Grapevine, a crop of global economic importance, is annually affected by diseases that can compromise the quality and quantity of the harvest, producing large economic losses. Downy mildew caused by Plasmopara viticola (Berk. & M.A. Curtis) Berl. & de Toni is one of the most important diseases in the vineyard. To fight this pathogen, winegrowers often rely on conventional chemical fungicides or copper-based formulations, whose use is determined to be reduced by the European Commission due to their environmental consequences. Hence, alternative plant protection products (PPP) in grapevine must be considered and studied. In this context, we selected several alternative commercial products, based on basic substances (BS) or low-risk active substances (LRAS), to evaluate their suitability to deal with P. viticola. We measured the preventive activity of the products, both in vitro and in planta, as well as their toxicity against the sporangia and zoospores of the pathogen. Results showed that four commercial products were effective against the pathogen directly and preventively, being composed of approved basic substances, more concretely, chitosan, Equisetum arvense, lecithins, and Salix cortex. Among those, the products composed of lecithins and Salix cortex were the most toxic and active preventively. Therefore, these basic substances should be promoted in the vineyard as an alternative to conventional treatments in order to transition to a more sustainable viticulture.

Antimicrobial resistance (AMR) is a significant public health threat, with the food production chain, and, specifically, fermented products, as a potential vehicle for dissemination. However, information about dairy products, especially raw ewe milk cheeses, is limited. The present study analysed, for the first time, the occurrence of AMRs related to lactic acid bacteria (LAB) along a raw ewe milk cheese production chain for the most common antimicrobial agents used on farms (dihydrostreptomycin, benzylpenicillin, amoxicillin and polymyxin B). More than 200 LAB isolates were obtained and identified by Sanger sequencing (V1-V3 16S rRNA regions); these isolates included 8 LAB genera and 21 species. Significant differences in LAB composition were observed throughout the production chain (P ≤ 0.001), with Enterococcus (e.g., E. hirae and E. faecalis) and Bacillus (e.g., B. thuringiensis and B. cereus) predominating in ovine faeces and raw ewe milk, respectively, along with Lactococcus (L. lactis) in whey and fresh cheeses, while Lactobacillus and Lacticaseibacillus species (e.g., Lactobacillus sp. and L. paracasei) prevailed in ripened cheeses. Phenotypically, by broth microdilution, Lactococcus, Enterococcus and Bacillus species presented the greatest resistance rates (on average, 78.2 %, 56.8 % and 53.4 %, respectively), specifically against polymyxin B, and were more susceptible to dihydrostreptomycin. Conversely, Lacticaseibacillus and Lactobacillus were more susceptible to all antimicrobials tested (31.4 % and 39.1 %, respectively). Thus, resistance patterns and multidrug resistance were reduced along the production chain (P ≤ 0.05). Genotypically, through HT-qPCR, 31 antimicrobial resistance genes (ARGs) and 6 mobile genetic elements (MGEs) were detected, predominating Str, StrB and aadA-01, related to aminoglycoside resistance, and the transposons tnpA-02 and tnpA-01. In general, a significant reduction in ARGs and MGEs abundances was also observed throughout the production chain (P ≤ 0.001). The current findings indicate that LAB dynamics throughout the raw ewe milk cheese production chain facilitated a reduction in AMRs, which has not been reported to date.

Grapevine downy mildew is a major disease in vineyards all around the world, caused by the oomycete Plasmopara viticola (Berk. & M. A. Curtis) Berl. & De Toni. Normally, its control depends almost exclusively on chemical and copper-based fungicides, especially in high-incidence areas with high relative humidity and mild temperatures. However, the European Union is determined to reduce the application of these phytochemicals by at least 50% by 2030, forcing winegrowers to seek alternative low-input strategies for proper sanitary maintenance. Basic substances (BSs), described in European Regulation (EC) 1107/2009, stand out as promising alternatives, but their molecular mechanism of action remains mostly unknown. In this context, this study analyzed the genetic effect in grapevine plants of several commercial products composed of BSs (chitosan, soy lecithin, Equisetum arvense and Salix cortex). All products exhibited promising results, triggering the induction of similar defence mechanisms, which included pathogenesis-related proteins (PRs), involved in direct pathogen repression; stilbenes, capable of producing antimicrobial compounds such as resveratrol and pterostilbene; several hormones, including oxylipins, ethylene, salicylic acid and terpenes, mediating immune signalling; and genes related to structural features of the plant, such as lignin, callose, cellulose and cuticular wax, constituting a first physiological barrier against P. viticola. Disease severity reduction differed among treatments, with Salix cortex showing the highest efficacy (58%), followed by BABA (38%) and LESOY (35%), while LECI and CHIT had minor effects (<9%). Gene expression analyses revealed that Salix cortex modulated the highest percentage of genes (41%), followed by natural infection without treatment (32%), LESOY (27%), BABA (26%), LECI (23%) and CHIT (23%). In terms of defence mechanisms, Salix cortex promoted the most pathways, LESOY induced eight, BABA and LECI seven and CHIT five. Overall, these results indicate that BSs can modulate several defence pathways in grapevine, supporting their potential use as sustainable alternatives for controlling downy mildew.

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