Unravelling the Function of the Sesquiterpene Cyclase STC3 in the Lifecycle of Botrytis cinerea.

The genome sequencing of Botrytis cinerea supplies a general overview of the map of genes involved in secondary metabolite synthesis. B. cinerea genomic data reveals that this phytopathogenic fungus has seven sesquiterpene cyclase (Bcstc) genes that encode proteins involved in the farnesyl diphosphate cyclization. Three sesquiterpene cyclases (BcStc1, BcStc5 and BcStc7) are characterized, related to the biosynthesis of botrydial, abscisic acid and (+)-4-epi-eremophilenol, respectively. However, the role of the other four sesquiterpene cyclases (BcStc2, BcStc3, BcStc4 and BcStc6) remains unknown. BcStc3 is a well-conserved protein with homologues in many fungal species, and here, we undertake its functional characterization in the lifecycle of the fungus. A null mutant ΔBcstc3 and an overexpressed-Bcstc3 transformant (OvBcstc3) are generated, and both strains show the deregulation of those other sesquiterpene cyclase-encoding genes (Bcstc1, Bcstc5 and Bcstc7). These results suggest a co-regulation of the expression of the sesquiterpene cyclase gene family in B. cinerea. The phenotypic characterization of both transformants reveals that BcStc3 is involved in oxidative stress tolerance, the production of reactive oxygen species and virulence. The metabolomic analysis allows the isolation of characteristic polyketides and eremophilenols from the secondary metabolism of B. cinerea, although no sesquiterpenes different from those already described are identified.

In Vitro Analysis of the Antagonistic Biological and Chemical Interactions between the Endophyte Sordaria tomento-alba and the Phytopathogen Botrytis cinerea.

Plant pathogenic infections causing substantial global food losses are a persistent challenge. This study investigates a potential biocontrol strategy against the necrotrophic fungus Botrytis cinerea using the endophytic fungus Sordaria tomento-alba isolated from Gliricidia sepium in Colombia. Today, synthetic fungicides dominate B. cinerea control, raising environmental and health concerns. S. tomento-alba exhibits notable in vitro effects, inhibiting B. cinerea growth by approximately 60% during co-culture and 50% in double disc co-culture. Additionally, it suppresses botryanes production and produces the compound heptacyclosordariolone, which has proven effective in inhibiting B. cinerea mycelial growth and spore germination in vitro. This biocontrol agent could be a potential eco-friendly alternative to replace synthetic fungicides. Our study provides insights into the chemical and biological mechanisms underpinning the antagonistic activity of S. tomento-alba, emphasizing the need for further research to understand its biosynthesis pathways and optimize its biocontrol potential. It also contributes molecular evidence of fungal interactions with implications for advanced forums in molecular studies in biology and chemistry, particularly in addressing plant pathogenic infections and promoting sustainable agriculture.

In Vitro Studies of Endophytic Bacteria Isolated from Ginger (Zingiber officinale) as Potential Plant-Growth-Promoting and Biocontrol Agents against Botrytis cinerea and Colletotrichum acutatum.

Agriculture currently confronts a multitude of challenges arising from the excessive utilization of chemical pesticides and the proliferation of phytopathogenic fungi strains that exhibit resistance to commonly employed active compounds in the field. Botrytis cinerea and Colletotrichum acutatum are phytopathogenic fungi that inflict substantial economic losses within agriculture and food due to their high impacts on crops both pre- and post-harvest. Furthermore, the emergence of fungal strains that are resistant to commercial fungicides has exacerbated this problem. To explore more environmentally sustainable alternatives for the control of these pathogens, an investigation into the endophytic bacteria associated with ginger (Zingiber officinale Rosc.) was conducted. The primary focus of this study involved evaluating their inhibitory efficacy against the fungi and assessing their potential for promoting plant growth. The endophytic bacteria genera Lelliottia, Lysinibacillus, Kocuria, Agrococcus, Acinetobacter, Agrobacterium, Zymobacter, and Mycolicibacterium were identified. All isolates showed remarkable in vitro antagonistic ability against B. cinerea (>94%) and C. acutatum (>74%). Notably, the Lelliottia amnigena J29 strain exhibited a notable proficiency in producing extracellular enzymes and indole compounds (IAA), solubilizing phosphate and potassium, and forming biofilm. Furthermore, the Lysinibacillus capsici J26, Agrococcus citreus J28, and Mycolicibacterium sp. J5 strains displayed the capacity for atmospheric nitrogen fixation and siderophore production. These findings underscore the agricultural and biotechnological potential of endophytic bacteria derived from ginger plants and suggest the feasibility of developing alternative approaches to manage these two phytopathogenic fungi.

