Combination of Sulfur Dioxide-Generating Pads Reduces Gray Mold Disease Caused by Botrytis cinerea in ‘BRS Vitoria’ Hybrid Seedless Grapes during Cold Storage

Abstract

This study aimed to assess the cold storage preservation of ‘BRS Vitoria’ seedless grape by combining SO₂-generating pads. ‘BRS Vitoria’ grapes were freshly harvested from a commercial vineyard in Marialva, PR, Brazil. The trial was carried out in a completely randomized design with four treatments and five replications, and each plot consisted of five bunches individually packaged in clamshells. Treatments included (a) control (without SO₂ pads), (b) field ultrafast SO₂-generating pad before packaging (FieldSO₂), (c) dual-release SO₂-generating pad during cold storage (DualSO₂), and (d) FieldSO₂ + DualSO₂. After being harvested, bunches subjected to FieldSO₂ treatments were placed in a 20 kg harvest box with perforated liners. Subsequently, the FieldSO₂ was placed on top of the grapes, and the liner was sealed for 4 h. Afterwards, bunches were packaged according to the treatments, and the boxes were kept in cold storage (1 °C ± 1 °C) for 60 days and at room temperature for 3 days. After 60 days of cold storage, by using a combination of FieldSO₂ and DualSO₂ pads, the cold preservation of ‘BRS Vitoria’ grape bunches can be extended up to 60 days with total absence of gray mold with fresh stems, with no negative impact on weight loss, shattered berries or even bleaching. This treatment can be used to optimize the cold storage period of ‘BRS Vitoria’, especially for export markets, where long periods of cold preservation are required.

1. Introduction

The grapevine (Vitis vinifera L.) is one of the main fruit plants cultivated worldwide. In 2022, the world grape production was 78 million tons, including approximately 32 million tons of table grapes [1]. This high acceptance of table grapes is due to their excellent sensory and nutritional properties [2,3].

Seedless grape production has gained increasing interest in recent years because of its favorable sensory and commercial characteristics for both producers and consumers [4]. Seedless table grapes have certain characteristics that make them high-quality fruits that are more accepted by consumers. Among them, ‘BRS Vitoria’ (Arkansas 1976 × (‘White Niagara’ × ‘Venus’) × ‘BRS Linda’) is a hybrid seedless grape with excellent growth performance in tropical and subtropical areas, being an excellent option for foreign and domestic markets due to its firmness and peculiar flavor [5].

In some subtropical areas, two harvest seasons of table grapes per year are possible because of the mild winter and the application of budbreak stimulators [6]. However, in this intensive production system, harvesting can occur under highly favorable conditions for the development of fungal diseases and rachis dehydration during the post-harvest period [7], which can further limit the transport of fresh grapes over long distances if control measures are not adopted.

Botrytis cinerea Pers. ex Fr., a pathogenic fungus that causes gray mold, is the most common post-harvest microorganism of table grapes worldwide [8,9] because of its high growth rate even at low cold storage temperatures [10]. Controlling gray mold is extremely difficult as post-harvest treatments with chemical fungicides are being increasingly limited because of environmental and toxicological risks [11]. Furthermore, dehydration of the rachis during the post-harvest period, in addition to negatively impacting the appearance of the bunches, can accelerate berry shattering and depreciate the product during commercialization [12,13].

The maintenance of post-harvest quality of fresh grapes requires packaging bunches in vented plastic clamshells, which is an innovative alternative that meets the requirements of the local and export markets [14]. Moreover, sulfur dioxide gas (SO₂) usage has demonstrated good results in preventing gray mold growth and prolonging stem freshness of the bunches due to its antioxidant action [15]. Among the different types of SO₂-generating pads available for the cold conservation of table grapes, the recently developed field ultrafast SO₂-generating pad (FieldSO₂), which is used immediately after harvesting the grapes and before packaging, is an alternative to better control the incidence of gray mold and keep the bunches fresh for prolonged periods [14,16,17].

It is important to mention that other methods of grape preservation can also be used, such as edible coatings. As an example, Guirao et al. [18] recently described that ‘Doña María’ table grapes treated with sorbitol–Ca on bagged bunches reduced berry drop at harvest and during post-harvest and increased calcium transport from the leaves to the berries.

