Antifungal activity of five plant-extracted essential oils

Biological Agriculture & Horticulture An International Journal for Sustainable Production Systems

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Antifungal activity of five plant-extracted essential oils against anthracnose in papaya fruit A. Sarkhosh, B. Schaffer, A. I. Vargas, A. J. Palmateer, P. Lopez, A. Soleymani & M. Farzaneh To cite this article: A. Sarkhosh, B. Schaffer, A. I. Vargas, A. J. Palmateer, P. Lopez, A. Soleymani & M. Farzaneh (2018) Antifungal activity of five plant-extracted essential oils against anthracnose in papaya fruit, Biological Agriculture & Horticulture, 34:1, 18-26, DOI: 10.1080/01448765.2017.1358667 To link to this article: https://doi.org/10.1080/01448765.2017.1358667

Published online: 27 Jul 2017.

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Date: 18 December 2017, At: 05:07

Biological Agriculture & Horticulture, 2018 VOL. 34, NO. 1, 18–26 https://doi.org/10.1080/01448765.2017.1358667

Antifungal activity of five plant-extracted essential oils against anthracnose in papaya fruit A. Sarkhosha,b, B. Schafferb, A. I. Vargasb, A. J. Palmateerb, P. Lopezb, A. Soleymanic and M. Farzanehd

Downloaded by [University of Florida] at 05:07 18 December 2017

a

Department of Primary Industry and Resources, Northern Territory Government, Katherine Research Station, Katherine, Australia; bTropical Research and Education Center, University of Florida, Homestead, FL, USA; cDepartment of Chemistry and Biochemistry, Florida International University, Miami, FL, USA; dDepartment of Agriculture, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran

ABSTRACT

Inhibitory effects of five different plant essential oils were assessed against papaya fruit decay caused by anthracnose. Essential oils were extracted from savory, thyme, cinnamon, mint, and lavender plants and chemical components of each oil were identified by GC-mass spectroscopy. The polymerase chain reaction (PCR) method using the internal transcribed spacer (ITS) primer confirmed the identity of the Colletotrichum gloeosporioides isolate. Then different concentrations of each oil were tested in vitro and in vivo. The in vitro results revealed a 100% reduction of mycelium growth for savory and thyme essential oils. In the in vivo experiment, savory and thyme oils at 2000 μL L−1 caused a 59.26 and 58.40% reduction in lesion diameter and a 64.07 and 54.82% of fruit decay, respectively. Application of savory or thyme oil resulted greater maintenance of fruit firmness than the application of the other essential oils after fruit were inoculated with the anthracnose fungus. Savory oil with the main chemical compound carvacrol (71.2%), and thyme oil with the main chemical constituent thymol (73.3%) were the most effective of the oils tested at controlling anthracnose in vitro and in vivo. The half maximal effective concentration (EC50) of savory oil was the lowest among the extracted oils tested, resulting in EC50 = 58.42 μL L−1 for the in vitro test and EC50 = 1507.19 μL L−1 for the in vivo experiment. This study showed that savory oil is effective as a natural fungicide for controlling anthracnose decay and prolonging the storage life of papaya fruit.

ARTICLE HISTORY

Received 20 February 2017 Accepted 19 July 2017 KEYWORDS

Colletotrichum gloeosporioides; in vitro; in vivo; mycelium growth; Satureja khuzistanica; Thymus daenensis

Introduction One of the most devastating diseases in tropical fruits and vegetables, including papaya, is anthracnose, caused by the fungus Colletotrichum gloeosporioides Penz. (Paull et al. 1997). Anthracnose infects fruit in the field while they are immature. Symptoms also appear on fruit after ripening, causing damage to the fruit during storage, transit, and marketing (Gonzalez-Aguilar et al. 2003). Generally, for most fruits and vegetables, producers apply a combination of hot water with synthetic fungicides such as benomyl, mancozeb, carbendazim or orthiabendazol to reduce potential infection by postharvest diseases (Couey et al. 1984; Barrera-Necha et al. 2008). Hot-water treatment can affect the nutritional value and sensory properties of fruits and vegetables, whereas continuous

