Antagonism of Bacillus subtilis strain AG1 against vine wood fungal pathogens

Summary. Antagonistic substances produced by a Bacillus subtilis strain (AG1), which were previously found to slow down the growth of esca fungi in vitro, were produced in an artifi cial medium, isolated from the cell-free medium by precipitation and acidifi cation (to less than pH 2.5) and extracted from the precipitate with 96% ethanol. The crude extract employed in antibiotic assays confi rmed, in vitro, the antagonism of B. subtilis against Phaeoacremonium aleophilum and Phaeomoniella chlamydospora, and also showed antifungal activity toward Verticillium dahliae and Botryosphaeria rhodina.

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 pathogen-infected 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 h…

Epidemiological investigations and molecular characterization of ‘candidatus phytoplasma solani’ in grapevines, weeds, vectors and putative vectors in western Sicily, (Southern Italy)

Bois noir is caused by &lsquo

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&ndash

INVESTIGATIONS ON ‘CANDIDATUS PHYTOPLASMA SOLANI’ IN WESTERN SICILY, SOUTHERN ITALY

‘Candidatus Phytoplasma solani’, belonging to the 16SrXII-A subgroup, is associated with grapevine Bois Noir (BN). It is usually transmitted by Hyalesthes obsoletus from a wide range of weeds to grapevine, which is considered a dead-end host. Moreover, other alternative vectors can play a role in the epidemiology of the disease. In Sicily, the presence of BN was reported in 1996, both on typical and on newly introduced cultivars. Furthermore, high BN incidence was observed on native grape cultivars grown close to ‘Chardonnay’ vineyards. In summer 2014 and 2015, the spread and the severity of BN were recorded in a 10 year-old vineyard located in San Giuseppe Jato (PA, Western Sicily), grown …

Pathogenicity and possible biological control of fungi associated to Botryosphaeria dieback of grapevine in Western Sicily

Botryosphaeria dieback is a widespread grapevine trunk disease caused by species of fungi within the family Botryosphaeriaceae. Symptoms of the disease include bud necrosis, leaf chlorosis, vascular discoloration of the wood, cankers and dieback. In 2013 typical symptoms of Botryosphaeria dieback was observed in Western Sicily and four species associated with the syndrome were isolated and identified as Diplodia seriata, Lasiodiplodia mediterranea, Neofusicoccum parvum and Neofusicoccum vitifusiforme. In order to fulfill Koch’s postulates, pathogenicity tests of eighteen isolates were performed on two-year grapevine cuttings of cv. Inzolia. Six months after inoculation all the Botryosphaeri…

An Optimized and Rapid DNA Extraction Method From Leaves of Grapevine Suitable for PCR-DGGE Based Analysis

Molecular techniques and genetic studies require a fair amount of DNA of high quality in order to produce reliable and clear results (Li et al., 2007). In grapevine, the yield and quality of DNA can be significantly affected by secondary metabolites such as polyphenols, polysaccharides and tannins, that may be consistently abundant during the different stages of leaf development (Iandolino et al., 2004). The development of a simple, rapid and reliable method for the extraction of genomic DNA from grape leaves collected at several stages during development, as well as from healthy grape leaves and leaves infected by pathogenic microorganisms, was the main objective of this study. The protoco…

Interaction between Acremonium byssoides and Plasmopara viticola in Vitis vinifera

The endophytic fungus Acremonium byssoides was isolated from the leaves of grapevine cv. Regina Bianca to determine whether it could act as an antagonistic endophyte of the downy mildew agent Plasmopara viticola. The occurrence of this fungus was ascertained in 34 grapevines (cultivars Regina Bianca, Catarratto and Insolia) representing all the grapevine plants in an experimental vineyard located in Sicily and which had never been treated with any fungicides. The isolation frequency of the endophytic Acremonium strains was assessed in several plant organs by means of monthly samplings over a 2-year period. The endophyte was found in all the grapevines, with higher frequencies in the cv. Reg…

