Effects of ethanol and ultraviolet-c treatments on inactivation of Rhizopus oryzae spores which cause postharvest rot

Ultraviolet light (UV) is classified based on wavelength as UVA (320-400 nm), UVB (280-320 nm), UVC (200-280 nm) and UVV (100-200 nm) (Guerrero-Beltrán & Barbosa-Cánovas, 2004). UV treatment is a physical method which produces neither undesirable by-products nor chemical residues (Keyser et al., 2008), therefore, this technology is widely used in the food industry for disinfection of air, control of contamination on the surface of plants and packaging materials or in post-harvest storage of fruits and vegetables (Begum et al., 2009). The mechanism of UV effect may vary according to species. UV radiation in the range of 250-260 nm is germicidal for most microorganisms, including fungi, yeast, bacteria, viruses, protozoa and algae (Begum et al., 2009).


Introduction
There are several methods to control postharvest diseases on fresh food and agricultural products.Chemical treatments are effective but restricted in most countries.On addition, consumers are looking for products free of chemical residues.Consequently, safe alternative control strategies, such as antagonists, natural compounds, and physical treatments have drawn attention (Cia et al., 2007).
Gamma and ultraviolet light irradiations are physical treatments that can be used for the control of postharvest diseases.Besides exhibiting fungicidal effects, these treatments can also induce resistance in fruits (Wilson et al., 1994).
Ultraviolet light (UV) is classified based on wavelength as UVA (320-400 nm), UVB (280-320 nm), UVC (200-280 nm) and UVV (100-200 nm) (Guerrero-Beltrán & Barbosa-Cánovas, 2004).UV treatment is a physical method which produces neither undesirable by-products nor chemical residues (Keyser et al., 2008), therefore, this technology is widely used in the food industry for disinfection of air, control of contamination on the surface of plants and packaging materials or in post-harvest storage of fruits and vegetables (Begum et al., 2009).The mechanism of UV effect may vary according to species.UV radiation in the range of 250-260 nm is germicidal for most microorganisms, including fungi, yeast, bacteria, viruses, protozoa and algae (Begum et al., 2009).
The UVC germicidal effect is mainly at the nucleic acid level and the mechanism of inactivation is by causing a physical shifting of electrons and breaking of bonds in the DNA (Lopez-Malo & Palou, 2005).
There are many studies about germicidal effect of UVC on fungi, especially those that cause decay on postharvest products.Botrytis cinerea, Monilinia fructigena, Monilinia fructicola, Penicillium digitatum were reported as postharvest spoilage fungi of some fruits like strawberries, cherries, peaches and tangerine, respectively (Marquenie et al., 2002;Stevens et al., 1997Stevens et al., , 1998)).Additionally, Liu et al. (1993) showed that Alternaria alternata, B. cinerea and Rhizopus stolonifer which cause tomato diseases, were effectively eliminated by UVC light treatment.
There are variations in the response of fungal species to UV radiation.The UV radiation induces the production of melanin in some fungal species.Melanin is a dark-photoprotective pigment found in animals, plants and microorganisms and increases the survival and longevity of fungal spores (Bell & Wheeler, 1986).Penicillium janthinellum produces slightly pigmented conidia which are more susceptible to UVC than dark pigmented Aspergillus niger (Valero et al., 2007).
Among chemical treatment methods, immersion of fruits in ethanol solutions has been reported to effectively control postharvest decay caused by fungi (Gabler et al., 2004).The major target of ethanol as a stress agent is the lipid membrane, but it has many other effects on fungal cells.Ethanol lowers the transition temperatures of fungal membrane lipids and increases their fluidity; these interactions are thought to be the mode of lethal action of ethanol (Russell & Furr, 1996).According to Cabeca-Silva et al. (1982), the primary sites of action of both heat and ethanol are mitochondrial membranes.Lichter et al. (2002) showed that B. cinerea on the table grapes was eliminated by 40% (v/v) ethanol.Yuen et al. (1995) found that ethanol vapor inhibited decay of oranges caused by Penicillium italicum and P. digitatum after 5 days of continuous exposure.The complete inhibition of germination of B. cinerea and A. alternata by 30s exposure to 30% (v/v) ethanol at ambient temperature was also reported (Gabler et al., 2004).
Ethanol already occurs in many food products and additives and is an approved substance for use as a disinfectant or sanitizer in organic crop production by USDA National Drganic Program (Lichter et al., 2002).
Combining different methods to control postharvest diseases creates a synergistic effect.For example, heat and ethanol treatments are used to control postharvest decay of strawberries caused by B. cinerea and R. stolonifer (Margosan et al., 1994), lemons caused by P. digitatum, and peaches and nectarines by Monilinia fructicola (Margosan et al., 1997;Smilanick et al., 1995).
Rhizopus oryzae (Rhizopus arrhizus) is a filamentous fungi which is mainly known as a lactic acid producer.Ot is also a well known severe postharvest pathogen causing serious problems like soft rot disease in potato (Amadioha, 2001).Also postharvest rot caused by R. oryzae has been recorded on various fruits including banana, apple (Kwon et al., 2011(Kwon et al., , 2012)).Another study reported postharvest root rot of Codonopsis lanceolata (a medicinal root crop native to East Asia) by R. oryzae (Park et al., 2014).
The studies on the inhibition of R. oryzae spores are very rare: Dne such study explores the antifungal effects of some leaf extracts against R. oryzae.Azadirachta indica (Neem), Cymbopogon citratus, and Ocimum gratissimum leaf extracts proved effective against the spore germination and mycelial growth of R. oryzae (Amadioha, 2001).
The aim of this research was to quantify the germicidal effect of UVC and ethanol treatments on R. oryzae spores.To the best of our knowledge, this is the first study to examine the effect of UVC on R. oryzae spore germination.

