In vitro and in vivo antimicrobial efficacy of essential oils and individual compounds against Phytophthora parasitica var. nicotianae

Authors


Correspondence

Lei Yao, Aromatic Plant R & D Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China. E-mail: lumin111@sjtu.edu.cn; yaolei@sjtu.edu.cn

Abstract

Aim

To evaluate the antimicrobial effects of essential oils (EOs) from cassia, basil, geranium, lemongrass, cumin and thyme, as well as their major components, against Phytophthora parasitica var. nicotianae; to investigate morphological changes in hyphae and sporangia in response to treatment with cinnamaldehyde; and to further evaluate potential biocontrol capacities against tobacco black shank under greenhouse conditions.

Methods and Results

The results revealed that the extent of mycelial growth inhibition was primarily dependent on the composition and concentration of the EOs and the structure of individual compounds. Cinnamaldehyde had a significantly higher inhibitory effect on mycelial growth, formation of sporangia, and production and germination of zoospores in P. parasitica var. nicotianae in vitro, achieving complete inhibition of these phenotypes at 72, 36, 36 and 18 mg l−1, respectively. Scanning electron microscopic observations revealed that cinnamaldehyde can cause considerable morphological degenerations of hyphae and sporangia such as cytoplasmic coagulation, shrivelled mycelia and sporangia aggregates and swelling and lysis of mycelia and sporangia walls. In vivo assays with cinnamaldehyde demonstrated that this compound afforded protective effect against tobacco black shank under greenhouse conditions in susceptible tobacco plants.

Conclusions

The results of in vitro and in vivo bioassays, together with SEM imaging of the microstructure of P. parasitica var. nicotianae supported the possibility of using cinnamaldehyde as a potent natural biofungicide in the greenhouse.

Significance and Impact of the Study

This study provides a theoretical basis for the potential use of cinnamaldehyde as commercial agents or lead compounds that can be exploited as commercial biofungicides in the protection of tobacco plants from P. parasitica var. nicotianae infection.

Introduction

Tobacco black shank, a disease caused by Phytophthora parasitica (Dast.) var. nicotianae (Breda de Haan) Tucker, is one of the most important diseases affecting tobacco (Nicotiana tabacum L.) production worldwide, especially in China (Erwin and Ribeiro 1996; Ma and Gao 2007). This pathogen can cause root, stem and crown rot, as well as fruit and foliar blights from splash dispersal of propagules from the soil, on many agronomic and horticultural plants in seed beds, nurseries, fields and landscape plantings (Bowers and Locke 2004). Symptoms of the disease develop rapidly in many hosts during periods of high soil moisture associated with prolonged, rainy weather or frequent irrigation. According to incomplete statistics, the plant disease may account for more than 10% of pre- and postharvest losses in tobacco crops in China (Su et al. 2011). Current control of plant diseases is primarily dependent on continued application of synthetic fungicides (Matheron and Porchas 1999). Although these synthetic fungicides are effective, their continued or repeated application has disrupted biological control by natural enemies and led to outbreaks of diseases, widespread development of resistance to various types of fungicides (Stack and Millar 1985), toxicity to nontarget organisms and environmental problems.

Because of these concerns, investigators have begun to develop more acceptable compounds that are more effective and safe for humans and the environment. The antimicrobial properties of plant products have been recognized and used to develop potentially efficacious products as commercial fungicides or lead compounds (Hedin et al. 1997). Among the various plant products being investigated, essential oils (EOs) are especially promising as safe natural products with potential antimicrobial applications. Many EOs and individual components of EOs have been classified as ‘generally regarded as safe’ (GRAS) by the United States Food and Drug Administration (FDA), making them potential natural fungicides due to their safety in eukaryotic systems (Burt 2004; Tolouee et al. 2010). In recent years, numerous studies have demonstrated the antimicrobial efficacy of EOs in controlling some plant pathogens in vitro and in vivo (Soylu et al. 2010; Tian et al. 2011a,b).

In addition, regarding research on the antimicrobial activity of EOs, most studies have investigated the effect of EOs as a whole. However, the individual compounds that make up EOs are structurally different and have varying levels of oxidative stability, which may affect the overall antimicrobial activity of these EOs (Lopes-Lutz et al. 2008; Liu and Yang 2012). Different studies have demonstrated the ability of active compounds to control or inhibit the growth of pathogenic micro-organisms, and these abilities have been shown to depend on the chemical structure and concentration of the active compound, as well as the number and type of micro-organism (Lee et al. 2008; Liu and Yang 2012). Therefore, a detailed investigation on the antimicrobial parameters of individual compounds will provide quantitative information concerning the relative importance of individual compounds to the overall antimicrobial activity of EOs. However, to our knowledge, the in vitro and in vivo efficacies of EOs and their major constituents against tobacco black shank caused by P. parasitica var. nicotianae infection have not been well studied, and only the inhibitory efficacies of formulated plant extracts and EOs on P. nicotianae in soil and periwinkle were described (Bowers and Locke 2004).