Primi studi su Biscogniauxia mediterranea in Sicilia occidentale

È stata condotta un’indagine su un diffuso deperimento delle querce, all’interno di un’area della riserva naturale orientata “Bosco della Ficuzza, Rocca Busambra, Bosco del Cappelliere e Gorgo del Drago”, nelle contrade Pulpito del Re e S. Barbara (Corleone, PA). Campioni di organi sinto-matici e di strutture miceliari associate alle alterazioni sono stati sottoposti alle opportune analisi. I saggi di laboratorio hanno consentito di isolare colonie fungine identificate con metodi tradizionali e molecolari come appartenenti a B. mediterranea. Tali ceppi hanno mostrato una variabilità morfologica in relazione all’ospite e all’epoca stagionale. I saggi di inoculazione artificiale hanno evidenz…

Fungal Assemblages Associated with Roots of Halophytic and Non-halophytic Plant Species Vary Differentially Along a Salinity Gradient

Structure of fungal communities is known to be influenced by host plants and environmental conditions. However, in most cases, the dynamics of these variation patterns are poorly understood. In this work, we compared richness, diversity, and composition between assemblages of endophytic and rhizospheric fungi associated to roots of two plants with different lifestyles: the halophyte Inula crithmoides and the non-halophyte I. viscosa (syn. Dittrichia viscosa L.), along a spatially short salinity gradient. Roots and rhizospheric soil from these plants were collected at three points between a salt marsh and a sand dune, and fungi were isolated and characterized by ITS rDNA sequencing. Isolates…

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 (1 and 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-tetrahydrofuran- 2-yloxy)-2,4-dimethyl-tetrahydro-furan-3-yl]-methanols by IR, 1D-and 2D-NMR, and HR-ESI-MS. Other four metabolites 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 hydroxylated s…

Pathogenicity bioassays of isolates of Beauveria bassiana on Rhynchophorus ferrugineus

BACKGROUND The control of Rhynchophorus ferrugineus (Olivier), the main palm pest in the Mediterranean Basin, is problematic because of its biology and the current restrictions in many European countries on the use of chemical insecticides in urban areas. Entomopathogenic fungi have been studied as potential biological control agents, but information on their natural incidence is limited. Strains of Beauveria bassiana (Balsamo) Vuillemin were isolated from symptomatic insects collected on dead palms, and their pathogenicity against different instars of R. ferrugineus was evaluated in the laboratory. RESULTS The overall percentage of infected insects found in Canary palms was 7%. In laborato…

Latifoglie in Sicilia: un laboratorio di casi fitopatologici

Si riportano alcuni casi fitopatologici osservati su differenti latifoglie, in vari ambienti boschivi siciliani. Le indagini, in particolare, sono state rivolte all’isolamento e all’identificazione dei mi-crorganismi fungini associati alle alterazioni. In alcuni casi, inoltre, mediante saggi d’inoculazione artificiale, è stata valutata la loro eventuale patogenicità. Tra i numerosi taxa identificati, alcuni, noti come parassiti di debolezza, hanno mostrato una spiccata attitudine fitopatogena su ospiti sani e stressati. Gli studi condotti, evidenziando l’incidenza e la severità di alcune malattie, accertano un generale stato di sofferenza delle latifoglie forestali, dovuto, probabilmente, a…

ChemInform Abstract: 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 γ-lactols, lasiolactols A and B (1 and 2), were characterized as (2S*,3S*,4R*,5R*,2'S*,3'S*,4'R*,5'R*)- and (2R*,3S*,4R*,5R*,2'R*,3'S*,4'R*,5'R*)-(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 four metabolites 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 hydroxylated se…

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 discernib…

Arthrinium phaeospermum , Phoma cladoniicola and Ulocladium consortiale , New Olive Pathogens in Italy

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…

Spatial pattern analysis of esca in different geographical areas of Italy.

