Identification of the Causal Agent of Aqueous Spot Disease of Sweet Cherries (Prunus avium L.) from the Jerte Valley (Cáceres, Spain).

The pre and postharvest disease named 'aqueous spot' is an emerging risk for sweet cherries growing in Jerte Valley (Cáceres, Spain). Early stages of the disease appear in the tree, but it is usually detected after harvesting, during the postharvest period. Symptoms include the appearance of skin discolouration and translucency in the shoulder areas. At the most advanced stages, a mycelium of white colour partially or completely covers the fruit. This manuscript provides a detailed description of the microbes involved in this disease, such as bacteria, yeasts, and moulds. Microbes of different cherry cultivars were studied during two consecutive seasons (2019 and 2020). The counts of bacteria and yeast in damaged tissues were higher (7.05 and 6.38 log10 CFU/g for total aerobic mesophilic microbes and yeasts, respectively) than sound tissues (6.08 and 5.19 log10 CFU/g, respectively). The Enterobacterales order dominated the bacteria population. Among yeasts, Yarrowia lipolytica, in 2019, and Metschnikowia pulcherrima and Metschnikowia viticola, in 2020, were consistently isolated from all samples. The presence of moulds was inconsistently detected at the early stage of this disease by plate counts. However, microscopic observations revealed the presence of hyphae in cherry flesh. Different pathogenic moulds were identified, although white mycelium, identified as Botrytis cinerea by molecular methods, was consistently isolated at later stages. Inoculation tests confirmed the involvement of white-mycelium B. cinerea in the development of this new postharvest disease in the Jerte Valley. Its combination with Enterobacterales enhanced the evolution of rotting, whereas the combination with yeasts decreased and delayed the symptoms. This work presents the first report of a consortia of microorganisms implicated in the development of 'aqueous spot', an emerging disease in sweet cherry cultivars in the Jerte Valley.

In Vitro Biological Control of Aspergillus flavus by Hanseniaspora opuntiae L479 and Hanseniaspora uvarum L793, Producers of Antifungal Volatile Organic Compounds.

Aspergillus flavus is a toxigenic fungal colonizer of fruits and cereals and may produce one of the most important mycotoxins from a food safety perspective, aflatoxins. Therefore, its growth and mycotoxin production should be effectively avoided to protect consumers' health. Among the safe and green antifungal strategies that can be applied in the field, biocontrol is a recent and emerging strategy that needs to be explored. Yeasts are normally good biocontrol candidates to minimize mold-related hazards and their modes of action are numerous, one of them being the production of volatile organic compounds (VOCs). To this end, the influence of VOCs produced by Hanseniaspora opuntiae L479 and Hanseniaspora uvarum L793 on growth, expression of the regulatory gene of the aflatoxin pathway (aflR) and mycotoxin production by A.flavus for 21 days was assessed. The results showed that both yeasts, despite producing different kinds of VOCs, had a similar effect on inhibiting growth, mycotoxin biosynthetic gene expression and phenotypic toxin production overall at the mid-incubation period when their synthesis was the greatest. Based on the results, both yeast strains, H. opuntiae L479 and H. uvarum L793, are potentially suitable as a biopreservative agents for inhibiting the growth of A. flavus and reducing aflatoxin accumulation.

Control of Penicillium glabrum by Indigenous Antagonistic Yeast from Vineyards.

Biocontrol is one of the most promising alternatives to synthetic fungicides for food preservation. Botrytis cinerea, Alternaria alternata, and Aspergillus section Nigri are the most concerning pathogens for grape development. However, frequently, other species, such as Penicillium glabrum in this study, are predominant in spoiled bunches. In this work, 54 native yeasts from vineyards were screened by direct confrontation in potato dextrose agar plates as antagonists against P. glabrum. Isolates of Pichia terricola, Aureobasidium pullulans, and Zygoascus meyerae were selected for their antagonist activity in vitro, plus isolates of Pichia kudriavzevii, Hormonema viticola, and Hanseniaspora uvarum were used as negative controls. However, in vivo, confrontations in wounded grapes showed disagreement with direct confrontation in vitro. P. terricola, P. kudriavzevii, H. viticola, Z. meyerae, and H. uvarum significantly reduced the incidence of P. glabrum on grapes. Nevertheless, P. terricola, H. viticola, and H. uvarum themselves spoiled the wounded grapes. Inhibitions were associated with different mechanisms such as the production of volatile organic compounds (VOCs), lytic enzymes, biofilm formation, and competition for nutrients. The isolates of P. kudriavzevii L18 (a producer of antifungal VOCs which completely inhibited the incidence of P. glabrum) and Z. meyerae L29 (with pectinase, chitinase and ß-glucanase activity and biofilm formation which reduced 70% of the incidence of P. glabrum) are proposed as suitable biocontrol agents against P. glabrum.