In the current study, we evaluated the antimicrobial parameters of EOs from cassia, basil, geranium, lemongrass, cumin and thyme, as well as their major components on mycelial growth, formation of sporangia, and production and germination of zoospores from P. parasitica var. nicotianae in vitro; used scanning electron microscopy (SEM) to investigate the morphological changes in hyphae and sporangia in response to treatment with the most effective compound, cinnamaldehyde; and further evaluated this compound for protective and curative effects against tobacco black shank under greenhouse conditions.

Materials and methods

Plant EOs and individual compounds

Cassia (Cinnamomum cassia Presl), basil (Ocimum basilicum L.), geranium (Pelargonium graveolens), lemongrass (Cymbopogon citratus), cumin (Cuminum cyminum) and thyme (Thymus vulgaris) were harvested from Shanghai, China. Based on the morphological features, these plant materials were indentified and then confirmed by Prof. Lei Yao at the School of Agriculture and Biology, Shanghai Jiao Tong University. The EOs were obtained by steam distillation for 3-6 h using a Clevenger-type apparatus (Ildam, Ankara) according to the procedures described in the European Pharmacopoeia. Analyses of these EOs were carried out by gas chromatography (GC) and gas chromatography/mass spectroscopy (GC-MS).

GC was performed with a fused-silica capillary column (HP-5MS: 30 m × 0·25 mm i.d., film thickness: 0·25 μm). The operating conditions were as follows: initial oven temperature of 60°C for 10 min, then raised to 220°C by increments of 4°C min−1 and held at 220°C for 10 min, then raised to 240°C by increments of 1°C min−1; injector and detector temperatures, 250 and 270°C, respectively; carrier gas, N2 (0·8 ml min−1); split ratio, 50 : 1; manual sample injection, 0·2 μl.

GC-MS was performed with a same fused-silica capillary column (HP-5MS: 30 m × 0·25 mm i.d., film thickness: 0·25 μm). Helium was used as the carrier gas. The mass spectrometer operating conditions were as follows: ionization voltage, 70 eV; ion source, 250°C. A mass range from 35 to 500 u was scanned at 1 scans s−1. The GC analysis conditions were as described above.

The major components of the above EOs are listed in Table 1 (Lu et al. 2013). Of these, β-pinene (≥98%), cuminaldehyde (≥99%), thymol (≥99%), citral-a (≥95%), p-cymene (≥99%), cinnamaldehyde (≥95%), linalool (≥98%) and geraniol (≥96%) were purchased from Sinopharm Chemical Reagent Company, LTD (Shanghai, China). Citronellol (≥98%), nerol (≥98%), 4-allylanisole (≥97%) and γ-terpinene (≥95%) were acquired from Tokyo Kasei (Tokyo, Japan).

Table 1. Main components of the investigated cassia (bark), geranium (twigs and leaves), cumin (seeds), thyme (twigs and leaves), basil (leaves) and lemongrass (leaves) essential oils (Lu et al. 2013)
ComponentaPeak area (%)
CassiaGeraniumCuminThymeBasilLemongrass
  1. The compounds were identified by comparison of retention indices and mass spectra with those of the NIST library.

  2. a

    The components with >10% peak area were shown.

β-Pinene14·07
p-Cymene16·8837·66
γ-Terpinene11·30
Linalool45·95
Geraniol20·78
4-Allylanisole35·06
Citronellol41·5514·19
Cuminaldehyde46·91
Nerol13·83
Citral-a26·00
Cinnamaldehyde80·40
Thymol39·77

Micro-organisms and cultural methods

The highly pathogenic P. parasitica var. nicotianae was obtained from a tobacco seeding greenhouse by harvesting the fungus from diseased stems and plating it on oatmeal agar (OA). Stock cultures obtained from a single spore were maintained on OA, kept at 16°C and subcultured once a month. P. parasitica var. nicotianae was grown for 10–12 days on OA at 26°C before use.

Screening for antimicrobial activities of EOs and individual compounds in vitro

The antimicrobial activities of EOs and individual compounds were assessed to determine their inhibitory effects towards mycelial growth of P. parasitica var. nicotianae as described previously (Dimitra et al. 2003; Soylu et al. 2010). EOs and individual compounds were dispersed as emulsions in water using Tween 80 (0·5% v/v) and added to OA immediately before plating on Petri dishes (90 × 20 mm in diameter) at a temperature of 45–50°C. Screening for antimicrobial activity was performed with different concentrations of EOs and individual compounds (9–864 mg l−1). The controls received the same amount of Tween 80 mixed with OA. Phytophthora parasitica var. nicotianae was inoculated immediately by plating on the Petri dishes with a 5-mm-diameter disc of the fungus. Three Petri plates were used per treatment. The Petri dishes were sealed immediately with Parafilm to prevent the loss of EOs and individual compounds and were incubated in the dark at 26°C. After 3 days, the mean radial mycelial growth of the pathogen was determined by measuring the diameter of the colony in two directions at right angles. Each experiment was repeated five times.

Mean growth values were obtained and then converted into the per cent inhibition of mycelial growth relative to the control treatment using the following formula: Inhibition rate (%) = [(Mc − Mt)/Mc] × 100, where Mc and Mt represent mycelial growth diameter in the control and treated Petri plates, respectively. The effective concentration causing a 50% reduction in the linear growth of the fungi on OA (ED50 value) was calculated by Probit analysis using the values of dose–response experiments.