However, little is known about the combined usage of FieldSO₂ with the dual-release SO₂-generating pad during cold storage (DualSO₂) regarding the post-harvest conservation of the ‘BRS Vitoria’ seedless grape. Furthermore, to the best of our knowledge, no other study has been conducted using FieldSO₂ alone for the post-harvest conservation of this seedless grape. In this context, the hypothesis of this study is that the post-harvest preservation of ‘BRS Vitoria’ seedless grapes subjected to the association of FieldSO₂ and DualSO₂ pads can be prolonged up to 60 days during cold storage.

2. Material and Methods

2.1. Experimental Area and Climate

Fresh bunches of ‘BRS Vitoria’ seedless grape were harvested from a commercial vineyard located in the municipality of Marialva, Paraná, Brazil, at coordinates 23°30′06′ S, 51°44′52′ W, and elevation of 591 m. According to the Köppen climate classification, the climate of this region is classified as Cfa (subtropical), with an average annual temperature of 20.7 °C and annual precipitation of around 1640.7 mm [19]. The bunches were harvested during the summer harvest season of 2021.

2.2. Treatments and Experimental Design

Right after being harvested, the bunches were placed in plastic boxes with a capacity of 20 kg, with or without the FieldSO₂ and perforated plastic liners with a 0.3% ventilation area (VA), where they remained for 4 h. Afterward, the bunches were transferred to a packing house for cleaning and packaging. Subsequently, the bunches treated, or not, with the FieldSO₂ were standardized and individually settled in vented plastic clamshells with a capacity of 0.5 kg, packaged according to the treatments.

A completely randomized design was used as the statistical model, consisting of four treatments and five replications, and each plot consisted of one corrugated cardboard box containing 5 bunches individually packaged in plastic clamshells (n = 25 bunches per treatment), with a total of 100 bunches in the whole experiment (n = 4 treatments × 5 bunches). Grapes were subjected to the following treatments: (a) control (without SO₂-generating pads); (b) field ultrafast SO₂-generating pad before packaging (FieldSO₂); (c) SO₂-generating pad during cold storage (DualSO₂); and (d) FieldSO₂ + DualSO₂.

2.3. Materials

The FieldSO₂, measuring 46 cm × 26 cm, contained 1.4 g of active ingredient (a.i.), whereas the DualSO₂, measuring 26 × 23 cm, contained 8 g of a.i. (1 g and 7 g for fast and slow phases, respectively). The SO₂-generating pads and perforated plastic liners (Grape Guard Uvas Quality^®, Santiago, Chile), as well as other packaging materials used, were provided by Suragra S.A. (San Bernando, Chile) and manufactured by IMAL SpA (Santiago, Chile).

The grape packaging steps were conducted as follows. Corrugated cardboard boxes with the dimensions of 38 cm × 31 cm × 10 cm with a storage capacity of five clamshells were internally lined with perforated plastic liners with 0.3% VA. A moisture-absorbing paper was placed above the plastic liner. Clamshells containing the grape bunches were set in a cardboard box, and a DualSO₂ was positioned above them, depending on the treatment. Finally, the liners were sealed with adhesive tape (Figure 1).

The cardboard boxes were placed in cold storage (1.0 ± 1.0 °C and 95% relative humidity) for 60 days. Afterward, the boxes were removed from cold storage and maintained at room temperature (22.0 ± 1.0 °C) for 3 days without perforated plastic liners or SO₂-generating pads to simulate the conditions of a supermarket.

Following 30, 45, and 60 days of cold storage, the characteristics assessed were the incidence of gray mold, stem or rachis browning, weight loss, and shattered berries. After 3 days at the room temperature, the characteristics assessed were the incidence of gray mold, stem or rachis browning, and shattered berries.

