INFLUENCE OF EIGHT CHOSEN ESSENTIAL OILS IN THE VAPOR PHASE ON THE GROWTH OF RHIZOPUS STOLONIFER AND RHIZOPUS LYOCOCCUS

This study aimed to evaluate the fungicidal effect of eight essential oils against five strains of the genus Rhizopus. Strains were obtained from various moldy foods, namely Rhizopus stolonifer KMi 383 from chestnut, R. stolonifer KMi 510 from strawberry, R. stolonifer KMi 511 from nectarine, R. stolonifer KMi 524 from cherry tomatoes, and R. lyococcus KMi 512 from blackberry. The essential oils (EO) used in this study were jasmine EO (extract from Jasminum officinale L.), bergamot EO (Mentha aquatica L. var. citrata (Her.) Fresen), bitter orange EO (from Citrus aurantium L.), grapefruit EO (Citrus paradisi Macfady), sweet flag EO (East Asian Calamus, from Acorus calamus L. var. angustatus Bes), star anise EO (from Illicium verum J.D.Hook), geranium EO (from Pelargonium graveolens), and lemongrass EO (from Cymbopogon citratus DC). The semi-quantitative composition of the essential oil samples was determined by gas chromatography coupled with mass spectrometry (GC-MS). The antifungal activity of essential oils against the strains of R. stolonifer and R. lyococcus was determined during 7 days, using the micro-atmosphere method (0.625 μL.mL-1 of air). Two essential oils, geranium and lemongrass, completely inhibited the growth of all isolates. Bitter orange essential oil inhibited the growth of all isolates of Rhizopus stolonifer, but isolate of Rhizopus lyococcus began to grow after four days of cultivation. In conclusion, certain essential oils are highly effective in the vapor phase. These could be used in further tests of their antifungal activity and could be used in the control of Rhizopus spp. or other fungal pathogens.


INTRODUCTION
Rhizopus species including R. stolonifer are naturally found in soil, debris, and air. Fungal dispersal mechanisms of R. stolonifer are wind, air currents, and some invertebrates such as mites and insects, among others (Bautista-Baños et al., 2014). Rhizopus stolonifer is a ubiquitous fungus and can be isolated from many kinds of foods. It grows rampantly at 25 o C, filling a Petri dish with sparse, dark mycelium in 2 days. It produces barely macroscopic aerial fruiting structures which are at first white, then become black. Given seven undisturbed days, it sheds dry black spores outside the Petri dish, providing an effective inoculum for a continuous chain of future contamination (Pitt and Hocking, 2009). Rhizopus rot is common on soft fruits, more abundant in warm humid climates than in cool climates. In several fruits and crops such as strawberries, peaches, avocados, tomatoes, cucumbers, and table grapes, Rhizopus rot causes soft rot during transport and storage (Kassemeyer and Berkelmann-Löhnertz, 2009). Infection usually starts from wounding after the cracking of fruits. At first, the lesions (soak with water) are rapidly softened and diseased lesions gradually expanded. The mycelia grow vigorously on the surface of fruits and formed stolons. Colonies are white cottony at first, becoming heavily speckled by the presence of sporangia and then brownish-black. They spread rapidly using stolons fired to various points of the substrate and attach by rhizoids. The color of sporangia is white at first and then turns black with many spores (Kwon et al., 2001). The postharvest handling operations are the main reason R. stolonifer succeeds in entering and infecting most horticultural commodities (Bautista-Baños et al., 2014).
Significant postharvest losses occur during the supply chain on fresh produce. Postharvest decay is one of the main factors that determine losses and compromises the quality of fruits and vegetables. Traditionally, postharvest decay control is achieved using chemical fungicides; however, the important concerns relating to environmental and human health require the development of novel methods for the control of postharvest decay (Mari, Bautista-Baños and Sivakumar, 2016). The growing awareness of consumers concerning the relationship between food and health revolutionized the food industry. New techniques such as high pressure, nanotechnology, irradiation, etc., are increasingly used to maximize the nutritional properties of foods, while new ingredients with functional properties contribute to improving health. The ''elimination" of additives used in a wide variety of foods is demanded, while ''natural" additives are seen as a benefit for both quality and safety (Viuda-Martos et al., 2008). Over the past few years, consumers demand safe, environmentally-friendly, and natural products. It has driven the search for preservation techniques that improve product quality and safety without causing nutritional or sensory losses. Natural antimicrobial essential oils have the potential to provide quality and safety benefits and have fewer impacts on human health ( The present research aimed to determine the inhibitory effect of chosen essential oils on the growth of different Rhizopus stolonifer and Rhizopus lyococcus strains.

Scientific hypothesis
The chosen essential oils can affect the growth of Rhizopus strains.