CONTACT A. Sarkhosh

sarkhosha@gmail.com, ali.sarkhosh@nt.gov.au

© 2017 Informa UK Limited, trading as Taylor & Francis Group


BIOLOGICAL AGRICULTURE & HORTICULTURE

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applications of synthetic fungicides can lead to the development of fungicide resistant strains of the pathogen. Furthermore, chemical residues on the fruit, because of fungicide application, may be harmful to consumers and the environment (Cutler and Cutler 1999; Maqbool et al. 2011; Sivakumar and Bautista-Baños 2014; Calo et al. 2015). The use of chemical fungicides to control postharvest decays is restricted in some countries, and there is a high demand by consumers for agricultural products that have not been treated with chemicals (Cutler and Cutler 1999; Serrano et al. 2005). Research has shown that application of essential oils is a non-toxic method of controlling postharvest fruit diseases including those caused by fungi (Regnault-Roger et al. 2012; Sivakumar and Bautista-Baños 2014; Calo et al. 2015). The use of non-hazardous products, such as plant essential oils for controlling anthracnose in papaya during storage, is a promising consumer-friendly and environmentally safe alternative to chemical fungicides (Calo et al. 2015). Papaya fruit are very perishable and susceptible to anthracnose. Some essential oils have been tested for controlling anthracnose in papaya fruit, but the results have been mixed. For example, Bosquez-Molina et al. (2010) found that thyme (Thymus vulgaris) oil was very effective at controlling C. gloeosporioides mycelial growth in vitro in papaya fruit. However, Barrera-Necha et al. (2008) found that cinnamon (Cinnamomum zeylanicum) and clove (Syzygium aromaticum) oils were more effective than thyme oil at inhibiting in vitro growth of this fungus and therefore only tested the efficacy of these two essential oils in vivo on papaya fruit. Of these two oils, they found clove oil to be the most promising essential oil for control of anthracnose in papaya fruit during storage. Volatile application of carvacrol, the main chemical component found in savory (Satureja khuzistanica) oil, was effective at controlling C. gloeosporiodes in pepper fruit (Hong et al. 2015), but to the authors’ knowledge, essential oil or its chemical components from this plant species has not been tested on papaya fruit. The objective of this study was to test and compare the effects of different concentrations of essential oils extracted from thyme, savory, mint, cinnamon, and lavender plants on inhibition growth of C. gloeosporioides in vitro and to evaluate effects of these essential oils for controlling decay of papaya fruit caused by this fungus in vivo.

Material and methods The plant-extracted essential oils from thyme (Thymus daenensis Celak.), savory (Satureja khuzistanica Jamzad.), mint (Mentha piperita Willd.), cinnamon (Cinnamomum zeylanicum Blume.), and lavender (Lavandula angustifolia Mill.) tested in this study were previously isolated and the major chemical components of each oil identified by gas chromatography-mass spectroscopy (GC-Mass Spec) analysis (Sarkhosh et al. 2017). Pathogen preparation and identification A single spore isolate of Colletotrichum gloeosporioides was obtained from samples of diseased mango fruit with anthracnose from the University of Florida’s Plant Diagnostic Clinic located at the Tropical Research and Education Center in Homestead. Colonies produce abundant conidia that are hyaline, one-celled, straight, cylindrical and average 14.7 × 5.0 μm with ranges of 12.5–17.5 × 3.8 to 7.5 μm. Cultural and morphological characteristics of the isolate matched those for C. gloeosporioides producing appressoria and characteristic lobed hyphopodia that average 10.5 (7.9–11.5) × 8.9 (7.0– 10.1) μm (Bailey and Jeger 1992). A sequence from Internal Transcribed Spacer (ITS) regions 1 and 4 for the mango isolate (GenBank Accession no KY447324) was nearly identical (99% homology) to C. gloeosporioides isolated from Cymbidium sinensis in China (Accession No. KC010549). Effects of the essential oils on mycelial growth in vitro Inhibitory effects of essential oils extracted from the five plant species were tested at different concentrations on fungal mycelia growth in petri dishes containing potato dextrose agar (PDA). The essential

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oils were disseminated individually as an emulsion in sterilized water containing the surfactant Tween 80 (0.05%) which was added to PDA medium immediately before it was poured into the petri dishes at a temperature of 45–50 °C. The concentrations of the essential oils were 0 (control), 65, 125, 250, 500, 1000, and 2000 μL L−1. A 5-mm diameter circular disk of each fungus species was cut with a sterile cork bore from the margin of the actively growing cultures on the PDA and placed in the center of each petri dish containing the essential oil treatment. Petri dishes were then incubated at 25 °C, and radial growth of mycelia was measured every 24 h after initially placing the fungal mycelium on the dish. The experiment was terminated when the petri dishes containing the control treatment were fully covered with mycelium. The efficacy of each treatment was evaluated by measuring fungal mycelial growth (mm) from the center of the colony to the edge of the colony. The means of two radiuses at 90° angle from each other were combined for each measurement dish.