Culturable Yeast Diversity of Grape Berries from Vitis vinifera ssp. sylvestris (Gmelin) Hegi.

Vitis vinifera L. ssp. sylvestris (Gmelin) Hegi is recognized as the dioecious parental generation of today's cultivars. Climatic change and the arrival of pathogens and pests in Europe led it to be included on the International Union for Conservation of Nature (IUCN) Red List of Threatened Species in 1997. The present work focused on the study of culturable yeast occurrence and diversity of grape berries collected from wild vines. Sampling was performed in 29 locations of Azerbaijan, Georgia, Italy, Romania, and Spain. In total, 3431 yeast colonies were isolated and identified as belonging to 49 species, including Saccharomyces cerevisiae, by 26S rDNA D1/D2 domains and ITS region sequencing. Isolates of S. cerevisiae were also analyzed by SSR-PCR obtaining 185 different genotypes. Classical ecology indices were used to obtain the richness (S), the biodiversity (H'), and the dominance (D) of the species studied. This study highlights the biodiversity potential of natural environments that still represent a fascinating source of solutions to common problems in winemaking.

Improving an Industrial Sherry Base Wine by Yeast Enhancement Strategies.

There is growing interest in yeast selection for industrial fermentation applications since it is a factor that protects a wine's identity. Although it is strenuous evaluating the oenological characteristics of yeasts in selection processes, in many cases the most riveting yeasts produce some undesirable organoleptic characteristics in wine. The aim of the present work is to improve an industrial yeast strain by reducing its hydrogen sulfide (H2S) production. To accomplish this, two different improvement approaches were used on said yeast: hybridization by mass mating and adaptive laboratory evolution, both performed through spore generation and conjugation, thus increasing genetic variability. Three evolved variants with lower H2S production were obtained and used as starters to carry out fermentation at an industrial level. Wine quality was analyzed by its principal oenological parameters and volatile aroma compounds, which were both corroborated by sensory evaluations. Significant differences between the produced wines have been obtained and a substantial improvement in aromatic quality has been achieved. Both hybrids were the most different to the control due to terpenes and esters production, while the evolved strain was very similar to the parental strain. Not only have organoleptic defects been reduced at an industrial level, more floral and fruitier wines have been produced.

Unravelling the Initial Triggers of Botrytis cinerea Infection: First Description of Its Surfactome.

Botrytis cinerea is a critically important phytopathogenic fungus, causing devastating crop losses; signal transduction cascades mediate the "dialogue" among the fungus, plant, and environment. Surface proteins play important roles as front-line receptors. We report the first description of the surfactome of a filamentous fungus. To obtain a complete view of these cascades during infection of B. cinerea, its surfactome has been described by optimization of the "shaving" process and LC-MS/MS at two different infection stages, and with both rapid and late responses to environmental changes. The best results were obtained using PBS buffer in the "shaving" protocol. The surfactome obtained comprises 1010 identified proteins. These have been categorized by gene ontology and protein-protein interactions to reveal new potential pathogenicity/virulence factors. From these data, the percentage of total proteins predicted for the genome of the fungus represented by proteins identified in this and other proteomics studies is calculated at 54%, a big increase over the previous 12%. The new data may be crucial for understanding better its biological activity and pathogenicity. Given its extensive exposure to plants and environmental conditions, the surfactome presents innumerable opportunities for interactions between the fungus and external elements, which should offer the best targets for fungicide development.

Deletion of the Bcnrps1 Gene Increases the Pathogenicity of Botrytis cinerea and Reduces Its Tolerance to the Exogenous Toxic Substances Spermidine and Pyrimethanil.

