogen-resistant populations. Replacing synthetic pesticides with sustainable natural compounds, for example those deriving from plants such as EOs, is therefore a priority for plant protection. Several EOs have been shown to effectively inhibit plant pathogens, thanks to their volatile nature. In addition, EOs have a short persistence on the plant, are easily degraded in the environment and many of them have limited negative drawbacks (Isman, 2000; Batish et al., 2008). Their spectrum of activity is also commonly quite large, being effective against different microorganisms, including bacteria, yeast and filamentous fungi (Delaquis et al., 2002; López et al., 2005). Mycorrhizal fungi can increase the concentration of effective compounds in the EO composition of some aromatic plants following root colonization (Kapoor et al., 2002, 2004; Copetta et al., 2006; Karagiannidis et al., 2011). For example, the EO concentration from dill (Anethum graveolens) colonised by

K. Karimi et al. / Industrial Crops and Products 90 (2016) 126–134

Glomus macrocarpum increased by up to 90% (Kapoor et al., 2002). Similarly such increasing in EOs content of mint and oregano plants using mycorrhization by Funneliformis mosseae has been observed (Khaosaad et al., 2006; Bharti et al., 2013) which was formerly known as Glomus mosseae (Schüßler and Walker, 2010). On dill, arbuscular mycorrhiza colonization increases chlorophyll content, land equivalent ratio and essential oil (EO) yield and composition as compared with non-inoculated plants (Weisany et al., 2015). An increase in antimicrobial secondary compounds in an EO can result in greater efficacy against pathogens and/or a lower dosage to achieve the same effect, thus decreasing the cost per hectare of treatments, which is still one of the main limiting factors for the commercial use of EO-based pesticides. In several researches, EOs extracted from various drug plants have been evaluated against different pathogens, including fungal post-harvest pathogens (López et al., 2005; Tian et al., 2011; Ma et al., 2015). Among fungal pathogens, Colletotrichum spp. seriously limit cultivation and production of various crops and are responsible for anthracnose and other diseases of a broad range of plant species (Cai et al., 2009). Strawberry (Fragaria × ananassa D.) is an economical and valuable crop for the income of many farmers in several regions all over the world and can be infected by some Colletotrichum spp. (Howard et al., 1992; Tehranifar and Sarsaefi, 2002). In fact, soft and juicy tissues of strawberry is easily attacked by a number of Colletotrichum spp., necessitating strict plant protection programmes, mainly based on the use of chemical fungicides. Three species of Colletotrichum, namely Colletotrichum acutatum, Colletotrichum gloeosporioides and Colletotrichum fragariae (Freeman and Katan, 1997; Freeman et al., 2004) are among the main fruit pathogens occurring worldwide. Two species of C. acutatum and C. gloeosporioides are currently known as complex species (Freeman et al., 2001; Damm et al., 2012; Weir et al., 2012) and based on recent multi-gene phylogeny analysis, C. fragariae is now considered to be synonymous with Colletotrichum theobromicola in C. gloeosporioides sensu lato (Weir et al., 2012). Colletotrichum acutatum sensu lato is frequently the main causal agent of strawberry anthracnose and infects different parts of strawberry plant, including the leaf, stem, root, crown, flower and fruit (Howard et al., 1992; Peres et al., 2005). A high damage rate and severe outbreaks of C. acutatum on the strawberry have previously been reported and under conducive conditions for the disease, yield loss can reach up to 40% (Freeman et al., 1997; Daugovish et al., 2009). Several members of C. acutatum sensu lato encourage anthracnose on the strawberry (Damm et al., 2012); among them, C. nymphaeae (Pass.) Aa is referred to as the most widespread and aggressive species (Baroncelli et al., 2015). Protection against anthracnose is normally based on the intensive application of synthetic chemical fungicides (Debode et al., 2015) and EOs may represent an alternative biological tool against this disease. In this context, based on the fact that mycorrhization of target plants may lead to modification of the EO concentration/composition (Kapoor et al., 2002; Copetta et al., 2006; Rapparini et al., 2008; Zubek et al., 2010; Rydlová et al., 2015; Weisany et al., 2015) and the lack of studies related to the impact of such modifications on the prevention of plant pathogens, the aim of this study was to assess the impact of root colonization of dill by F. mosseae on the EO efficacy against C. nymphaeae under in vitro conditions.

