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Eucalyptus staigeriana essential oil in the control of postharvest fungal rots and on the sensory analysis of grapes

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Introduction
The major fact causing fruit loss is the postharvest decay in the supply chain, which results in significant economic impact for the food industry. That is the fruit marketing chain's case, due to previously established infections that come from injuries during harvesting operations (Prusky, 2011;Pedrotti et al., 2019a).
Bunch rots in grapes are caused by Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. and Greeneria uvicola (Berk. & M.A. Curtis) Punith., responsible for ripe rot and bitter rot, respectively (Steel et al., 2007;Greer et al., 2011). Bunch rots are the most frequent and severe diseases of grape berries in Serra Gaúchain the subtropical highlands of the state of Rio Grande do Sul (RS), Brazil -which has grape production for both processing and in natura consumption (Longland & Sutton, 2008;Greer et al., 2011;. Chemical agents are typically used to reduce fruit decay. However, due to growing concerns over food safety containing chemical additives, natural antifungal products for fruit preservation are attracting increasing attention. Some essential oils (EOs) could be an alternative for chemical fungicides because they are biodegradable natural products, with antifungal properties, low toxicity, and low environmental impact (Pandey et al., 2017).
Eucalyptus genus belongs to the Myrtaceae family and comprises about 900 species extensively spread worldwide (Brooker & Kleing, 2006;Dhakad et al., 2018). This genus contains plants with volatile oils in their leaves and it has been commercially used to produce EOs in the pharmaceutical, toiletry, cosmetics, and food industries. Previous studies have reported the antifungal properties of Eucalyptus EOs against various phytopathogens in the postharvest of fruits (Jhalegar et al., 2015;Abd-El-Latif, 2016;Pedrotti et al., 2021).
The objective of this work was evaluate the effect of E. staigeriana essential oil on C. gloeosporioides and G. uvicola mycelial growth and conidia germination on grapes, as well as its potential for the control of postharvest rot diseases and its effect on the organoleptic properties of grapes.

Strains
of Colletotrichum gloeosporioides (A021/17C) and Greeneria uvicola (A021/17B) were isolated from grapes collected in the municipality of Bento Gonçalves, RS, Brazil, and maintained at 25±2ºC in potato dextrose agar (PDA) medium. For the molecular identification, total DNA was extracted from fungal mycelia according to Tappia-Tussell et al. (2006), then used in the PCR amplification of internal transcribed sequence (ITS-5.8S rDNA), and its products were sequenced as described by Echeverrigaray et al. (2020). The DNA sequences were compared with those deposited in the GeneBank Database using the nBLAST algorithm (NCBI). Both isolates were classified by the sequencing of large subunit RNA gene (GenBank codes A21/17C -MN759013 and A21/17B -MW582304).
Leaves of E. staigeriana were collected in the municipality of Caxias do Sul, RS, Brazil, in September 2017. During the month of collection, the climatic conditions showed 17°C average temperature, 182 mm precipitation, and 79.7% relative humidity. Leaves were oven-dried at 30ºC until a constant mass was obtained.
The extraction of E. staigeriana leaf EO was performed from dried plant leaves, using the steam distillation method in a Clevenger-type apparatus for 1 hour, according to Pedrotti et al. (2021). For the identification and quantification of EO compounds, the method described in Pedrotti et al. (2019b) was used. The chromatographic peaks of each component were analyzed and identified by comparison between the obtained spectra and those from the Wiley library, and by comparison of the calculated linear retention indexes (LRIs) and the literature references (Adams, 2005). The LRI values were calculated with the Van den Dool & Krats (1963) equation and a standard solution of C8-C26 hydrocarbons.
