New Potential Biological Limiters of the Main Esca-Associated Fungi in Grapevine.

The strains Trichoderma harzianum TH07.1-NC (TH), Aphanocladium album MX95 (AA), Pleurotus eryngii AL142PE (PE) and Pleurotus ostreatus ALPO (PO) were tested as biological limiters against Fomitiporia mediterranea Fme22.12 (FM), Phaeoacremonium minimum Pm22.53 (PM) and Phaeomoniella chlamydospora Pc22.65 (PC). Pathogens were obtained from naturally Esca-affected 'Nero di Troia' vines cropped in Grumo Appula (Puglia region, Southern Italy). The antagonistic activity of each challenge organism was verified in a dual culture. TH and PO completely overgrew the three pathogens. Partial replacement characterized PE-FM, PE-PM, PE-PC and AA-PC interactions. Deadlock at mycelial contact was observed in AA-FM and AA-PM cultures. The calculated antagonism index (AI) indicated TH and PE as moderately active antagonists (10 < AI < 15), while AA and PO were weakly active (AI < 10). The maximum value of the re-isolation index (s) was associated with deadlock among AA-PM, AA-PC and PE-FM dual cultures. The tested biological limiters were always re-isolated when PO and TH completely replaced the three tested pathogens. TH and AA confirmed their efficiencies as biological limiters when inoculated on detached canes of 'Nero di Troia' in dual combination with FM, PC and PM. Nevertheless, additional experiments should be performed for a solid conclusion, along with validation experiments in the field.

Chitinolytic Enzymes of the Hyperparasite Fungus Aphanocladium album: Genome-Wide Survey and Characterization of A Selected Enzyme.

The filamentous fungus Aphanocladium album is known as a hyperparasite of plant pathogenic fungi; hence, it has been studied as a possible agent for plant protection. Chitinases secreted by A. album have proven to be essential for its fungicidal activity. However, no complete analysis of the A. album chitinase assortment has been carried out, nor have any of its chitinases been characterized yet. In this study, we report the first draft assembly of the genome sequence of A. album (strain MX-95). The in silico functional annotation of the genome allowed the identification of 46 genes encoding chitinolytic enzymes of the GH18 (26 genes), GH20 (8 genes), GH75 (8 genes), and GH3 (4 genes) families. The encoded proteins were investigated by comparative and phylogenetic analysis, allowing clustering in different subgroups. A. album chitinases were also characterized according to the presence of different functional protein domains (carbohydrate-binding modules and catalytic domains) providing the first complete description of the chitinase repertoire of A. album. A single chitinase gene was then selected for complete functional characterization. The encoded protein was expressed in the yeast Pichia pastoris, and its activity was assayed under different conditions of temperature and pH and with different substrates. It was found that the enzyme acts mainly as a chitobiosidase, with higher activity in the 37-50 °C range.

Preliminary Studies on the In Vitro Interactions Between the Secondary Metabolites Produced by Esca-Associated Fungi and Enological Saccharomyces cerevisiae Strains.

Esca-affected vines alter the carbohydrate metabolism, xylem transport of water and photosynthesis and show regular grapes (but berries do not reach maturity), and phenolic compounds are reduced in concentration, oxidate and polymerizate. Pullulan and a mixture of scytalone and isosclerone (9:1; w/w), secondary metabolites produced in vitro and in planta by Phaeoacremonium minimum (syn. P. aleophilum) and Phaeomoniella chlamydospora, were assayed against the strains Byosal HS1 and IOC 18-2007 in microvinifications with synthetic grape must. The presence of pullulan and pentaketides mix affects the growth and metabolism of the tested Saccharomyces cerevisiae strains. Assays at 100 and 1000 µg mL-1 inhibited the growth of both strains, while no effects were recorded when evaluated at 1 and 5 µg mL-1. In comparison with the controls, pullulan and the scytalone/isosclerone mixture at 10 µg mL-1 had a growth reduction, a lower alcohol yield, reduced the concentration of tartaric acid and malic acid; and slowed down the production of lactic acid, acetic acid and total polyphenol content of the tested S. cerevisiae strains. These metabolites could be applied as an alternative to the sulfite addition in the early stages of vinification to support the action of selected Saccharomyces. Appealing is the subtractive action of pullulan against tartaric acid. Further data are needed to confirm and validate the enological performance in freshly pressed grape juice.

Protein Sources Alternative to Meat: State of the Art and Involvement of Fermentation.

