Accepted Manuscript

Essential oil composition and antifungal activity of Melissa officinalis originating from
north-Est Morocco, against postharvest phytopathogenic fungi in apples

Y. El Ouadi, M. Manssouri, A. Bouyanzer, L. Majidi, H. Bendaif, H. Elmsellem, M.A.

Shariati, A. Melhaoui, B. Hammouti

PII: S0882-4010(17)30275-9
DOI: 10.1016/j.micpath.2017.04.004
Reference: YMPAT 2203

To appear in: Microbial Pathogenesis

Received Date: 15 March 2017

Revised Date: 1 April 2017
Accepted Date: 3 April 2017

Please cite this article as: El Ouadi Y, Manssouri M, Bouyanzer A, Majidi L, Bendaif H, Elmsellem
H, Shariati MA, Melhaoui A, Hammouti B, Essential oil composition and antifungal activity of Melissa
officinalis originating from north-Est Morocco, against postharvest phytopathogenic fungi in apples,
Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.04.004.

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 ACCEPTED MANUSCRIPT
Essential oil composition and antifungal activity of
Melissa Officinalis originating from North-Est Morocco,
against postharvest phytopathogenic fungi in apples

Y. El Ouadi1, M. Manssouri2, A. Bouyanzer1, L. Majidi2, H. Bendaif3, H.

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Elmsellem1*, M.A. Shariati4, A. Melhaoui3 and B. Hammouti1

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1
Laboratoire de chimie analytique appliquée, matériaux et environnement (LC2AME),
Faculté des Sciences, B.P.717, 60000 Oujda, Morocco

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2
Laboratoire des Substances Naturelles & Synthese et Dynamique Moléculaire, Faculté
des Sciences et Techniques, Errachidia, Morocco

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LCOMPN-URAC25, Faculty of Sciences, laboratory of Organic Chemistry,
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Macromolecular and Natural Products, University Mohamed 1st University, BP 524,
60000 Oujda, Morocco
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Research Department, LLC «Science & Education», Russia & Researcher, All Russian
Institue of Phtopatology, Moscow Region, Russia
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*Corresponding authors: E-mail: h.elmsellem@gmail.com

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Phone: (+212)0670923431
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ABSTRACT

To investigate biological control methods against post-harvest

phytopathogenic fungi in apples, tests on the antifungal activity of essential oil of

Melissa officinalis were carried out. The essential oil, obtained by hydrodistillation,

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was analyzed by gas chromatography coupled with mass spectrometry (GC-MS).

Analysis of the essential oil was able to detect 88.7% of the components. The

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main components are P-mentha-1,2,3-triol (13.1%), P-menth-3-en-8-ol (8.8%),

pulegone (8.8%), piperitynone oxide (8.4%) and 2-piperitone oxide (7.3%).

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The determination of the antifungal activity of the essential oil of M.

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officinalisis carried out in vitro using the technique of poison food (PF) and the
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volatile activity test (VA). To carry out these two tests, three phytopathogens that

cause the deterioration of apples have been selected: Botrytis cinerea, Penicillium
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expansum and Rhizopus stolonifer.

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The overall results of this study suggest that M. officinalis essential oil has
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potential as a bio-antifungal preservative for the control of post-harvest diseases

of apple.
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Keywords: Antifungal activity, Melissa officinalis, Essential oil, GC-MS analysis,

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Apple.
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1. INTRODUCTION

The therapeutic virtues of aromatic essences have been known since

antiquity. However, interest in the scientific study of the power of aromatic and

medicinal plants has only increased in recent years, with the aim of seeking

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alternatives to chemicals that present risks to human health and to the

environment.

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Several studies have demonstrated the different biological activities of

aromatic and medicinal plants, especially antifungal [1], antibacterial [2, 3],

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antioxidant [4] and insecticidal powers [5]. Thus, essential oils could be used as a

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preservative for foodstuffs. In this context, numerous studies have shown that
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extracts of certain aromatic plants have an inhibitory action on the growth and

toxinogenesis of several bacteria and fungi responsible for food infections [6, 7].
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The third most cultivated fruit crop (5280 Mha) and the third in production

(59 059 Mt) in 2004 was the apple (Malus domestica), according to Food and
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Agricultural Organization Statistics [8]. However, the quality of the apple is

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influenced by post-harvest diseases, such as blue mold caused by Penicillium