Effects of EOs and individual compounds on sporangia formation in vitro

The inhibitory effects of EOs and individual compounds on sporangia formation were assessed as described previously, with some modifications (Matheron and Porchas 1999). Three agar discs (5 mm in diameter, aged 7–10 days) of P. parasitica var. nicotianae were removed from the edge of an actively growing OA culture of each isolate and placed in a series of plastic Petri dishes (60 × 20 mm in diameter) containing 7 ml of 0·1% KNO3 containing one of the test EOs or individual compounds to give a final concentration of 9–216 mg l−1. In addition, control plates containing 0·1% KNO3 solution, without the addition of EOs or individual compounds, were prepared. After a 3-day incubation period at 26°C in the dark, the liquid was decanted from each Petri dish, and the agar discs were stained and fixed with acid fuchsin in 85% lactic acid. The number of sporangia along the margins of each agar disc was counted under a light microscope (Nikon Eclipse 50i, Tokyo, Japan). Each experiment was repeated five times.

The mean number of sporangia were obtained, and these numbers were then converted into inhibition rates relative to the control treatment using the following formula: Inhibition rate (%) = [(Sc − St)/Sc] × 100, where Sc and St represent the mean number of sporangia in the control and treated Petri plates, respectively.

Effects of EOs and individual compounds on zoospore production and germination in vitro

The effects of EOs and individual compounds on the production and germination of zoospores were evaluated following the method of Matheron and Porchas (1999). To examine the inhibitory effects of EOs and individual compounds on zoospore production, sporangia were produced as described above by placing 8 OA discs (5 mm in diameter) of P. parasitica var. nicotianae into a series of plastic Petri dishes (60 mm in diameter) containing 7 ml of 0·1% KNO3 solution mixed with one of the test EOs or individual compounds to give a final concentration of 9–216 mg l−1. The controls contained 0·1% KNO3 solution without EOs or individual compounds. After 3 days of incubation at 26°C, sporangia were induced to release zoospores by chilling at 12°C for 30 min and then rewarming at 26°C for 20 min. Agar discs and attached mycelia were subsequently removed from each Petri dish, and the number of zoospores remaining in the suspension was determined using a blood count board under a light microscope (Nikon Eclipse 50i). Each experiment was repeated five times.

To examine the inhibitory effects of EOs and individual compounds on zoospore germination, zoospore suspensions (106 CFU ml−1) of P. parasitica var. nicotianae were prepared as described above. Afterwards, aqueous mixtures of each concentration of EOs or individual compounds (9–216 mg l−1) were added to equal volumes of zoospore suspensions, give final concentrations of 9–216 mg l−1. Control zoospore suspensions received equal volumes of water only. Following a 12-h incubation period at 26°C in darkness, the number of germinating zoospores was estimated under a light microscope (Nikon Eclipse 50i), with five replicates for each treatment and counting a minimum of 100 zoospores in each replicate.

The mean numbers of zoospores produced and germinating were obtained, and these numbers were then converted into inhibition rates relative to the control treatment using the following formula: Inhibition rate (%) = [(Zc − Zt)/Zc] × 100 where Zc and Zt represent the mean number of zoospores produced and germinating in control and treated Petri plates, respectively.

SEM imaging analysis of the effects of cinnamaldehyde on the morphology of Phytophthora parasitica var. nicotianae

For the analysis of morphological changes in the mycelia, thin layers (1 mm) of agar discs (5 mm in diameter) containing mycelium were placed in the centre of OA plates containing 72 mg l−1 cinnamaldehyde; the controls received the same quantity of Tween 80 mixed with OA. For the analysis of morphological changes in the sporangia, agar discs (5 mm in diameter) were placed in plastic Petri dishes containing 7 ml of 0·1% KNO3 containing 36 mg l−1 cinnamaldehyde; control samples received only 7 ml of 0·1% KNO3. These discs were incubated in darkness at 26°C for 3 days. Subsequently, treated agar discs were fixed with 2·5% glutaraldehyde in 0·1 mol l−1 phosphate buffer (pH 7·2) for 6 h at room temperature and were then washed twice, for 10 min each time, in the same buffer. Afterwards, the discs were dehydrated in a graded series of ethanol (50, 60, 70, 80, 90 and 100%) for a period of 20 min in each alcohol dilution. The last step was performed three times for 30 min each time. After dehydration, the samples were dried at critical point in liquid carbon dioxide in a drying apparatus (Leica EM-CPD 030; Leica, Wetzlar, Germany). The fixed specimens were then mounted on stubs using double-sided carbon tape and coated with gold–palladium using a sputter coater system with a high vacuum chamber (Hitachi-1045 Ion Sputter, Japan) for 3–5 min. Finally, the samples were scanned and photographed using a Nova NanoSEM 230 microscope at an accelerating voltage of 5 kV.