Incidence of gray mold (% of diseased berries) was calculated by using the equation below [20]:

$$\mathrm{I}\mathrm{n}\mathrm{c}\mathrm{i}\mathrm{d}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e}\left(\%\right)={\displaystyle \frac{\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{d}\mathrm{i}\mathrm{s}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{e}\mathrm{d}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{r}\mathrm{i}\mathrm{e}\mathrm{s}}{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{r}\mathrm{i}\mathrm{e}\mathrm{s}}}\times 100$$

(1)

Stem browning was evaluated by visual assessment, according to Ngcobo et al. [21], assigning the following scores corresponding to the levels of stem or rachis browning: (1) fresh and green, (2) light browning, (3) significant browning, and (4) severe browning.

Bunch weight loss was calculated by weighing the bunches at the initial time of storage and during each evaluation by using the equation below [22]:

$$\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{l}\mathrm{o}\mathrm{s}\mathrm{s}\left(\mathrm{\%}\right)={\displaystyle \frac{\mathrm{i}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}-\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{x}\mathrm{a}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{d}\mathrm{a}\mathrm{t}\mathrm{e}}{\mathrm{i}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}}}\times 100$$

(2)

The incidence of shattered berries was assessed by counting the loose berries from the bunch inside the clamshells expressed as a percentage [23].

2.4. Statistical Analysis

The data were subjected to Anova, and the means were compared by Fisher’s least significant difference (p ≤ 0.05) by applying the R Studio version 4.4.1 software.

3. Results and Discussion

The most important characteristic of table grapes during cold storage is the incidence of decay, mainly caused by gray mold. After 30 days of cold storage, bunches subjected to treatment with SO₂-generating pads had a lower incidence or even a total absence of this disease (

Table 1. Incidence of gray mold disease (% of diseased berries) in ‘BRS Vitoria’ seedless grape at 30, 45, and 60 days of cold storage (CS) (1.0 ± 1.0 °C) and 3 days at room temperature (RT) (22.0 ± 1.0 °C), individually packaged in clamshells and subjected to different treatments with SO₂-generating pads.

Treatments	Incidence of Gray Mold Disease (%)
	30 d at CS	45 d at CS	60 d at CS	3 d at RT
Control a	1.60 a	2.47 a	3.67 a	4.47 a
FieldSO2	0.40 b	1.33 b	2.53 a	5.33 a
DualSO2	0.00 b	0.07 c	0.07 b	1.47 b
FieldSO2 + DualSO2	0.00 b	0.00 c	0.00 b	1.87 b
F-value	9.74 **	23.86 **	30.44 **	9.26 **
CV (%)	24.91	19.39	20.82	19.61

Mean values followed by the same letter within columns are not significantly different according to Fisher’s test (p ≤ 0.05). **: significant (p ≤ 0.01). ^a: Without SO₂-generating pads; FieldSO₂: field ultrafast SO₂-generating pad before packaging; DualSO₂: dual-release SO₂-generating pad during cold storage.

). During this period, no substantial difference was found between the treatments in which SO₂-generating pads were used, whether associated or not, but all treatments performed better than the control treatment, and the latter had the highest incidence of the disease. After 45 and 60 days of cold storage, the treatments in which the DualSO₂ was used, with or without the FieldSO₂, had the lowest incidence or even complete absence of gray mold on berries when used in combination. Accordingly, the use of the FieldSO₂ + DualSO₂ kept the bunches completely healthy during 60 days of cold storage. However, the isolate used in the FieldSO₂ was not sufficient to control the disease and did not differ from the control treatment.

After 3 days of storage at room temperature without SO₂-generating pads, the development of the disease was found for all treatments, which demonstrates that the continuous release of SO₂ by the pads plays an essential role in suppressing the development of the fungus during the cold-storage period (Figure 2). However, the highest incidence of gray mold disease was found in control treatment bunches and those subjected only to the FieldSO₂. During this period, the DualSO₂ used alone or in combination with the FieldSO₂ reduced the incidence of gray mold on berries by approximately 70 and 60%, respectively.

