Commercial grape varieties (Bordô and Niágara Rosada) from conventional cultivation were purchased at local markets in Mariópolis, PR, Brazil. The Niágara Rosada and Bordô cultivars were selected due to their prevalence in the Pato Branco region. Uniform bunches were selected based on size (40–45 berries per bunch, with an average berry diameter of approximately 2.4 cm), firmness, colour, and absence of disease or damage, all at the same degree of ripeness. These bunches were placed in plastic trays to retain moisture and kept at an average temperature of 25 °C under a 12-hour photoperiod. The bunches were disinfected with 70 % alcohol and placed in plastic trays to create a humid chamber, where they were then sprayed with different treatments: control (Water), β-D-1,6-glucan (lasiodiplodian, 200 mg L⁻¹) (Figure 1), and Acibenzolar-S-Methyl (ASM, Bion® 500 WG, 100 mg L⁻¹) as a positive control. Treatments were applied under two conditions: with and without induced injury. Injuries were made at random, and the experiment was structured as a two-factorial design (treatment and injury) with samples arranged in a randomised block design and five replicates (one bunch per replicate). β-D-1,6-glucan (Figure 1) was produced following the protocol of Cunha et al. (2012), with purification conducted via dialysis using a membrane (1.3 inches, MW 11331, Sigma-Aldrich) at 4 °C for seven days.

The grape berries in the wounded treatments were punctured with an entomological pin (2 mm deep punctures on 10berries). Additionally, 12 hours after treatment application, the grapes were inoculated by spraying a suspension of Glomerella cingulata conidia (10⁵ conidia mL^-1).

The fungus used in this experiment was obtained through indirect isolation from a symptomatic leaf of the Niagara Rosada grape cultivar, collected in a commercial vineyard in Mariópolis, Paraná, Brazil (26° 21′ 58″ S, 52° 33′ 32″ W) in February 2019. Following collection, a monospore isolation procedure was performed, and the isolate was maintained on potato dextrose agar (PDA) at 25 °C in darkness. It was subsequently preserved in the phytopathology laboratory of the Instituto Federal do Paraná (IFPR), Palmas, Paraná, Brazil, until the study was conducted. The number of infected berries per bunch was monitored over 10 days.

The total phenolic content was determined on the 10th day after elicitation, using 3 berries per bunch, including skin and pulp, were collected, rapidly immersed in liquid nitrogen (–180 °C), macerated into powder, and stored in Eppendorf tubes in an ultra-freezer (–80 °C, Coldlab, CL 800-86 V, Brazil). The extraction of phenolic compounds was carried out with 1 g of macerated fruit and 5 mL of extraction buffer (sodium borate buffer) with a final concentration of 0.2 mg mL^-1. For quantification, the Folin–Ciocalteu spectroscopic method was used at 740 nm (Shimadzu, UV-1800, Japan), and the results were expressed in mg GAE g^-1 (GAE: gallic acid equivalent) according to the methodology proposed by Singleton et al. (1999).

Fruit firmness (pulp and fruit) was measured using 3 berries per bunch, chosen at random, on the 10th day after elicitation, using a benchtop digital texture analyzer (Stable Micro Systems, TA.XT plusC, UK) with an 8 gauge probe. mm, the results were expressed in N/m².

Disease severity was assessed daily after the first symptoms appeared by counting the number of infected berries per bunch over ten days to calculate the Area Under the Disease Progress Curve (AUDPC). The AUDPC quantifies disease intensity over time by averaging disease severity at consecutive time points and multiplying it by the time intervals between observations. This final value reflects accumulated disease throughout the study, allowing for comparison across conditions, such as locations or treatments (APS, 2007). The AUDPC was calculated using the equation from Campbell and Madden (1990):

$AUDPC= \sum _{i=1}^n\left[\right(Y_{i+1}\text{+}{Y}_i\text{)/2]*[(}{T}_{i+1}-T_i\left)\right] \left(1\right)$

where:

The colour of the berries was measured using a Konica Minolta Chroma Meter CR-400 (Japan), which provided the L*, a*, and b* coordinates. ºBrix measurements were corrected to 25 °C. The pH was determined using a digital pH meter.

We conducted ANOVA (analysis of variance) and Tukey’s mean comparison test at a significance level of 5 %. The Gene software (version 1990.2023.45) was employed for the analyses, while the OriginPro software (version 10.1.0.178) was used for graph construction.

Figure 1. Micrographs (SEM) of native Lasiodiplodan (β-D-1,6-glucan) at 329× magnification. Grape bunches of the varieties Bordô and Niagara, treated with β-D-1,6-glucans and without (water), were challenged by Glomerella cingulata. NW = no injury.