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2. Materials and methods 2.1. Mycorrhizal fungus, pathogen and plant material The fungus, F. mosseae strain of BEG 119, was obtained from the culture collection of the University of Tabriz, Iran. The pathogen, C. nymphaeae strain CCh32, was isolated from infected plants in a strawberry field in Iran in 2014. In order to carry out the experimental investigations, the C. nymphaeae strain CCh32 was grown on potato dextrose agar (PDA; Oxoid LTD, UK) at 26 ◦ C for 10 days until acervuli were formed. Anethum graveolens plants inoculated and not inoculated with F. mosseae were grown in an experimental farm in the Agriculture and Natural Resources Research Centre of Kurdistan Province, Iran in 2014. The plot size was 4 × 5 m for both inoculated and non-inoculated plants 30 g of soil-based inoculum (containing 10 endomycorrhizal spores g-1 soil) along with 300 mg of chopped AM-colonized Zea mays roots was added to each plot at sowing time, just below the seeds. For dilution and suitable distribution of inoculum in the soil, it was intermingled with colonized root fragments, sand, AM hyphae, and spores (Vázquez et al., 2002). In control plot non-mycorrhizal Z. mays roots were applied. A randomized block design with three replicated plots of 4 m × 5 m (each with 8 rows) per treatment was used. Three seeds were manually sown at a distance of 10 cm on the row. After emergence, seedlings were thinned and only one plant was left in each sowing point. The plants were managed according to organic farming practices, without any application of pesticides or mineral fertilizers. No mechanical weeding was performed after sowing. Sowing took place on April 4, 2014 and the seeds were harvested 15 weeks later. Soil composition was analysed (Table 1) and meteorological data were recorded over the growing season 2014 (Supplementary Fig. S1 in the online version at DOI: 10.1016/j.indcrop.2016.06.024). 2.2. Mycorrhization rate of dill plants The root samples of A. graveolens were extracted using a cylindrical corer (10 mm). The soil was removed by soaking the roots in water and gently washing them to ensure that all the thinner roots and tips remained intact. Root samples were stained according to Vierheilig et al. (2005) with the following slight modifications. The roots were cut into small pieces (1 cm) and soaked in a solution of 10% KOH in a beaker for 60 min at 65 ◦ C in a water bath. The roots were then rinsed with tap water and acidified with lactic acid (5%) at room temperature for 12 h. Finally, the roots were stained with a solution containing lactic acid (875 mL), glycerol (63 mL), tap water (63 mL) and acid fuchsine (0.1 g) for 30 min at 70 ◦ C and then de-stained in the laboratory using lactic acid for 15 min. Ten root segments were mounted onto glass slides and observed at 100× to 400× magnification under a YS100 microscope (Nikon, Japan). The percentage of root colonization by the mycorrhizal fungus was calculated (McGonigle et al., 1990). 2.3. Essential oil extraction At the end of experiment, dill seeds were harvested in each plot and the EOs were obtained by hydro distillation in H2 O (500 mL) in a Clevenger apparatus for 2 h. They were separated using diethyl–ether as a solvent (1:1, v:v) and dried over anhydrous

Table 1 Main physical and chemical properties of the soil in the experimental field (values refer to the pooled soil samples from the plots). Texture

Organic Carbon (%)

pH (1:2.5)

Electrical Conductivity (dSm−1 ) (1:2.5)

K

P

Ca

Na

Zn

Mn

Fe

1150.1

450.2

0.476

7.054

6.97

(␮g kg−1 soil) Sandy clay loam

1.14

7.12

0.072

131

12.2

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Table 2 Chemical composition (%) of EOs extracted from the seeds of non-inoculated and inoculated dill plants grown in Iran in 2014. Compounds (Synonymous)

Non-inoculated

Inoculated

Confirmed by

␣-Pinene ␣-Phellandrene Limonene ␤-Phellandrene Dill ether (3,9-epoxy-1-p-menthene; Anethofuran) N-Dihydrocarvone (trans-Dihydrocarvone) Iso-Dihydrocarvone (cis-Dihydrocarvone) Carvone Neoiso-dihydrocarveol Iso-dihydrocarveol trans-Carveol Isopiperitenone cis-Carveol Thymol Carvacrol Elemicin Myristicin Dill apiole