Protective and curative effects of cinnamaldehyde on disease development in vivo

To assess the potential protective and curative effects of cinnamaldehyde against tobacco black shank caused by P. parasitica var. nicotianae in vivo, N. tabacum cv. K326 plants were selected and treated with different concentrations of cinnamaldehyde following the method described by Keller et al. (1999).

Two-month-old tobacco plants were cultivated in an insect-free greenhouse. Different concentrations of cinnamaldehyde (20, 40, 80, 160 and 320 mg l−1) were prepared by dissolving the requisite amounts in sterile Tween 80 (0·5% v/v) solutions. To determine the protective effects of cinnamaldehyde, the stem epidermis of the tobacco plant was scratched with a blade and covered with a cotton ball soaked in 10 ml of cinnamaldehyde at varying concentrations. After 24 h, the wounded regions were inoculated with 5-mm OA discs of P. parasitica var. nicotianae. To determine the curative effects of cinnamaldehyde, the wounded regions of tobacco plants stems were pre-inoculated with 5-mm P. parasitica var. nicotianae discs for 24 h and then covered with cotton balls soaked in different concentrations of cinnamaldehyde. Control plants were pretreated uniformly with 10 ml aqueous solution of sterile Tween 80 (0·5% v/v) or the same mycelium disc used in negative or positive control groups, respectively. For comparison, metalaxyl, a commercial fungicide used to treat tobacco black shank, was also evaluated in both protective and curative assays at the recommended concentration (100 mg l−1). Twenty plants were used per treatment; each experiment was repeated five times.

Control and cinnamaldehyde-treated plants were assessed 6 days after treatment. The disease index of tobacco black shank, based on the stem girth, was rated on a scale of 0–9 (0 = no disease symptom; 1 = ≤1/3; 3 = 1/3–1/2; 5 = 1/2–2/3; 7 = ≥2/3; and 9 = rotted away) as the percentage of diseased stem girth (Ma et al. 2010). The efficacy of cinnamaldehyde was calculated according to the following formula: Disease control (%) = (Dc − Dt)/Dc × 100 where Dc and Dt represent the mean disease index in the positive control and the relevant treatment, respectively.

Statistical analyses

All measurements were taken three times for each treatment, and the data are reported as means ± standard deviations. Significant differences between mean values were determined by Duncan's multiple range test (P < 0·05), following analysis with one-way anova. All statistical analyses were performed using statistical software (SPSS 18.0, Chicago, IL, USA).

Results

Screening for antimicrobial activities of EOs and individual compounds against mycelial growth

The results of our screening for inhibitory activities of EOs and individual compounds against the mycelial growth of P. parasitica var. nicotianae are shown in Fig. 1. Of these, EOs of cassia, cumin, thyme, lemongrass and cuminaldehyde, citral-a, cinnamaldehyde and thymol were found to be the most effective at inhibiting mycelia growth, and the ED50 value were 36·8, 72·6, 70·0, 67·7, 31·7, 35·4, 35·4 and 59·3 mg l−1, respectively (Fig. 1). However, Other EOs and individual compounds exhibited a moderate action against mycelial growth of P. parasitica var. nicotianae.

Figure 1.

The screening on antimicrobial activity of essential oils (EOs) and individual compounds against Phytophthora parasitica var. nicotianae in vitro. The ED50 was calculated by Probit analysis using the values of dose–response experiments. The mean of ED50 value was a concentration of the EOs and individual compounds causing a 50% reduction in the linear growth of P. parasitica var. nicotianae on oatmeal agar.

Effect of EOs and individual compounds on mycelial growth

We examined the dose-dependent inhibitory effects of cassia, cumin, thyme and lemongrass EOs, as well as cuminaldehyde, citral-a, cinnamaldehyde and thymol on the mycelial growth of P. parasitica var. nicotianae (Table 2). All of these EOs and individual compounds exhibited dose-dependent inhibitory effects on mycelia growth. Complete inhibition of mycelial growth was observed for cuminaldehyde, cinnamaldehyde and cassia EO at 72 mg l−1, thymol at 144 mg l−1, and citral-a, cumin EO, thyme EO and lemongrass EO at 216 mg l−1 (Table 2).

Table 2. The effects of different concentrations of essential oils and individual compounds on mycelial growth, sporangia formation, zoospore production and zoospore germination
Inhibition rate (%)Concentration (mg l−1)EOsIndividual compounds
CuminCassiaThymeLemongrassCuminaldehydeCinnamaldehydeThymolCitral-a
  1. Values were expressed as mean (n = 5) ± standard deviations. Mean values followed by different large or small letters within the each column and line indicate significant differences between various EOs and individual compound according to Duncan's multiple range test (P ≤ 0·05).

  2. a

    Results were expressed as inhibition rate percentage of mycelial growth relative to control.

  3. b

    Results were expressed as inhibition rate percentage of sporangium formation relative to control.

  4. c

    Results were expressed as inhibition rate percentage of zoospore production relative to control.

  5. d

    Results were expressed as inhibition rate percentage of zoospore germination relative to control.