In general, the AUDPC values showed a tendency to be lower for fruits treated with β-D-1,6-Glucan, regardless of the variety (Bordô or Niagara Rosada) and whether the fruits were wounded or not (Figure 2A, 2C, and 2D). For wounded Niagara fruits, ASM showed a tendency for lower AUDPC values up to the 8th day of evaluation (Figure 2B); however, by the 10th day, β-D-1,6-Glucan exhibited the lowest AUDPC value (Figure 2B). These AUDPC values align with the visual symptoms observed in Figure 1, where both varieties treated with water show more pronounced disease symptoms compared to those treated with β-D-1,6-Glucan. American cultivars Bordô and Niágara Rosada resist G. cingulata, which harms fruit and vegetable quality (Bueno Júnior et al., 2022; Wang et al., 2015). However, extensive cultivation has increased disease incidence. Natural compounds, an alternative to synthetic fungicides, form a biofilm on fruits that stimulates defence compound production, inhibiting fungi and pathogens (Matrose et al., 2021). β-D-1,6-Glucan has immunomodulatory and antioxidant properties (Kagimura et al., 2015), stimulating enzymes like phenylalanine ammonia-lyase (PAL) involved in phenolic and flavonoid biosynthesis, which protect against storage-related fungal stress (Chen et al., 2006; Kong et al., 2024).

Figure 2. Area Under the Disease Progress Curve (AUDPC) in grape fruits treated by Acibenzolar-S-methyl (ASM), β-D-1,6-Glucan, and Water, and challenged by Glomerella cingulata: Niagara grapes without wounds (A) and with wounds (B); Bordô grapes without wounds (C) and with wounds (D).

As shown in Figure 3, the highest Total Phenol Content (TPC) values were also found for the elicitor β-D-1,6-Glucan. Niagara fruits treated with β-D-1,6-Glucan exhibited the greatest pulp firmness compared to those treated with ASM and water (Figure 4). Lignin deposition strengthens tissues against microbes. In studies by our group, applying elicitors (Harpin and ASM) to strawberries enhanced post-harvest defences by boosting PAL enzyme activity (Scariotto et al., 2021), crucial for phenol production. This elevated PAL activity induced defence responses, leading to increased pulp firmness via lignin deposition (Danner et al., 2008; Alamino et al., 2013; Tomazeli et al., 2016). Natural elicitors like β-D-1,6-Glucan, a pathogen-associated molecular pattern (PAMP), enhance plant immunity. Our group also found β-D-1,6-Glucan reduced disease severity, elevated antioxidant enzyme activity (e.g., superoxide dismutase), and promoted phenol accumulation in grapes and apples (Suchoronczek et al., 2018). The physicochemical analysis showed no statistically significant differences among treatments. The overall averages were: Brix 12.6, pH 3.4, and colour coordinates L* 17.54, a* –0.44, and b* 3.1.

Figure 3. Total Phenol Content (TPC) in grape fruits treated by Acibenzolar-S-methyl (ASM), β-D-1,6-Glucan, and Water, and challenged by Glomerella cingulata. Histograms show Niagara grapes without wounds (A) and with wounds (B), and Bordô grapes without wounds (C) and with wounds (D). Means followed by different letters differ significantly according to Tukey’s test at the 5 % significance level. *ns = not significant.

Figure 4. Pulp Firmness (averages of wound and not wound) in grape fruits of Bordô and Niagara varieties treated by Acibenzolar-S-methyl (ASM), β-D-1,6-Glucan and Water, and challenged by Glomerella cingulata. Means followed by different letters differ significantly according to Tukey’s test at the 5 % significance level. *ns = not significant.

Fruits treated with β-D-1,6-Glucan consistently showed a tendency for lower AACPD values across all conditions (Bordô and Niágara Rosada, both wounded and unwounded). For wounded Niágara grapes, ASM initially showed a tendency to reduce AACPD until the 8th day, but β-D-1,6-Glucan had the lowest values by the 10th day. β-D-1,6-Glucan also produced the highest TPC values, regardless of variety or injury, leading to greater pulp firmness compared to ASM and water in Niágara grapes. This study highlights β-D-1,6-Glucan's potential as a natural alternative to synthetic fungicides for enhancing grape resistance to G. cingulata, extending shelf life, and minimising environmental and health impacts. Future studies should explore its role in stimulating plant immunity, as our results suggest β-D-1,6-Glucan may provide effective, low-impact fungal disease resistance.

We extend our sincere gratitude to the funding agencies for their financial support: the National Council for Scientific and Technological Development (CNPq), the Coordination for the Improvement of Higher Education Personnel (CAPES), the Araucária Foundation for the Support of Scientific and Technological Development of Paraná (FUNDAÇÃO ARAUCÁRIA) / SETI / NAPI Sudoeste (#282/2022 PDI), and the Federal University of Technology—Paraná (UTFPR). We also wish to thank Syngenta Crop Protection for generously providing ASM.