Chemical composition of essential oils
The relative composition of essential oils was determined, and the compounds were identified by gas chromatography with mass spectrometry (GC-MS). Essential oils were diluted in hexane to a concentration of l μL.mL -1 . Analyses were carried out using an Agilent 7890A GC coupled to an Agilent MSD5975C MS detector (Agilent Technologies, Palo Alto, CA, USA) with an HP-5MS column (30 m × 0.25 mm, 0.25 mm film thickness). One microliter of the sample was injected in split mode 1:12, at an injector temperature of 250°C and electron ionization energy of 70 eV. Analysis were measured in SCAN mode, the mass range was 40 -400 m/z. Starting at 60 °C, the oven temperature was increased at a rate of 3 °C/min to a maximum of 231 °C, where it was kept constant for 10 min. The identification of constituents was based on a comparison of their mass spectra and relative retention indices (RI) against the National Institute of Standards and Technology Library (NIST, USA), as well as authentic analytical standards and data from the literature. Relatively proportion of EO constituents were assessed by Agilent 6890 GC-FID (Agilent Technologies, Palo Alto, CA, USA) with RTX5 column (Restek, Bellefonte, PA; 20 m × 0.18 mm, 0.2 µm film thickness). The same method for GC-MS was used. Relative proportions were calculated by dividing individual peak area by total area of all peaks. The response factor was not taken into account. Only compounds over 0.1% were included. The used standards are listed in Table 1.

Antifungal activity of essential oils
The micro atmosphere method was used to study the effect of essential oils on the growth of strains of Rhizopus sp. The test was performed in sterile plastic Petri dishes (Ø 90 mm) containing 15 mL of potato dextrose agar (PDA, HIMEDIA India). 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 sterile filter paper (cca1.5 x 1.5 cm) was placed in the lid of the Petri dish and 50 μL of essential oil was pipetted by micropipette to the paper. Dishes were kept in an 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 on the center of Petri dishes with 5 µL of spore's suspension (10 5 spores in 1 mL). 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 on the 2 nd , 4 th , and 7 th day with a digital caliper.

Inhibition of mycelial growth
According to (1): Where: C is the mean of six replicates of hyphal extension (mm) of controls; T is the mean of six replicates of hyphal extension (mm) of plates treated with either essential oil.

Minimum inhibitory doses (MIDs)
Essential oils that completely inhibit the growth of all strains of R. stolonifer or strain of R. lyococcus were used to determine their minimum inhibitory doses (MIDs). EOs dissolved in dimethylsulfoxide (DMSO) were prepared at different concentrations (500, 250, 125, 63, 31.25, and 15.63 μL.L -1 of air). For each fungal strain, a conidial spore suspension of 10 6 spore in mL -1 was prepared. The EVE TM Automatic cell counter (NanoEnTek, Korea) was used to determine the number of spores. Petri dishes (Ø 90 mm, three-sector, two replicates) containing 15 mL of PDA were inoculated by 5 µL spore suspension. Cultivation was carried out at 25± 1 °C and measured after 7 and 14 days. The MID (expressed as microliters of EOs per volume unit of the atmosphere above the organism growing on the agar surface) was defined as the lowest concentration of the oil which did not permit any visible growth after 7 or 14 days in comparison with control sets.

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 the control group was evaluated too. The results were mathematically processed using the Microsoft Excel program and statistically evaluated by SAS/9.3 (2010). A used statistical model can be written in the following form: yij = μ + ISOLATEi /TREATMENTj/ + eij Where: 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).

Probit analyses
The ability of strains to grow in the presence of EO was coded to a binomial scale (1 -growth observed, 0without growth). Such data were processed by probit analysis in Statgraphics Centurion XV (Statgraphics) software. Doses that inhibit the growth in 50% respectively 90% of cases (MID50 and MID90) were reversely predicted from the regression equation.

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 the production of essential oils (Bhattacharya, 2016). In this research, we evaluated the antifungal properties of 8 essential oils from families Oleaceae (jasmine EO), Rutaceae (bergamot EO, bitter report significantly higher antimicrobial activity of some essential oils, which we observed in the vapor phase. In our research, we also used the vapor phase to test the effect of selected essential oils on Rhizopus growth. The antifungal activity of 7 essential oils against the Rhizopus stolonifer (4 strains) and Rhizopus lyococcus (1 strain) were determined, using the micro-atmosphere method (625 μL.L -1 of air). The results are shown in Table 2 and Figure 1.
Two essential oils: lemongrass (Cymbopogon citratus DC) and geranium (Pelargonium graveolens) completely inhibited the growth of all strains. Lemongrass (Cymbopogom citratus DC) essential oil is known due to its broad-spectrum antimicrobial activity (Leimann et al.,

