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Revista mexicana de fitopatología

On-line version ISSN 2007-8080Print version ISSN 0185-3309

Rev. mex. fitopatol vol.36 n.1 Texcoco Jan./Apr. 2018

http://dx.doi.org/10.18781/r.mex.fit.1707-4 

Phytopathological notes

Effect of natural oils against Mycosphaerella fijiensis under in vitro conditions and detection of active plant chemicals

Eduardo Gutiérrez-Jiménez1 

Aurelio Pedroza-Sandoval2  * 

Luciano Martínez-Bolaños3 

José Alfredo Samaniego-Gaxiola4 

Fabián García-González5 

1 Universidad Autónoma Chapingo, Departamento de Parasitología Agrícola, Km. 38.5 Carretera México-Texcoco, CP. 56230, Chapingo, Estado de México.

2 Universidad Autónoma Chapingo, Unidad Regional de Zonas Áridas, Carretera Gómez Palacio - Ciudad Juárez Km 40, CP. 35230, Bermejillo, Durango.

3 Universidad Autónoma Chapingo, Unidad Regional Sur Sureste, Km 7 Carretera Teapa - Vicente Guerrero, CP. 86800, Teapa, Tabasco.

4 Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias. Blvd. José Santos Valdéz No. 1200, Centro, CP. 27440, Matamoros, Coahuila.

5 Universidad Autónoma Chapingo, Unidad Regional de Zonas Áridas. Km 40 Carretera Gómez Palacio - Ciudad Juárez, CP. 35230, Bermejillo, Durango.

Abstract:

Chemical control of black sigatoka in banana Mycosphaerella fijiensis has increased the selection pressure on the pathogen, a fact which in turn creates an environmental impact. The objective of this study was to evaluate the effect of essential Pimenta dioica, Piper auritum, Sysygium aromaticum, Cinnamomum zeylanicum, Origanum vulgare, Artemisia ludoviciana, and Origanum majorana oils on M. fijiensis growth under in vitro conditions, and to identify their active metabolites. The oils were obtained by hydrodistillation. M. fijiensis was isolated and developed on PDA culture medium. Thin layer chromatography (TLC) and column chromatography (CC) were used to identify the metabolites of each essential oil. Five concentrations: 50, 100, 500, 1000 y 5000 ppm of each oil were used. P. dioica, C. zeylanicum and O. vulgare oils had a significant effect with lower M. fijiensis mycelial growth at a concentration of 500 ppm, whereas the effect on O. majorana and A. ludoviciana was observed at a concentration of 1000 ppm. Cinnamic aldehyde was the main metabolite detected in C. zeylanicum species, and eugenol and carvacrol in P. dioica y O. vulgare species.

Key words: plants extract; secondary metabolites; fungi inhibition; biological control

The use of chemicals to control crop pests is a useful tool to fulfill productivity and sustainability objectives, provided that such tool is combined with proper management technologies (Wheeler, 2002). This is necessary because most of the phytopathogen microorganisms have become resistant to active ingredients of chemical fungicides (Chávez-Solís et al., 2014). An efficient and inexpensive alternative for controlling diseases is the use of plant extracts with fungicidal properties (Guerrero et al., 2007). Plant extracts and essential oils are biodegradable and have a limited environmental impact (Bravo et al., 2000). Based on this, there is an increased interest in using essential spice and other plants oils as natural antimicrobials in food and agricultural crops (Celis et al., 2012). Lambert et al. (2001) state that nowadays there is an increasing demand to precisely obtain the values of the inhibitory minimum concentration (CMI) of diverse essential oils so as to achieve a balance between sensory acceptation and antimicrobial effectiveness, which can be attained by conducting in vitro and in vivo studies.

In Mexico, the most used fungicides against M. fijiensis are: mancozeb and chlorothalonil, as well as systemic ingredients from the groups of benzimidazoles, triazoles, strobirulins and anilopyrimidines (Martínez-Bolaños et al., 2012). Chemical management of black sigatoka requires from 10 to 45 fungicide applications per year (mainly mancozeb, propiconazole and tridemorph), but there is the risk that the pathogen may develop resistance to those fungicides (Mena-Espino and Couoh-Uicab, 2015). The objective of this study was to evaluate the biological effectiveness of different essential oils extracted from native plants on M. fijiensis mycelial growth under in vitro conditions, and identify their main active chemical compounds.

Essential oils were obtained from momo (Piper auritum), cinnamon (Cinnamomum zeylanicum), oregano (Origanum vulgare), estafiate (Artemisia ludoviciana), marjoram (Origanum majorana) leaves, and pepper (Pimenta dioica) and clove (Syzygium aromaticum) fruits. Plant samples were collected in the municipalities of Teapa, Tabasco, and Pichucalco, Chiapas, Mexico. The samples were dried under shade and grinded using a manual mill. The obtained powder was subjected to hydrodistillation for two hours in a proportion of 30 g in 500 ml of water (Domínguez, 1979), and the oil was collected using a Clevenger-type equipment (Muñoz et al., 2001). The oil was poured in amber jars and kept in refrigeration.