The EO antifungal effect was assessed both for its contact and volatile phase effects against mycelial growth of the phytopathogens. The contact phase effect of EO was determined using PDA medium according to Pedrotti et al. (2019b). For that, different concentrations of EO (0.0, 0.25, 0.5, 1.0, and 1.5 µL mL -1 ) were emulsified with Tween 20 (1:1) and added to the PDA culture medium. These emulsions were poured into 9 cm (⌀) Petri dishes (20 mL volume) and inoculated with 5 mm (⌀) agar disks colonized Pesq. agropec. bras., Brasília, v.57, e02782, 2022 DOI: 10.1590/S1678-3921.pab2022.v57.02782 by C. gloeosporioides or G. uvicola obtained from seven-day-long pre-cultures. Tests were carried out in triplicate with fifteen plates for each treatment. Incubation was conducted in a growth chamber at 25±2ºC in 12-hour photoperiod. The evaluation was recorded on the 3 rd , 5 th , 7 th , 10 th , and 14 th days by measuring the diameter of the mycelial growth.
The EO volatile phase effect on the mycelial growth of the phytopathogens was evaluated using the method adopted by Pedrotti et al. (2019b), as follows: agar disks with 5 mm (⌀) colonized by C. gloeosporioides or G. uvicola were placed at the center of Petri dishes containing PDA medium (20 mL); aliquots of EO (100 μL) at 12.5, 25, and 50 µL, emulsified with 0.1% Tween 20, and pure EO (100 µL, devoid of Tween 20) were applied onto a cotton ball attached to the inner face of a Petri dish lid. The control treatment consisted of 100 μL of 0.1% Tween 20 solution. Tests were carried out in triplicate with fifteen plates for each treatment. Incubation was conducted in a growth chamber at 25±2ºC in 12-hour photoperiod. The evaluation was recorded on the 3 rd , 5 th , 8 th , 10 th , 13 th , and 15 th days by measuring the diameter of the mycelial growth.
Transfer experiments were performed according to Pedrotti et al. (2019a), to distinguish the fungicidal and fungistatic effects of EO.
The antifungal activity of EO on conidia germination was evaluated on the basis of a protocol described by Pedrotti et al. (2019b). Conidia of C. gloeosporioides and G. uvicola were harvested from 14-day-old fungal colonies grown in PDA (25±2ºC, in 12-hour photoperiod). Conidial suspensions at concentration of 1x10 6 conidia mL -1 were used. The effect of different EO concentrations (0.0, 0.25, 0.5, 1.0, and 1.5 µL mL -1 ) were evaluated. The evaluations were performed after 6, 12, and 24 hours. Tests were conducted in triplicate with fifteen replicates per treatment.
For the in vivo evaluation of antifungal activity in the postharvest period, 'Isabella' grapes (Vitis labrusca × Vitis vinifera) were obtained from a vineyard in the municipality of Bento Gonçalves, RS, Brazil. The collection of grapes was followed by surface-sanitization with 1.5% sodium hypochlorite (3 min), then fruit were rinsed with sterile distilled water. Conidia suspension of C. gloeosporioides and G. uvicola were obtained as above described. Conidial suspensions at 1x10 6 conidia mL -1 concentration were used. The treatments were as follows: an absolute control (nontreated grapes); EO control [EO emulsified with Tween 20 (Synth) (1:1)] and added to sterile water at 1.0, 2.0, and 3.0 µL mL -1 concentrations; inoculated control (grapes inoculated with conidia suspension); and preventive and curative treatment with E. staigeriana EO (EO emulsified with Tween 20 (1:1) and added to the sterile water at 1.0, 2.0, and 3.0 µL mL -1 concentrations. The EO antifungal activity on grapes was evaluated according to the method described by Pedrotti et al. (2019a). Wounds (approximately 2 mm deep) were made on ten berries in grape clusters. In the curative treatment, a conidia suspension of C. gloeosporioides or G. uvicola (10 µL in each wound) was inoculated, and after 24 hours, grape clusters were sprayed with E. staigeriana EO at different concentrations. In the preventive treatment, the same EO concentrations were sprayed on grape clusters, and after 24 hours, these clusters were inoculated with a conidia suspension of C. gloeosporioides or G. uvicola (10 µL in each wound). Grapes were placed in plastic boxes and incubated at 25±2ºC / 80% relative humidity in 16-hour photoperiod. The incidence was evaluated for the presence or absence of symptoms of the diseases, and severity was evaluated, using a scale according to the berry area affected by the disease (Pedrotti et al., 2021). The experiment consisted of four treatments with 12 grape clusters for each treatment. This assay was repeated three times.
