**1. Introduction**

Released in 2012, the new hybrid seedless table grape 'BRS Vitoria' is recommended for cultivation in tropical and subtropical areas. This cultivar has good development and production, and is tolerant to downy mildew (*Plasmopara viticola*), one of the major grape diseases in humid areas. Due to its excellent flavor and firmness, this cultivar is a good option not only for the internal market, but also

for export [1]. Therefore, it is necessary to develop techniques that allow postharvest conservation of this grape in cold storage for long periods.

The fungus *Botrytis cinerea*, which causes gray mold, is considered one of the most damaging postharvest pathogens to the quality of table grapes during storage and transport over long distances. The control of this fungus is particularly important during refrigerated storage, as it also develops at low temperatures (−0.5 ◦C) and spreads rapidly through the berry clusters [2–4].

Sulfur dioxide (SO2) is the main fungicide treatment used to retard the growth of this fungus in refrigerated chambers, and the purpose of its use is to inhibit fungus development and to allow the storage and transport of table grapes for long periods of time [5]. There are two main concepts for packaging grapes in prolonged storage: packing with SO2 pads and fumigation of refrigeration chambers by repeated application of this gas [6,7]. Despite its effectiveness, there are restrictions for the fumigation of SO2, as this may compromise fruit flavor, cause damage to the berries, and result in excessive sulfite residues [8].

Thus, the use of SO2-generating pads was developed because they provide good and efficient control and lower risk than fumigation. Depending on the grape cultivar, different types of pads were developed, such as slow release and dual phase release, with different concentrations of SO2 [9]. The SO2-generating pads contain sodium metabisulfite, which when in contact with moisture inside the grape packaging, reacts by releasing SO2 gas [10].

The choice of SO2-generating pads should be judicious, in order to maintain the quality of the grape to its final destination, and to that end, the level of the active ingredient should be specific for each table grape cultivar. The main import markets for fresh grapes, such as the European Community and the United States, have established levels of tolerance to the use of SO2 in postharvest management, aiming at greater protection of the consumer and also of the environment, since a high gas concentration and/or residue may be harmful to man and the environment [11].

Therefore, the evaluation of techniques to avoid grape losses during postharvest storage are needed to maintain quality and profit. In this context, the objective of this work was to evaluate different types of SO2 generator pads that prevent the incidence of gray mold of 'BRS Vitoria' seedless grape, as well to avoid other grape injuries during cold storage.

#### **2. Materials and Methods**

Grape bunches were obtained from a commercial field of 'BRS Vitoria' seedless table grape grafted on 'IAC 766' rootstock, located at Marialva, state of Parana (PR) (South Brazil) (23◦29 S, 51◦47 W, elevation 570 m). Samples were collected from regular crops in 2018. According to the Köppen classification, the climate is type Cfa, i.e., subtropical with an average temperature in the coldest month below 18 ◦C, and average temperature in the warmest month above 22 ◦C. The maximum temperature is 31 ◦C, and the average annual rainfall is 1596 mm, with a tendency for concentrated rainfall in summer. The field was selected because of its history of gray mold incidence.

*Botrytis cinerea*, used in this study, was isolated from infected grapes showing typical gray mold symptoms, purified and identified morphologically and molecularly [12]. The isolates were maintained on potato dextrose agar (PDA) slants and stored at 4 ◦C for further use. Fungal conidia were harvested from 2-week-old PDA cultures of *Botrytis cinerea* grown at 23 ± 1 ◦C. A volume of 5 mL of distilled water, containing 0.05% (*v*/*v*) Tween 80, was added to a Petri plate culture. The conidia were gently dislodged from the surface with a distilled glass rod, and suspensions were filtered through three layers of cheesecloth to remove any adhering mycelia. The suspensions were diluted with sterile water and the concentration was determined with a hemacytometer. Further dilutions with sterile water were made to obtain the desired conidial concentrations. *Botrytis cinerea* suspension (10<sup>6</sup> conidia mL−1) was used for grape inoculation.

Grapes were harvested at full maturity when soluble solids content reached around 16◦Brix, and then, bunches were subjected to the following treatments: (a) control; (b) SO2 slow release pad; (c) SO2 dual release pad; (d) SO2 dual release–fast reduced pad; (e) SO2 slow release pad with grapes

inoculated with *Botrytis cinerea*; (f) SO2 dual release pad with grapes inoculated with *Botrytis cinerea*; (g) SO2 dual release–fast reduced pad with grapes inoculated with *Botrytis cinerea*. The inoculation was carried out by spraying a conidial suspension (10<sup>6</sup> conidia mL−1). A completely randomized experimental design was used with seven treatments and four replicates, with five bunches per plot.