Effects of essential oils on fruit decay and firmness in vivo Papaya fruit (Carcia papaya Linn. cv. Red Lady) were harvested at the mature-green stage from a local conventional farm (non-organic production) in Homestead, Florida, USA. Harvested fruit were sorted based on size, shape and maturity and free from any indication of mechanical injury, insect or pathogen infection. Before applying the treatments, fruit were washed with tap water and sodium hypochlorite (1%), rinsed with deionized water and dried at room temperature. Each fruit was artificially wounded (with a sterile toothpick) in three separate locations on the fruit and sprayed (with a hand-held spray bottle) with a conidial suspension of 1 × 106 conidia ml−1. After 24 h, essential oils were sprayed on the fruit at the following concentrations: 0, 500, 1000, or 2000 μL L−1 fruit. The treated fruit were packed in plastic zip-lock bags and stored for three weeks at 12 ± 1 °C followed by one week at room temperature (24 ± 1 °C). Disease severity evaluation in fruit The diameter (mm) of the lesion around each inoculation point (wound hole) of each fruit was measured with a digital caliper (Anyi Instrument, Guangxi, China) immediately after three weeks storage at 12 ± 1 °C followed by one week at room temperature (24 ± 1 °C). After taking a digital photograph of each fruit, ImageJ software (Abràmoff et al. 2004) was used to measure the affected (discoloured) area of the fruit. The severity of anthracnose was scored based on ImageJ calculations using the following scale: 1 = 0–5%; 2 = 6–15%; 3 = 16–30%; and 4 = > 30% of the total fruit area that appeared discoloured and rotten. Fruit firmness was measured based on kilogram force (Kgf) with a Wagner FT 30 Penetrometer (Greenwich, CT, USA). Half maximal effective concentrations (EC50) The half maximal effective concentration (EC50) was determined for each essential oil based on inhibition of each oil on radial growth of Colletotrichum gloeosporioides myclieum (in vitro) and diameter of lesion on papaya fruit caused by the fungus (in vivo). Statistical analyses The experiment was arranged as a completely randomized design (CRD) with a factorial combination of treatments and five single-fruit replications of each treatment combination. Treatments were the essential oil from each plant and application rates of each oil. Data were analysed by two-way analysis of variance (ANOVA) to test for main treatment effects and interactions. Mean treatment differences among essential oils were tested by Tukey’s studentized range test. Differences among essential oil application rates were determined by linear and quadratic regression analyses. ANOVA, mean separations, and regression analyses were done with SAS, version 9.3 statistical software (SAS Institute,


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Cary, NC, USA). The EC50 for each essentail oil was determined using disease severity as the dependent variable. EC50 were determined by probit analysis at 95% confidence limits using SPSS 10.0 statistical software (SPSS Inc., Chicago, IL, USA).