0.07b ± 0.00 1.21b ± 0.04 3.66b ± 0.14 0.31b ± 0.01 0.54a ± 0.02 5.00a ± 0.07 9.03b ± 0.16 26.48b ± 0.09 0.31a ± 0.01 0.47a ± 0.02 0.11b ± 0.00 0.04b ± 0.00 0.06b ± 0.00 0.01b ± 0.00 0.04b ± 0.00 0.15b ± 0.01 0.49b ± 0.02 46.10a ± 0.27

0.14a ± 0.00 2.42a ± 0.05 5.44a ± 0.07 0.38a ± 0.00 0.50a ± 0.02 4.07a ± 0.04 10.01a ± 0.15 30.32a ± 0.08 0.22a ± 0.00 0.46a ± 0.01 0.12a ± 0.00 0.06a ± 0.00 0.08a ± 0.00 0.03a ± 0.00 0.15a ± 0.00 0.21a ± 0.00 0.67a ± 0.01 39.38b ± 0.17

STD, MS STD, MS STD, MS RI, MS RI, MS STD, MS STD, MS STD, MS STD, MS STD, MS STD, MS RI, MS STD, MS STD, MS STD, MS RI, MS STD, MS RI, MS

STD: confirmed by injection of standard and by mass spectra library; MS: tentative identification, confirmation only by mass spectra library; RI: confirmed by n-alkanes retention index by mass spectra library. Percentages of compounds are the average of three replications (extractions) ± standard deviation. For each compound different letters indicate significant difference between essential oils obtained from inoculated and non-inoculated dill (ANOVA; p < 0.05).

sodium sulphate. The organic layer was then concentrated at 35 ◦ C using a Vigreux column and the EOs stored at 4 ◦ C prior to analysis. The percentage of EO content referred to 100 g of dry weight. 2.4. Gas chromatography–mass spectrometry Gas chromatography–mass spectrometry (GC–MS) analysis was performed on the extracts originated from the plots (three replicates) of inoculated and non-inoculated dill samples by mycorrhizal fungus of F. mosseae using a Trace GC Ultra gas chromatograph coupled with a TSQ Quantum Tandem mass spectrometer, upgraded to the XLS configuration. A Dura Brite IRIS ion source with pre-filter was installed to improve the performance of the spectrometer. The system was equipped with a Triplus auto sampler (Thermo Electron Corporation, Waltham, USA). The injection volume was 1 ␮L, post injection dwell time 4s, tray temperature 7 ◦ C. GC separation was performed on a 30 m VF-WAXms capillary column with an internal diameter of 0.25 mm and a film thickness of 0.25 ␮m (Varian, Inc. USA). Temperature programming: 40 ◦ C hold for 4 min after injection, 6 ◦ C min−1 up to 250 ◦ C hold for 5 min. Injection parameters were: split injection, split ratio 100:1, inlet temperature 250 ◦ C, the carrier gas was helium 5.5, constant flow: 1.2 mL min−1 . Mass spectrometry was used in scan mode in the range of 40–400 mz−1 with a scan time of 0.200 s. The ionization mode was Electron Impact and the source temperature was kept at 250 ◦ C. The concentration of single compounds analysed in the EO was expressed as a percentage of the sum of the areas of the compounds reported in Table 2. 2.5. Contact fungicide activity bioassay After pooling the three replicates, the essential oils obtained from seeds of inoculated and non-inoculated dill by F. mosseae were dissolved separately for each concentration in 3 mL of 0.5% ethanol (99.96%) and 0.1% (v/v) tween 80 (required volume; 60 mL) to better emulsify them in the growth medium and the EOs. The pooled EOs from inoculated and non-inoculated dill were added to 57 mL melted PDA when the temperature reached 45 ◦ C to obtain the final concentrations of 0 (control), 0.05, 0.10, 0.25, 0.50 and 1.00 ␮l mL−1 correspond to 0 to 1000 mg/L of the original EOs respectively. The medium (20 mL) was then plated in Petri dishes by a sterile pipette. Petri plates (three for each EO concentration) were then inoculated by inserting a sterile needle in the conidial ooze which had formed

