Antifungal metabolites produced by Bacillus amyloliquefaciens\ud AG1, previously isolated from wood of grapevine\ud with “esca syndrome”, were studied. The crude protein\ud extract (CPE) obtained from culture supernatant fluid by\ud precipitation with ammonium sulfate was assayed against\ud many grapevine fungal pathogens. B. amyloliquefaciens strain\ud AG1 showed a broad spectrum of antifungal activity, inhibiting\ud mycelial growth in vitro of all tested fungal microorganisms.\ud The metabolites contained in CPE were heat stable and\ud remained active over a wide pH range (2–10). Their activity\ud was not affected by proteolytic or glycolytic enzymes. Tricine-\ud SDS-polyacrylamide gel electrophoresis revealed a single\ud band within the range of 2,510–3,480 Da, that showed inhibitory\ud activity when used in the antifungal assay. Mass spectrometry\ud analysis of this band allowed the substances involved\ud in antibiosis to be identified as two tryptic peptides that\ud correspond to the N-terminal sequence of subtilisin BPN’.\ud These results suggest a potential role of B. amyloliquefaciens\ud AG1 as a biocontrol agent

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The genomic grapevine (Vitis vinifera L.) DNA extraction is difficult because of secondary metabolites that interfere with DNA isolation procedures and subsequent applications. We developed a simple, rapid and efficient method for the extraction of genomic DNA from asymptomatic and pathogeninfected grape leaves. The protocol reported, based on a modified cetyl trimethylammonium bromide (CTAB) extraction procedure, allowed the rapid DNA extraction from little amounts of leaf material without employment of liquid nitrogen for initial tissue grinding. The protocol included polyvinylpyrrolidone (PVP) to bind phenolic compounds, β-mercaptoethanol to inhibit the oxidation of polyphenols, and a high concentration of NaCl (2.5 M) to increase the solubility of polysaccharides, thus reducing their co-precipitation with DNA. Final DNA solution did not contain polysaccharides, polyphenols and other major contaminants. The purity of genomic DNA was confirmed by A 260/280 and A 260/230 ratios calculated from the spectrophotometric readings. In addition, the quality of the DNA extracted from asymptomatic, Oidium tuckeri-and Plasmopara viticola-infected leaves of V. vinifera L. was evaluated in polymerase chain reaction (PCR) analyses by using different set of primers to be able to amplify vegetal, fungal and bacterial DNA.

In recent years, leaf necrosis and twig dieback in the olive crop have been detected in Sicily (Italy). In this article, we identify the predominant fungal species associated with symptomatic leaves and twigs, using morphological features and DNA sequencing of the internal transcribed spacer (ITS) region, as Alternaria alternata, Arthrinium phaeospermum, Phoma cladoniicola and Ulocladium consortiale. The pathogenicity of these four species was tested on olive plants cv. Biancolilla. All species were pathogenic on leaves, but only U. consortiale produced cortical lesions on twigs, thus suggesting its main role in the Olea europaea twig dieback. To our knowledge, this is the first report of A. phaeospermum, P. cladoniicola and U. consortiale as olive pathogens.