Meat represents an important protein source, even in developing countries, but its production is scarcely sustainable, and its excessive consumption poses health issues. An increasing number of Western consumers would replace, at least partially, meat with alternative protein sources. This review aims at: (i) depicting nutritional, functional, sensory traits, and critical issues of single-cell proteins (SCP), filamentous fungi, microalgae, vegetables (alone or mixed with milk), and insects and (ii) displaying how fermentation could improve their quality, to facilitate their use as food items/ingredients/supplements. Production of SCP (yeasts, filamentous fungi, microalgae) does not need arable land and potable water and can run continuously, also using wastes and byproducts. Some filamentous fungi are also consumed as edible mushrooms, and others are involved in the fermentation of traditional vegetable-based foods. Cereals, pseudocereals, and legumes may be combined to offer an almost complete amino acid profile. Fermentation of such vegetables, even in combination with milk-based products (e.g., tarhana), could increase nutrient concentrations, including essential amino acids, and improve sensory traits. Different insects could be used, as such or, to increase their acceptability, as ingredient of foods (e.g., pasta). However, insects as a protein source face with safety concerns, cultural constraints, and a lack of international regulatory framework.

Phytotoxic metabolites produced by Verticillium dahliae Kleb. in olive wilting: a chemical and spectroscopic approach for their molecular characterisation.

Most of the symptoms associated with Verticillium wilt disease in olive cultivation are due to complexes excreted by Verticillium dahliae. In this study chemical and physico-chemical techniques were combined to investigate how the molecular structure of phytotoxins isolated from two pathotypes of Verticillium dahliae, defoliating, D, and non-defoliating, ND, grown on two different media, Verticillium-dahliae-Medium (VdM) and Simulated Xylem-fluid-Medium (SXM), can affect their aggressiveness. Data obtained highlight important structural differences, both in term of elemental composition and in functional groups distribution. Such peculiarities strongly affect their solubility, resulted higher for the phytotoxins from D pathotype. This property likely induces serious modifications of the conformational state of the proteinaceous component, making tyrosine residues accessible to the phosphate ion. A phosphorylation mechanism would thus be promoted, that is going to interfere with the function of the involved proteins in intracellular signalling networks, likely by altering its role in modulating the plant's response.

Plant Health and Rhizosphere Microbiome: Effects of the Bionematicide Aphanocladium album in Tomato Plants Infested by Meloidogyne javanica.

The artificial introduction in the soil of antagonistic microorganisms can be a successful strategy, alternative to agrochemicals, for the control of the root-knot nematodes (Meloidogyne spp.) and for preserving plant health. On the other hand, plant roots and the associated rhizosphere constitute a complex system in which the contribution of microbial community is fundamental to plant health and development, since microbes may convert organic and inorganic substances into available plant nutrients. In the present study, the potential nematicidal activity of the biopesticide Aphanocladium album (A. album strain MX-95) against the root-knot nematode Meloidogyne javanica in infected tomato plants was investigated. Specifically, the effect of the A. album treatment on plant fitness was evaluated observing the plant morphological traits and also considering the nematode propagation parameters, the A. album MX-95 vitality and population density. In addition, the treatment effects on the rhizosphere microbiome were analysed by a metabarcoding procedure. Treatments with A. album isolate MX-95 significantly decreased root gall severity index and soil nematode population. The treatment also resulted in increased rhizosphere microbial populations. A. album MX-95 can be favourably considered as a new bionematicide to control M. javanica infestation.

Study of defense-related gene expression in grapevine infested by Colomerus vitis (Acari: Eriophyidae).

Real-time quantitative polymerase chain reaction was used to study the expression of some marker genes involved in the interaction between grape (Vitis vinifera L.) and the erineum mite Colomerus vitis Pagenstecher (Acari: Eriophyidae). Potted vines of cultivars Atabaki (resistant to C. vitis), Ghalati (susceptible to C. vitis) and Muscat Gordo (moderately resistant to C. vitis) were infested at the six-leaf stage. The expression of protease inhibitor (PIN), beta-1,3-glucanase (GLU), polygalacturonase inhibitor (PGIP), Vitis vinifera proline-rich protein 1 (PRP1), stilbene synthase (STS), and lipoxygenase (LOX) genes was assessed on young leaves collected 96, 120 and 144 h after mite infestation (hami). As a control, non-infested leaves collected 24 h before mite infestations were used. Differences were detected in expression of the selected genes during the C. vitis-grapevine interaction. The resistant cultivar Atabaki increased the expression of LOX, STS, GLU, PGIP and PRP1 genes during the first 120 hami. On the contrary, in the susceptible Ghalati, all selected genes showed an expression level similar or lower than non-infested leaves. Muscat Gordo increased the expression of all selected genes in comparison with non-infested leaves, but it was lower than in Atabaki. Significant transcript accumulation of PIN gene was detected for Muscat Gordo whereas it was slightly up-regulated in Ghalati and Atabaki. LOX, STS, PIN, GLU, PGIP and PRP1 genes were clearly expressed in response to C. vitis infestation. We therefore infer that expression of PGIP, PIN and PRP1 genes could represent a defense strategy against C. vitis infestations in grapevine leaves.