During the infection of grapevine (Vitis vinifera) by the fungus Botrytis cinerea, the concentration of polyamines, which are toxic substances for the phytopathogen, increases in the grape. Nine NRPS genes have been identified in the genome of B. cinerea, yet the function of five of them remains unknown. For this reason, we have studied the expression of the 9 NRPS genes by RT-qPCR in a medium supplemented with sublethal concentrations of three polyamines (1,3-diaminopropane (1,3-DAP), spermidine (SPD), and spermine (SPM)). Our results show that the presence of polyamines in the culture medium triggered the overexpression of the Bcnrps1 gene in the pathogen. Deleting Bcnrps1 did not affect mycelial growth or adaptation to osmotic stress, and we show that its expression is not essential for the cycle of infection of the B. cinerea. However, mutating the Bcnrps1 gene resulted in overexpression of the Bcnrps6 gene, which encodes for the excretion of siderophores of the coprogen family. Moreover, gene deletion has reduced the tolerance of B. cinerea B05.10 to toxic substances such as the polyamine SPD and the fungicide pyrimethanil, and its virulence has increased. Our findings provide new insights into the function of the Bcnrps1 gene and its involvement in the tolerance of B. cinerea against exogenous toxic compounds.

Endophytic Bacteria Bacillus subtilis, Isolated from Zea mays, as Potential Biocontrol Agent against Botrytis cinerea.

Plant diseases are one of the main factors responsible for food loss in the world, and 20-40% of such loss is caused by pathogenic infections. Botrytis cinerea is the most widely studied necrotrophic phytopathogenic fungus. It is responsible for incalculable economic losses due to the large number of host plants affected. Today, B. cinerea is controlled mainly by synthetic fungicides whose frequent application increases risk of resistance, thus making them unsustainable in terms of the environment and human health. In the search for new alternatives for the biocontrol of this pathogen, the use of endophytic microorganisms and their metabolites has gained momentum in recent years. In this work, we isolated endophytic bacteria from Zea mays cultivated in Colombia. Several strains of Bacillus subtilis, isolated and characterized in this work, exhibited growth inhibition against B. cinerea of more than 40% in in vitro cultures. These strains were characterized by studying several of their biochemical properties, such as production of lipopeptides, potassium solubilization, proteolytic and amylolytic capacity, production of siderophores, biofilm assays, and so on. We also analyzed: (i) its capacity to promote maize growth (Zea mays) in vivo, and (ii) its capacity to biocontrol B. cinerea during in vivo infection in plants (Phaseolus vulgaris).

Culturable Yeasts as Biofertilizers and Biopesticides for a Sustainable Agriculture: A Comprehensive Review.

The extensive use of synthetic fertilizers and pesticides has negative consequences in terms of soil microbial biodiversity and environmental contamination. Faced with this growing concern, a proposed alternative agricultural method is the use of microorganisms as biofertilizers. Many works have been focused on bacteria, but the limited literature on yeasts and their potential ability to safely promote plant growth is gaining particular attention in recent years. Thus, the objective of this review is to highlight the application of yeasts as biological agents in different sectors of sustainable agricultural practices through direct or indirect mechanisms of action. Direct mechanisms include the ability of yeasts to provide soluble nutrients to plants, produce organic acids and phytohormones (indole-3-acetic acid). Indirect mechanisms involve the ability for yeasts to act as biocontrol agents through their high antifungal activity and lower insecticidal and herbicidal activity, and as soil bioremediating agents. They also act as protective agents against extreme environmental factors by activating defense mechanisms. It is evident that all the aspects that yeasts offer could be useful in the creation of quality biofertilizers and biopesticides. Hence, extensive research on yeasts could be promising and potentially provide an environmentally friendly solution to the increased crop production that will be required with a growing population.

Development of the "Applied Proteomics" Concept for Biotechnology Applications in Microalgae: Example of the Proteome Data in Nannochloropsis gaditana.

Most of the marine ecosystems on our planet are still unknown. Among these ecosystems, microalgae act as a baseline due to their role as primary producers. The estimated millions of species of these microorganisms represent an almost infinite source of potentially active biocomponents offering unlimited biotechnology applications. This review considers current research in microalgae using the "omics" approach, which today is probably the most important biotechnology tool. These techniques enable us to obtain a large volume of data from a single experiment. The specific focus of this review is proteomics as a technique capable of generating a large volume of interesting information in a single proteomics assay, and particularly the concept of applied proteomics. As an example, this concept has been applied to the study of Nannochloropsis gaditana, in which proteomics data generated are transformed into information of high commercial value by identifying proteins with direct applications in the biomedical and agri-food fields, such as the protein designated UCA01 which presents antitumor activity, obtained from N. gaditana.