Mycelial growtha216100·0 ± 0·0F100·0 ± 0·0D100·0 ± 0·0F100·0 ± 0·0F100·0 ± 0·0D100·0 ± 0·0D100·0 ± 0·0E100·0 ± 0·0F
14489·3 ± 1·6aE100·0 ± 0·0dD95·5 ± 1·6cE95·2 ± 1·3cE100·0 ± 0·0Dd100·0 ± 0·0dD100·0 ± 0·0dE93·5 ± 1·6bE
7261·9 ± 1·5aD100·0 ± 0·0fD70·4 ± 3·3cD67·0 ± 3·2bD100·0 ± 0·0fD100·0 ± 0·0fD74·8 ± 2·5dD83·6 ± 2·6eD
3650·2 ± 2·8bC56·0 ± 4·9cC47·9 ± 3·5bC50·2 ± 2·1bC88·2 ± 2·5eC62·6 ± 2·6dC44·1 ± 1·9aC43·7 ± 3·3aC
1823·9 ± 5·2abcB25·4 ± 2·2bcB19·7 ± 3·5abB26·0 ± 6·5cB24·7 ± 4·4bcB27·7 ± 2·9cB18·8 ± 4·7aB29·6 ± 5·0cB
90·0 ± 0·0A0·0 ± 0·0A0·0 ± 0·0A0·0 ± 0·0A0·0 ± 0·0A0·0 ± 0·0A0·0 ± 0·0A0·0 ± 0·0A
Sporangium formationb216100·0 ± 0·0bE100·0 ± 0·0bC100·0 ± 0·0bC100·0 ± 0·0bD0·0 ± 0·0a100·0 ± 0·0bC100·0 ± 0·0b100·0 ± 0·0bD
14481·3 ± 5·0bD100·0 ± 0·0cC100·0 ± 0·0cC100·0 ± 0·0cD0·0 ± 0·0a100·0 ± 0·0cC100·0 ± 0·0c93·7 ± 7·3cC
7260·8 ± 5·3dC95·0 ± 8·2eC100·0 ± 0·0eC51·8 ± 6·7cC0·0 ± 0·0a100·0 ± 0·0eC100·0 ± 0·0e27·7 ± 4·7bB
3625·3 ± 4·6bB68·0 ± 7·0 dB59·9 ± 7·1cB22·2 ± 4·7bB0·0 ± 0·0a100·0 ± 0·0eC100·0 ± 0·0e0·0 ± 0·0aA
180·0 ± 0·0aA0·0 ± 0·0aA0·0 ± 0·0aA0·0 ± 0·0aA0·0 ± 0·0a24·7 ± 5·6bB0·0 ± 0·0a0·0 ± 0·0aA
90·0 ± 0·0A0·0 ± 0·0A0·0 ± 0·0A0·0 ± 0·0A0·0 ± 0·00·0 ± 0·0A0·0 ± 0·00·0 ± 0·0A
Zoospore productionc216100·0 ± 0·0bD100·0 ± 0·0bD100·0 ± 0·0bC100·0 ± 0·0bC67·8 ± 7·6aD100·0 ± 0·0b100·0 ± 0·0bD100·0 ± 0·0bD
14480·5 ± 8·7bC100·0 ± 0·0aD100·0 ± 0·0aC100·0 ± 0·0aC53·1 ± 6·6aC100·0 ± 0·0c100·0 ± 0·0cD78·5 ± 8·6bC
7239·2 ± 8·6bB79·2 ± 7·8dC100·0 ± 0·0eC65·0 ± 6·7cB24·0 ± 4·4aB100·0 ± 0·0e100·0 ± 0·0eD25·1 ± 6·1aB
360·0 ± 0·0aA34·5 ± 7·0bB36·5 ± 6·9bB0·0 ± 0·0aA0·0 ± 0·0aA100·0 ± 0·0d86·5 ± 7·4cC0·0 ± 0·0aA
180·0 ± 0·0aA0·0 ± 0·0aA0·0 ± 0·0aA0·0 ± 0·0aA0·0 ± 0·0aA0·0 ± 0·0a27·5 ± 7·1bB0·0 ± 0·0aA
90·0 ± 0·0A0·0 ± 0·0A0·0 ± 0·0A0·0 ± 0·0A0·0 ± 0·0A0·0 ± 0·00·0 ± 0·0A0·0 ± 0·0A
Zoospore germinationd216100·0 ± 0·0C100·0 ± 0·0D100·0 ± 0·0C100·0 ± 0·0100·0 ± 0·0C100·0 ± 0·0100·0 ± 0·0100·0 ± 0·0
144100·0 ± 0·0C100·0 ± 0·0D100·0 ± 0·0C100·0 ± 0·0100·0 ± 0·0C100·0 ± 0·0100·0 ± 0·0100·0 ± 0·0
7250·6 ± 2·9bB100·0 ± 0·0cD100·0 ± 0·0cC100·0 ± 0·0c24·0 ± 4·4aB100·0 ± 0·0c100·0 ± 0·0c100·0 ± 0·0c
360·0 ± 0·0aA60·7 ± 4·2bC81·4 ± 4·3cB0·0 ± 0·0a0·0 ± 0·0aA100·0 ± 0·0d100·0 ± 0·0d0·0 ± 0·0a
180·0 ± 0·0aA22·7 ± 3·4bB0·0 ± 0·0aA0·0 ± 0·0a0·0 ± 0·0aA100·0 ± 0·0c100·0 ± 0·0c0·0 ± 0·0a
90·0 ± 0·0A0·0 ± 0·0A0·0 ± 0·0A0·0 ± 0·00·0 ± 0·0A0·0 ± 0·00·0 ± 0·00·0 ± 0·0