Microorganism and growth conditions
On this study, R. oryzae (ATCC 9363) was used.Ot was sporulated on streaked agar plates containing Potato Dextrose Agar (PDA) for 4-5 days at 30 °C.After sporulation, the plates were stored at 4 °C until preparation of spore suspension; spore suspension was prepared by washing the spores with sterile distilled water.Spore concentration in the suspension was determined by counting the spores on hemocytometer.

Ethanol treatment and UV-C irradiation
Spores were collected as described previously.Test tubes were prepared that contained 9 mL of solution each, to make final concentrations of 0, 10, 20, and 30% (v/v) ethanol after 1 mL of spore suspension containing 3.58.10 7spores/mL of spores was added.The tubes were incubated for 5 minutes at 25 °C, then the solutions were centrifuged for 5 minutes at 25 °C and 5000 rpm.The supernatant was discarded and the spores were washed with sterile distilled water to remove any ethanol residue.After washing, the spores were resuspended in 10 mL of sterile distilled water.
For UVC irradiation studies 10 mL of the spore suspensions was carefully poured aseptically in empty sterile petri plates, which had 90 mm.diameter, flat bottom and no lid, then they were placed under a germicidal UV lamp (2.5 × 88 cm).The lamp used was rated at 30W power and its peak emission was at 254 nm wavelength.The distance between the lamp and the petri plates was 20 cm.The petri plates were irradiated for various time periods (0, 1, 2, 5, 10 minutes).Under these conditions the average dose rate was 3.18 W/m 2 and the radiant exposure (dosage) varied from 0.19 to 1.91 kJ/m 2 (Liu et al., 1993;Stevens et al., 1997).After the exposure, they were kept in dark for two hours to prevent photoreactivation (Stevens et al., 1998).
On order to examine the combined effect of ethanol and UVC on the survival ratio of the spores, the treatments mentioned above applied sequentially (first ethanol, followed by UVC) to the spore suspensions of R. oryzae.

Survival ratio determination
For determination of the survival ratio, spores were inoculated on agar plates for colony formation.For this purpose, serial dilutions of the treated spore suspensions were made.To obtain a homogeneous suspension between dilutions, the suspensions were shaken for 15 s using a vortex mixer.Sterile distilled water was used for dilution.Dne millilitre of each spore suspension was plated on PDA and they were incubated at 30 °C.Formed colonies were counted on 5th day after the inoculation.There were three replicates of plates for each condition, and the experiments were done twice.The control treatment in each experiment was water alone at 25 °C.
Survival ratio of the spores (S) was calculated by Equation 1 (Peleg & Cole, 2000): where N 0 is the spores' initial number and N is the number of surviving spores after the exposure to the UVC and/or ethanol treatment.

Statistical analysis
The effects of ethanol and UVC exposure times on spores from R. oryzae were statistically analysed using Microsoft Excel (ANDVA two-factor with replication).
To compare the sensitivity of the spores to the treatments, LD50 values which are the concentration of ethanol or duration of UVC treatment that prevented germination of 50% of fungal spores were calculated.

