First report of Diplodia africana on Grevillea robusta

Branch cankers and dieback were observed on silky-oak trees (Grevillea robusta) along some streets of Palermo (Sicily, Italy). Fungi isolated from symptomatic branches were identified as Diplodia africana and D. seriata by morphological charac-ters and phylogenetic analyses of combined ITS and translation elongation factor 1-α sequences. Pathogenicity was verified by inoculating twigs of 3-y-old silky-oak plants. This is the first report of D. africana on G. robusta and the first record of D. seriata on this host in the northern hemisphere

Fungal pathogens associated with grapevine trunk diseases in young vineyards in Sicily

After the first report of grapevine decline caused by Botryosphaeriaceae in Sicily in 2007, epidemiological studies carried out in mature vineyards until 2011 confirmed the widespread occurrence of “Botryosphaeria dieback” and the “Esca complex” disease. Dieback symptoms were also recently observed in two young vineyards in Partanna and Castellammare del Golfo in western Sicily (Trapani province). Declining vines were inspected for grapevine trunk disease (GTD) symptoms, and were uprooted and submitted for analyses. Fungal isolates were collected and identified using culturing and molecular analyses. One isolate per identified species was inoculated to three grapevine shoots to evaluate pathogenicity and fulfil Koch’s postulates. Several GTD Botryosphaeriaceae pathogens in the genera Cadophora, Ilyonectria, Neonectria, Phaeoacremonium and Phaeomoniella were isolated from the symptomatic young vines. Artificial inoculation confirmed the pathogenicity of these fungi. In addition, virulence variability was observed among the isolates, with P. chlamydospora causing the largest lesions. The different species were associated with specific symptoms and/or host vine parts, especially in the roots and around the grafting areas. Several fungi associated with Petri disease and black foot were shown to be responsible of young vine decline

Sull'antagonismo in vivo ed in vitro di Acremonium byssoides, endofita in Vitis vinifera, nei confronti di Plasmopara viticola

Lo studio dell\u2019interazione fra Acremonium byssoides, Vitis vinifera e Plasmopara viticola, condotto nell\u2019ultimo decennio, ha evidenziato in vitro e in vivo l\u2019attivit\ue0 antagonistica dell\u2019ifomicete, endofita negli organi verdi di alcune cultivars di vite, nei confronti del patogeno. In particolare, \ue8 stato accertato che sospensioni conidiche, filtrati colturali, estratti grezzi e metaboliti di A. byssoides riducono sensibilmente la germinazione delle spore agamiche e gamiche di P. viticola, limitando la produzione di propaguli. Inoltre, l\u2019uso di un microscopio laser confocale e l\u2019impiego di un\u2019opportuna tecnica di decolorazione dei tessuti fogliari, seguita da colorazione di contrasto, ha consentito di visualizzare l\u2019ifomicete, latente nelle nervature di foglie sane e iperparassita dell\u2019oomicete in foglie infette. In queste ultime, infatti, A. byssoides, dopo aver prodotto metaboliti secondari tossici per P. viticola, ne invade e degrada micelio, rami sporangiofori e spore gamiche. Tale attivit\ue0 antagonistica, determinando il contenimento sia della diffusione che della sopravvivenza del patogeno, pu\uf2 assumere, quindi, un ruolo rilevante nella definizione di strategie di difesa biologica contro la peronospora della vite

A cytological and ultrastructural study on the maturation and germination of oospores of Plasmopara viticola from overwintering vine leaves

Observations on cytological and ultrastructural changes in Plasmopara viticola oospores were carried out during the overwintering period. Three types of oospores were observed. Type I, characterized by a thin inner oospore wall (IOW), large lipid globules and two nuclei, was recovered only in samples collected in October. These oospores were considered to be immature. Maturation occurred during November and involved a noticeable increase in thickness of the IOW, fusion of nuclei, formation of an ooplast and break up of large lipid globules into smaller ones (type II oospores). A few oospores (type III) showed abnormal organization with very large lipid globules and less frequently discernible nuclei. IOW solubilization, dissolution of the ooplast and lipid globules and nuclear division were the first detectable events during oospore germination. Germinating oospores produce a germ tube which was terminated by a sporangium. In its young stage, the sporangium had a thick wall and an unusual multi-layered membrane. During this phase, nuclear divisions took place in the sporangium. While sporangium development progressed, the ribosome density in the cytoplasm decreased and mitochondria, initially roundish with evident cristae, became their usual tubular profile. The plasma membrane had a typical structure and storage organelles, such as finger print vacuoles and lipid globules, became more numerous in the cytoplasm. Larger vacuoles contained the flagella of differentiating zoospores.Observations on cytological and ultrastructural changes in Plasmopara viticola oospores were carried out during the overwintering period. Three types of oospores were observed. Type I, characterized by a thin inner oospore wall (IOW), large lipid globules and two nuclei, was recovered only in samples collected in October. These oospores were considered to be immature. Maturation occurred during November and involved a noticeable increase in thickness of the IOW, fusion of nuclei, formation of an ooplast and break up of large lipid globules into smaller ones (type II oospores). A few oospores (type III) showed abnormal organization with very large lipid globules and less frequently discernible nuclei. IOW solubilization, dissolution of the ooplast and lipid globules and nuclear division were the first detectable events during oospore germination. Germinating oospores produce a germ tube which was terminated by a sporangium. In its young stage, the sporangium had a thick wall and an unusual multi-layered membrane. During this phase, nuclear divisions took place in the sporangium. While sporangium development progressed, the ribosome density in the cytoplasm decreased and mitochondria, initially roundish with evident cristae, became their usual tubular profile. The plasma membrane had a typical structure and storage organelles, such as finger print vacuoles and lipid globules, became more numerous in the cytoplasm. Larger vacuoles contained the flagella of differentiating zoospores

A simple and rapid DNA extraction method from leaves of grapevine suitable for polymerase chain reaction analysis.

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 A260/280 and A260/230 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

Lasiolactols A and B Produced by the Grapevine Fungal Pathogen Lasiodiplodia mediterranea

A strain of Lasiodiplodia mediterranea, a fungus associated with grapevine decline in Sicily, produced several metabolites in liquid medium. Two new dimeric c-lactols, lasiolactols A and B (1and 2), were characterized as (2S*,3S*,4R*,5R*,20S*,30S*,40R*,50R*)- and (2R*,3S*,4R,5R*,20R*,30S*,40R*,50R*)-5-(4-hydroxymethyl-3,5-dimethyl-tetrahydro-furan-2-yloxy)-2,4-dimethyl-tetrahydro-furan-3-yl]-methanols by IR, 1D- and 2D-NMR, and HR-ESI-MS. Other fourmetabolites were identified as botryosphaeriodiplodin, (5R )-5-hydroxylasiodiplodin, (–)-(1R,2R)-jasmonic acid, and (–)-(3S,4R,5R)-4-hydroxymethyl-3,5-dimethyldihydro-2-furanone (3-6, resp.). The absolute configuration (R) at hydroxylatedsecondary C-atom C(7) was also established for compound 3. The compounds 1–3,5,and 6, tested for their phytotoxic activities to grapevine cv. Inzolia leaves at different concentrations (0.125, 0.25, 0.5, and 1 mg/ml) were phytotoxic and compound 5 showed the highest toxicity. All metabolites did not show in vitro antifungal activity against four plant pathogens