Effect of EOs and individual compounds on sporangia formation

The inhibitory effects of EOs and individual compounds on sporangia formation in P. parasitica var. nicotianae are presented in Table 2. The results showed that sporangia were significantly inhibited by the different concentrations of all EOs and individual compounds, with the exception of cuminaldehyde, which had no effect on sporangia formation at any concentrations. Total inhibition of sporangia formation was achieved by cinnamaldehyde and thymol at 36 mg l−1, thyme EO at 72 mg l−1, cassia and lemongrass EOs at 144 mg l−1, and citral-a and cumin EO at 216 mg l−1, respectively. Thus, cinnamaldehyde and thymol were found to be the most effective inhibitors of sporangia formation in this study than other EOs and individual compounds (< 0·05; Table 2).

Effect of EOs and individual compounds on zoospore production and germination

The effects of different concentrations of EOs and individual compounds on the production of zoospores are shown in Table 2. Cinnamaldehyde had a significantly higher inhibitory effect on zoospore production than other EOs and individual compounds (< 0·05), with complete inhibition observed at 36 mg l−1. Thymol and thyme EO exhibited the second highest inhibitory effects, with 100% inhibition observed at 72 mg l−1. Total inhibition of zoospore production was achieved by cassia and lemongrass EOs at 144 mg l−1 and by citral-a and cumin EO at 216 mg l−1.

Table 2 shows the inhibitory effects of EOs and individual compounds on zoospore germination in P. parasitica var. nicotianae. As the concentration of EOs and individual compounds increased, a prominent reduction in the percentage of germinating zoospores was observed, while all zoospores germinated after 12-h incubation at 26°C in control samples. Cinnamaldehyde and thymol were found to be the most effective inhibitors of zoospore germination (< 0·05), with total inhibition observed at 18 mg l−1. In addition, citral-a, cassia EO, thyme EO and lemongrass EO showed 100% inhibition at 72 mg l−1, followed by cuminaldehyde and cumin EO at 144 mg l−1 (Table 2).

SEM examination of the effects of cinnamaldehyde on the morphology of Phytophthora parasitica var. nicotianae

To investigate the morphological changes induced by cinnamaldehyde in P. parasitica var. nicotianae, the morphologies of the mycelium and sporangia were observed by SEM (Fig. 2). SEM imaging confirmed that cinnamaldehyde caused considerable morphological alterations in the tested micro-organism. In imaging analysis of mycelia, untreated control samples showed a shock, anfractuous and robust morphology, and the hyphal structure was intact and had a smooth surface (Fig. 2a,b). In contrast, mycelia treated with 72 mg l−1 cinnamaldehyde exhibited morphological abnormalities, including shrivelled hyphal aggregates, swelled and lysed mycelial walls, and collapsed and flattened hyphal structures (Fig. 2c,d). In addition, sporangia treated with 0·1% KNO3 solution showed a normal morphology, with a spherical or approximately spherical shape, conspicuous papillate, plump appearance, deciduous characteristics and a smooth surface (Fig. 2e,f). However, after treatment with 36 mg l−1 cinnamaldehyde, sporangia underwent considerable morphological changes, exhibiting shrivelled sporangia aggregates, lysis of sporangia walls, flattened and a collapsed, atrophic, rugged structure (Fig. 2g,h).

Figure 2.

Scanning electron microscopy illustrated the morphological changes in Phytophthora parasitica var. nicotianae by cinnamaldehyde. Plates were incubated at a temperature of 26°C for 3 days. (a and b) Healthy hyphae in a control Petri plates; (c and d) effects of 72 mg l−1 concentration cinnamaldehyde on hyphal morphology; (e and f) healthy sporangia in a control Petri plates containing 0·1% KNO3 solution; (g and h) effects of 36 mg l−1 concentration cinnamaldehyde on morphology of sporangia.

Protective and curative effects of cinnamaldehyde on disease development in vivo

As cinnamaldehyde exhibited the most potent effects on the pathogen in vitro, we selected this compound for greenhouse trials to assess its efficacy against tobacco black shank in vivo.

The disease index and disease control efficacy of different concentrations of cinnamaldehyde against tobacco black shank are presented in Fig. 3. As the dose of cinnamaldehyde increased, P. parasitica var. nicotianae infection was obviously suppressed. Comparing the curative and protective activities of cinnamaldehyde on P. parasitica var. nicotianae infections, we observed the greatest effects on the disease index and disease control of the pathogen in protective activity. In protective treatments, there were no significant differences in the disease index and disease control between cinnamaldehyde at 160 mg l−1 (disease index and disease control were 1·4 and 84·3%, respectively) and metalaxyl at 100 mg l−1 (disease index and disease control were 1·1 and 88·0%, respectively; > 0·05). In addition, all concentrations of cinnamaldehyde and the recommended concentration of metalaxyl caused significant decreases in disease severity in tobacco plants relative to positive controls (< 0·05). In contrast, cinnamaldehyde had very little curative activity at all concentrations, in comparison with the same concentration used in experiments examining protective activities (Fig. 3).