Before packing, bunches were subjected to forced air precooling, cleaned, and the damaged berries were removed. Then, they were standardized according to their appearance and mass (~0.5 kg), and arranged individually in a plastic clamshell of 0.5 kg capacity, measuring 20 × 10 cm. The process of packaging the grapes followed several steps: arrangemen<sup>t</sup> of plastic micro perforated liner films in 4.5 kg-capacity carton boxes measuring 50 × 30 × 10 cm; deposition of a sheet of moisture-absorbing paper, measuring 37 × 28 cm on the bottom of the liner; placement of plastic clamshells with grapes; arrangemen<sup>t</sup> of the SO2 pad on top; and sealing of liner. The cartons boxes were then placed in a cold chamber at 2 ◦C with high relative humidity for 50 days followed by one week of room temperature at 22 ± 2 ◦C. For treatments with SO2, one generator pad measuring 37 × 28 cm per box provided with slow release, dual release, or dual release–fast reduced phases of sodium metabisulfite (Na2S2O5) (Uvasys®, Cape Town, South Africa) was used, containing 3.85 g; 4.50 g, and 4.25 g of active ingredient, respectively. The dual release–fast reduced pad was designed for SO2-sensitive cultivars, since it releases 60% of the active ingredient during the first phase, and 40% during the subsequent fast reduced phase.

The incidence of gray mold on grapes was evaluated at 50 days after the beginning of cold storage and at 7 days at room temperature after the end of cold storage. The disease incidence was then obtained by the formula: disease incidence (%) = (number of infected bunches/total number of bunches) × 100 [12].

Stem browning development was measured by using the following scoring system: (1) fresh and green; (2) some light browning; (3) significant browning; and (4) severe browning, calculated by the weighted average of the scale value and number of bunches at each level, ranging between 1 and 4 [13] (Figure 1). The shattered berries incidence of was determined by counting the separated berries from the bunch stem inside the clamshell, and were expressed as a percentage of the total number of berries.

**Figure 1.** Stem browning scores of the 'BRS Vitoria' table grapes. 1: fresh and green; 2: light browning; 3: moderate browning; and 4: severe browning.

The bunch mass loss (%) during postharvest storage was determined by periodic weighing, and calculated by dividing the mass change during storage by the original mass: mass loss (%) = [(mi − ms)/mi] × 100, where mi = initial mass and ms = mass at examining time [14]. The berry firmness or maximum compression force was performed with a texture analyzer TA.XT*Plus* (Stable Micro Systems, Surrey, UK), analyzing the equatorial position of 10 berries with pedicels per plot. Each berry was placed on the base of the equipment and compressed using a cylindrical probe with a diameter of 35 mm parallel to the base. A constant force of 0.05 N at a speed of 1.0 mm s<sup>−</sup><sup>1</sup>

was applied to deform the berry to 20% of its equatorial diameter. The berry firmness (N) was then determined [15].

Berry color was analyzed using a colorimeter Minolta® CR-10 to obtain the following variables from the equatorial portion of berries (*n* = 2 measurements per berry): *L\** (lightness), *C\** (chroma), and *h*◦ (hue angle) [16]. Lightness values may range from 0 (black) to 100 (white). Chroma indicates the purity or intensity of color, the distance from gray (achromatic) toward a pure chromatic color and is calculated from the *a\** and *b\** values of the CIELab scale system (International Comission on Illumination, Vienna, Austria), starting from zero for a completely neutral color, and does not have an arbitrary end, but intensity increases with magnitude. Hue refers to the color wheel and is measured in angles; green, yellow, and red correspond to 180◦, 90◦, and 0◦, respectively [17–19].

For chemical analysis, 15 berries were collected from each plot. The juice was used to determine soluble solids (SS) and titratable acidity (TA). SS was determined with a digital refractometer (Krüss, Hamburg, Germany) at 20 ◦C, and the results were expressed in ◦Brix. The pH of the juice was recorded using a Jenway 3510 bench pH meter (Cole-Parmer, Staffordshire, UK) and then TA was determined by potentiometric titration with NaOH 0.1 N up to pH 8.2, using 10 mL of diluted juice in 40 mL distilled H2O, and the results were expressed in % of tartaric acid [20]. The grape physicochemical analysis was performed at 50 days of cold storage and 7 days of room temperature.

All data were subjected to analysis of variance using Sisvar software (UFLA, Lavras, Brazil). Mean values of treatments were compared by using Scott Knott's test and judged at *p* ≤ 0.05 levels.
