EFFECT OF ESSENTIAL OILS OF LAMIACEAE PLANTS ON THE RHIZOPUS SPP

The aim of this study was to evaluate the fungicidal effect of eleven essential oils against six isolates of the genus Rhizopus. Isolates were obtained from various moldy foods (chestnut, bread, strawberry, nectarine, blackberry and cherry tomatoes). The essential oils used in this study were extracts of basil (Oscimum basilicum L.), hyssop (Hyssopus officinalis L.), lavender (Lavandula angustifolia MILLER.), marjoram (Origanum majorana L.), mint (Mentha piperita L.), oregano (Origanum vulgare L.), rosemary (Rosmarinus officinalis L.), sage (Salvia officinalis L.), summer savory (Satureja hortensis L.), thyme (Thymus vulgaris L.) and wild thyme (Thymus serpyllum L.). Semi-quantitative composition of the essential oil samples was determined by gas chromatography coupled with mass spectrometry (GC-MS). The GC-MS analyses of the essential oils led to identification of 139 compounds, of which 49 were presented in ≥1% amount in at least one essential oil. The antifungal activity of essential oils against the Rhizopus spp. was determined, using microatmosphere method (0.625 μL.ml of air), during 7 days. Seven essential oils: thyme, mint, summer savory, lavender, marjoram, oregano and wild thyme completely inhibited the growth of all isolates. Other essential oils have different effects on the growth of isolates. Basil essential oil stimulated growth of two isolates on the second day of cultivation. The growth of other isolates was, by contrast, inhibited by this essential oil in the same time of cultivation. Hyssop essential oil completely inhibited growth of two isolates, other 4 isolates were inhibited to fourth day of cultivation. In conclusion, certain essential oils are highly effective in vapour phase and can be used in another test of their antifungal activity and could be used in control of Rhizopus spp. or other fungal pathogens.


INTRODUCTION
Due to an increasing risk of chemical contamination upon the application of synthetic fungicides, to preserve fresh fruits and vegetables, essential oils are gaining increasing attentions (Farzaneh, 2015).Today, it is very important to find out the protection of products of natural origin as an alternative to synthetic fungicides.The promising alternative is the use of the essential oils.Essential oils from plants have great potential as a new source of fungicide to control the pathogenic fungi (Císarová et al., 2016, Nikkhah et al., 2017).In the past decade, due to concerns regarding safety of the chemical control measures, particular attention has been given to the potential applications of essential oils as alternative (Nikkhah et al., 2017).
Essential oils are odiferous, highly volatile substances present in plants.Because of their volatility, these substances can be isolated by means of steam distillation from an aromatic plant of a single botanical species and can be detected by both smell and taste.Individual essential oils are known by the name of the plant from which they are derived and the odor is similar to that of the part of the plant from which they are obtained, although the aroma is generally more intense (Ríos, 2016).
The use of essential oils is extremely diverse depending on the source, quality, extraction procedure, ect.Essential oils have proven industrial applications in the manufacture of perfumes, cosmetics, soaps, shampoos, or cleaning gels.Another interesting aspect of these oils is their potential as therapeutic agens in aromatherapy or as active principles or excipients of medicine.Another significant application of essential oils is in the agrofood industry, both for producing beverages and for flavoring foods (Ríos, 2016).
Fruit and vegetables are rich in essential vitamins, minerals, fibre, and health promoting compound, and the consumption thereof increased during the past years.
Consumers have a right to good quality produce that is safe for consumption and therefore they are increasingly interested in the nutritional value, good taste and flavour of the fruits and vegetables they consume.According to consumers, the term "quality" can be defined as a fruit with a perfect shape, size, colour, aroma, and an absence of defects such as cuts, bruises or decay (Sivakumar and Bautista-Baños, 2014

Scientific hypothesis
The aim of the present research was to determine the inhibitory effect of eleven essential oils to growth of different Rhizopus isolates.

