Antifungal potential of essential oils from two varieties of Citrus sinensis (lima orange and bahia navel orange) in postharvest control of Rhizopus stolonifer (Ehrenb.: Fr.) Vuill.

Brazil, the world’s largest citros producer, yields around 19 million tons per year and exports most part of its production as orange juice. Essential oils (EDs) extracted from leaves, fruit and flowers of many species of the genus Citrus have been widely used as the result of their promising bioactivities. The fungus Rhizopus stolonifer (Ehrenb.) Vuill., an agent which causes soft rot in fruit, has been considered one of the main factors that cause postharvest diseases, thus, leading to major economic losses in agribusiness. This research aimed at evaluating the chemical composition and in vitro antifungal effect of EDs from two varieties of fresh Citrus sinensis (lima orange and bahia navel orange) peel on mycelial growth of R. stolonifer . EDs were obtained by hydrodistillation, which was carried out by a Clevenger-type apparatus, while their chemical composition was analyzed by gas chromatography-flame ionization detection (GC-FOD) and gas chromatography-mass spectrometry (GC-MS). Limonene was the major monoterpene identified in EDs from lima orange (95.2%) and bahia navel orange (93.2%) peel. EDs from lima orange and bahia navel orange inhibited 91.95% and 80.05% mycelial growth of R. stolonifer , respectively, at the highest dose under evaluation (100 µL). This study revealed the biotechnological potential of EDs extracted from fruit peel of two varieties of citros which may applied to biofilm preparation, so as to coat and preserve different types of fruit.


Introduction
Citriculture is the branch of fruit farming that has stood out worldwide and leads to high production of citros fruit. Ot was introduced in Brazil when the country was a Portuguese colony and, since then, it has been very important to consumption habits of its population. However, the country only became a global leader in orange production after 1960 and has been the world's main producer of this fruit since the mid 80's (Couto & Canniatti-Brazaca, 2010). Despite the high production of tropical fruit, there are also many losses, which correspond to 30% of total production, on average. Postharvest losses may be the result of several causes, such as diseases caused by different types of fungi (Dantas et al., 2003).
Onfestation of fungal pathogens in citros in the postharvest period usually results from inadequate field and postharvest practices, transportation and/or packaging. Control of postharvest diseases is based on balanced fertilization of groves, pruning for cleaning -and elimination of fruit with rot -and spraying of fungicides in groves from blooming to fructification (Junqueira & Junqueira, 2014).
On the postharvest period, cold storage (4-7 °C) has been recommended to treat fruits and delay rot development. The literature has reported postharvest efficiency with the use of both fungicides strobilurin and prochloraz (Fischer et al., 2011). However, some countries do not allow postharvest treatment with prochloraz anymore, as the result of increasing restrictions to the use of certain fungicides in the postharvest period (Fischer et al., 2011). A sustainable alternative which aims at the use of natural products has led to recent interest in biofilms produced from EDs to coat fruit (Sousa et al., 2019). This alternative improves fruit appearance and increases preservation periods, a fact that may be explained by decrease in the transpiratory rate and metabolic activity of fungi (Fischer et al., 2011).
The fungus Rhizopus stolonifer (Ehrenb.) Vuill., an agent which causes soft rot in fruit, has been considered one of the main factors that cause postharvest diseases; it is often responsible for about 50% of loss of fruit that would be commercialized (Bassetto et al., 2007). Even though thermal and chemical treatments are the most common ones to control R. stolonifer and other fungi in food, the search for new antimicrobial agents based on plants has been intense due to microorganism resistance to synthetic products (Elizei et al., 2016).
The use of biodegradable films which have essential oils incorporated into them has recently become an alternative which is capable of offering benefits regarding the maintenance of food Antifungal potential of essential oils from two varieties of Citrus sinensis (lima orange and bahia navel orange) in postharvest control of Rhizopus stolonifer (Ehrenb.: Fr.) Vuill.
properties and of helping decrease the microbial load found on its surface (Prasad et al., 2018). Ot should be highlighted that the incorporation of substances into films not only offers the opportunity for films to interact with food, but also modifies film properties (São José et al., 2019). On addition, several studies reinforce the fact that this active packaging promotes highly efficient protection. Therefore, this study advocates that essential oils from C. sinensis may be used for manufacturing this type of packaging in the near future to protect certain kinds of food against contamination by R. stolonifer.
Considering much controversy over the use of synthetic fungicides in agriculture, due to risks posed to health and the environment, this study aimed at finding some application and/or use for fruit peel of two varieties of citros, which are considered waste in orange juice industries. Therefore, essential oils (EDs) were extracted from fruit peel of two varieties of Citrus sinensis (lima orange and bahia navel orange) and their chemical composition and in vitro antifungal activity against R. stolonifer were determined.