2009). According to Abdulazeez, Abdullahi and James (2016), Božik et al. (2017), Císarová et al. (2020)
lemongrass oil has been also shown to be an effective fumigant for stored food commodities due to its bioactivity in the vapor phase. Lemongrass EO was found to significantly reduce colony development against key postharvest pathogens: Colletotrichum coccodes, Botrytis cinerea, Cladosporium herbarum, Rhizopus stolonifer, A. flavus, A. parasiticus, A. ochraceus, A. westerdijkiae, and Aspergillus niger in vitro. Lemongrass essential oil at concentrations of 0.5% and 1.0% was incorporated into 0.5% and 1.0% chitosan solution and evaluated as means of controlling anthracnose of bell pepper in vitro and in vivo. Note: a -identification confirmed by co-injection of authentic standard, GS-MS -gas chromatography coupled with mass spectrometry, B -bergamot, BO -bitter orange, GP -grapefruit, GR -geranium, L -lemongrass, J -jasmine, AN -star anise, S -sweet flag.
Fungal growth was effectively controlled by 0.5% and 1.0% EO in vitro (Ali, Noh and Mustafa, 2015). Moore-Neibel et al. (2011) described the antimicrobial activity of lemongrass oil against Salmonella directly on the leafy greens, romaine, and iceberg lettuces, and both mature and baby spinach described.
As mentioned above, the geranium EO completely inhibited the growth of the Rhizopus strains used in this study, as well. This essential oil has the potential to be used in the food industry to prolong the shelf life of fresh and processed foods (Verma, Chandra Padalia and Chauhan, 2016). Bouzenna and Krichen (2013) tested the antifungal activity Pelargonium graveolens Eo against Rhizoctonia solani, and results showed that the essential oil was highly active at a dose of 12.5 µL.20 mL -1 of PDA. Naeini, Nazeri and Shokri (2011) reported that P. graveolens EO has considerable anti-Malassezia activities.
Bitter orange (Citrus aurantium L.) essential oil completely inhibited the growth of Rhizopus stolonifer strains. But we recorded growth on day seven of cultivation at the Rhizopus lyococcus strain, in the presence of this essential oil. Bitter orange oil has been reported to possess various pharmacological properties. Properties of bitter orange oil for food preservation are discussed too (Anwar et al., 2016). In contrast with bitter orange EO, grapefruit essential oil inhibited the growth of R. lyococcus completely but only weakly Rhizopus stolonifer. Ng et al. (2016) show that grapefruit oil (Citrus paradisi) exhibits an array of activities encompassing insecticidal and antimicrobial activities. The strains of the test species responded differently to the presence of bergamot EO. Significant antifungal activities were observed in all Rhizopus stolonifer strains. The strain of Rhizopus lyococcus began to grow intensively after two days of cultivation and no growth inhibition was found on the fourth day of cultivation. Some authors e.g. Avila-Sosa et al. (2016), report that bergamot essential oil may be very helpful when applied to food preservation systems. Jasmine (Jasminum officinale L.) and sweet flag (Acorus calamus L. var. angustatus Bes) essential oils did not inhibit the effect on the growth of tested strains genus Rhizopus. Apart from other uses of jasmine essential oil, it is active against various gram-negative, gram-positive bacteria and fungi. This property of jasmine oil allows it to be used in food preservation. It also possesses antioxidant activity ( Table 4. The most effective tested essential oil was lemongrass, less effective bitter orange EO for R. stolonifer strains, and grapefruit EO for R. lyococcus strain. The specific compounds isolated from the oils may be non-fungi toxic in nature. Different components of the oils as such may also check the development of races of fungi during their application due to more than one site of action. Fungi can easily develop resistant races against a single component due to its specific mode of action. The exploitation of the essential oils as such would be more economical than a single component as a fungitoxicant (Tripathi and Shukla, 2009). The safe use of EO ingredients is also supported by their self-limiting properties as flavoring substances in food resulting in low levels of use; their rapid absorption, metabolic detoxication, and excretion in humans and other animals; the wide margins of safety between the conservative estimates of intake and the no observed-adverse effect levels determined from subchronic and chronic studies and the lack of significant genotoxic, developmental and teratology potentials (Adams et al., 2011).

CONCLUSION
In our research, we evaluated the antifungal properties of jasmine EO (extract from Jasminum officinale L.), bergamot EO (Mentha aquatica L. var. citrata (Her.) Fresen), bitter orange EO (from Citrus aurantium L.), grapefruit EO (Citrus paradisi Macfady), sweet flag EO (East Asian Calamus, from Acorus calamus L. var. angustatus Bes), star anise EO (from Illicium verum J.D.Hook), geranium EO (from Pelargonium graveolens), and lemongrass EO (from Cymbopogon citratus DC), two essential oils: geranium and lemongrass completely inhibited the growth of all isolates. Bitter orange essential oil inhibited the growth of all isolates of Rhizopus stolonifer, but isolate of Rhizopus lyococcus began to grow after four days of cultivation. In conclusion, certain essential oils are highly effective in the vapor phase. These could be used in further tests of their antifungal activity and could be used in the control of Rhizopus spp. or other fungal pathogens. In further research, we plan to test the effect of selected essential oils in in vitro (on selected fruits). In the next part we will test the impact of EOs on sensory quality of selected fruits.