Ormosils loaded with SiO2 nanoparticles functionalized with Ag as multifunctional superhydrophobic/biocidal/consolidant treatments for buildings conservation.

The alarming increase of pollution has significantly increased buildings maintenance. Nowadays, the economic figures associated to repairing activities are even more relevant than those corresponding to new construction works, especially on heritage buildings. Since the degradation of building materials is the result of a complex combination of physical, chemical and biological agents, the development of multifunctional protective treatments remains a significant challenge. We report a simple strategy to produce a versatile biocidal/superhydrophobic/consolidant treatment by incorporating biocidal Ag nanoparticles (AgNPs) grafted to functionalized SiO2NPs into a silica sol, which can be applied by simple procedures such as spraying. The use of an Ag-SiO2 coupling agent increases biocidal effectiveness up to >90% values due to: (1) an increase of the AgNPs stability; (2) a hierarchical roughness due to the formation of Ag/SiO2NPs clusters; and (3) an enhanced contact with the cell walls. In addition, the synergistic effect allows for an easier removal of the dead cells, increasing the durability of the treatment.

Phenotypic Effects and Inhibition of Botrydial Biosynthesis Induced by Different Plant-Based Elicitors in Botrytis cinerea.

Botrytis cinerea is considered a model organism for the study of plant-pathogen interaction showing great genetic diversity and a high degree of morphological variability depending on environmental conditions. The use of new compounds and plant-based elicitors may trigger the expression of different B. cinerea genes, providing new sources of virulence factors. This work is focused on elucidating the phenotypic effect in B. cinerea of different carbon sources such as glucose, cellulose and tomato cell walls (TCW). Production of botrydial and dihydrobotrydial toxins was evaluated using thin-layer chromatography (TLC), proton nuclear magnetic resonance spectroscopy (1H-NMR) and mass spectrometry (UPLC-HRESIMS). Expression of the toxin biosynthesis gene BcBOT2 was followed using RT-qPCR. Results show an inhibition of the toxin biosynthesis pathway when TCW are present as a sole carbon source, suggesting that the toxin is only produced when rich molecules, like glucose, are available for fungal metabolism. That suggests a connection between gene expression of virulence factors and environmental conditions, where the silent genes can be induced by different culture conditions.

Wild Grape-Associated Yeasts as Promising Biocontrol Agents against Vitis vinifera Fungal Pathogens.

The increasing level of hazardous residues in the environment and food chains has led the European Union to restrict the use of chemical fungicides. Thus, exploiting new natural antagonistic microorganisms against fungal diseases could serve the agricultural production to reduce pre- and post-harvest losses, to boost safer practices for workers and to protect the consumers' health. The main aim of this work was to evaluate the antagonistic potential of epiphytic yeasts against Botrytis cinerea, Aspergillus carbonarius, and Penicillium expansum pathogen species. In particular, yeast isolation was carried out from grape berries of Vitis vinifera ssp sylvestris populations, of the Eurasian area, and V. vinifera ssp vinifera cultivars from three different farming systems (organic, biodynamic, and conventional). Strains able to inhibit or slow the growth of pathogens were selected by in vitro and in vivo experiments. The most effective antagonist yeast strains were subsequently assayed for their capability to colonize the grape berries. Finally, possible modes of action, such as nutrients and space competition, iron depletion, cell wall degrading enzymes, diffusible and volatile antimicrobial compounds, and biofilm formation, were investigated as well. Two hundred and thirty-one yeast strains belonging to 26 different species were isolated; 20 of them, ascribed to eight species, showed antagonistic action against all molds. Yeasts isolated from V. vinifera ssp sylvestris were more effective (up to 50%) against B. cinerea rather than those isolated from V. vinifera ssp vinifera. Six strains, all isolated from wild vines, belonging to four species (Meyerozyma guilliermondii, Hanseniaspora uvarum, Hanseniaspora clermontiae, and Pichia kluyveri) revealed one or more phenotypical characteristics associated to the analyzed modes of antagonistic action.

