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

Fungal contaminants in Sicilian livestock feeds and first studies on the enzymatic activity of Aspergillus isolates

The purposes of this study were 1) to determine the total fungal contamination in Sicilian raw materials and livestock, 2) to evaluate the occurrence of Aspergillus spp., Penicillium spp. and Fusarium spp., 3) to identify fungi belonging to the genus Aspergillus and 4) to determine their ability to produce cellulolytic enzymes. Fourteen feed samples were collected in a feed mill near to Palermo (Sicily, Italy). Analysis of the total mycobiota was performed on Sabourad Dextros Agar (SAB) and Potato Dextrose Agar (PDA) and total fungal counts were expressed as CFU/g. Aspergillus spp. isolates were selected on the basis of the frequency of isolation and identifed using micro and macro-morphological characteristics and ITS sequence analysis. The ability of the Aspergillus isolates to produce cellulolytic enzymes was tested qualitatively by in vitro assay at two temperature, 25 and 30 °C, and in static and shaking condition. Total fungal population ranged from 1.11x106 to 1.31x108 and from 1.11x103 to 1.58x106 CFU/g on PDA and SAB, respectively. All feed samples showed the recurrent presence of colonies belonging mostly to the ubiquitous genera Aspergillus, Fusarium and Penicillium. Eight isolates of Aspergillus spp. were obtained and identifed as A. amstelodami, A. awamori, A. flavus, A. niger, A. oryzae and A. tubingensis. Between them, A. awamori, A. niger and A. tubingensis showed the highest enzymatic activity. The presence of potential mycotoxigenic isolates of Aspergillus spp. in the analysed feeds represents a risk for animal health; moreover their ability to produce cellulolytic enzymes can seriously affect feed quality

Cultivable fungal endophytes in roots, rhizomes and leaves of Posidonia oceanica (L.) Delile along the Coast of Sicily, Italy

The presence of endophytic fungi in the roots, rhizomes, and leaves of Posidonia oceanica was evaluated in different localities of the Sicilian coast. Samples of roots, rhizomes, and leaves were submitted to isolation techniques, and the obtained fungal colonies were identified by morphological and molecular (rRNA sequencing) analysis. Fungal endophytes occurred mainly in roots and occasionally in rhizomes and leaves. Lulwoana sp. was the most frequent of the isolated taxa, suggesting a strong interaction with the host. In addition, eight other fungal taxa were isolated. In particular, fungi of the genus Ochroconis and family Xylariaceae were identified as endophytes in healthy plants at all sampling stations, whereas Penicillium glabrum was isolated at only one sampling station. Thus, several organs, especially roots of Posidonia oceanica, harbor endophytic fungi, potentially involved in supporting the living host as ascertained for terrestrial plants

Fatty acids produced by Neofusicoccum vitifusiforme and N. Parvum, fungi associated with grapevine botryosphaeria dieback

There is evidence that secondary metabolites are involved in the fungal pathogenicity and virulence of Neofusicoccum spp. Fatty acids may also influence the plant–pathogen interaction but, so far, no information is available on their production by species of Neofusicoccum associated with Botryosphaeria dieback, which is a well-known syndrome of several plants with a complex etiology. In the present paper, the production of fatty acids in liquid medium, by strains of N. vitifusiforme and N. parvum associated with declining Sicilian vine plants, was evaluated. Data, acquired via gas chromatography–mass spectrometry (GC/MS), show the presence of linoleic acid as the most abundant fatty acid produced by both examined strains. In addition, the pathogenicity of N. vitifusiforme was tested on 2-year-old grapevine plants of cv. Inzolia

Identification of Acremonium isolates from grapevines and evaluation of their antagonism towards Plasmopara viticola