on the acervuli of the C. nymphaeae for 1 mm and then inserting it in the centre of the Petri plate, thus ensuring that the same quantity of conidia were inoculated. Petri plates were then incubated at 26 ◦ C for 10 days in the dark. Mycelium growth was measured after three, six and ten days. The percentage of inhibition (Soylu et al., 2006) was calculated with the following formula: Tc − Tt/Tc × 100 Where Tc is the diameter of mycelium in the untreated control plate; Tt is mycelium diameter of each treatment dilution. 2.6. Volatile fungicide activity bioassay To determine the fungicide efficacy of the volatile fraction of the EOs, PDA plates were inoculated as described above. Then sterile filter papers (6 mm diameter, Whatman no.1) were placed in the inner space of the inverted lid of the Petri plates (80 mm) and soaked with 500 ␮L of the five EO concentrations. Then the Petri plates were immediately sealed with two layers of parafilm to avoid any loss of volatile compounds (Soylu et al., 2006). The Petri plates were incubated at 26 ◦ C for 10 days in darkness, and radial mycelia growth was measured after three, six and ten days. The percentage of inhibition was calculated with the above-mentioned formula. 2.7. Conidia germination inhibition assay To evaluate the effect on germination of the pathogen’s conidia, the required amount of EO was diluted in 0.5 mL L−1 tween 80 (0.1%) solution and each vial of diluted EO was added to Falcon tubes (Sigma-Aldrich, USA) containing 4.5 mL PDA to obtain oil concentrations of 0 (control), 50, 100, 250, 500 and 1000 mg/L. A conidial suspension with 1 × 106 conidia mL−1 was prepared by counting with haemocytometer and 200 ␮L was added to each Falcon tube and incubated at 25 ◦ C for 10 h, with shaking at 120 rpm. Drops of the suspensions were placed on microscope glass slides and after fixation and staining with lacto-phenol-cotton blue, were covered by cover glass and the germinated conidia were assessed. A total of 250 conidia in five replications for each concentration were scored to assess the percentage of germination under a light microscope (Nikon eclipse 80i, Japan) and among them germinated conidia were counted to calculate the percentage of conidial germination (Chen et al., 2014).

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2.8. Morphological changes and appressorium formation in the presence of dill essential oils In this test, the slide culture technique (Arzanlou et al., 2007) with some modifications was used to study the effects of different concentrations of EOs on the deterioration of morphological structures. Briefly, Petri plates containing the glass slide and cover were sterilized, and then three PDA plugs were cut out using a sterile cork borer and placed on the glass slide. The plugs were inoculated with the conidial ooze of C. nymphaeae in four directions using a narrow sterile needle and covered by the cover slide. The glass slides were kept on a U-shaped glass rod and 10 mL of sterile distilled water plus tween 80 (0.1% v/v), containing different concentrations of essential oil at 0 (control), 50, 100, 250, 500 and 1000 mg/L was added to the bottom of each plate. The Petri plates were firmly sealed with a double layer of parafilm and incubated at 26 ◦ C for two weeks. As a control, sterile distilled water plus 0.1% tween 80 (v/v) was used instead of EOs. The cover slides were then removed and placed on lactic acid on a clean glass slide and morphological changes and appressoria formation were examined under a light microscope at 400× and 1000× magnification. 2.9. Statistical analysis Data were analysed using SAS software (SAS institute, Inc., 2003). The experiments were repeated and carried out with three replications. One representative experiment is presented. The effects of the experimental factors including inoculated and noninoculated dill essential oil and essential oil concentrations (50, 100, 250, 500 and 1000 mg/L) were analyzed by two-way ANOVA and some data (contact and volatile assay) were normalized by arcsine transformation. Then, the data were analysed as three independent treatments using one-way ANOVA, followed by the Least Significant Difference (LSD) approach to mean comparison. Differences in growth inhibition with inoculated and non-inoculated dill EOs at each concentration were analysed with the t-test. Trend lines and related R2 in the volatile and fungicide activity bioassays were calculated with Excel 2010 (Microsoft). 3. Results 3.1. Mycorrhization rate of dill plants Based on microscopic observation, 80.6% of the roots of plants artificially inoculated with F. mosseae were shown to be colonised by this mycorrhizal fungus (Fig. 1) and the level was significantly higher as compared with naturally occurring colonization of the roots of uninoculated plants (F = 273.24; df = 1; p < 0.01), where colonization was only 7.1%.