Plant material
Fruit of both varieties of Citrus sinensis (lima orange and bahia navel orange) were purchased on February 12th, 2018, in Rio Verde, Goiás, Brazil. Fruits were washed with water, dried and peeled so that fresh peel could be collected. Both varieties of Citrus sinensis were identified by the botanist Erika Amaral and samples were deposited at the Herbarium Jataiense Professor Germano Guarim Neto at exsiccate numbers HJ #151 (lima orange) and HJ #152 (bahia navel orange).

Extraction of EOs
Samples of fresh C. sinensis (lima orange and bahia navel orange) peel were subjected to hydrodistillation for 2 hours by a Clevenger-type apparatus. On order to carry out the analysis, 300 g plant material was divided into three 100-g samples and 500 mL distilled water was added to each sample. After manual collection of samples of EDs, traces of remaining water in the oils were removed with anhydrous sodium sulfate and then filtered. Osolated oils were stored under refrigeration up to the analysis and testing.

Identification of chemical composition of EOs
Gas chromatography (GC) analyses were performed by a Shimadzu GC2010 Plus gas chromatograph equipped with an ADC-20s autosampler and fitted with FOD and a data-handling processor. An Rtx-5 (Restek Co., Bellefonte, PA, USA) fused silica capillary column (30-m x 0.25-mm i.d.; 0.25-μm film thickness) was employed. Dperation conditions were as followsI: the column temperature was programmed to rise from 60 to 240 °C at 3 °C/min and, then, to hold at 240 °C for 5 min; carrier gas was He (99.999%), at 1.0 mL/min; injection modeI:injection volume, 0.1 µL (split ratio of 1I:10); and injector and detector temperatures were 240 and 280 °C, respectively. Relative concentrations of components were obtained by peak area normalization (%). Relative areas were the average of triplicate GC-FOD analyses.
GC-MS analyses were carried out by a Shimadzu QP2010 Plus (Shimadzu Corporation, Kyoto, Japan) system equipped with an ADC-20i autosampler. The column was an RTX-5MS (Restek Co., Bellefonte, PA, USA) fused silica capillary column (30 m × 0.25 mm i.d. × 0.25 µm film thickness). Electron ionization mode occurred at 70 eV. Helium (99.999%) was employed as the carrier gas at constant flow of 1.0 mL/min. Onjection volume was 0.1 µL (split ratio of 1I:10). Onjector and ion-source temperatures were set at 240 and 280 °C, respectively. The oven temperature program was the same as the one used for GC. Mass spectra were taken at scan intervals of 0.5 s, in the mass range from 40 to 600 Da.
Odentification of volatile components of fresh C. sinensis (lima orange and bahia navel orange) peel (Table 1 and Table 2) was based on their retention indices on an Rtx-5MS capillary column under the same operating conditions as the ones in the case of GC relative to a homologous series of n-alkanes (C 8 -C 20 ). Structures were computer-matched with Wiley 7, NOST 08 and FFNSC 1.2 spectra libraries and their fragmentation patterns were compared with literature data (Adams, 2007).

In vitro antifungal activity of EOs from fresh C. sinensis (lima orange and bahia navel orange) peel against phytopathogen R. stolonifer
Pathogenic isolates of R. stolonifer were collected in November 2018, by direct isolaton of fungal structures of infected grapes. Assays were carried out in the agricultural microbiology laboratory at OF Goiano -Campus Rio Verde and the antifungal activity of EDs from C. sinensis fruit peel were evaluated in agreement with the disc-diffusion method described by Xavier et al. (2016), at 25-100 µL doses of EDs (Figure 1 -lima orange and Figure 2 -bahia navel orange). Negative controls were dishes with no addition of EDs (witness) whereas the positive control was the fungicide Carboxin + Thiram, at 25 µg/mL of active ingredient. Petri dishes were sterilized and prepared with PDA culture medium. After  (Adams, 2007); RA% = relative area (peak area in relation to the total peak area in the GC-FID chromatogram). medium solidification, EDs, at the previously mentioned doses, were added and smeared on the surface of the dish with the help of a Drigalski spatula. Afterwards, 5 mm diameter PDA medium discs with 10-day-old mycelia were placed in the center of the dishes. Then, they were incubated at 28 ± 2 °C. Mycelial growth was measured daily, until the fungus had fully grown on the control dishes. The treatment was carried out in quadruplicate and the experimental design was thoroughly randomized. Data were submitted to the analysis of variance (ANDVA) and the means of the treatments were evaluated by the Scott-Knott test at 5% significance level by the ASSOSTAT software.
Percentage of inhibition of mycelial growth (OMG) was calculated by the following Formula 1I: (1)

