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.

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.

Basidiomycetes Are Particularly Sensitive to Bacterial Volatile Compounds: Mechanistic Insight Into the Case Study of Pseudomonas protegens Volatilome Against Heterobasidion abietinum.

Volatile organic compounds (VOCs) play an important role in the communication among organisms, including plants, beneficial or pathogenic microbes, and pests. In vitro, we observed that the growth of seven out of eight Basidiomycete species tested was inhibited by the VOCs of the biocontrol agent Pseudomonas protegens strain CHA0. In the Ascomycota phylum, only some species were sensitive (e.g., Sclerotinia sclerotiorum, Botrytis cinerea, etc.) but others were resistant (e.g., Fusarium oxysporum f. sp. cubense, Verticillium dahliae, etc.). We further discovered that CHA0 as well as other ten beneficial or phytopathogenic bacterial strains were all able to inhibit Heterobasidion abietinum, which was used in this research as a model species. Moreover, such an inhibition occurred only when bacteria grew on media containing digested proteins like peptone or tryptone (e.g., Luria-Bertani agar or LBA). Also, the inhibition co-occurred with a pH increase of the agar medium where the fungus grew. Therefore, biogenic ammonia originating from protein degradation by bacteria was hypothesized to play a major role in fungus inhibition. Indeed, when tested as a synthetic compound, it was highly toxic to H. abietinum (effective concentration 50% or EC50 = 1.18 M; minimum inhibitory concentration or MIC = 2.14 M). Using gas chromatography coupled to mass spectrometry (GC/MS), eight VOCs were found specifically emitted by CHA0 grown on LBA compared to the bacterium grown on potato dextrose agar (PDA). Among them, two compounds were even more toxic than ammonia against H. abietinum: dimethyl trisulfide had EC50 = 0.02 M and MIC = 0.2 M, and 2-ethylhexanol had EC50 = 0.33 M and MIC = 0.77 M. The fungus growth inhibition was the result of severe cellular and sub-cellular alterations of hyphae occurring as early as 15 min of exposure to VOCs, as evidenced by transmission and scanning electron microscopy observations. Transcriptome reprogramming of H. abietinum induced by CHA0's VOCs pointed out that detrimental effects occurred on ribosomes and protein synthesis while the cells tried to react by activating defense mechanisms, which required a lot of energy diverted from the growth and development (fitness cost).