Conidia germination inhibition (%)

Fig. 1. Colonised root of Anethum graveolens by Funneliformis mosseae (A). The root of A. graveolens in the untreated control (B).

e

100 90

c

80

e

d

70 b b

60 50 40 30 20 10

a

a

a 0

a

0 NC

50

100

250

500

1000

EO concentrations (mg/L) Fig. 2. Germination inhibition of Colletotrichum nymphaeae strain CCh32 under the effect of non-inoculated () and inoculated ( ) dill essential oils in comparison to the negative control (NC). The histogram is the average inhibition at all concentrations. Different letters show a significant difference (Fisher test; ␣ = 0.05).

3.2. Gas chromatography mass spectrometry analysis As expected GC–MS analysis revealed that Dill apiole had the highest concentration in the EOs from both treatments, although its content was lower in inoculated dill EOs (Table 2). The next most abundant components were carvone, iso-dihydrocarvone, both a little higher in the mycorrihized sample, as well as limonene, whose concentration also increased in the EOs of seeds from inoculated plants. N-dihydocarvone was higher in non-inoculated samples. Other minor volatile compounds were also detected in the EO, with concentrations below 3% (Table 2). 3.3. Conidia germination assay Multifactorial ANOVA indicated a significant reduction of conidial germination of C. nymphaeae after 10 hrs of incubation of EO from inoculated dill in comparison to non-inoculated dill (F = 4.24; df = 1; p = 0.0469). However, one way ANOVA carried out for each single concentration revealed a significant effect of mycorrhization only at the concentration of 500 mg/L (F = 2272.85; df = 10; p = < 0.0001). Conidia germination was completely suppressed by applying 1000 mg/L of EOs from both inoculated and non-inoculated plants (Fig. 2). 3.4. Contact and volatile fungicide activity bioassay The EOs extracted from the seeds of both non-inoculated and inoculated dill plants inhibited C. nymphaeae mycelium growth in vitro, both in the volatile and contact bioassays (Fig. 3). The maximum inhibitory effect was noticed at the highest EO concentration

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Fig. 3. Efficacy of non-inoculated () and inoculated ( ) dill essential oils on the radial mycelium growth inhibition of the Colletotrichum nymphaeae strain CCh32 in the volatile (left) and contact fungicide activity bioassay (right) and after 3 (A) and (D), 6 (B) and (E) and 10 (C) and (F) days of incubation. Best-fit trend lines and related R2 are presented (upper and lower equations refer to the efficacy of inoculated and non-inoculated oils). (*) Indicates a significant difference at the specific EO concentration between non-inoculated and inoculated EOs (t-test; p < 0.01).

tested (1000 mg/L), although the maximum growth inhibition percentage of the volatile fraction (41.875 ± 1.78%) was significantly (F = 757.57; df = 1; p < 0.01) lower compared with the contact effect (91.667 ± 0.263%). The differences between the volatile fraction of the EOs from non-inoculated and inoculated plants in term of growth inhibition become significant only after six days of exposure and at the highest concentration (Fig. 4B and C). However, after 10 days the significant difference between EOs from non-inoculated and inoculated plants in terms of the volatile inhibitory effect was noticed at all the concentrations tested (F = 55.51; df = 1; p < 0.0001) (Fig. 3C). In the contact bioassay the differences between EOs from non-inoculated and inoculated plants were already present after three days of incubation and at the lowest concentrations (from 50 to 500 mg/L) and

tended to disappear at the highest concentration (1000 mg/L), but also at 500 mg/L after 10 days of incubation (Fig. 4D–F). 3.5. Essential oils effect on morphological characters The vegetative growth responses of C. nymphaeae to different concentrations of EO from both non-inoculated and inoculated dill plants using the slide culture method were completely different in comparison to the untreated control (Fig. 4I). The morphological changes to the vegetative organs of the pathogen in both non-inoculated and inoculated dill EOs were the same. Appressorium formation was not observed at concentrations of 250, 500 and 1000 mg/L in either non-inoculated or inoculated dill EOs (Fig. 4C–E). At low concentrations the morphological changes were