Some endophytic fungal genera in Vitis vinifera, including Acremonium, have been reported as antagonists of Plasmopara viticola. Endophytic Acremonium isolates from an asymptomatic grapevine cultivar Inzolia from Italy were identified by morphological features and multigene phylogenies of ITS, 18S and 28S genes, and their intra-specific genomic diversity was analyzed by RAPD analysis. Culture filtrates (CFs) obtained from Acremonium isolates were tested in vitro for their inhibitory activity against the P. viticola sporangia germination. Among 94 isolates, 68 belonged to the Acremonium persicinum and 26 to the Acremonium sclerotigenum. RAPD analysis grouped the A. persicinum isolates into 15 clusters and defined 31 different strains. The A. sclerotigenum isolates, instead, were clustered into 22 groups and represented 25 strains. All A. persicinum CFs inhibited sporangia germination of P. viticola, while not all those of A. sclerotigenum had inhibitory effect. A different degree of inhibition was observed between strains of the same species, while some strains of different species showed identical inhibitory effect. No correlation was found between RAPD groups and inhibitory activity in both Acremonium species

Naphthalenone polyketides produced by Neofusicoccum parvum, a fungus associated with grapevine Botryosphaeria dieback

A strain of Neofusicoccum parvum isolated from declining vines was pathogenic to grapevine cultivar Inzolia in Sicily. This strain produced some metabolites in liquid medium. Crude extract, through a bio-guided purification process, yielded four naphthalenone polyketides. They were identified by comparison with spectroscopic data and optical proprieties reported in literature as: (3S, 4S)-7-ethyl-3,4,8-trihydroxy-6-methoxy-3,4-dihydro- 1-(2H)-naphthalenone, (3S*, 4S*)-3,4-dihydro-3,4,8-trihydroxy-7-(1-hydroxyethyl)-6-methoxy-1-(2H)-naphthalenone, (4S)-3,4-dihydro-4,8-dihydroxy-1-(2H)-naphthalenone, named botryosphaerones D and A, isosclerone, respectively, and (3S*, 4S*)-3,4,5-trihydroxy-1-tetralone (1-4). Phytotoxic activity of the isolated compounds (1-4) was tested on grapevine leaves at using the leaf puncture assay. All tested compounds were phytotoxic, with botryosphaerone D showing the greatest activity. The phytotoxic effects decreased when treated leaves were exposed to light. All of the metabolites did not show in vitro antifungal activity against Diplodia seriata, Lasiodiplodia mediterranea, Neofusicoccum vitifusiforme, or Phytophthora citrophthora. This is the first report of in vitro production of botryosphaerones D and A, and 3,4,5-trihydroxy-1-tetralone by N. parvu

Epidemiological survey on 'Candidatus Phytoplasma solani' and putative vectors in Western Sicily

Bois noir (BN) associated with ‘Candidatus Phytoplasma solani’ is an important grapevine yellows disease widespread in Europe and Mediterranean regions. During a two years survey (2014-15) carried out in a 10-year-old vineyard located in San Giuseppe Jato (PA), symptoms of Bois noir were observed on cvs Chardonnay, Pinot Noir and Nero d’ Avola. Incidence of the disease increased over the two years, reaching values of about 34 % on cv Chardonnay. Ca. ‘P. solani’ was detected on symptomatic leaf samples from all cultivars with the highest percentage on cv Chardonnay followed by Pinot noir and Nero d’Avola. Further surveys were performed in order to highlight the epidemiology of the disease by monitoring wild herbaceous species and potential vectors and by molecular characterization of Bois noir phytoplasma isolates. Herbaceous weed samples were collected within and at the border of the vineyard, whereas plant- and leafhoppers individuals were monitored from June to August of each year, using yellow sticky traps replaced at weekly intervals. Eleven weed species and about 2000 insect specimens were collected and identified at species or genus level by morphological feature. No Hyalesthes obsoletus was found. ‘Ca P. solani’ was detected in 5 weed species and nine out twenty-two taxa of the different insect specimens: Anaceratagallia laevis, Empoasca decipiens, E. vitis, Empoasca/Zigina sp., Hauptipia provincialis, Neoaliturus fenestratus, Selachina apicalis, Zygina rhamni and Z. serpentine. RLFP analysis of the tuf gene of phytoplasma isolates recovered from symptomatic grapes, weeds and insect specimens revealed the presence of only Ca P. solani’ tuf type-b