MATERIAL AND METHODOLOGY Fungal culture
Isolates from moldy foods were transfer on the potato dextrose agar (PDA, HIMEDIA India) and after incubation (25 ±1 °C, 7 days) were identificated to the genus according

Chemical composition of essential oils
Semi-quantitative composition of the essential oil samples was determined by gas chromatography coupled with mass spectrometry (GC-MS) using an Agilent 7890B oven coupled with Agilent 5977A mass detector (Agilent Technologies Inc., Palo Alto, CA, USA) and CombiPal autosampler 120 (CTC Analytics AG, Zwingen, Switzerland).Prior to the analysis, essential oil samples were diluted in hexan (HPLC ≥97%, Sigma Aldrich GmbH, Germany) to a concentration of 10 µL.mL -1 .One microliter of diluted sample was injected in inlet operated in split mode (1 : 10; 250 °C).Separation was achieved using a ZB-WAXplusTM capillary column (10 m × 0.1 mm × 0.10 µm) (Phenomenex Inc., Torrance, CA, USA) and the following oven temperature programme: 50 °C for the first 5 minutes, increased to 240 °C at the rate of 3 °C min -1 , where it was kept constant for 2 minutes.Helium was used as carrier gas at the constant flow (1.2 mL.min -1 ).The mass detector parameters were as follows: ionization energy of filament: 70 eV, transfer line temperature: 250 °C, MS source temperature: 230 °C, quadrupole temperature: 150 °C.The mass spectrometer was programmed under electron impact (EI) in a full scan mode at m/z 40 -400.The identification of compounds was carried out by comparing of mass spectra (over 80% match) with a commercial database NIST® 2014 and retention times of reference standards (nerol, linalool, geraniol, citral, α-pinene and β-pinene).Semi-quantitative content of determined compounds were calculated by dividing individual peak area (excluded by solvent peak area) by total area of all peaks.Peaks under 0.1% were not counted.

Antifungal activity of essential oils
The antifungal activity of selected essential oils was investigated by microatmosphere method.The test was performed in sterile plastic Petri dishes (Ø 90 mm) containing 15 mL of PDA.Evaluation by filter paper was made by the method adapted from Guynot et al. ( 2003).Essential oils were tested in concentration 0.625 μL.cm -3 of air.A round sterile filter paper (Ø 9 cm) was placed in the lid of Petri dish and 50 μL of essential oil was pipetted by micropipette to the paper.Dishes were kept in inverted position.Filter paper discs impregnated with sterilized distilled water were used as a control to confirm no solvent effect of bioactivity.Each isolate was inoculated in the center of Petri dishes with needle.Dishes were tightly sealed with parafilm and incubated for seven days at 25 ±1 °C (three replicates were used for each treatment).Diameters (Ø mm) of the growing colonies were measured at the 2nd, 4th and 7th day with a digital capiler.

Inhibition of mycelial growth
According where, C is the mean of six replicates of hyphal extension (mm) of controls and T is the mean of six replicates of hyphal extension (mm) of plates treated with either essential oil.

Statistical analysis
The size of colonies of isolates (mm) for each day of cultivation within treatment was evaluated.Also the size of colonies of isolate for each treatment to the same isolate in control group was evaluated too.The results were mathematically processed using the Microsoft Excel program and statistically evaluated by SAS/9.3 ( 2010).Used statistical model can be written in the following form: yij = μ + ISOLATEi /TREATMENTj/ + eij, yij = the measurements for size of colonies, μ = overall mean, ISOLATEi = the fixed effects of isolates (i = 1 to 6), TREATMENTj = the fixed effect of treatment (j = 1 to 5), eijk = random error, assuming eijkl ~ N(0, I σe2).

RESULTS AND DISCUSSION
According to market data, there are about 400 species, from 67 plant families, which are cultivated on a large commercial scale for production of essential oils.The most important families from this point of view are Asteraceae (syn.Compositae), Lamiaceae (syn.Labiatae), and Table 1 Essential oils tested for the fungicidal effect and their compounds (%)* determined by gas chromatography coupled with mass spectrometry (GC-MS).The antifungal activity of 11 essential oils against the Rhizopus spp. was determined, using micro-atmosphere method (0.625 μL.cm -3 of air).Seven essential oils: thyme (Thymus vulgaris L.), mint (Mentha piperita L.), summer savory (Satureja hortensis L.), lavender (Lavandula angustifolia MILLER.),marjoram (Origanum majorana L.), oregano (Origanum vulgare L.) completely inhibited the growth of all isolates.Other essential oils (Table 2, Figure 2) have different effects on the growth of Rhizopus isolates.Basil essential oil stimulated growth of two isolates (KMi 511and KMi 512) on the second day of cultivation.The growth of other isolates was, by contrast, inhibited by this essential oil in the same time of cultivation.Hyssop essential oil completely inhibited growth of two isolates (KMi 510 and KMi 512), other 4 isolates were inhibited to fourth day of cultivation.2008) tested addition of different concentrations of marjoram essential oils in sausage on the aerobic heterotrofic bacteria´s growth.A significant reduction in the number of CFU was noted during the first time step (10 days) after a 35-day storage period.At the end of the storage period it was observed that the lowest oil concentration exerted not only antimicrobial activity but also a bactericidal effect, which may as account for the extended shelf-life of certain products.Michalczyk et al. (2012) reported that the most signicicant benefit of applying essential oils (coriander and hyssop) to stored ground beef was to inhibit undesirable sensory changes and the growth of Enterobacteriaceae bacteria (by 1 -2 log cycles), especialy at 6 ±1 o C. Based on our results and the results of other authors, it is important to test not only different species but also different isolates of the same species of moulds to gain important insights into the antifungal properties of essential oils.