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Fig. 4. Morphological responses of Colletotrichum nymphaeae to the effects of inoculated and non-inoculated dill essential oil using the slide culture method. (A) and (B) Mycelia swelling at concentrations of 250 (inoculated) and 100 mg/L (non-inoculated) respectively. (C) Conidia fused to vegetative mycelia and each other, and twisted mycelium at concentrations of 250 mg/L (inoculated). (D) and (E) Thickened mycelia and vesicle bodies at a concentration of 500 mg/L (non-inoculated and inoculated respectively). (F) Conidia in control treatment (Upper image) and plasmoplized conidia (beneath image) in 1000 mg/L (inoculated). (G) and (H) Collapsing and cytoplasmic content leakage (non-inoculated and inoculated respectively). (I) Mycelia shape and appressorium formation in the control treatment. Scale bars represent 10 ␮m except scale bar of (D) image which represents 100 ␮m.

mostly limited to the creation of mycelial swelling, a lot of twisted mycelia and conidia fused to each other and vegetative hyphae (Fig. 4A–C); while at high concentrations (500 mg/L) in addition to the formation of mycelial swelling, vesicle-like body formations, a reduction in hyphal length, mycelial thickening and cytoplasmic

content leakage were visible (Fig. 4D, E, G and H). Vesicle-like bodies were also abundantly observed along the fungal hyphae, when the concentration of EO was increased (Fig. 4E). Plasmolized conidia were evident at a 1000 mg/L concentration of non-inoculated and inoculated dill EOs (Fig. 4F).

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4. Discussion The high rate of root colonization of dill plants confirmed the compatibility between the dill plants and the strain of F. mosseae used in the present study. Indeed, the tendency of plants to be colonised by mycorrhizal fungi depends on the plant species and the strain of mycorrhizal fungi (Rydlová et al., 2015). Analysis performed with gas chromatography mass spectrometry revealed that the EO constituents extracted from both non-inoculated and inoculated plant seeds were the same. This result was in agreement with several previous studies, where dill plants were inoculated with Rhizophagus irregularis (Rydlová et al., 2015) and Glomus macrocarpum (Kapoor et al., 2002) and confirms the results of Weisany et al. (2015). Generally, it appears that at least for dill, the mycorrhization mechanism acts as an enhancer or reducer of compounds and does not have any effect in terms of changing the number or type of compounds. For instance, the concentration of some components, such as carvone, iso-dihydrocarvone and limonene, which are known to have antimicrobial, acaricidal and insecticidal properties, and along with dill apiole, was higher in inoculated EO in agreement with previous studies (Table 2) (Aggarwal et al., 2002; Ibrahim et al., 2001; Nazzaro et al., 2013; Tabassum and Vidyasagar, 2013; Weisany et al., 2015). Carvone and limonene have been frequently seen individually or in combination with other inhibitor substances as a synergistic composition against different fungal pathogens. For instance their antifungal activity against various species of pathogenic fungal genera, such as Fusarium, Aspergillus, Alternaria, Penicillium, Colletotrichum and Lasiodiplodia, have been documented (du Plooy et al., 2009; Combrinck et al., 2011; Morcia et al., 2012). Carvone is also used as potato sprouting inhibitor (under the commercial name Talent) (de Carvalho and da Fonseca, 2006). Commercial products based on the patented use of limonene and dihydrocarvone have also been developed against various pathogenic fungi and yeasts (Whitehurst and Titusville, 1973; Chastain et al., 1994). The discrepancy in the percentage composition of the obtained compounds in various studies is related to the species of inoculated plant and mycorrhizal fungus. For instance, the mycorrhizal fungus Gigaspora rosea increased the EO concentration of Ocimum basilicum var. Genovese, but in contrast Gi. margarita decreased the EO concentration (Copetta et al., 2006). Similarly, the amounts of chemical compounds in the EOs of dill inoculated with F. mosseae in this study were different from those inoculated with R. irregularis (Rydlová et al., 2015). Several other factors, such as geographical origin, harvesting time, growth conditions and the extraction procedure may affect the EOs major component profiles (Pino et al., 1995; Ghassemi-Golezani et al., 2008; Vokk et al., 2011); therefore extensive studies are needed to precisely quantify the expected change in the concentration of the different components following mycorrhization of dill plants under different environmental conditions. Based on results it can be postulated that the application of inoculated dill seed EO in particular, in high concentrations might lead to a deficiency in spore germination and subsequently result in efficient disease control. Obviously spore germination plays a pivotal role in the establishment of the pathogen on the host surface and disease development. Suppression of conidia germination has previously been demonstrated when high concentrations of EOs are applied to combat different species of the Colletotrichum genus, including Colletotrichum musae, C. gloeosporioides and C. acutatum (Maqbool et al., 2011; Duduk et al., 2015). In both the volatile and contact bioassays we observed an increase in mycelium growth inhibition when increasing the EO dosage (dose effect); this increase followed a linear trend line in the volatile assay, while the trend line was logarithmic in the contact bioassay (Fig. 3). In particular, in the contact bioassay with 250 mg/L