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expansum, Bull’s-eyexrot caused by Alternaria species and Rhizopus soft rot

caused by Rhizopus stolonifer [9]. Developing countries suffer from the survival of
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these pathogenic fungi in food, which leads to deterioration in the quality of food
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products. Mycotoxins that can be mutagenic, teratogenic or carcinogenic and

cause feed refusal and emesissin humans or animalssare also produced by these

fungal agents [10]. Recently, many researchers have demonstrated that essential

oils, due to their low toxicity and environmental effects and wide public

acceptance, should be used as a promising alternative to synthetic fungicides

[11].
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Melissa officinalisis a shrub of the family Lamiaceae.This Moroccan

medicinal plant is known locally as Mchechtrou. However, the antifungal activity of

essential oil of M. officinalis against post-harvest phytopathogenic fungi in apples

has not been studied previously.

The aim of this study is to test the antifungal activity of the essential oil of M

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officinalis against the following fungi: Botrytis cinerea, P expansum and

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Rstolonifera. The motivation of this studyis the wish to have natural substances

that are active for possible biological control against the deterioration of apples.

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2.2. MATERIALS AND METHODS

2.2.1. Materials
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The aerial part of M. officinalis was harvested in January 2013 in the wild in
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Taza in the Nord-East of Morocco. A voucher specimen was deposited in the

herbarium of the Faculty of Sciences, Oujda, Morocco. The dried plant material
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was stored in the laboratory at room temperature (4°C) and in the shade before
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extraction.
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Essential oil isolation

The dried vegetal material (100 g) was water-distillated (3 h) using a

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Clevenger-type apparatus. Anhydrous sodium sulfate was used to dry the

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resulting essential oil, which was then stored at 4°C in the dark before analysis.

The average yield of essential oils was 1%.

2.2.2. Hydrodistillation apparatus

The Deryng or Clevenger apparatus was used to carry out the

hydrodistillation. The extraction of essential oil from the leaves of M. officinalis

was carried out by hydrodistillation using a Clevenger type apparatus [1, 12].
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2.2.3. Characterization and chemical composition of essential oils

The GC analysis was performed using a Perkin-Elmer Auto System XL GC

apparatus (Waltham, MA, USA) equipped with a dualflame ionization detector

(FID) and fused silica system capillary columns (film length 60 m, internal

diameter 0.22 mm, film thickness 0.25 µm), Rtx-1 (polydimethylsiloxane) and

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RTX-wax (polyethylene glycol). The furnace temperature was programmed to

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increase from 60°C to 230°C at a rate of of 2°C/min and then was isothermally

maintained at 230°C for 30 min. The temperature of the injector and the detector

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was maintained at 280°C. Moreover the oil samples w ere injected using the split

mode (1/80) using helium as the vector gas (1 mL/min) and an injection volume of

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0.1 µL of pure oil. The retention (IR) indices of the compounds were determined
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with relation to the retention time of n-alkanes (C5-C30) (Restek, Lisses, France)
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by linear interpolation using the method of Van den Dool and Kratz (1963) and the

Perkin-Elmer software equation.

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The samples were analyzed with a GC/MS Perkin-Elmer turbo

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(quadrupole) mass detector coupled to a Perkin-Elmer AutoSystem XL equipped

with Rtx-1 and RTX-wax fused silica capillary. Helium was used as the carrier
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gas(1 mL/min). The temperature of the ion source was 150°C; oven temperature

programmed to increase from 60°C to 230 °C at 2°C/m in and then was

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isothermally maintained at 230°C (for 35 min); the injector temperature was

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280°C; ionization energy was 70 eV; mass spectra of the ionization electron were

acquired over the mass range 35-350 Da; the injection split was 1/80; andthe

injection volume was 0.2 µL of pure oil.

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2.2.4. Fungal strains isolation

Various fungal species were isolated and identified from the rotten apple

fruit of different refrigeration stations and local markets of the major apple

production areas in Morocco. Species included R stolonifer, Alternaria alternata,

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Fusarium oxysporum, Aspergillus fumigatus, P expansum and B cinerea [13, 14].

In the following, we limit the study to three fungal species: R stolonifer, P

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expansum and B cinerea.