Figure 3.

Disease index (a) and disease control (b) efficacy of cinnamaldehyde on the control of tobacco black shank by Phytophthora parasitica var. nicotianae on Nicotiana tabacum cv. K326 plants (in vivo). Metalaxyl represents the chemical fungicide used at the concentration of 100 mg l−1. Positive and negative controls indicate artificial inoculation of the pathogen and water treatment, respectively. Bars, for each effect, with the same small or large letters represent values that are not significantly different according to Duncan's multiple range test (< 0·05). (image_n/jam12208-gra-0001.png) protective; (image_n/jam12208-gra-0002.png) curative.

Discussion

Several strategies are currently used to control infection and spread of P. parasitica var. nicotianae, including chemical treatments with azoxystrobin, dimethomorph, fluazinam, metalaxyl and dimethomorph, and biocontrol micro-organisms (Matheron and Porchas 1999; Ma et al. 2010). However, these methods require sophisticated equipment and expensive chemicals or reagents. Therefore, it is important to find a practical, cost-effective and nontoxic method to prevent fungal infections. EOs, aromatic volatile products of plant secondary metabolism, are the basis of many applications in flavouring and preservation industries. In recent years, research has focused on the potential applications of EOs and individual compounds in the prevention of phytopathogens in vitro and in vivo (Bowers and Locke 2004; Liu et al. 2009; Sharif et al. 2010; Soylu et al. 2010; Tian et al. 2011a,b).

In the present study, EOs and individual compounds exhibited high antimicrobial efficacy against mycelial growth in P. parasitica var. nicotianae. Overall, the extent of inhibition of mycelial growth was widely dependent upon the composition of EOs and structure of individual compounds. Moreover, we found that EOs containing phenolic and aldehyde compounds (thymol, cinnamaldehyde, cuminaldehyde and citral-a) possessed more potent antimicrobial activities than others EOs containing alcohol and terpene compounds against P. parasitica var. nicotianae. These results are also in agreement with studies by Kurita and Koike (1982), Lee et al. (2008), Liu and Yang (2012), Lu et al. (2013), who reported that the antimicrobial activities of EOs were based on their major components, following the rule: phenols, aldehydes > alcohols > ketones, esters > hydrocarbons.

An interesting observation in our bioassays was the inhibition of sporangia formation and zoospore production and germination in relation to the inhibition of mycelial growth. The marked reduction in sporangia formation and zoospore production induced by EOs and individual compounds might reflect the effects of volatile components on surface mycelial development and/or the perception/transduction of signals involved in the switch from vegetative to reproductive development (Tzortzakis and Economakis 2007). Similar results have been reported for pathogenic fungi treated with the EOs of Origanum syriacum L. var. bevanii (Soylu et al. 2010) and Cicuta virosa L. var. latisecta Celak (Tian et al. 2011a,b). In addition, the calculated per cent inhibition of sporangia formation and zoospore production and germination was obviously higher than mycelial growth inhibition at the same concentration. Therefore, the EOs and individual compounds were more effective in a liquid medium than in a solid medium. In summary, the EOs and individual compounds tested in the current study significantly restricted mycelial growth, formation of sporangia, and production and germination of zoospores in a dose-dependent manner.

The structure–activity relationships of certain plant compounds against microbial infections have been well studied. Moleyar and Narasimham (1986) reported that unsaturated aldehydes (citral, cinnamaldehyde and citronellal) and unsaturated alcohols, such as geraniol, were more effective against Aspergillus niger, Fusarium oxysporum and Penicillium digitatum than hydrocarbons, such as camphene, limonene and a-terpinene. In our study, there was a significant difference in antimicrobial activity among different functional groups. Aldehydes compounds (cinnamaldehyde, cuminaldehyde and citral-a) and phenolic compounds (thymol) were general more toxic than alcohols and hydrocarbons. Among the aldehydes tested, cinnamaldehyde was more active than cuminaldehyde and citral-a when applied at the same concentration. This result suggested that the position of the aldehyde group and the structure of the benzene ring and alkene were related to the antimicrobial activity of the compound. In addition, it has been reported that conjugation of an aldehyde group to a carbon–carbon double bond is a highly electronegative arrangement that may interfere with electron transfer during biological processes (Moleyar and Narasimham 1986). In addition, citronellol, nerol, geraniol, menthol and linalool, classified as alcohol terpenoids, exhibited moderate antimicrobial effects against P. parasitica var. nicotianae in this study. As with other bactericidal alcohols, the antibacterial mechanisms of these alcohols and alcohol terpenoids may be attributed to protein denaturation or dehydration in the vegetative cells (Dorman and Deans 2000).