Dataset of the Botrytis cinerea phosphoproteome induced by different plant-based elicitors.

Phosphorylation is one of the main post-translational modification (PTM) involved in signaling network in the ascomycete Botrytis cinerea, one of the most relevant phytopathogenic fungus. The data presented in this article provided a differential mass spectrometry-based analysis of the phosphoproteome of B. cinerea under two different phenotypical conditions induced by the use of two different elicitors: glucose and deproteinized Tomate Cell Walls (TCW). A total 1138 and 733 phosphoproteins were identified for glucose and TCW culture conditions respectively. Raw data are deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier (PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD003099). Further interpretation and discussion of these data are provided in our research article entitled "Phosphoproteome analysis of B.cinerea in response to different plant-based elicitors" (Liñeiro et al., 2016) [1].

The F-actin capping protein is required for hyphal growth and full virulence but is dispensable for septum formation in Botrytis cinerea.

Filamentous (F-) actin is an integral part of the cytoskeleton allowing for cell growth, intracellular motility, and cytokinesis of eukaryotic cells. Its assembly from G-actin monomers and its disassembly are tightly regulated processes involving a number of actin-binding proteins (ABPs) such as F-actin nucleators and cross-linking proteins. F-actin capping protein (CP) is an alpha/beta heterodimer known from yeast and higher eukaryotes to bind to the fast growing ends of the actin filaments stabilizing them. In this study, we identified the orthologs of the two CP subunits, named BcCPA1 and BcCPB1, in the plant pathogenic fungus Botrytis cinerea and showed that the two proteins physically interact in a yeast two-hybrid approach. GFP-BcCPA1 fusion proteins were functional and localized to the assumed sites of F-actin accumulation, i.e. to the hyphal tips and the sites of actin ring formation. Deletion of bccpa1 had a profound effect on hyphal growth, morphogenesis, and virulence indicating the importance of F-actin capping for an intact actin cytoskeleton. As polarized growth - unlike septum formation - is impaired in the mutants, it can be concluded that the organization and/or localization of actin patches and cables are disturbed rather than the functionality of the actin rings.

Modifications of fungal membrane proteins profile under pathogenicity induction: A proteomic analysis of Botrytis cinerea membranome.

Botrytis cinerea is a model fungus for the study of phytopathogenicity that exhibits a wide arsenal of tools to infect plant tissues. Most of these factors are related to signal transduction cascades, in which membrane proteins play a key role as a bridge between environment and intracellular molecular processes. This work describes the first description of the membranome of Botrytis under different pathogenicity conditions induced by different plant-based elicitors: glucose and tomato cell wall (TCW). A discovery proteomics analysis of membrane proteins was carried out by mass spectrometry. A total of 2794 proteins were successfully identified, 46% of them were classified as membrane proteins based on the presence of transmembrane regions and lipidation. Further analyses showed significant differences in the membranome composition depending on the available carbon source: 804 proteins were exclusively identified when the fungus was cultured with glucose as a sole carbon source, and 251 proteins were exclusively identified with TCW. Besides, among the 1737 common proteins, a subset of 898 proteins presented clear differences in their abundance. GO enrichment and clustering interaction analysis revealed changes in the composition of membranome with increase of signalling function in glucose conditions and carbohydrate degradation process in TCW conditions. All MS data have been deposited in the ProteomeXchange with identifier PXD003099 (http://proteomecentral.proteomexchange.org/dataset/PXD003099).

Phosphoproteome analysis of B. cinerea in response to different plant-based elicitors.