B cinerea, P expansum and R stolonifer, three fungi responsible for apple

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rot, were isolated directly from rottenapples from different parts of Midelt

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(Morocco). All isolated fungal species were transferred to sterile, three-reagent, 9
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cm Petri dishes containing fresh potato dextrose medium (PDA) in the presence

of an amount of streptomycin to stop the growth of the bacteria. Plates were

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incubated in the dark for 7 days at 25 ± 2°C. Purif ication and identification of

developing fungal colonies up to species level was done by microscopic

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examination, using the following references [15]. The isolates collected were
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maintained on PDA at 4°C.

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2.2.4.1. Blue mold (Penicillium rot)

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The Penicillium rot, caused particularly by P expansum, is the main fungal

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disease of apples stored after harvest [16]. It causes awet rot that is generally

circular in shape with sharp contours of light brown; the mold is first white, and

then bluish green appears at the surface of the mold (Fig.1).

Fig.1. Apples contaminated by P expansum.

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2.2.4.2. Gray mold (Botrytis)

Gray mold, caused by the fungus B cinerea, is a fungal disease known

since antiquity. It has the characteristic of being extremely polyphagous. It is

capable of developing both its prophyte on plant debris (cankers, dried leaves and

rotten fruit). (Fig.2)

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Fig.2. Grey mold (B cinerea) on an apple.

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2.2.4.3. Rhizopus rot

Rhizopus rot or soft rot is caused by R stolonifer fungus (Fig.3). It is one of the

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most important post-harvest diseases that attack fruits like apples, pears and
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peaches [17]. Moreover, it causes severe economic losses.
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Fig.3. Symptoms of Rhizopus rot on apples

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2.2.5. Antifungal activity assay

We used the poisoned food technique (PF) [18] and volatile activity assay
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(VA) [19], with some modifications, to determine the antifungal activity of the

essential oil of M. officinalisagainst mycelial growth of the fungi that were isolated.
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The PF technique requires the dispersion of the essential oil as an

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emulsion in a sterile agar suspension (0.2%), added to PDA immediately before

being emptied into the glass Petri dishes (90 × 20 mm diameter) at a temperature

of 40-45°C. The levels tested were 0.25 to 2 µL/mL. The concentrations tested

were 0.25 to 2 µL/mL. The controls had the same quantity of sterile agar

suspension (0.2%) mixed with PDA. Moreover, the tested fungi were inoculated
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with 6 mm mycelial plugs from 7-day-old cultures cut with a sterile cork and

incubated for 60 hours for Rstolonifer, 7 days for Pexpansum, and 11 days for

Bcinera at 25±2°C.

In VA, the Petri dishes (90 ×20 mm) were filled with 20 mL of PDA medium

and then seeded with a mycelial disk (6 mm diameter) cut from the periphery of

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the 7-day mycelium culture of the fungi tested. The Petri dishes (having 80 mL air

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spaces after addition of 20 mL agar media) were inverted, and sterile filter paper

discs (9 mm in diameter) impregnated with different levels of essential oil (10, 20,

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40, 80 and 160 µL/disc) were deposited on the inverted lid and incubated for 10

days for Bcinera, 6 days for Pexpansum and 48 hours for Rstolonifer at 25±2°C.

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Water was poured onto sterilized paper filters for each corresponding control.
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According to the two techniques described above, for each treatment
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(fungi/quantity), three replicate plates were inoculated, and the experiment was

repeated three times. The mycelial growth was trackedviathe measurement of the
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diameter following two perpendicular lines passing through the center of the dish.
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The fungitoxicity of the essential oil is expressed as percent inhibition of mycelial

growth (I%) and evaluated according to the formula of Pandey et al. [20]:
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where Dt and Di represent mycelial growth diameter in control and treated Petri

plates, respectively.

Tests were carried out to evaluate the minimum inhibitory concentration

(MIC) (the lowest concentration of the essential oil that will stop the visible growth

of a microorganism after incubation at night) and the EC50 values (the

concentration at which 50% of mycelial growth inhibition ison control media).The

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EC50 value was calculated as a function of the relationship between the essential

oil concentrations and the percent inhibition of mycelial growth. The fungistatic-

fungicidal nature of the essential oil was determined by observation of resumption

of growth of the inhibited mycelial disk after its transfer to the untreated

PDA.When there is no growth, we talk about the fungicidal effect; if there is

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growth, we talk about the fungistatic effect.