Results and discussion
Figure 1 shows the effect of UVC exposure after ethanol treatment on the survivability of R. oryzae spores.On all cases, a rapid inactivation was observed after one minute of UVC exposure, then the rate of reduction in survivability of spores decreased.
The effect of only UVC exposure can also be seen in Figure 1, the topmost line represents the case where spores were not immersed in the ethanol solution.Here, exposure of R. oryzae spores to UVC resulted in 1.50 log 10 reduction after 1 minute of exposure, and a total of 2.20 log 10 reduction after 10 minute of exposure.
On a comparable study, it has been shown that UV irradiation was very effective at inactivating the spores of A. flavus, P. corylophilum and E. rubrum and was also effective against A. niger, although to a lesser extent (Begum et al., 2009).
Another study reported a comparable effect of UVC radiation on five different fungi, the survival fractions were found to decay exponentially (Valero et al., 2007).
First data points of all lines in Figure 1 show the samples where UVC was not applied (UVC exposure time = 0), and demonstrate the effect of only ethanol treatment on the survivability of R. oryzae spores.Every 10% (v/v) ethanol concentration increase resulted in an average of 0.29 log 10 reduction, 30% (v/v) ethanol treatment resulted in 0.88 log 10 reduction in survivability of spores.For 50% (v/v) ethanol concentration fungal growth was not observed (result not shown).
Dur findings are supported by previous literature, it was reported that, at 25 °C, 30% (v/v) ethanol significantly reduced the percentage of germinated spores of B. cinerea and A. alternata, while higher temperatures were required to inhibit the more resistant spores such as R. stolonifer and A. niger (Gabler et al., 2004).
Ot should also be pointed that in fruit postharvest, spores would be more protected by the fruit itself and those treatments may not yield as much inactivation as reported in this study.Dn the other hand, UVC is a hormetin (stimulatory agent in subharmful doses) which induces resistance to pathogens in postharvest products (Stevens et al., 1997).Therefore the combined fungicidal and hormetic effects of these agents need to be assessed in further applications.
Combination of UVC exposure and ethanol treatment were more effective for reducing the spore viability (Figure 1).Moreover, total efficiency of spore reduction increased when both methods were applied indicating a synergistic effect.For example, one minute UVC exposure alone resulted in 1.50 log 10 decrease and 10% (v/v) ethanol treatment alone resulted in 0.35 log 10 decrease, whereas 10% (v/v) ethanol treatment followed by one minute UVC exposure resulted in 1.92 log 10 decrease, which is higher than the sum of both methods.This effect is valid for all 12 combinations of UVC and ethanol treatments tested, moreover the efficiency difference increased as the UVC exposure time and ethanol concentrations increased.
This effect was illustrated in Table 1, where for all 12 combinations of UVC and ethanol treatments tested, the difference in efficiency was tabulated.Here, the difference was calculated by Equation 2: The difference value was positive in all cases with an average of 0.27 log 10 , which shows that the combination of UVC and ethanol is more lethal to the spores than expected.Moreover, the efficiency difference increased up to 0.8 log 10 as the UVC exposure time and ethanol concentrations increased.
Such synergistic effect was reported in a previous study, in which the combined application of UVC and heat treatment improved on the results found for each treatment separately and reduced the percentage of spore germination to the greatest extent (Pan et al., 2004).Under the tested conditions, the highest reduction of spore survivability was obtained after 30% (v/v) ethanol treatment followed by UVC exposure: Here, 5 minutes of exposure resulted in 3.71 log 10 reduction, and increasing the UVC exposure to 10 minutes improved reduction only marginally to 3.83 log 10 , which corresponds to 0.015% survival ratio for the spores.Figure 2a shows the LD50 values of UVC treatment durations at various ethanol concentrations and Figure 2b shows the LD50 values of ethanol concentration at various UVC treatment durations.For every 1 minute increase in UVC treatment, the LD50 value for concentration of ethanol decreased by 0.3%, and for every 10% (v/v) increase in ethanol concentration, the LD50 value for the duration of UV-C treatment decreased by 0.35 second.
The combined effect of the two factors can also be seen here, the lines have a negative slope, that is, increase in one factor decreases LD50 (increases lethality) of the other factor.
Both F and P-values show that ethanol content, UVC treatment time and their interactions all were highly significant at the 99% level of confidence for spores suspended in aqueous medium.

Conclusions
Ethanol treatment and UVC irradiation were used for the inactivation of R. oryzae spores, both individually and in combination.A rapid inactivation of fungal spores was observed after one minute of UVC exposure, then the rate of reduction in survivability of spores decreased, whereas inactivation due to ethanol treatment was directly proportional to the ethanol concentration.The combination of UVC exposure and ethanol treatment enhanced the benefits of applying each treatment separately.The benefits increased as the UVC exposure time and ethanol concentrations increased.Oncrease in one factor decreases LD50 (increases lethality) of the other factor.
The results of this work support the use of ethanol treatment and UVC exposure as a possible strategy to inactivate R. oryzae spores to prevent postharvest rot.

Figure 2 .
Figure 2. (a) UVC durations that inhibited the germination of 50% of the spores (LD50) of R. oryzae; (b) Ethanol concentrations that inhibited the germination of 50% of the spores (LD50) of R. oryzae.

Table 1 .
Efficiency difference of log 10 decrease in spore survivability for combined EtDH and UVC treatment.