CONCLUSION
In this study, we evaluated the antifungal properties of basil (Oscimum basilicum L.), rosemary (Rosmarinus officinalis L.), thyme (Thymus vulgaris L.), mint (Mentha piperita L.), savory (Satureja hortensis L.), sage (Salvia officinalis L.), lavender (Lavandula angustifolia MILLER.),marjoram (Origanum majorana L.), wild thyme (Thymus serpyllum L.), oregano (Origanum vulgare L.) and hyssop (Hyssopus officinalis L.) essential oils.Seven essential oils: thyme, mint, summer savory, lavender, marjoram, oregano and wild thyme completely inhibited the growth of all isolates.Other essential oils demonstrated different effects on the growth of isolates.Basil essential oil stimulated growth of two isolates on the second day of cultivation.The tested antifungal activity (against the Rhizopus spp.) of essential oils can be presented as: lavender = marjoram = mint = oregano = savory = thyme = wild thyme ˃hyssop ˃sage ˃rosemary ˃basil.In conclusion, certain essential oils are highly effective in vapour phase and can be used in another test of their antifungal activity and could be used in control of Rhizopus spp. or another fungal pathogens.
Samson et al. (2010), Pitt and Hocking (2009).These isolates belong to the collection of microorganisms at the Department of Microbiology of the Slovak Agricultural University in Nitra.
to Cakir et al. (2005) and Kordali et al. (2008) growth inhibition of treated samples (T) against control (C) was calculated by percentage of growth inhibition using the following equation: % of inhibition =  −    100

Mari et al., 2016; Sivakumar and Bautista-Baños, 2014).
). Postharvest diseases are one of the major causes for the postharvest loss of horticultural fresh produce during the supply chain (Rhizopus stolonifer is one of the most common and fastest-growing species in the Zygomycota phylum.Disease caused by this fungus is known as soft rot, black mould and Rhizopus rot (

Bautista-Baños et al., 2014).
Rhizopus rot is common on soft fruits, more abundant in warm, humid climates than in cool climate.In several fruits and crops such as strawberry, peaches, avocados, tomato, cucumber and table grapes Rhizopus rot causes soft rot during transport and storage (

Ben Farhat, et al., 2016; Méndez-Tovar et al., 2016; Dušková et al., 2016), the
growing seasons, different growth stage of plants, climatic conditions of each years in terms of the essential oil content and composition were proven.Based on the above, we also focused on the composition of the essential oils used.The GC-MS analyses of the essential oils led to

Table 2
Among all oils tested, savory, oregano and thyme oils proved to be the best inhibitors of all tested pathogenes.These three essential oils completely inhibited the growth of Rhizopus isolates in our experiment as well.As in our experiment, they did not notice a significant effect of basil oil on the testing fungi.Rattanapitigorn et at.(2006) reported that although the antifungal activity of plant essential oils is documented, the dose response of the inhibition effect varies widely, depending on the type of essential oils, extraction method, and antifungal test Effect of essential oils (treatment) on the growth of Rhizopus isolates.

savory Oregano Wild thyme KMi 383 KMi 392 KMi 510 KMi 512 KMi 524
different letters are significant within treatment at the level p <0.05.Asignificant difference (p <0.05) of the same isolate within all treatments to the same isolate in control.No.1/2018 method.Influence of essential oils addition to the food tested several scientists.Busatta et al.(