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2.2.6. Transfer experiments
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In both typessof experiments, to make a distinction between fungistatic or
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fungicidal effects of the essential oil on the target organism (minimumxfungicidal

concentration [MFC] and minimum fungicidal quantity [MFQ]), the discs were
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transferred from the Petri dish where inhibition by essential oil was conducted,
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and total error of data was calculated using SAS software (SAS for Windows.

version 9.0). The separation of means was done by using the least significant
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difference test at p<0.05.

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2.2.7. Data analysis 2.2.7. Data analysis

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The inhibitory effect of essential oil on mycelial growth was analyzed by an

analysis of variance (ANOVA). Mean and standard error of data were calculated
using SAS software (SAS for Windows. version 9.0). The separation of means
was done by using the Least Significant Difference (LSD) test at p<0.05.

The inhibitory effect of essential oil on mycelial growth was analyzed by an

analysis of variance (ANOVA). Mean and standard error of data were calculated
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using SAS software (SAS for Windows. version 9.0). The separation of means

was done by using the Least Significant Difference (LSD) test at p<0.05.

3. RESULTS

3.1. Essential oil composition

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Using GC/FID and GC-MS, the chemical composition of the essential oil of

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M. officinaliswas analyzed. Thus, 55 components representing 88.7% of total

essential oil were identified by comparing their EI-mass spectra and retention

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indices with those of our own authentic compound library (Table 1).

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Table 1. Chemical constituents of Melissa Officinalis oil (%) [21].
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3.2. In vitro antifungal activity

Table 2 summarizes the effects of M. officinalis essential oil concentrations

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on mycelial growth after an 11-day incubation period for Bcinerea, 7days for P
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expansum and 70hours for R stolonifer at 25 ± 2°C, using the PF technique.

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Table 2. Percentages of inhibition of mycelial growth of Bcinera, P expansum and

R stolonifer at various concentrations of M. officinalis essential oil.

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With different concentrations of essential oil of M. officinalis, the percentage

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inhibition of mycelial growth was remarkable for all strains tested. This result

suggests that essential oil has significant activity (p <0.05) and inhibits mycelial

growth of all strains. It was clear that P expansum and Rstolonifer showed a high

sensitivity to the essential oil of M. officinalis at a concentration of 2 µL/mL. The

inhibition rate was 73.2% for P expansum at 0.25 µL/mL and 100% at 1 µL/mL,
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whereas inhibition was 16.27% for R stolonifer at 0.25 µL/mL and 100% at 2

µL/mL, indicating that these latter concentrations were the MIC of M. officinalis

essential oil against P expansum and Rstolonifera.While exhibiting high antifungal

activity against B cinerea, percent inhibition increases moderately with

concentration and reaches its maximum value of 76.81% at 2 µL/mL,

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demonstrating that its MIC is greater than 2 µL/mL. This can be attributed to the

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resistance of B cinerea against various substances present in the essential oil.The

pathogens studied can be classified according to their sensitivities to essential oil

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in the following order:

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P expansum>R stolonifer>Bcinera
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Therefore, it is necessary to know the fungitoxic nature of the essential oil

at 2 µL/mL forP expansum and R stolonifer. Indeed, the transfer of a disk of

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mycelium from the plates containing both a PDA medium and 2 µL/mL of essential
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oil on fresh PDA (without oil) showed that the mycelium of P expansum had not

grown after incubation for 11days, indicating a fungicidal effect of this oil on this
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strain (no fungistatic activity) at 2 µL/mL and a fungistatic effect at1 µL/mL.For R

stoloniferafter 60 hincubation, no mycelial growth was found at 2 µL/mL (fungicidal

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effect at 2 µL/mL) (Table 3).

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Table 3. Evolution of mycelial growth (in cm) after transfer of discs where

inhibition was complete (2 µL/mL) during 11 days of incubation of P expansum

and 60 h of Rstolonifer. Values are means (n = 3) ± standard deviations.

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Using the micro-atmosphere technique, the results of the effect of essential

oil vapor on the growth of the three strains mycelium after a 10-day incubation

period for B cinera, 6 days for P expansum and 48h for R stolonifer, at 25±2°C,

are summarized in Table 4.

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Table 4. Percentage inhibition of growth of Bcinera mycelium, P expansum and R

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stolonifer according to the quantity of essential oil of M. officinalis.

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The data indicate that the percentage inhibition increases with an

increasing quantity of essential oil forall the fungi tested, suggesting that the

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essential oil of M. officinalis inhibits the growth of all strains. In addition, the
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essential oil has antifungal activity that is significant for mycelial growth of all
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strains (p <0.05).