Other authors demonstrated that the use of FieldSO₂ alone was effective in controlling gray mold disease in other grape cultivars. An 85% reduction in disease incidence was found in the berries of the ‘BRS Nubia’ table grape [16] and a 75% reduction in ‘Italia’ table grapes [14] subjected to FieldSO₂ treatment. Both observations were made after 3 days at room temperature after 45 days of cold storage. In ‘Benitaka’ table grapes, it was found that the utilization of the FieldSO₂ associated with other materials increased their efficiency, further reducing the incidence of the disease. Reductions of 78% and 87% in the incidence of the disease were observed by associating the FieldSO₂ with the DualSO₂ and bio-based liner, respectively. Notably, this control was not found for these materials when isolated [17].

The total absence of gray mold symptoms on berries throughout the cold storage period through the FieldSO₂ + DualSO₂ can be explained by the ability of the FieldSO₂ to eradicate Botrytis cinerae spores from the berry skins [14]. In addition to the eradicating effect promoted by FieldSO₂, the continuous release of SO₂ by DualSO₂ during cold storage allowed the suppression of any fungal structure growth that was latent inside the berries and that had not previously been controlled by the FieldSO₂ for up to 60 days; thus, when a combination of these materials was used, a better control of gray mold was achieved.

The freshness of the stem, based on the stem browning score, is also an important feature of cold-preserved table grapes. In our trials, no statistical differences were found during the cold storage period, with the bunch stems from all treatments remaining fresh and green until 60 days of cold storage (

Table 2. Stem browning in the ‘BRS Vitoria’ seedless grape at 30, 45, and 60 days of cold storage (CS) (1.0 ± 1.0 °C) and 3 days at room temperature (RT) (22.0 ± 1.0 °C), individually packaged in clamshells and subjected to different treatments with SO₂-generating pads.

Treatments	Stem Browning b
	30 d at CS	45 d at CS	60 d at CS	3 d at RT
Control a	1.00 a	1.00 a	1.08 a	1.08 b
FieldSO2	1.00 a	1.00 a	1.00 a	1.04 b
DualSO2	1.00 a	1.00 a	1.00 a	1.16 b
FieldSO2 + DualSO2	1.00 a	1.00 a	1.00 a	1.76 a
F-value	1.00 ns	1.00 ns	2.67 ns	29.89 **
CV (%)	0.00	0.00	5.37	10.94

Mean values followed by the same letter within columns are not significantly different according to Fisher’s test (p ≤ 0.05). ^ns: not significant. **: significant (p ≤ 0.01). ^a: Without SO₂-generating pads; ^b: stem browning scores: (1) fresh and green, (2) light browning, (3) significant browning, and (4) severe browning [20]. FieldSO₂: field ultrafast SO₂-generating pad before packaging; DualSO₂: dual-release SO₂-generating pad during cold storage.

).

During cold storage, water loss and stem dehydration are usually expected, which are aspects associated with stem or rachis browning and deteriorating grape quality [16]. However, in this study, the bunches were well preserved during the 60 days of cold storage, even those in the control treatment, demonstrating that ‘BRS Vitoria’ seedless grape presents good post-harvest conservation under these conditions.

Bunches with slight stem browning were found only after 3 days at room temperature and when the treatment FieldSO₂ + DualSO₂ was applied, and this treatment differed from the others with the highest stem browning score; however, it was considered low, represented only by a slight darkening of the stem. Although the bunches were no longer in contact with the SO₂-generating pads during this evaluation period, the amount of gas released by the combination of both pads probably resulted in a slight injury.

No substantial differences in bunch weight loss were found among the assessed treatments at any evaluation period (

Table 3. Bunch weight loss (%) in ‘BRS Vitoria’ seedless grape at 30, 45, and 60 days of cold storage (CS) (1.0 ± 1.0 °C) individually packaged in clamshells and subjected to different treatments with SO₂-generating pads.

Treatments	Weight Loss (%)
	30 d at CS	45 d at CS	60 d at CS
Control a	2.20 a	3.18 a	4.14 a
FieldSO2	2.17 a	3.13 a	4.20 a
DualSO2	2.26 a	3.25 a	4.36 a
FieldSO2 + DualSO2	2.37 a	3.67 a	4.79 a
F-value	0.09 ns	0.64 ns	0.96 ns
CV (%)	12.33	9.23	6.95

Mean values followed by the same letter within columns are not significantly different according to Fisher’s test (p < 0.05). ^ns: not significant. ^a: Without SO₂-generating pads; FieldSO₂: field ultrafast SO₂-generating pad before packaging; DualSO₂: dual-release SO₂-generating pad during cold storage.