For the sensory analysis, mature, and asymptomatic 'Isabella' grapes were used in the experiments. Berries were removed from the rachis, but peduncles and pedicels were kept. The treatments consisted of: a control (washed with distilled and autoclaved water); and a treatment with E. staigeriana EO [EO was emulsified with Tween 20 (1:1) and added to sterile water at 1 µL mL -1 concentration]. Berries of both treatments were dried at room temperature (24±2ºC). Samples were prepared 24 hours before the sensory evaluation. A panel of 50 untrained consumers participated in the sensory test. The consumers were conducted to individual booths at room temperature, under white light. The samples were provided in a transparent plastic container with three berries, accompanied with mineral water (22±2°C) to clean the palate between the samples. The containers were coded with three-digit random numbers. Two testing sessions were carried out in sequence, and consumers filled out a sensory form for each one. In the first stage, the samples were Pesq. agropec. bras., Brasília, v.57, e02782, 2022 DOI: 10.1590/S1678-3921.pab2022.v57.02782 served in a blind test, asking the consumer to evaluate the sample. Consumers tasted each sample and evaluated flavor, appearance, aroma, and color using a 10-point hedonic scale (10 = liked very much, 1 = disliked very much). In the second stage, new samples were given to consumers under the same conditions above described, but the samples were identified. The consumers evaluated the characteristics (better, equal, or worse than the control), and the degree of difference (none, weak, moderate, high, or extreme) between the treatment with E. staigeriana EO (unidentified) and the control (identified). Moreover, consumption intent was also evaluated using a 5-point hedonic scale (1 = certainly would not buy, 5 = certainly would buy).
All statistical analysis was performed using SPSS 22.0 software. The Kolmogorov-Smirnov test was used to determine the data normality, and the homogeneity of variances was determined using Levene's test. The data were subjected to analysis of variance, and the threshold for statistical significance was set at 5% probability. In the case of statistical significance, the Tukey's or Dunnett's T3-test was applied to compare the means, at 5% probability.
The antifungal evaluation of E. staigeriana EO showed that the inhibitory effect on the mycelial growth increased proportionally with oil concentrations; mycelial growth was also affected by treatment duration. The mycelial growth of C. gloeosporioides (Figure 1 A) and G. uvicola (Figure 1 B) in the contact phase resulted in the complete inhibition from the EO application at 1 µL mL -1 concentration. Fungicidal action was confirmed by the transfer experiment, in which no mycelial growth was observed for both fungi species. For the treatments at 0.25 and 0.5 µL mL -1 EO against C. gloeosporioides, there was a significant mycelial growth inhibition, in comparison with the control, until the 3 rd and 5 th days, respectively (Figure 1 A). For G. uvicola, a significant mycelial growth inhibition could be observed until the 7 th day, at 0.25 µL mL -1 EO concentration. EO at 0.5 µL mL -1 significantly inhibited the mycelial growth until the 14 th day (Figure 1 B).
In its volatile phase, the effect of EO concentration at 12.5 µL on the mycelial growth of C. gloeosporioides guaranteed a significant inhibition until the 10 th day (Figure 2 A). However for higher concentrations, such effect could be observed until the 15 th day. Against G. uvicola, 12.5 µL and 25 µL EO allowed of significant inhibition until the 10 th and the 15 th days, respectively. At the concentration of 50 µL EO, no growth was observed, and the transfer test confirmed the fungicidal activity (Figure 2 B).
The germination of C. gloeosporioides and G. uvicola conidia was completely inhibited at 0.5 µL mL -1 EO concentration in all times of evaluation (Figure 3 A and B). The 0.25 µL mL -1 EO treatment showed a significant reduction of C. gloeosporioides conidia germination in comparison with the control; for G. uvicola, conidia germination could only be observed at 24 hours after EO application.
These results show that E. staigeriana EO is efficient in the in vitro control of C. gloeosporioides and G. uvicola. Similarly to these results, Tomazoni et al. (2017Tomazoni et al. ( , 2018 and Pedrotti et al. (2019b) showed that E. staigeriana EO had fungicidal activity on the mycelial growth and conidia germination of Alternaria solani, Stemphylium solani, Botrytis cinerea, and Colletotrichum acutatum.