Presence of endophytic bacteria in Vitis vinifera leaves as detected by Fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) in combination with confocal laser scanning microscopy (CLSM) was applied to detect and localize bacterial colonies in leaf tissues of Vitis vinifera. Leaves were cleared to minimize the autofluorescence of plant fragments. The use of fluorescently labeled bacterial probe EUB338 on discolored grapevine leaf disks allowed the estimation of the spatial distribution of different bacterial colonies. In particular, bacterial colonies were found in veins, cells, hairs, intercellular spaces, and in cut edges of leaf disks of both non-Acremonium byssoides-colonized and A. byssoides-colonized leaves of five different cultivars. Furthermore, CLSM confirmed th…

Botryosphaeriaceae species causing canker and dieback of English walnut (Juglans regia) in Italy

Digestion of DNA regions to discriminate ochratoxigenic and non-ochratoxigenic strains in the Aspergillus niger aggregate

Abstract Aspergillus strains belonging to the Aspergillus niger aggregate, either isolated from Italian grapes or received from public collections, were analysed in order to discriminate between the ochratoxin A (OTA) producing and the non-producing strains by means of the analysis of Internal Transcribed Spacers (ITS), Intergenic Spacers (IGS) and of a β-tubulin gene portion. A. niger and Aspergillus awamori were identified observing the macro- and microscopic features of the colonies and the strains ochratoxigenicity was evaluated through Thin Layer Chromatography and/or High Performance Liquid Chromatography. PCR amplification of ITS, IGS and β-tubulin gene portion produced 600, 440 and …

Endophytic Fungi in Asymptomatic Vitis vinifera L. and their Effects on Plasmopara viticola

During a multi-year study on the life cycle of Plasmopara viticola to assess the typical epidemiology of downy mildew on grapevine (Vitis vinifera) in west Sicily (Italy), several leaves on which the downy mildew had collapsed were found and observed in a vineyard located in Marsala, in Trapani Province. An antagonistic fungus was isolated from these leaves and named A20; through its morphology, it was identified as a hyphomycete belonging to the genus Acremonium. The antagonism of A20 against the asexual and sexual spores of P. viticola, as well as endophytic nature of the genus Acremonium in V. vinifera were confirmed by further studies which have been carried out over the past decade. In…

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 1…

Detection of Bacterial Endophytes in Vitis vinifera L. and Antibiotic Activity against Grapevine Fungal Pathogens

This chapter presents the results of studies conducted to (i) detect and localize endophytic bacteria in symptomless and symptomatic leaf tissues of grapevine (Vitis vinifera) using fluorescence in situ hybridization (FISH) in combination with confocal laser scanning microscopy (CLSM); and (ii) test the antagonistic activity of Bacillus amyloliquefaciens AG1, previously isolated from grape wood affected by the esca syndrome, against grapevine fungal pathogens: Alternaria alternata, obtained from grapevine leaves showing necrotic concentric spots; Aspergillus carbonarius, A. ochraceus and Penicillium verrucosum, isolated from grapes affected by secondary rot; Botrytis cinerea, isolated from …

Grapevine decline in Italy caused by Lasiodiplodia theobromae

The first report of a dieback of grapevine caused by Lasiodiplodia theobromae in Sicily (Italy) is given. About twelve per cent of the vines in the cv. Insolia vineyard surveyed, showed spur dieback, retarded growth and wood necrosis. Isolation trials and pathogenicity tests are briefl y reported, together with morphological, cultural and molecular characters on which identification was based.

First report of Neofusiccoccum vitifusiforme and presence of other Botryosphaeriaceae species associated with Botryosphaeria dieback of grapevine in Sicily (Italy)

Summary. Since 2007, when a grapevine decline caused by Lasiodiplodia theobromae was reported for the first time in Sicily, vines showing similar declining symptoms have been also found in other grape-growing areas of western and central Sicily. We report the result of a study on fungi associated with grapevine decline in Sicily, with particular regard to those belonging to the Botryosphaeriaceae. Four species were found to be associated with declining vines, namely Diplodia seriata, Lasiodiplodia sp., Neofusicoccum parvum and Neofusicoccum vitifusiforme, the latter species reported for the first time on Vitis vinifera in Italy.