Mycosphaerella fijiensis was directly isolated from infected tissue from infected banana plants collected at the producing zones of the municipalities of Teapa, Tabasco. Bits of around 1 cm2 from diseased-plant tissue were cut and stuck to filter paper discs. The infected tissue was superficially disinfected in a 3% hypochlorite solution and then washed three times with distilled water. The discs were placed on Petri dish covers that contained solid culture medium (agar-water). Three days after the fungus started to grow, it was transferred to PDA culture medium for development (Stover, 1963).

Different concentrations of each oil were prepared in assay tubes with 10 mL of sterile distilled water: 50, 100, 500, 1000 and 5000 ppm. The content of each tube was mixed in flasks containing PDA medium (30 mL) and then the content of each flask was distributed among four Petri dishes (10 mL). PDA plates without treatment were used as control. Essential oils extracted from seven plant species were evaluated at the five concentrations (50, 100, 500, 1000 and 5000 ppm), except for S. aromaticum that was not evaluated at the concentration of 50 ppm. For inoculation, small discs of around 0.4 mm2 from the culture medium containing the M. fijiensis isolate were cut and placed in the middle of each Petri dish.

In this study, a completely randomized design with four replications was used, where the treatments were the five concentrations of each plant oil plus an absolute control (PDA).

The measured variables were the percentage of radial growth inhibition (PICR) as well as that of M. fijiensis own growth. To evaluate the efficacy of the treatments, the fungal colony development was measured (in mm) until the Petri dishes containing the control were completely covered with mycelium. The PICR was calculated according to the following formula:

PICR %=RC-RTRC100

where RC= mycelium radius in the control, and RT= mycelium radius in the treatments.

Data were analyzed using the SAS version 9.0 software. To determine the effect of the treatment, a variance analysis and Tukey’s multiple range test of means were carried out.

After the antifungal activity of the plant oils ended, those showing the highest biological activity were selected to know their approximate chemical composition using the thin-layer chromatography (CCF) (Randerath, 1970) and column chromatography (CC) techniques (Abbot and Andews, 1970). Before using chromatography, the literature was reviewed to know what type of metabolites were present in the raw extracts obtained, as well as their biological activity, and then the corresponding relation was established with the phytochemical results achieved in this study.

According to the percentage of M. fijensis radial growth inhibition (RGI) and mycelial growth, the oils with the best inhibitory effect on Mycosphaerella fijiensis development were those extracted from C. zeylanicum, O. vulgare and P. dioica, whose inhibitory effect was observed at a dose of 100 ppm, with inhibition values of 90.5, 71.5 and 58%, respectively. When the oils were used at a concentration of 500 ppm, 100% inhibition was achieved. The other oils showed lower values: S. aromaticum: 10.5%, P. auritum: 22.5%, A. ludoviciana: 38% and O. majorana: 45.9%. A. ludoviciana and O. majorana oils had a significant inhibitory effect at a concentration of 1000 and 5000 ppm. S. aromaticum did not show any inhibitory effect on the fungus (Table 1).

Table 1 Percentage of radial growth inhibition (PRGI) of Mycosphaerella fijiensis by the effect of essential oils five days after inoculation. 

Especie Concentración (ppm)
Control 50 100 500 1000 5000
Origanum vulgare 0 D 23 C b 71.5 B b 100 A a 100 A a 100 A a
Artemisia ludoviciana 0 D 13.5 C c 17 C e 38 B cd 100 A a 100 A a
Cinnamomum zeylanicum 0 D 32.5 C a 90.5 B a 100 A a 100 A a 100 A a
Piper auritum 0 D 9.5 D cd 8.5 D ef 22.5 C def 49.5 B c 90.0 A a
Pimenta dioica 0 D 14.5 C bc 58 B c 100 A a 100 A a 100 A a
Syzygium aromaticum 0 A 3.5 A de 10.5 A ef 10.5 A efg 10 A de 10.5 A c
Origanum majorana 0 D -------- 36.9 C d 45.9 B c 94.7 A a 98.0A a
Control ---- 0 f 0 f 0 g 0 e 0 d

Figures with the same letter in the rows are not statistically different (Tukey’s grouping test, p≤0.01). Capital letters: grouping among concentration of each oil (row). Lowercase letters: grouping among oil concentrations (column).

On the first day of exposure, C. zeylanicum, O. vulgare and P. dioica oils at a dose of 100 ppm showed 100% PICR with no M. fijiensis growth. The same effect lasted up to three days when essential C. zeylanicum oil was used. On day five of exposure, the same oils showed minimum M. fijiensis mycelial growth, with values from 2 to 9 mm (Figure 1A), but when the three oils were used at a concentration of 500 ppm, fungal growth inhibition was 100% (Figure 1B).

Figure 1 Mycosphaerella fijiensis growth (in mm) in essential oils obtained from different plant species. a) at 100 ppm and b) at 500 ppm. DAI= days after inoculation. 