Several authors have reported the antimicrobial activity of EOs and individual compounds against Phytophthora. A study by Lee et al. (2008) demonstrated that the antimicrobial activity of Myrtaceae EOs and their major components against P. cactorum and the presence of citronellol, neral, geraniol and geranial were responsible for the antimicrobial effects of these EOs. Bajpai et al. (2008) investigated the antimicrobial activities of EOs and organic extracts from Nandina domestica Thunb. against Phytophthora capsici, and complete inhibition of mycelial growth was achieved at 500 mg l−1 of the EO. Lee et al. (2009) assessed the antimicrobial activity of 40 commercially available EOs and components from Liquidambar orientalis against P. capsici and demonstrated that the presence of cinnamaldehyde and benzaldehyde were responsible for the antimicrobial effects of these EOs. In the current study, EOs from cassia, cumin, thyme and lemongrass showed prominent antifungal activity against P. parasitica var. nicotianae, supporting the hypothesis that the activities of these EOs could mainly be attributed to the presence of characteristic components (i.e. cinnamaldehyde, cuminaldehyde, thymol and citral-a, respectively).

SEM imaging of the microstructure of mycelia and sporangia from P. parasitica var. nicotianae revealed some of the antimicrobial mechanisms of cinnamaldehyde. Degenerative alterations in hyphal and sporangia morphology, such as shrivelled aggregates, rugged surface, swelling and lysis of hyphal and sporangia walls, and flattened hyphal and sporangia structures, were commonly observed in cinnamaldehyde-treated mycelia and sporangia. These results were similar to those from previous studies in which the microstructures of fungi treated with EOs were studied (Soylu et al. 2007; Rasooli et al. 2008; Liu et al. 2009; Soylu et al. 2010; Tolouee et al. 2010; Tian et al. 2011a,b; Lu et al. 2013). The induction of these morphological changes may be related to the interference of cinnamaldehyde with enzymatic reactions required for cell wall synthesis, suggesting an alteration in the normal assembly of the wall components after treatment with high doses of EOs, which prevents the correct arrangement of different parietal compounds, such as chitin, glucans and glycoproteins, during cell wall construction, thereby influencing morphogenesis and growth (Bartnicki 1999; Romagnoli et al. 2005). In addition, Oussalah et al. (2006) and Gill and Holley (2004, 2006) indicated that cinnamaldehyde causes a decrease in intracellular ATP by inducing ATPase activity without apparent changes to the cell membrane in Escherichia coli, E. coli O157:H7 and Listeria monocytogenes. In previous studies, cinnamaldehyde and EOs of oregano and fennel causes increased membrane permeability and leakage of cytoplasm (Kim et al. 1995; Soylu et al. 2007). Interaction of cinnamaldehyde with the cell membrane may cause disruption of the membrane, dispersing the proton gradient by leakage of small ions or inhibiting the enzymes necessary for amino acid biosynthesis (Wendakoon and Sakaguchi 1995). In our study, the cell wall, cell membrane and organelles may be important targets of cinnamaldehyde. Moreover, cinnamaldehyde may affect these targets, causing an imbalance in intracellular osmotic pressure, blockage of enzymatic reactions and leakage of cytoplasmic contents, potentially inducing cell necrosis.

In vitro studies on cinnamaldehyde have indicated its potential as an ideal antimicrobial agent against P. parasitica var. nicotianae. Thus, further investigations into its efficacy as a botanical pesticide for the control of tobacco black shank in vivo are necessary. Both treatments were effective in reducing microbial infection, and the greatest control of infection was consistently achieved when cinnamaldehyde applications were made 24 h before inoculation (i.e. protective efficacy). Bortrytis fabae and the rust fungus Uromyces fabae were also controlled in vivo, with basil EO and methyl chavicol and linalool, significantly reducing infections of broad bean leaves (Oxenham et al. 2005). The most effective control of these fungal infections was achieved if treatments were applied 3 h postinoculation. In our case, this suggested that cinnamaldehyde exerted its greatest effect on microbial development of the stem, for example, mycelial growth, formation of sporangia, and production and germination of zoospores. In addition, higher concentrations of EOs are required in vivo experiments than in laboratory media, possibly because nutritional and moisture conditions promote microbial growth in vivo.

In conclusion, the results of in vitro and in vivo bioassays, together with SEM imaging of the microstructure of P. parasitica var. nicotianae, supported the possibility of using cinnamaldehyde as a potent natural biofungicide in the greenhouse. However, further studies are needed to evaluate the cost and efficacy of cinnamaldehyde in the treatment of a wide range of diseases in commercial greenhouses conditions. In addition, the mechanism through which cinnamaldehyde alters the morphology of P. parasitica var. nicotianae hyphae and restricts the development of sporangia and zoospores should be further evaluated to fully understand the antimicrobial activities of cinnamaldehyde.

Acknowledgements

This work was supported by the China National Tobacco Company (CNTC) project no. 2010YN47. The authors would like to thank to research fellow Yun Xu, Agricultural Environment & Resource Research Institute, Yunnan Academy of Agricultural Sciences, for providing the seeds of Nicotiana tabacum cv. K326.

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