Fungal control is one of the main challenges faced by winegrowers, as phytopathogenic fungi reduce vineyard productivity and negatively affect the quality of grapes. Botrytis cinerea, Colletotrichum acutatum and species of the genus Aspergillus are among the fungi of greatest concern for viticulture. These fungi may infect berries during ripening or storage, and they are a problem in all wine-growing regions of the world. B. cinerea causes the disease called gray rot, which, like the rot of ripe grapes caused by C. acutatum and the diseases caused by Aspergillus spp., results in great damage to global viticulture (Welke, 2019).

Furthermore, some Aspergillus species such as Aspergillus niger and Aspergillus westerdijkiae can synthesize ochratoxins, while Aspergillus flavus can produce aflatoxins. In fresh grapes, the ochratoxigenic potential of A. niger has been reported in the literature (Ferranti et al., 2018; Mondani et al., 2020), while aflatoxins produced by A. flavus appear to be more frequent in overripe and/or dehydrated grapes (Khashaba et al., 2018; Tópor et al., 2023). Although the production of ochratoxins by A. westerdijkiae has been barely reported in the literature, there is an emerging concern regarding the fungal ability to produce mycotoxins in response to climate changes (Casu et al., 2024). These mycotoxins pose a health concern, since the International Agency for Research on Cancer (IARC) classifies ochratoxin A (OTA) as possibly carcinogenic to humans (group 2B) (IARC, 1993), and aflatoxins (AFB1, AFB2, AFG1 and AFG2) as carcinogenic to humans (group 1) (IARC, 2012).

The main strategy for controlling fungi in agriculture is the use of synthetic fungicides (Fenta and Mekonnen, 2024). However, the emergence of fungal resistance to widely used products, environmental contamination, the impact on biodiversity and the toxic effect of these products are of particular concern (Khan et al., 2020). Interest in biocontrol techniques has grown as a means of improving the safety and sustainability of agricultural production (Fenta and Mekonnen, 2024). Antagonistic microorganisms, such as species of the genus Bacillus, have shown promising action for controlling diseases caused by fungi. Bacillus spp. are attractive for the industrial production of bioproducts due to their rapid growth, ability to produce a variety of antifungal substances (such as enzymes, lipopeptides, and volatile compounds), and ability to stimulate plant defense mechanisms to prevent the spread of disease (Veras et al., 2023). Furthermore, Bacillus spp. form endospores, which provide resistance and ensure their survival in unfavorable conditions (Etesami et al., 2023).

In a previous study, nine strains of Bacillus spp. isolated from the Brazilian Amazon were evaluated regarding their antagonistic potential against toxigenic fungi (Veras et al., 2016). Four Bacillus velezensis strains (P1, P7, P11, and P45) were the most effective at inhibiting the growth of 13 fungi, including A. flavus and Aspergillus carbonarius, as well as preventing the synthesis of aflatoxin B1 and ochratoxin A in the culture medium. Promising results were also found when these four strains were used as a biological control strategy for A. carbonarius in grapes, which also inhibited the synthesis of OTA and other ochratoxins (Silveira et al., 2022). It is important to mention that several strains of the B. subtilis clade (in which B. velezensis is included) have received “Generally Recognized as Safe” (GRAS) status by the United States Food and Drug Administration (FDA, 2022a, FDA, 2022b). Additionally, B. velezensis has “Qualified Presumption of Safety” (QPS) status according to the European Food Safety Authority (EFSA, 2020).

The objective of this study was to evaluate the effect of B. velezensis strains P1, P7, P11 and P45 on the growth of B. cinerea, C. acutatum, A. flavus, A. niger and A. westerdijkiae, both in vitro and in grape berries. The ability of the bacterial strains to reduce spore germination and mycelial growth of these five fungi was also investigated, as well as the potential of inhibiting the production of mycotoxins in grapes, including the assessment of possible synergism between the investigated strains. Additionally, an in silico approach by genomic analysis was carried out to search for genes related to the biosynthesis of antifungal secondary metabolites.