The antifungal capacity of the essential C. zeylanicum oil partially coincides with that reported by Barrera and García (2008), who found a growth inhibitory effect of 70% in Fusarium spp isolated from papaya at a concentration of 150 µg mL-1. In the case of O. vulgare oil, Soylu et al. (2006) found Phytophthora infestans total growth inhibition at concentrations from 0.3 to 6.4 ug ml-1. Cáceres et al. (2013) reported that an O. vulgare extract at a concentration of 200 ppm caused total growth inhibition of Fusarium oxysporum and Aspergillus niger, whereas in the case of Alternaria alternata, Geotrichum candidum, Trichoderma spp. and Penicillum digitatum, growth inhibition started at a concentration of 0.25 ᶙL mL-1. Ramírez et al. (2011)reported that Moniliophthora roreri development in culture medium was totally inhibited by the same oil obtained as a hydrolate by distillation at 50% (volume-volume). Also, Ramírez et al. (2016) state that hydrodistillates and C. zeylanicum, S. aromaticum and P. dioica oils obtained by microwaving can efficiently inhibit A. solani and C. gloesporioides development.

Origanum majorana oil had a 95% inhibition effect at a concentration of 1000 ppm. The fungistatic effect of this species was reported by Gamboa et al. (2003), who found that the methanol extract of marjoram inhibited 100% of P. infestans mycelial growth at 8000 ppm, and from 59-80% Rhizoctonia solani growth at 2000 and 4000 ppm, respectively. Damian et al. (2010) observed that the active principle of the extract of A. ludoviciana was dichloromethanol-methanol (1:2 v/v), which inhibited 100% of Phytophthora cactorum, P. capsici, P. cinnamomi and P. mirabilis growth, and 60% of P. infestans growth, at a concentration of 100 ppm. In this study, A. ludoviciana oil inhibited 100% of M. fijiensis mycelial development at a concentration of 1000 ppm.

The main active chemical components identified were cinnamic aldehyde and eugenol in essential C. zeylanicum oils (50 and 10%, respectively); P. dioica and O. vulgare. P. dioica and O. vulgare had the highest concentration of eugenol (72.6 and 76.3%), followed by caryophyllene and limonene. The other components were reported in lower concentrations; for example, safrole was present only in C. zeylanicum oil; myrcene and O-cimene were present only in P. dioica oil; and limonene and carvacrol were present only in O. vulgare oil (Table 2).

Table 2 Main active chemical compounds as ingredients of essential oils from three plant species. 

Compuestos Concentración (%)
Cinnamomum
zeylanicum
Pimenta
dioica
Origanum
vulgare
Mirceno 4.17
Felandreno 1.11 0.06
Limoneno 6.19
O-cimeno 0.98
Carvacrol 1.06
Safrol 8
Eucaliptol 3.4 0.24
Aldehído cinámico 50 0.82 0.39
Terpineno 0.47 0.06
Terpinoleno 1.39 2.20
Alpha-terpineol 1.07 2.20
Eugenol 10 72.6 76.31
Cariofileno 6.13 0.21

Barrera and García (2008) reported that cinnamic aldehyde and carvacrol inhibited completely Fusarium spp fungal mycelial growth at 100 µg mL-1. Wang et al. (2010) reported that Fusarium moniliforme, Sclerotinia sclerotiorum, Cercospora beticola, Mycogone perniciosa, Macrophoma kawatsukai, Thanatephorus cucumeris, Alternaria alternata and Botrytis cinereal were highly sensible to eugenol applications, and from these B. cinerea and S. sclerotiorum were the most sensible to such active principle, with values of CE50 of 38.6 and 39.9 ᶙg mL-1, respectively.

Gill and Holley (2006) evaluated eugenol, carvacrol and cinnamic aldehyde to observe ATP changes and cell variability in Escherichia coli, Listeria monocytogenes and Lactobacillus sakei. Their results show that eugenol and carvacrol action mechanism is a disruption of the membrane cytoplasmic activity, which increases their non-specific permeability. The absence of extracellular ATP of cells treated with cinnamic aldehyde suggests that eugenol and carvacrol have inhibitory activity from ATPase, which contains no cinnamic aldehyde. These results suggest that the effect of C. zeylanicum, P. dioica and O. vulgare oils inhibited M. fijiensis mycelial growth because of the presence of eugenol and cinnamic aldehyde, as well of carvacrol in O. vulgare oil.

Conclusions

Essential C. zeylanicum, O. vulgare and P. dioica oils had the best inhibitory effect on M. fijiensis mycelial growth under in vitro conditions at a concentration of 100 ppm, with 100% inhibition at 500 ppm. A. ludoviciana and O. majorana oils showed an inhibitory effect at concentrations of 1000 ppm; P. auritum at 5000 ppm; S. aromaticum did not show any inhibitory effect. Cinnamic aldehyde was the main chemical compound principle detected in C. zeylanicum; eugenol in P. dioica and O. vulgare. The latter also showed small concentration of carvacrol.

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Received: July 25, 2017; Accepted: October 19, 2017

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