The phytopathogen Botrytis cinerea is a ubiquitous fungus with a high capacity to adapt its metabolism to different hosts and environmental conditions in order to deploy a variety of virulence and pathogenicity factors and develop a successful plant infection. Here we report the first comparative phosphoproteomic study of B. cinerea, aimed to analyze the phosphoprotein composition of the fungus and its changes under different phenotypical conditions induced by two different carbon sources as plant based elicitors: glucose and deproteinized tomato cell wall (TCW). A total of 2854 and 2269 different phosphosites (2883 and 1137 phosphopeptides) were identified in glucose and TCW respectively, which map to 1338 phosphoproteins in glucose and 733 in TCW. Out of the identified phosphoproteins, 173 were exclusively found when glucose was the only carbon source and 11 when the carbon source was TCW. Differences in the pattern of phosphorylation-sites were also detected according to the carbon source. Gene ontology classification of the identified phosphoproteins showed that most of the characteristic proteins of the different carbon sources were related to signalling and transmembrane transport, thus highlighting the importance of these processes in the fungal adaptation to the surrounding conditions. BIOLOGICAL SIGNIFICANCE: The characterization of the B. cinerea phosphoproteome under different induction conditions reported here is the first comparative phosphoproteomic approach in this model phytopathogenic fungus. The identified phosphopeptides contribute to expand the map of known phosphoproteins in this pathogen and the observed changes according to the used carbon source contribute to understand the adaptation of the fungus to the environment changes. This knowledge improves the understanding of the adaptation mechanism, defines the role of the phosphoproteins involved in this process, and enables the advance in the design of novel strategies against the fungi.

Analysis of temperature-mediated changes in the wine yeast Saccharomyces bayanus var uvarum. An oenological study of how the protein content influences wine quality.

Saccharomyces bayanus var. uvarum plays an important role in the fermentation of red wine from the D.O. Ribera del Duero. This is due to the special organoleptic taste that this yeast gives the wines and their ability to ferment at low temperature. To determine the molecular factors involved in the fermentation process at low temperature, a differential proteomic approach was performed by using 2D-DIGE, comparing, qualitatively and quantitatively, the profiles obtained at 13 and 25°C. A total of 152 protein spots were identified. We detected proteins upregulated at 13°C that were shown to be related to temperature stress, the production of aromatic compounds involved in the metabolism of amino acids, and the production of fusel alcohols and their derivatives, each of which is directly related to the quality of the wines. To check the temperature effects, an aromatic analysis by GC-MS was performed. The proteomic and "aromatomic" results are discussed in relation to the oenological properties of S. bayanus var. uvarum.

Acetyltransferase SAS2 and sirtuin SIR2, respectively, control flocculation and biofilm formation in wine yeast.

Cell-to-cell and cell-to-environment interactions of microorganisms are of substantial relevance for their biotechnological use. In the yeast Saccharomyces cerevisiae, flocculation can be an advantage to clarify final liquid products after fermentation, and biofilm formation may be relevant for the encapsulation of strains of interest. The adhesion properties of wine yeast strains can be modified by the genetic manipulation of transcriptional regulatory proteins, such as histone deacetylases, and acetylases. Sirtuin SIR2 is essential for the formation of mat structures, a kind of biofilm that requires the expression of cell-wall protein FLO11 as its deletion reduces FLO11 expression, and adhesion of cells to themselves and to agar in a commercial wine strain. Deletion of acetyltransferase GCN5 leads to a similar phenotype. A naturally flocculant wine yeast strain called P2 was characterized. Its flocculation happens only during grape juice fermentation and is due to the presence of a highly transcribed version of flocculin FLO5, linked to the presence of a δ sequence in the promoter. Deletion of acetyltransferase SAS2 enhances this phenotype and maltose fermentation even more. Therefore, the manipulation of acetylation/deacetylation machinery members is a valid way to alter the interaction of industrial yeast to their environment.

Proteomic analysis of conidia germination in Colletotrichum acutatum.

Colletotrichum acutatum is an important phytopathogenic fungus causing anthracnose in commercially important fruit crops, such as strawberry. The conidia produced by the fungus are survival structures which play a key role in host infection and fungal propagation. Despite its relevance to the fungal life cycle, conidial biology has not been extensively investigated. Here, we provide the first proteomic description of the conidial germination in C. acutatum by comparing the proteomic profiles of ungerminated and germinated conidia. Using two-dimensional electrophoresis combined with MALDI-TOF/TOF mass spectrometry, we have identified 365 proteins in 354 spots, which represent 245 unique proteins, including some proteins with key functions in pathogenesis. All these proteins have been classified according to their molecular function and their involvement in biological processes, including cellular energy production, oxidative metabolism, stress, fatty acid synthesis, protein synthesis, and folding. This report constitutes the first comprehensive study of protein expression during the early stage of the C. acutatum conidial germination. It advances our understanding of the molecular mechanisms involved in the conidial germination process, and provides a useful basis for the further characterization of proteins involved in fungal biology and fungus life cycles.