The application of E. staigeriana EO results on the control of disease development in postharvest of grapes are presented ( Table 2). All evaluated EO concentrations (1, 2, and 3 µL mL -1 ) were efficient for the incidence reduction of the disease caused by  Values are the mean of 15 replicates per treatment ± standard deviation. Means followed by equal letters, among the different essential oil concentrations evaluated in each day, do not differ by Dunnett's T3-test, at 5% probability.
Pesq. agropec. bras., Brasília, v.57, e02782, 2022 DOI: 10.1590/S1678-3921.pab2022.v57.02782 C. gloeosporioides in the preventive treatment by 57, 65, and 75%, at 1.0, 2.0, and 3.0 µL mL -1 , respectively, and in the curative treatment (reduction of 83, 85, and 86% at 1.0, 2.0, and 3.0 µL mL -1 , respectively). In the preventive and curative treatments (in all evaluated concentrations), the severity of the disease was equal to that of the inoculated control (1%), which shows that the EO had no effect on the severity of the ripe rot. The EO also reduced the incidence of the disease caused by G. uvicola only in the curative treatment (by 54, 33, and 23%, at 1.0, 2.0, and 3.0 µL mL -1 , respectively). Thus, E. staigeriana EO appears to be more efficient for controlling the incidence of disease caused for C. gloeosporioides in the preventive and curative treatments than in the control of the incidence of G. uvicola, which was effective only in the curative treatment. Its low efficacy in the control of bitter rot (G. uvicola) may be associated with the high severity of this disease. This result shows that E. staigeriana EO was efficient for the control of postharvest incidence of fungal rots diseases, mainly in the curative treatment, and it can be applied in the postharvest chain for the storage of table grapes. The present study corroborates the findings by Pedrotti et al. (2021), who reported that the efficacy of E. staigeriana EO reduced the incidence and severity of disease caused by B. cinerea and the incidence of disease caused by C. acutatum, in preventive and curative treatments.
Considering the effectivity and the antifungal properties of many EO types, previous studies (Zuzarte et al., 2012;Cabral et al., 2013;Scariot et al.,   2020) suggest plasma membrane disruption as one of the main mechanisms of action of EOs, which leads to the loss of membrane integrity, and necrotic death of fungi cell (Sharifi-Rad et al., 2017). Moreover, the effect of EOs on the inhibition of conidial germination is associated with a loss of membrane integrity, a decrease of cell metabolism, and accumulation of reactive oxygen species . The sensory attributes evaluated (flavor, appearance, aroma, and color) showed no statistical difference between grapes of the control and those of the EO treatment (Table 3). The average score of all samples ranged between 6 and 7 in the hedonic scale, corresponding to the items "liked it slightly" and "liked it moderately", respectively.
Regarding the degree of differences between the control and EO-treated samples, the consumers presented the following evaluations for differences perceived between the treatments with EO: 24%, slight differences; 36%, moderate; 24%, considerable; 8% extreme differences; and 8% could not perceive any difference between both samples. The consumers presented consumption intention as high, with 4.60 (on a 5-point hedonic scale) which corresponds to "very often" consumption of grapes treated with EO. Abdollahi et al. (2012) and Frankova et al. (2016) used different EO types for the postharvest control of grape and apple diseases, respectively, and, in both studies, it was observed that the treatments with EOs showed a minimal adverse effect on their sensory profile. According to Romanazzi et al. (2012), the ideal alternative treatment for controlling postharvest diseases should be affordable and easy to implement, and should not negatively influence the fruit, the environment, or human health, and should be under food safety norms.

Conclusions
1. Eucalyptus staigeriana essential oil (EO) at low concentrations results in the complete inhibition of mycelial growth and conidia germination of Colletotrichum gloeosporioides and Greeneria uvicola and show fungicidal action.
2. E. staigeriana EO is efficient in the control of postharvest incidence of fungal rots diseases, mainly in curative treatment.
3. The OE applied on grapes shows no negative effects on the sensory properties (flavor, appearance, aroma, and color) of fruit; therefore, the consumption intention of this product is high and ranked as "very often" for grapes treated with EO.