Results


In vitro experiment The results of the effect of essential oils on mycelial growth of C. gloeosporioides in vivo are shown in Table 1. At a concentration of 65 μL L−1, savory oil showed stronger inhibitory activity than other essential oils, decreasing mycelial growth by 68.1% compared to the control. At this concentration thyme oil decreased mycelial growth by 53.3% compared to the control. The essential oils extracted from the other three plant species reduced mycelial growth at this low application rate, but to a lesser extent than savory or thyme oil and mycelial growth on plates treated with these three essential oils were not significantly different from the non-treated controls. At an application rate of 125 μL L−1, thyme and savory oils completed inhibited mycelial growth, whereas the other three essential oils completely inhibited mycelial growth at a higher application rate (1000 μL L−1). Therefore, the minimum complete inhibitory concentration (MCIC) was determined to be 125 μL L−1 for savory and thyme essential oils and 1000 μL L−1 for the essential oils extracted from mint. All of the essential oils tested inhibited mycelial growth to some extent at an application rate of 500 μL L−1 but savory and thyme oils were the most effective, totally inhibiting mycelial growth while cinnamon oil was very effective but did not total inhibit growth of the mycelium in vitro. There was a significant positive linear relationship between application rate and mycelial growth for cinnamon (R2 = 0.81) and lavender (R2 = 0.80) oils, whereas as there was a significant positive quadratic relationship between application rate and mycelial growth for mint oil (R2 = 0.94). The MCIC of, cinnamon, mint and lavender essential oils against mycelial growth was determined at 1000 μL L−1. In vivo experiment Effect of essential oils on fruit lesion diameter Compared to the control treatment, application of savory or thyme oil at the lowest application rate (500 μL L−1) resulted in a 26.5 or 25.1% reduction, respectively in lesion diameter around the wounds on fruit inoculated with C. gloeosporioides (Table 2). The essential oils extracted from the other plant species were less effective than savory or thyme oils at reducing lesion diameter at this application rate. There were strong positive quadratic relationships between application rate and lesion diameter for savory (R2 = 0.96) and thyme (R2 = 0.97) oils, whereas the quadratic relationship between these variables was significant but not as strong for cinnamon (R2 = 0.74) and lavender (R2 = 0.52) oils

Table 1. Effect of different concentrations of essential oils extracted from five different plant species on Colletotrichum gloeosporioides mycelium growth (mm day−1) in vitro. Application rate (μL L−1) Essential oil Savory Thyme Cinnamon Mint Lavender

0 8.30az 8.30a 8.30a 8.30a 8.30a

65 2.66a 3.88b 6.71c 7.26c 6.88c

125 0.00a 0.00a 6.88b 7.08b 6.60b

250 0.00a 0.00a 5.74c 6.66b 6.30c

500 0.00a 0.00a 4.84bc 5.32b 5.96c

750 0.00a 0.00a 3.64c 4.48b 5.38d

1000 0.00a 0.00a 0.00a 0.00a 0.00a

2000 0.00a 0.00a 0.00a 0.00a 0.00a

Sig. – – L*y Q* L*

R2 – – 0.81 0.94 0.80

Notes: ZDifferent letters, within columns, indicate significant differences between essential oils within each application rate according to Tukey’s HSD Test (p ≤ 0.05). y *indicates that the linear (L) or quadratic (Q) regression model was significant at p ≤ 0.01 for the application rate of each essential oil.

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Table 2. Effect of essential oils on diameter (mm) of lesions on papaya (Carica papaya) fruit caused by Colletotrichum gloeosporioides after 2 weeks of fruit storage at 12 ± 1 °C followed by one week at ambient temperature (24 ± 1 °C). Application rate (μL L−1) Essential oils Savory Thyme Lavender Cinnamon Mint

