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).

Physiological features were examined of a 20-year-old Vitis vinifera ‘Italia’ table grape vineyard cropped in Apulia, Italy. Healthy vines with no foliar symptoms and any indications of wood or berry alterations, vines with natural wood infections by Phaeoacremonium minimum (syn. P. aleophilum) and Phaeomoniella chlamydospora showing brown wood streaking symptoms, and vines naturally infected with P. minimum, P. chlamydospora and Fomitiporia mediterranea with brown wood streaking and white rot symptoms, were surveyed. Bleeding xylem sap, collected at bud-break from healthy vines showed the greatest total ascorbic acid level, while vines with brown wood streaking and white rot had the greatest viscosity coefficient, glutathione concentration, and plant growth regulator activities. Compared to healthy vines, leaves of wood affected vines, sampled during the unfolded leaf, fruit setting, cluster closing and bunch ripening vine growth stages, had reduced fresh and dry weights, total chlorophyll concentrations, and increased leaf surface area. Low ascorbic acid and reduced glutathione concentrations, weak redox state, and moderate levels of dehydroascorbic acid and oxidized glutathione were also detected in these vines. Analyses also detected reduced activities of dehydroascorbate reductase, ascorbate free radical reductase and glutathione reductase in diseased vines. The cell membrane damage, associated with lipid peroxidation, was coupled with high hydrogen peroxide concentrations. These changes could contribute to the cell death of leaves and foliar symptom development. The ascorbate-glutathione cycle supports grapevine susceptibility to Esca complex-associated fungi.

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This study explored the role of Aphanocladium album (strain MX95), Pleurotus ostreatus (strain ALPO) and Pleurotus eryngii (strain AL142PE) as potential biological limiters. MX95, ALPO and AL142PE were screened under laboratory studies against Phytophthora nicotianae (PN), Fusarium oxysporum f. sp. radicis-lycopersici (FORL), Fusarium oxysporum f. sp. lycopersici (FOL), Fusarium solani (FS), Sclerotinia minor (SM), Sclerotinia sclerotiorum (SS), Athelia (Sclerotium) rolfsii (AR) and Verticillium dahliae (VD). The radial growth inhibition and the over-growth of potential antagonists on the target organisms were used to assess the interactions in the in vitro dual culture plate assay. The antagonistic ability of each challenge isolate was evaluated by calculating an index of the antagonism (AI) based on the interaction type in the dual cultures. MX95, reducing the growth of SS (20%) and FS (40%), displayed deadlock at mycelial contact against FOL and FORL, deadlock at distance versus VD and completely over-grew PN and SM. ALPO reduced (43 to 88%) the mycelial growth of tested pathogens except FORL and replaced PN and VD. AL142PE reducing (53 to 67%) SS, VD, FS and FOL mycelial growth and completely over-grew PN. AR showed combative ability against all the experienced biological limiters. Based on the results of the AI values, MX95 (AI = 16.5) was considered an active antagonist, while ALPO (AI = 11.5) and AL142PE (AI = 12.0) were moderately active antagonists. Strains MX95, ALPO and AL142PE were suitable as environment-friendly potential biocontrol agents to manage some of the main soil-borne agents of foot, root, soft rot and wilt diseases. These results are the first step in the assessment of the potential capacity of these organisms as biological limiters. Nevertheless, additional experiments should be performed for the translation to the field conditions in plant protection against soil-borne plant pathogens. In particular, the optimisation of dose and application time validation should be performed for a solid conclusion about the competitive ability of MX95, ALPO and AL142PE and the usefulness of potential biological limiters.