Abstract
Postharvest spoilage fungi, such as Botrytis cinerea, are considered the main cause of losses of fresh fruit quality and vegetables during storage, distribution, and consumption. The current control strategy is the use of SO2 generator pads whose application is now largely under observation. A high quantity of SO2 can be deleterious for fresh fruits and vegetables and it is not allowed in organic agriculture. For this reason, great attention has been recently focused on identifying Biological Control Agents (BCA) to implement biological approaches devoid of chemicals. In this direction, we carried out our study in isolating five different non-Saccharomyces yeast strains from local vineyards in the South of Italy as possible BCA. We performed both in vitro and in vivo assays in semi-commercial conditions on detached grape berries stored at 0 °C, simulating the temperature normally used during cold storage, and obtained relevant results. We isolated three M. pulcherrima strains and one L. thermotolerans strain able to largely antagonize the development of the B. cinerea, at both in vitro and in vivo conditions. In particular, we detected the ability of the three isolates of M. pulcherrima strains Ale4, N20/006, and Pr7 and the L. thermotolerans strain N10 to completely inhibit (100% in reduction) the mycelial growth of B. cinerea by producing fungistatic compounds. We found, using an extracellular lytic enzymes activity assay, that such activity could be related to lipid hydrolyzation, β-1,3-glucanase and pectinase activity, and pectinase and protease activity, depending on the yeasts used. Results from our in vitro assays allowed us to hypothesize for M. pulcherrima strains Ale4 and N20/006 a possible combination of both the production of soluble metabolites and volatile organic compounds to antagonize against B. cinerea growth. Moreover, in semi-commercial conditions, the M. pulcherrima strain N20/006 and L. thermotolerans strain N10 showed relevant antagonistic effect also at low concentrations (with a significantly reduction of ‘slip skin’ incidence of 86.4% and 72.7%, respectively), thus highlighting a peculiar property to use in commercial development for organic agriculture and the handling process.
Highlights
Italy: two isolates, coded N10 and N20/006 (Table S1), were isolated from new table grape crossbreeds (Turi, Bari, Italy), Ale4 and Ale5 were isolated from Aleatico grapes, and Pr7 was isolated from Primitivo variety
The color and morphology of each isolate were analyzed on WL semisolid plates, by using the morphological traits proposed by Pallmann et al, 2001 [23] (Figure S1, Table S2)
Overall data revealed that the five isolates belonged to three genera: Metschnikowia, Lachancea, and Hanseniaspora, and, by combining sequencing both of 18S and 5.8S rDNA, this allowed us to assign each isolate to a species: the three isolates Ale4, N20/007, and Pr7 matched with M. pulcherrima with a percentage of sequence identity >97%, we performed multi-alignment analysis among M. pulcherrima sequences and data showed few differences among these three isolates sequences (Supplementary Materials)
Summary
Without appropriate conditions, symptoms of infections start with small necrosis on the skin, which enlarges in brown spots In those areas, the fungus produces macerating enzymes below the skin, that separate the cuticle from the flesh (slip skin). Especially for export purposes, the EU countries proposed 10 ppm as the maximum level of SO2 , in order to protect consumers and the environment For these reasons, several alternative strategies have been proposed in the postharvest management of B. cinerea rots, including chemical [10], biological [11], and physical means [11,12]. In line with environmental-friendly approaches, ‘organic viticulture’ continues to increase globally
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