0 35.1az 35.1a 35.1a 35.1a 35.1a

500 25.8a 26.3ab 29.5abc 30.8bc 32.5c

1000 19.8a 20.8a 30.2b 28.7b 28.2b

2000 14.3a 14.6a 28.8b 27.5b 25.7ab

Sig. Q*y Q* Q* Q* L*

R2 0.96 0.97 0.52 0.74 0.66



(Table 2). There was a positive linear relationship between application rate and lesion diameter for mint oil (R2 = 0.66). Effect of essential oils on fruit decay At the lowest application rate of 500 μL L−1, savory oil had a significantly greater effect on reducing the amount of fruit decay than oils extracted from the other four plant species (Table 3). At application rates of 1000 and 2000 μL L−1, savory and thyme oil were each significantly more effective at reducing the amount of fruit decay than the other treatments and there was no significant difference in effectiveness between savory and thyme at this concentration (Table 3). There was a significant positive linear relationship between essential oil application rate and the amount of fruit decay for savory (R2 = 0.78) and mint, but the coefficient of determination was much lower for mint (R2 = 0.25) (Table 3). There was a strong positive quadratic relationship between the application rate and the amount of fruit decay for thyme (R2 = 0.81). There was a relatively weak positive quadratic relationship between application rate and fruit decay for both cinnamon and mint (R2 = 0.30 for each species). At the highest application rate of 2000 μL L−1 all oils were somewhat effective at reducing fruit decay, but savory and thyme oils were the most effective as can be seen in Figure 1. Effect of essential oils on fruit firmness Application of each essential oil helped fruit maintain firmness after inoculation with C. gloeosporioides, but for each oil, fruit firmness was greatest at the highest application rate of 2000 μL L−1 (Table 4). At this application rate, savory and thyme oils were significantly more effective at maintaining fruit firmness after inoculation than the other three essential oils tested. There was a very strong positive linear relationship between application rate and fruit firmness for both savory and thyme (R2 = 0.93) (Table 4). Fruit treated with 2000 μL L−1 of savory and thyme Table 3. Effect of different application rates of essential oils extracted from five difference plant species on disease severity (rating of the amount of fruit decay) on papaya fruit caused by Colletotrichum gloeosporioides after 2 weeks of fruit storage at 12 ± 1 °C followed by one week at ambient temperature (24 ± 1 °C). Application rate (μL L−1) Essential oils Savory Thyme Cinnamon Lavender Mint

0 3.4az 3.4a 3.4a 3.4a 3.4a

500 2.6a 3.0b 3.0b 3.0b 3.2b

1000 2.0a 2.0a 3.0b 3.0b 3.0b

2000 1.2a 1.4a 3.0b 3.0b 2.8b

Sig. L*y Q* Q* Q* L*

R2 0.78 0.81 0.30 0.30 0.25




(a)

(b)

(c)

(d)

(e)

(f)

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Figure 1. Effect of essential oil treatments: (a) untreated control, treatment with (b) lavender (Lavandula angustifolia Mill.) oil, (c) mint (Mentha piperita Willd.) oil, (d) cinnamon (Cinnamomum zeylanicum Blume.) oil, (e) thyme (Thymus daenensis Celak. oil, or (f) savory (Satureja khuzistanica Jamzad.) oil at an application rate of 2000 μL L−1 for controlling anthrachnose caused by C. gloeosporioides Penz. in artificially inoculated papaya (Carcia papaya Linn. cv. Red Lady) fruit after 3 weeks of storage at 12 ± 1 °C followed by 1 week at room temperature (24 ± 1 °C).

Table 4. Effects different application rates of essential oils extracted from five different plant species on fruit firmness (Kgf) of papaya fruit artificially inoculated with Colletotrichum gloeosporioides after 2 weeks of fruit storage at 12 ± 1 °C followed by one week at ambient temperature (24 ± 1 °C). Application rate (μL L−1) Essential oils Savory Thyme Mint Lavender Cinnamon

0 5.8az 5.8a 5.8a 5.8a 5.8a

500 6.9a 6.9a 5.5a 6.0a 5.8a

1000 11.8a 11.8a 7.4b 6.1b 5.8b

2000 20.4a 17.6a 11.0b 8.2bc 7.6c

Sig. y

L* L* Q* Q* Q*

R2 0.93 0.93 0.64 0.77 0.40

Notes: ZDifferent letters within columns indicate significant differences among essential oils within each application rate according to Tukey’s HSD Test (p ≤ 0.05). y *indicates that the linear (L) or quadratic (Q) regression model was significant at p ≤ 0.01 for the application rate of each essential oil.

oils had fruit that were 2.5 or 2.0-fold more firm, respectively compared to the non-treated controls. (Table 4). There was a weak but significant quadratic relationship between essential oil application rate and fruit firmness for cinnamon oil (R2 = 0.40), whereas as the quadratic relationship between these variable for mint (R2 = 0.64) and lavender (R2 = 0.77) oils were stronger. Half maximal effective concentrations (EC50) In the in vitro experiment, the results indicated the lowest EC50 for savory oil (58.42 μL L−1) and thyme oil (63.69 μL L−1), while the EC50 for cinnamon, mint and lavender were 379.49, 524.78, and > 750 μL L−1 respectively (Table 5). In vivo, the lowest EC50 based on reduction of lesion diameter on the fruit was observed after application of savory (1507.19 μL L−1) and the thyme (1555.41 μL L−1) oils. The EC50 for cinnamon, mint, and lavender was > 2000 μL L−1 for each of those essential oils.