We also found that all the strains tested are sensitive to the vapor of the
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essential oil of M. officinalis. This volatile fraction completely suppresses the

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growth of B cinera at 40 µL/disc (QMI = 40 µL/disc), P expansum at 80 µL/disc

(QMI = 80 µL/disc) and R stolonifer at 160 µL/disc (QMI = 160 µL/disc).It is clear
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that B cinera is very sensitive to essential oil, followed by P expansum, then by R

stolonifer.In addition, M. officinalis's essential oil inhibits the three fungi better
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when tested using the micro-atmosphere technique than when using the direct
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contact technique, especially in the case of B cinera.

The mycelial disks were transferred to a PDA medium without the essential

oil after inhibition of growth with M. officinalis oil. The results for the three strains

tested are shown in Table 5.

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Table 5. Evolution of mycelial growth (in cm) after transfer of disks where

inhibition is complete after 10 days’ incubation for Bcinera, 6 days for Pexpansum

and 48 h for R stolonifer. Values are averages (n = 3) ± standard deviation.

According to Table 5, the growth of B cinera begins onthe fourth day at a

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concentration of 40 µL/disc; beyond this concentration, no growth of the mycelium

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is observed for 10 days. Thus, essential oil of M. officinalisshowed a fungistatic

activity at 40 µL/disc (QMF = 40 µL/disc) and fungicidal activity at 80 µL/disc

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(QML = 80 µL/disc).

For P expansum, at the end of the fifth day, there was mycelial growth at

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80 µL/disc; no mycelial growth was observed after treatment with essential oil at a
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concentration of 160 µL/disc. Thus, essential oil of M. officinalis showed a
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fungicidal activity against P expansum at 160 µL/disc (QML = 160 µL / disc) and

fungistatic activity at 80 µL/disc (QMF = 80 µL/disc).Finally, R stolonifer revealed

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mycelial growth on the second day at a concentration of 160 µL/disc, which

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means that the essential oil of M. officinalis has a fungicidal activity at greater than

160 µL/disc.
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4. Discussion
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Currently, synthetic fungicides are used as the primary means of controlling

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plant diseases. However, alternative control methods have become necessary,

due to the negative effect of chemicals synthesized, increased resistance of

fungal pathogens to fungicides and the high cost of creatingnew chemicals [23].

Essential oils are rich in bioactive chemicals, so they can provide possible

alternatives to currently used agents.

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The use of essential oils to control post-harvest fruit diseases has been

studied [24]. Generally, the chemical composition of the essential oil is

responsible for the degree of antifungal activity. However, it is likely that this

activity also depends on the compounds thatact in a synergistic or antagonistic

manner. Thus, all the results obtained in the present study show that the essential

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oil of M. officinalis presents an important antifungal activity against the fungi

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tested.

The major compounds of the essential oil of M. officinalis are P-mentha-

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1,2,3-triol (13.1%), P-menth-3-en-8-ol (8.8%), piperitenone oxide (8.4%) and Z-

piperitone oxide (7.3%) [21]. Previous studies have reported that monoterpenoid

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ketones derived from para-menthane have a broad spectrum of antifungal activity,
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although even small amounts of mentone may have very good antifungal
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properties [25]. Piperitone and piperidenone have antifungal activity against B

cinerea [26]. All of these compounds are believed to act synergistically [22].
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In the present work, we observed that the volatile fraction of the essential
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oil studied shows better antifungal activity against the phytopathogens tested

compared to the direct contact method on agar medium. This result is in good
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agreement with the bibliographic data. Indeed, it has been shown that the volatile

fraction of the essential oils of Rosmarinus offcinalis and Eucalyptus globulus

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strongly inhibits the growth of the mycelium of Mortierellahyalina var. hyaline,

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Penicillium roquefortii and Phomaexiqua isolated from apples [27]. Similarly, the

volatile fraction of essential oils of Origanum syriacum var. bevanii, Lavandula

stoechas var. stoechas, and R. officinalis has remarkable toxicity on B cinerea as

the direct contact liquid fraction [19]. Sharma et al, showed that the vapor of the
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essential oil of Citrus sinensis has a fungicidal action on three pathogens of the

apple, namely P expansum, Ulocladium chartarum and Alternaria mali [24].