and higher concentrations of EO, the growth inhibition reached a plateau. Totally, the volatile fraction was significantly lower compared with the contact effect in terms of the maximum growth inhibition percentage (Fig. 3). This reaction could be explained by a lower concentration of the effective components in the volatile fraction compared with that present in the media of the contact bioassay. Another possible explanation is the faster accumulation of inhibiting components in the mycelium over the incubation time, which could be more efficient in the contact assay, where there is direct contact between EOs and fungal mycelium. When the pathogen grown at higher concentrations was transferred on a medium without EOs normal regrowth was observed, therefore the EO mechanism of action was postulated to be fungistatic instead of fungicidal. This is in agreement with the evaluation of three EOs containing thyme, cinnamon bark and clove bud against C. acutatum sensu lato, where the effect of the EOs was found to be fungistatic (Duduk et al., 2015). Similar results have been observed in inhibitory assay of dill EO on four food pathogens including Aspergillus niger, A. flavus, A. oryzae and Alternaria alternata (Tian et al., 2011). It appears that based on morphological observations the main mechanism of dill EOs in this study against C. nymphaeae was vegetative body malformation at low concentrations and the collapsing of vegetative mycelia and prevention of appressorium formation at high concentrations. Ergosterol synthesis inhibition, an increase in malate dehydrogenase, succinate dehydrogenase activities and external medium acidification was determined to be an effect of the dill seed EO mechanism against Sclerotinia sclerotiorum (Ma et al., 2015). In some studies, reactive oxygen species accumulation has been reported as an important mediator of the antifungal action of EOs (Tian et al., 2012; Chen et al., 2013). The totally hydrophobic nature of EOs enables them to permeate into the fungal cell wall, which is followed by leakage of intracellular content and enzymatic activity disorders (Sikkema et al., 1995; Lambert et al., 2001). It appears that swollen mycelia and the zigzag shape formation on vegetative organs are associated with the effect of EOs on ergosterol content, because it plays a significant role in maintaining cell function and integrity (Rodriguez et al., 1985; Tian et al., 2012). Similar morphological malformations have previously been exhibited by different EOs in some literatures (Soylu et al., 2007; Souza et al., 2010), supporting our observations. A mixed fraction of cilantro, coriander and dill EOs has shown a synergistic effect on dill essential oil, which led to rise in dlimonene in the mixed EOs and a positive increase in antimicrobial activity, although the reactions of pathogenic microorganism have been variable under the effect of the same mixed EOs and even pure dill EO (Delaquis et al., 2002). In a recent study on the effects of dill EO against S. sclerotium on rapeseed (Ma et al., 2015), it was postulated that antifungal activity of the carvone compound was stronger than limonene and this performance was attributed to the carbonyl group on the p-menthane skeleton, based on its structures. In our study, the carvone content in inoculated treatments was approximately six times higher than limonene and apparently had a greater role in suppressing C. nymphaeae growth. Generally it seems that the interaction between different compounds in an EO is decisive in terms of its efficacy.

5. Conclusion To the best of our knowledge, this study is the first report assessing the effects the interaction between the AM fungus and an aromatic plant in term of increased antifungal effects of the related EOs against a plant pathogen and proving that mycorrhization can be used to improve the fungicide efficacy of EOs. Indeed, mycorrhization of dill plants not only led to a rise in the