Discussion The major chemical components of each essential tested in this study were previously isolated and identified by gas chromatography-mass spectroscopy (GC-Mass Spec) (Sarkhosh et al. 2017). In the present study, savory oil with the main chemical compound carvacrol (71.2%), and thyme oil with the

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Table 5. The half maximal effective concentrations (EC50) essential oils extracted from five different plant species based on inhibition of radial growth of Colletotrichum gloeosporioides myclieum in vitro and diameter of lesion papaya fruit caused by the fungus (in vivo). Essential oil (μL L−1) Condition In vitro In vivo

Savory 58.42* 1507.19

Thyme 63.69 1555.41

Cinnamon 379.49 >2000

Mint 524.78 >2000

Lavender >750 >2000


Notes: *Probit analysis at 95% confidence limits using SPSS 10.0 statistical software.

main chemical constituent thymol (73.3%) were the most effective oils of those tested at controlling anthracnose of papaya fruit caused by C. gloesoporioides in vitro and in vivo. Essential oils have been reported to inhibit postharvest fungi, mostly under in vitro conditions (Bishop and Reagan 1998; de Billerbeck et al. 2001; Hidalgo et al. 2002), whereas the in vivo efficacy and practical activity of only a few essential oils have been studied. The antifungal and antibacterial activities exhibited by essential oils extracted from Thymus spp. have been demonstrated by several researchers (Karaman et al. 2001; Rasooli and Mirmostafa 2003; Rota et al. 2008). In papaya, inhibitory effects of thyme and Mexican lime essential oils against C. gloeosporioides and Rhizopus stolonifer in vitro and in vivo have been reported (Bosquez-Molina et al. 2010). In that study, the combination of both extracted oils in a mesquite gum-based coating were efficacious at controlling both of these pathogens and resulted in a longer shelf-life of oil-treated fruit. Also, a combination of 10% gum arabic with 0.4% cinnamon oil as a bio-fungicide to control postharvest anthracnose in banana and papaya was also reported (Maqbool et al. 2011). However, in that study phytotoxic effects were observed on the fruit when the oil was applied on fruit without gum arabic. In a study aimed at reducing postharvest decay from anthracnose in mango fruit, Abd-Alla and Haggag (2013) found that application of orange or lemon oil were more efficient for reducing the fruit decays than applications of basil or mustard oils. Prakash et al. (2015) reported fumigant toxicity of Boswellia carterii essential oil against aspergillus growth and aflatoxin contamination, which could be used in the food system as plantbased preservative. Previously, thyme (Bosquez-Molina et al. 2010) and cinnamon (Barrera-Necha et al. 2008) oils have been shown to be effective at controlling C. gloeosporioides in vitro and in stored papaya fruit, whereas mint oil was moderately effective at controlling this fungus (Barrera-Necha et al. 2008). We have found no reports of the effects of savory (Satureja khuzistanica) oil on postharvest management of anthracnose in papaya fruit. However, strong antimicrobial properties of essential oils isolated from various species of Satureja spp. on controlling different fungal pathogens on different crops than those tested in the present study have been reported (Ciani et al. 2000; Dorman and Deans 2000; Güllüce et al. 2003). Farzaneh et al. 2015 reported in vitro antifungal effects of essential oils extracted from three species of Satureja (S. hortensis, S. spicigera, and S. khuzistanica) on controlling the main pathogens of strawberry fruit; Penicillium digitatum, Botrytis cinerea and Rhizopus stolonifer. In that study S. khuzistanica oil exhibited the strongest activity against those pathogens at an application rate of 300 μL L−1. In the present study, savory oil had the lowest EC50 values (58.42 in vitro and 1507.19 in vivo) of the essential oils tested, indicating that savory oil was even more effective than cinnamon, thyme or mint oil for controlling C. gloeosporioides mycelial growth and preventing anthracnose in stored papaya fruit, and thus helping to maintain fruit firmness during storage. The biological activity of essential oils depends on their chemical composition, which is determined by plant genotype and influenced by environmental and agronomic conditions (Sivakumar and Bautista-Baños 2014). The antifungal and antibacterial activity of carvacrol and thymol, the main components of Thymus spp. and Satureja speices, respectively have been reported in other studies (Cosentino et al. 1999). The sensitivity of fungal species to essential oils are dependent on oil type and application dose, and individual compounds can exhibit antifungal activity alone or in synergy with other compounds (Plotto 2003). Ademe et al. (2013) evaluated some plant extracts against anthracnose