). There was an increase in the weight loss of the bunches throughout the cold storage period because of the natural respiratory processes of the bunches [24]. However, even after 60 days of cold storage, the mean weight loss was insignificant and remained below 5%.

Weight loss that is mainly caused by stem dehydration relies on several aspects, such as the grape cultivar, handling conditions during harvest, cold room temperature, storage period, types of packaging, and other factors, as observed in other studies, in which mass losses of 4.8% were recorded for ‘Crimson Seedless’ grapes stored at 0 °C for 60 days [25].

Weight loss is a critical factor in determining table grape quality; the greater the amount of water lost, the worse the quality of the produce, as it can cause a loss of firmness and shattered berries. ‘BRS Vitoria’ has a crunchy flavor, and the loss of firmness of the berries is not a desirable characteristic. However, the bunch weight loss recorded in this study was small and did not negatively impact the quality of the bunches.

Regarding the shattered berries, no notable differences were found among the treatments during the cold storage period or at room temperature (

Table 4. Shattered berries in the ‘BRS Vitoria’ seedless grape at 30, 45, and 60 days of cold storage (CS) (1.0 ± 1.0 °C) and 3 days at room temperature (RT) (22.0 ± 1.0 °C), individually packaged in clamshells and subjected to different treatments with SO₂-generating pads.

Treatments	Shattered Berries (%)
	30 d at CS	45 d at CS	60 d at CS	3 d at RT
Control a	1.80 a	3.87 a	6.20 a	8.40 a
FieldSO2	1.20 a	4.40 a	8.67 a	10.87 a
DualSO2	2.73 a	5.87 a	10.07 a	13.27 a
FieldSO2 + DualSO2	2.40 a	4.93 a	7.87 a	10.40 a
F-value	1.48 ns	0.62 ns	0.84 ns	0.86 ns
CV (%)	20.08	24.89	24.27	23.03

Mean values followed by the same letter within columns are not significantly different according to Fisher’s test (p ≤ 0.05). ^ns: not significant. ^a: Without SO₂-generating pads; FieldSO₂: field ultrafast SO₂-generating pad before packaging; DualSO₂: dual-release SO₂-generating pad during cold storage.

). However, a development of proportionally shattered berries was found for the bunches in all treatments as the cold storage period progressed. This indicates that the ‘BRS Vitoria’ grape presents this progression of detachment of the berries from the stem naturally, a common characteristic among some seedless cultivars.

Various factors may be responsible for the occurrence of shattered berries, including stem dehydration, incidence of gray mold, and sensitivity to SO₂. As described, ‘BRS Vitoria’ is a seedless grape that loosens berries more easily, and in this case, the utilization of clamshells is relevant for the commercialization of quality bunches, as they serve as a physical barrier to contaminating agents and water losses [26]. Therefore, in addition to providing a combination of practicality, protection, and commercialization for table grapes, clamshells reduce losses in the commercialization of such grapes in retail markets, as any loose berries may remain inside the packaging [27]. In addition, vented clamshells permit the circulation of the SO₂ gas, and better preservation of grapes is achieved.

It is also known that excessive accumulation of SO₂ released by pads may result in berry discoloration (bleaching) or off-flavors, especially when the amount of the active ingredient in the pads is too high. However, none of these situations were found in our trials, and the safety of the combination of FieldSO₂ and DualSO₂ was maintained during the assessments.

To summarize, by using a combination of FieldSO₂ and DualSO₂ pads, we demonstrated in our trials, for the first time, that the cold preservation of ‘BRS Vitoria’ grape bunches can be extended up to 60 days with a total absence of decay caused by gray mold. In addition, the stems remained fresh, and this treatment had no negative impact on bunch weight loss, shattered berries, or even bleaching. This treatment can be used to optimize the cold storage period of ‘BRS Vitoria’, especially for export markets, where long periods of cold preservation are required.