Target-gene amplicon sequencing is the most exploited high-throughput sequencing application in microbial ecology. The targets are taxonomically relevant genes, with 16S rRNA being the gold standard for bacteria. As for fungi, the most commonly used target is the internal transcribed spacer (ITS). However, the uneven ITS length among species may promote preferential amplification and sequencing and incorrect estimation of their abundance. Therefore, the use of different targets is desirable. We evaluated the use of three different target amplicons for the characterization of fungal diversity. After an in silico primer evaluation, we compared three amplicons (the ITS1-ITS2 region [ITS1-2], 18S ribosomal small subunit RNA, and the D1/D2 domain of the 26S ribosomal large subunit RNA), using biological samples and a mock community of common fungal species. All three targets allowed for accurate identification of the species present. Nevertheless, high heterogeneity in ITS1-2 length was found, and this caused an overestimation of the abundance of species with a shorter ITS, while both 18S and 26S amplicons allowed for more reliable quantification. We demonstrated that ITS1-2 amplicon sequencing, although widely used, may lead to an incorrect evaluation of fungal communities, and efforts should be made to promote the use of different targets in sequencing-based microbial ecology studies. IMPORTANCE Amplicon-sequencing approaches for fungi may rely on different targets affecting the diversity and abundance of the fungal species. An increasing number of studies will address fungal diversity by high-throughput amplicon sequencing. The description of the communities must be accurate and reliable in order to draw useful insights and to address both ecological and biological questions. By analyzing a mock community and several biological samples, we demonstrate that using different amplicon targets may change the results of fungal microbiota analysis, and we highlight how a careful choice of the target is fundamental for a thorough description of the fungal communities.

The use of sulfur dioxide (SO ₂ ) as wine additive is able to ensure both antioxidant protection and microbiological stability. In spite of these undeniable advantages, in the last two decades the presence of SO ₂ in wine has raised concerns about potential adverse clinical effects in sensitive individuals. The winemaking industry has followed the general trend towards the reduction of SO ₂ concentrations in food, by expressing at the same time the need for alternative control methods allowing reduction or even elimination of SO _2. In the light of this, research has been strongly oriented toward the study of alternatives to the use of SO ₂ in wine. Most of the studies have focused on methods able to replace the antimicrobial activity of SO ₂ . This review article gives a comprehensive overview of the current state‐of‐the‐art about the chemical additives and the innovative physical techniques that have been proposed for this purpose. After a focus on the chemistry and properties of SO ₂ in wine _, as well as on wine spoilage and on the conventional methods used for the microbiological stabilization of wine, recent advances on alternative methods proposed to replace the antimicrobial activity of SO ₂ in winemaking are presented and discussed. Even though many of the alternatives to SO ₂ showed good efficacy, nowadays no other physical technique or additive can deliver the efficacy and broad spectrum of action as SO ₂ (both antioxidant and antimicrobial), therefore the alternative methods should be considered a complement to SO ₂ in low‐sulfite winemaking, rather than being seen as its substitutes.

Aims: The microbial community of different types of unripened Pasta Filata cheese was investigated by culture‐independent methods with the aim of rapidly achieving knowledge about cheese microbiota ...

Two types of white wheat bread (high- and low-type loaves) were investigated for rope spoilage. Thirty of the 56 breads tested developed rope spoilage within 5 days; the high-type loaves were affected by rope spoilage more than the low-type loaves. Sixty-one Bacillus strains were isolated from ropy breads and were characterized on the basis of their phenotypic and genotypic traits. All of the isolates were identified as Bacillus subtilis by biochemical tests, but molecular assays (randomly amplified polymorphic DNA PCR assay, denaturing gradient gel electrophoresis analysis, and sequencing of the V3 region of 16S ribosomal DNA) revealed greater Bacillus species variety in ropy breads. In fact, besides strains of B. subtilis, Bacillus licheniformis , Bacillus cereus , and isolates of Bacillus clausii and Bacillus firmus were also identified. All of the ropy Bacillus isolates exhibited amylase activity, whereas only 32.4% of these isolates were able to produce ropiness in bread slices after treatment at 96°C for 10 min. Strains of lactic acid bacteria previously isolated from sourdough were first selected for antirope activity on bread slices and then used as starters for bread-making experiments. Prevention of growth of approximately 10 ⁴ rope-producing B. subtilis G1 spores per cm ² on bread slices for more than 15 days was observed when heat-treated cultures of Lactobacillus plantarum E5 and Leuconostoc mesenteroides A27 were added. Growth of B. subtilis G1 occurred after 7 days in breads started with Saccharomyces cerevisiae T22, L. plantarum E5, and L. mesenteroides A27.