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in papaya and found that Echinops sp.’s extract was the most effective of those tested at reducing disease development and maintaining the overall quality of papaya fruit at an acceptable level. In the present study, C. gloeosporioides was very susceptible to savory and thyme essential oils which had completely inhibited mycelium growth at a concentration of 125 μL L−1 in petri dishes. Both savory and thyme essential oils exhibited strong anti-fungal activity, totally eliminating decay caused by this fungus at an application rate of 2000 μL L−1 for each oil. Similar to the observations in this study with papaya, in a previous study of anthracnose in avocado fruit (Sarkhosh et al. 2017), it was observed that both savory and thyme essential oils demonstrated a strong anti-fungal activity at an application rate of 2000 μL L−1 by reducing fruit decay initiated by this fungus. Although these oils were tested individually, it is possible that if both of these essential oils were tested in combination they might exhibit additive or synergistic effects on controlling mycelial growth and fruit decay at lower concentrations. During last few decades, some studies have been conducted on safety assessment of plant essential oils as antimicrobial and antioxidant substitutes, and in most cases the results exhibited no toxicity to the mammals (Prakash et al. 2015). In the present study, carvacrol and thymol were identified as the main compounds in savory and thyme, respectively. The European Commission has already registered them for use as additive compounds in the food industry (Prakash et al. 2015). However, there is a need to consider that in a few cases essential oils have showed significant cytotoxicity on mammalian cells (Unlu et al. 2010). Future studies are warranted to test savory and thyme oils in combinations, and at varying and different applications rates of each of these oils, for controlling anthracnose caused by C. gloeosporioides in papaya fruit and for prolonging shelf life.

Acknowledgments This research was supported by the Australian Government (Department of Education) and the Department of Primary Industry and Resources of the Northern Territory. Thanks to the Tropical Research and Education Center at the University of Florida for providing facilities for this study. Special thanks go to Dr. Jonathan H. Crane, Ms. Pamela Moon, Ms. Tina Dispenza, and Dr. Georgina Sanahuja for their assistance during laboratory and field experiments. This work was support, in part, by an Endeavour Fellowship from the Australian Government.

Disclosure statement No potential conflict of interest was reported by the authors.

Funding This work was supported by the Australian Government (Endeavour Fellowship, Department of Education) and Department of Primary Industry and Resources of the Northern Territory.

References Abd-Alla M, Haggag WM. 2013. Use of some plant essential oils as post-harvest botanical fungicides in the management of anthracnose disease of mango fruits (Mangifera indica L.) caused by Colletotrichum gloeosporioides (penz). Int J Agric For. 31:1–6. Abràmoff MD, Magalhães PJ, Ram SJ. 2004. Image processing with ImageJ. Biophotonics Int. 11:36–42. Ademe A, Ayalew A, Woldetsadik K. 2013. Evaluation of antifungal activity of plant extracts against papaya anthracnose (Colletotrichum gloeosporioides). J Plant Pathol Microbiol. 4:10. Bailey JA, Jeger MJ. 1992. Colletotrichum: biology, pathology and control. Wallingford: CAB Internacional; p. 388. Barrera-Necha LL, Bautista-Baños S, Flores-Moctezuma HE, Estudillo AR. 2008. Efficacy of essential oils on the conidial germination, growth of Colletotrichum gloeosporioides (Penz.) Penz. and Sacc and control of postharvest diseases in papaya (Carica papaya L.). Plant Pathol J. 7:174–178. Bishop CD, Reagan J. 1998. Control of the storage pathogen Botrytis cinerea on Dutch white cabbage ( Brassica oleracea var. capitata ) by the essential oil of Melaleuca alternifolia. J Essent Oil Res. 10:57–60. Bosquez-Molina E, Ronquillo-de Jesús E, Bautista-Baños S, Verde-Calvo J, Morales-López J. 2010. Inhibitory effect of essential oils against Colletotrichum gloeosporioides and Rhizopus stolonifer in stored papaya fruit and their possible application in coatings. Postharvest Biol Technol. 57:132–137.


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