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concentration of some EO components, but also increased its inhibitory efficacy against C. nymphaeae. Although the use of dill EO would be still very expensive for open field application, the proof of efficacy of its volatile fraction against C. nymphaeae can be the basis for possible future practical applications, for example to prevent post-harvest decay of stored fruits. Because of the observed increase in term of antifungal activity of the EO extracted form inoculated dill plants compared with non-inoculated ones were limited only to certain concentrations, further studies in term of impact of each specific compound of the EOs on the whole fungitoxic effect and the effect of mycorrhization on each of them are needed. In addition, once optimised, mycorrhization may offer an advantage in terms of decreasing of the effective dosages of EOs in fungicide treatments and possibly reducing the cost of treatments. In addition extensive studies of pre and/or post-harvest treatments of strawberry fruits under commercial-like conditions of production are needed to prove the real advantaged of the mycorrhization of A. graveolens in terms of increase of efficacy. Conflict of interest The authors declare no conflict of interest. Acknowledgments The authors would like to thank the Research Deputy of the University of Tabriz, Iran for financial support and the Agriculture and Natural Resources Research Center of the Kurdistan province for their contribution. Cesare Lotti is acknowledged for GC–MS analysis. References Aggarwal, K.K., Khanuja, S.P.S., Ahmad, A., Santha Kumar, T.R., Gupta, V.K., Kumar, S., 2002. Antimicrobial activity profiles of the two enantiomers of limonene and carvone isolated from the oils of Mentha spicata and Anethum sowa. Flavour Fragr. J. 17, 59–63. Arzanlou, M., Groenewald, J.Z., Gams, W., Braun, U., Shin, H.-D., Crous, P.W., 2007. Phylogenetic and morphotaxonomic revision of Ramichloridium and allied genera. Stud. Mycol. 58, 57–93. Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M., 2008. Biological effects of essential oils. Rev. Food Chem. Toxicol. 46, 446–475. Baroncelli, R., Zapparata, A., Sarrocco, S., Sukno, S.A., Lane, C.R., Thon, M.R., Vannacci, G., Holub, E., Sreenivasaprasad, S., 2015. Molecular diversity of anthracnose pathogen populations associated with UK strawberry production suggests multiple introductions of three different Colletotrichum species. PLoS One 10 (6), e0129140. Batish, D.R., Singh, H.P., Kohli, R.K., Kaur, S., 2008. Eucalyptus essential oil as a natural pesticide. For. Ecol. Manage. 12, 2166–2174. Bharti, N., Baghel, S., Barnawal, D., Yadav, A., Kalra, A., 2013. The greater effectiveness of Glomus mosseae and Glomus intraradices in improving productivity, oil content and tolerance of salt-stressed menthol mint (Mentha arvensis). J. Sci. Food Agric. 93, 2154–2161. de Carvalho, C.C.C.R., da Fonseca, M.M.R., 2006. Carvone: why and how should one bother to produce this terpene. Food Chem. 95, 413–422. Cai, L., Hyde, K.D., Taylor, P.W.J., Weir, B.S., Waller, J., Abang, M.M., Zhang, J.Z., Yang, Y.L., Phoulivong, S., Liu, Z.Y., Prihastuti, H., Shivas, R.G., McKenzie, E.H.C., Johnston, P.R., 2009. A polyphasic approach for studying Colletotrichum. Fungal. Divers. 39, 183–204. Chastain, D.E., Sanders, W.E., Christine, J.R., Sanders, C., 1994. Method of killing yeast or fungi with dihydrocarvone. US Patent US5308871 A. Chen, Y., Zeng, H., Tian, J., Ban, X., Ma, B., Wang, Y., 2013. Antifungal mechanism of essential oil from Anethum graveolens seeds against Candida albicans. J. Med. Microbiol. 62, 1175–1183. Chen, Q., Xu, S., Wu, T., Guo, J., Sha, S., Zheng, X., Yu, T., 2014. Effect of citronella essential oil on the inhibition of postharvest Alternaria alternata in cherry tomato. J. Sci. Food Agric. 94, 2441–2447. Combrinck, S., Regnier, T., Kamatou, G.P.P., 2011. In vitro activity of eighteen essential oils and some major components against common postharvest fungal pathogens of fruit. Ind. Crop. Prod. 33, 344–349. Copetta, A., Lingua, G., Berta, G., 2006. Effects of three AM fungi on growth distribution of glandular hairs, and essential oil production in Ocimum basilicum L. var. Genovese. Mycorrhiza 16, 485–494. Damm, U., Cannon, P.F., Woudenberg, J.H.C., Crous, P.W., 2012. The Colletotrichum acutatum species complex. Stud. Mycol. 73, 37–114.

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