These results can be attributed to the low solubility of essential oils in water

and hence in the agar medium due to their hydrophobic character. Thus, their

volatile and hydrophobic character renders these oils more absorbable by the

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fungal mycelium than by direct contact on agar. This behavior may be due, on the

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one hand, to the lipophilic nature of the fungal tissue and, on the other hand, to

the high water content in the agar [25]. In the direct contact phase, relatively high

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concentrations of essential oils are necessary to inhibit mycelial growth [28].

However, this higher concentration may involve effects on the organoleptic

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properties of fruit (natural taste and flavor). Thus, the use of essential oils in the
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vapor phase seems to be a promising control protocol that could be applied in
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fumigation for the control of certain post-harvest diseases in the agro-food sector

[29].
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Given the diversity of molecules present in essential oil, antifungal activity

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appears to result from a combination of several modes of action, involving

different cellular targets: For example, some compounds appear to increase

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membrane permeability, destroying the external membrane of the fungi [30, 31],

and other compounds lead to a decrease in the size of the fungi and thus a
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modification of their cell morphology [32-33].

5. CONCLUSION

The present results confirm that the essential oil of M. officinalis is an

effective antifungal agent against Bcinera, Pexpansum and Rstolonifer. Because

this type of essential oil is biodegradable and does not persist in the environment,

essential oil of M. officinalis can be considered a potential alternative to synthetic

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fungicides for the protection of apples from phytopathogenic fungi and may also

prevent the deterioration of other food products during storage. The determination

of the antifungal activity of essential oil of M. officinalis against phytopathogenic

fungi in vivo is necessary before itcan be marketed.

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Table 1. Chemical constituents of Pelargonium oil (%) [22].

Composés Ir l Ir a Ir p %
α-pinene 936 929 1019 0.3
oct-1-en-3-ol 962 961 1440 0.5
Sabinene 973 964 1117 0.4
β-pinene 978 969 1107 0.5

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Myrcene 987 980 1155 1.1
α-terpinene 1013 1008 1175 0.3
p-Cymene 1015 1011 1263 0.2

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Cineole 1,8 1024 1020 1207 0.9
Limonene 1025 1020 1196 0.6
(Z)β-Ocimene 1029 1025 1220 0.8

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(E)β-Ocimene 1041 1035 1234 0.1
γ-terpinene 1051 1048 1240 0.7
E-hydrate de sabinene 1053 1056 1457 4.6

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P-Mentha-3,8-diene 1059 1059 1259 0.4
Terpinolene 1082 1078 1277 0.2
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Z-hydrate de sabinene 1082 1083 1536 0.5
1-octen-3-ol acetate 1093 1094 1371 1.8
3-octenyl acetate 1110 1107 1328 0.2
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P-Menth-3-en-8-ol 1136 1602 8.8

Menthone 1136 1136 1483 1.0
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isomenthone 1146 1140 1457 2.7

Neomenthol 1156 1154 1637 2.4
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Terpinen-4-ol 1164 1166 1598 3.5

α-Terpineol 1176 1175 1665 0.5
Pulegone 1215 1221 1639 8.8
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Z-piperitone oxyde 1230 1235 1717 7.3

Pseudodisphenol 1245 1248 0.2
Isopulegylacetate 1 1263 1258 1631 0.2
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Neomenthylacetate 1263 1263 1524 1.9

Thymol 1266 1263 2159 0.3
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Bornylacetate 1270 1270 1572 0.3

Diosphenol 1276 1279 1790 1.0
Menthylacetate 1280 1279 1557 1.0
Piperitenone 1318 1313 1900 1.6
Piperitenone oxyde 1335 1341 1939 8.4
E-jasmone 1364 1369 1890 0.2
α-Copaene 1379 1375 1483 0.1
Nepetalactone 1360 1379 1978 1.2
P-Mentha-1,2,3-triol 1395 1880 13.1
α-Gurjunene 1413 1409 1528 0.1
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E-Caryophyllene 1421 1420 1591 2.8
Cadina-3,5-diene 1448 1441 0.3
Trans-β-farnesene 1446 1448 1661 0.2
α-Humulene 1455 1450 1665 0.3
Cis Muurola-4(14),5-diene 1462 1458 0.7
Germacrene D 1479 1478 1706 3.4
Bicyclogermacrene 1494 1491 1725 0.4
γ-Cadinene 1507 1505 1756 0.1

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Calamenene 1517 1509 1817 0.3
δ-Cadinene 1520 1514 1752 0.1
Spathulenol 1572 1564 2101 0.4

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Caryophyllene oxyde 1578 1569 1965 0.5
Viridiflorol 1592 1581 2064 0.3

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1,10-di-epi-Cubenol 1615 1603 2040 0.4
α-cadinol 1643 1638 2209 0.4
Total 88.7
IR/apol = indice retention on the apolar column (Rtx-1)

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% apol= relative percentages of components
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Table 2. Percentages of inhibition of mycelial growth of B.Cinera , P. expansum and R.

stolonifer at various concentrations of Melissa Officinalis essential oil.