Results and discussion
GC-MS and GC-FOD identified 13 chemical constituents in EDs from bahia navel orange peel, corresponding to 100%, while ten were identified in EDs from lima orange peel, corresponding to 100%. Retention times, identified compounds, retention indexes and relative percentages (%) are shown in Table 1 (bahia navel orange) and Table 2 (lima orange). The major component found in EDs from fresh fruit peel was limonene (95.2% in lima orange; 93.2% in bahia navel orange).
On general, EDs draw attention because they are composed of mixtures of complex and volatile substances, which bestow specific aroma on plants. Besides, they act as a defense system in the vegetable kingdom, since they are sources of chemical agents that have antibacterial, insecticidal and antifungal activity; this is the case of EDs extracted from orange peel (Ferronatto & Rossi, 2018). Regarding the enormous amount of orange peel generated by food industries, their concern is that this waste may become a problem when it is badly managed and may pose risks to the environment and the population's health. Law no. 12.305, which was issued in August 2010 by the Brazilian Policy on Residues and Solids, says that a residue is only considered refuse when all alternatives of use have been used up (Ferronatto & Rossi, 2018). Ferronatto & Rossi (2018) also stated that EDs from Citrus sinensis had high contents of limonene (91.4%), a fact that corroborates the ones found by the study reported in this paper. They added that both compounds myrcene and linalool were considered major constituents, by comparison with the other ones, even though they were found at lower amounts. Velázquez-Nuñez et al. (2013) investigated components of EDs from orange peel and found limonene (96.6%) as the major one, followed by other terpenes, such as myrcene (1.72%) and β-pinene (0.53%). Espina et al. (2011) carried out a study of Spanish orange peel and identified 56 components; the major ones were limonene (85.5%), cis-limonene oxide (1.03%) and myrcene (0.92%). Ot should be highlighted that some studies have already shown the influence of genotypes on chemical compositions of EDs from citros; in all findings, limonene was a major constituent (Hosni et al., 2010).
Antifungal potential of EDs against postharvest phytopathogens has increasingly drawn researchers' attention worldwide (Znini et al., 2013), since these oils may act as biofungicides  (Adams, 2007); RA% = relative area (peak area in relation to the total peak area in the GC-FID chromatogram).  and replace chemical fungicides. Therefore, in vitro antifungal activity of EDs from fresh C. sinensis peel was evaluated against the phytopathogenic fungus R. stolonifer. EDs inhibited mycelial growth of R. stolonifer in a dose-dependent manner. Percentages of inhibition of mycelial growth (OMG) by EDs from fresh lima orange and bahia navel orange peel are shown in Figures 1 and 2. Figures 1 and 2 show data on in vitro antifungal potential of EDs from both varieties of citros peel. The fungistatic and/or antifungal effect of EDs under study may be related to their major chemical components. Lorenzetti et al. (2011) stated that EDs may contain chemical components at different concentrations, i. e., usually a major component and others, at lower concentrations, working together, may exhibit antifungal activity through a synergic effect.
According to Viuda-Martos et al. (2008), EDs from C. sinensis were found to inhibit in vitro growth of four fungal species that deteriorate food. Their highest inhibitory activity was registered against Aspergillus niger, which was kept up to the seventh day of evaluation. Reddy et al. (1998) submitted strawberries to the activity of volatile compounds of Thymus vulgaris and got satisfactory inhibitory activity against R. stolonifer. Vu et al. (2011) also showed that EDs may act against phytopathogens effectively when they studied the activity of EDs from Cymbopogon citratus against R. stolonifer. An important issue was described by Fisher & Phillips (2008), who state that EDs from species of Citrus are "Generally Recognized as Safe" (GRAS) by the Food and Drug Administration (FDA) as food additives, a fact that enables them to be used in several food matrices.
The promising in vitro anti-Rhizopus stolonifer activity of EDs from fresh C. sinensis peel may be justified by their major chemical constituent, i. e., limonene. Ot may also be implied that the high antifungal activity exhibited by EDs from lima orange results from the high concentration of limonene, which was 95.2% (Table 1). This monoterpene has already shown promising activity against several types of fungi, such as different species of Candida (Viriato, 2014). Dn the other hand, Chee et al. (2009) described limonene as a potent antifungal against Trichophyton rubrum. On general, Jing et al. (2014) intelligently concluded that EDs from Citrus have been widely used in food and pharmaceutical industries because of their antifungal activity.
Mechanisms of action used by EDs to inhibit either microbial proliferation or even cell lysis have not been fully understood yet, since there are few studies of these mechanisms in fungi. However, Nazzaro et al. (2017) stated that the activity of EDs may take place through changes in the integrity, composition and permeabilty of cell membranes, oxidative stress, inhibition of intracell processes of ion transport and rupture of cell membranes.

Conclusions
On sum, oranges are actually part of Brazilian consumers' diet as important sources of vitamins and fibers. EDs from citros have been mainly acknowledged as compounds that carry secondary metabolites whose biological functions act either synergically together or alone. EDs from both varieties of Citrus exhibited high contents of limonene, a monoterpene that may be related to the promising in vitro antifungal activity against Rhizopus stolonifer. Ot should be highlighted that the antifungal activity of pure limonene against R. stolonifer must be evaluated in the future.