Strain B.cinerea P. expansum R. stolonifer

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Concentration Incubation Times
(µL/mL)

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11 Days 7 Days 60 Hours
25 ± 2 °C 25 ± 2°C 25 ± 2 °C

76.81±2.27Aa 100 ±0.00 Aa 100 ±0.00 Ba

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42.59±3.40Ab 100 ±0.00 Aa 85.35±3.97 Bb
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35.18±1.60Ac 35.02±5.55Bc
Ab
0,50 84.46±4.87
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9.03±3.04 Ad
0,25 73.20±4.29Ab 16.27±4.57Bd
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Table 3. Evolution of mycelial growth (in cm) after transfer of discs where inhibition was
complete (2 µL/mL) during 11 days of incubation of P. expansum and 60 h of R.stolonifer.
Values are means (n = 3) ± standard deviations.

Strain P. expansum
EO

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quantity Incubation Times (Days)
(µL/mL)
1 2 3 4 5 6 7 8 9 10 11
2 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00.00 0.00±0.00

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1 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 1.56±0.36 2.36±1.12 3.56±2.03 5.56±1.23 6.23±3.21 7.11±3.89

Strain R. stolonifer

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EO
quantity Incubation Times (Hours)
(µL/mL)
12 24 36 48 60

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Table 4. Percentage inhibition of growth of B.cinerea mycelium, P. expansum and R. stolonifer

according to the quantity of EO of Melissa Officinalis.
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Strains B.cinerea P. expansum R. stolonifer

EO
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Concentration
Incubation Times
(µL/Disc)
10 Days 6 Days 48 Hours
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25 ± 2 °C 25 ± 2°C 25 ± 2 °C

160 100 ±0.00Ba 100 ±0.00Aa 100 ±0.00Ba

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80 100 ±0.00Ba 100 ±0.00Aa 61.10 ±3.67 Bb

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40 100 ±0.00Ba 67.44 ±4.56Ab 57.47±7.30Bc

20 85.46±4.87 Bb 64.85 ±7.01Ab 39.26 ±1.78Bd

10 79.15±2.01 Bb 47.52±12.37Ab 19.69±2.17 Be

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Table 5. Evolution of mycelial growth (in cm) after transfer of disks where inhibition is
complete after 10 days incubation for B.cinera, 6 days for P.expansum and 48 h for R.
stolonifer. Values are averages (n = 3) ± standard deviation.

Strain B. Cinera

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EO
quantity
Incubation Times (Days)
(µL/disc)

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1 2 3 4 5 6 7 8 9 10
40 0.00±0.00 0.00±0.00 0.00±0.00 1,22±0.56 2,33±1.23 3,56±0.19 4,02±1.00 4,21±2.00 4,68±1.87 5,22±0.24

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80 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00

160 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00

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Strain P. Expansum
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EO
quantity
Incubation Times (Days)
(µL/disc)
1 2 3 4 5 6
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80 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 1.25±0.36 2.33±0.00

160 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00

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Strain R. Stolonifer
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HEO
quantity
Incubation Times (Hours)
(µL/disc)
12 24 36 48
EP

160 0.00±0.00 0.00±0.00 1.21±1.01 3.98± 1.02

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Fig.1. Apples contaminated by Penicillium expansum.

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Fig.2. Grey mold (Botrytis cinerea) on an apple.
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Fig.3. Symptoms of Rhizopus rot on apples
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• The main detected components in essential oil of M officinalis were P-mentha-1,2,3-triol

(13.1%), P-menth-3-en-8-ol (8.8%), pulegone (8.8%), piperitynone oxide (8.4%) and 2-piperitone
oxide (7.3%) as detected antifungal compounds.
• the essential oil of M officinalis is an effective antifungal agent against Bcinera, Pexpansum and
Rstolonifer.

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