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In previous laboratory tests the culture filtrates and the crude extracts obtained from Acremonium byssoides were shown to completely inhibit Plasmopara viticola sporangia germination. A study was thus undertaken in order to investigate on the possible inhibiting activity of the hyphomycete towards pathogenesis of the oomycete in grapevine. A. byssoides was cultivated on malt-extract agar (MEA) to obtain pure colonies and on maltextract broth (MEB) to obtain culture filtrate and crude extract. Healthy leaves of grapevine were put in Petri dishes containing sporangia suspended in: deionized sterile water, malt-extract broth, dimethylsulfoxide (DMSO), culture filtrate, crude extract and a suspension of A. byssoides germinated conidia. After 6 days from inoculations, the bioactivity of A. byssoides was evaluated on the basis of P. viticola evasion percentage. The results show that the higher percentage of pathogen evasion occurred in leaves inoculated with sporangia suspended in distilled water, in MEB and in DMSO; lower values of pathogen evasion were noted when leaves were treated with culture filtrate and crude extract; no evasion occurred in presence of A. byssoides germinated conidia, showing either antibiotic or hyperparasitic activity.

Gli Autori riportano i risultati del Protocollo AIEOP-91 presentando igli 88 casi registrati in 15 Centro AIEOP dal gGennaio 1991 al Dicembre 193

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.

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. Regina Bianca in the buds, and in the cooler months of the year. All the strains collected from the leaves, and most of the strains from the buds of cv. Insolia were identified as A. byssoides by conventional means. The presence and spatial distribution of A. byssoides was determined in asymptomatic or P. viticola-infected leaves of the grapevine cv. Insolia using an optimised staining technique and differential interferential contrast light microscopy, as well as laser scanning microscopy. A. byssoides was a natural colonizer of grapevines and actively parasitized P. viticola. Culture filtrates and the crude extract of A. byssoides obtained from a cv. Insolia foliar strain, completely inhibited P. viticola sporangia.

In diversi vigneti della Sicilia occidentale è stata monitorata, su uve sane e infestate, la dinamica di presenza di specie di Aspergillus e Penicillium, alcune delle quali note produttrici di ocratossina A, la principale micotossina della filiera vitivinicola. Gli studi condotti dall’allegagione alla raccolta evidenziano la variabilità quali-quantitativa delle popolazioni di aspergilli e penicilli, in relazione alle condizioni ambientali. Le analisi tossicologiche, accertando la presenza occasionale della micotossina, confermano l’assenza di correlazione tra il livelli di OTA nelle uve e la presenza dei funghi in vigneto.

This work addresses questions about the embodied narratives during a collaborative inquiry involving 4 groups of women and professionals in two residential mother-and-child and whose outcomes were unforeseeable. In the epistemological framework of Embodied Cognition (Shapiro, 2010), the body is seen as a center for the researcher to give a shape to her own knowing and feeling. So, knowing through sense brings to an aesthetic positioning, redefining both reflexivity and encounter, in searching with subjects and in contexts. The desire to be more sensitive to the body, and to the embodied and embedded relationships that are constantly built in Adult Education, brought me - a doctoral student – to share a common path of research, whose scope is to interrogate the role of the researcher in the learning experience

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.

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.

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 secondary 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.

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 grapes with grey mould; Cladosporium viticola, agent of grapevine leaf spots; Fomitiporia mediterranea, obtained from grapevines with white rot symptoms; Fusarium oxysporum, isolated from the grapevine rhizosphere; Lasiodiplodia theobromae, isolated from vine wood with cortical cankers; Phaeoacremonium aleophilum and Phaeomoniella chlamydospora, both isolated from grapevine showing esca symptoms; Phoma glomerata from grape leaves showing necrotic spots; and Verticillium dahliae, obtained from decaying vine. The FISH/CLSM approach was successfully applied to visualize endophytic bacteria in grapevine leaves, including also those that it is not possible to cultivate in sterilized medium. Also, B. amyloliquefaciens AG1 showed a potential role as a biological control agent against trunk fungal pathogens as well as other fungal phytopathogens.

Posidonia oceanica is the most common, widespread and important monocotyledon seagrass in the Mediterranean Basin, and hosts a large biodiversity of species, including microorganisms with key roles in the marine environment. In this study, we ascertain the presence of a fungal endophyte in the roots of P.oceanica growing on different substrata (rock, sand and matte) in two Sicilian marine meadows. Staining techniques on root fragments and sections, in combination with microscope observations, were used to visualise the fungal presence and determine the percentage of fungal colonisation (FC) in this tissue. In root fragments, statistical analysis of the FC showed a higher mean in roots anchored on rock than on matte and sand. In root sections, an inter- and intracellular septate mycelium, producing intracellular microsclerotia, was detected from the rhizodermis to the vascular cylinder. Using isolation techniques, we obtained, from both sampling sites, sterile, slow-growing fungal colonies, dark in colour, with septate mycelium, belonging to the dark septate endophytes (DSEs). DNA sequencing of the internal transcribed spacer (ITS) region identified these colonies as Lulwoana sp. To our knowledge, this is the first report of Lulwoana sp. as DSE in roots of P.oceanica. Moreover, the highest fungal colonisation, detected in P.oceanica roots growing on rock, suggests that the presence of the DSE may help the host in several ways, particularly in capturing mineral nutrients through lytic activity.

Recently, a study on the Quercus ilex L. decay caused by biotic and abiotic agents has hypothesized alterations on endophytic and mycorrhizal fungal populations. Since holm oak is among the most representative Mediterranean species, that occurs widely in both urban and rural areas, in this work we preliminarly report these symbiotic fungal communities in asymptomatic seedlings, in order to compare these populations with those found in seedlings subjected to water and saline stress. Endophytic fungi were isolated from twigs and leaves and identified by morphological and molecular analysis. The ectomycorrhizal status was evaluated on root samples randomly selected to calculate the percentage of not vital and vital ectomycorrhizal root tips. Anatomical structure of the mantle was observed to assign each mycorrhiza to a morphotype. A total of 11 fungal species were isolated and isolation frequency (IF) ranged between 0,4% and 16,7%: Alternaria alternata, Biscogniauxia nummularia, Bjerkandera adusta, Diaporthe chamaeropis, Gnomoniopsis sp., Neosetophoma samarorum, Peziza varia, Phoma pinodella, Septoria sp., Stagonosporopsis cucurbitacearum and a Mycelia sterilia with the highest IF. Among these, A. alternata, B. adusta and the sterile mycelium were isolated from both organs, while the other were found only on twigs or on leaves, suggesting a possible organ specificity. A total of 393 colonized root tips were present in analyzed roots, of which only the 32% was vital. All examined tips were assigned to 2 different morphotypes. Further studies are also needed to understand the possible variability and role of these fungal communities on health status of host.

The first monitoring on the CTV occurrence in central-western Sicily carried out during spring 2011 in asymptomatic citrus groves, has allowed to report CTV- free citrus-growing areas (province of Caltanissetta and Trapani), the first focus in province of Agrigento and two new others in province of Palermo. Molecular analysis (RT-PCR of p18, p20 and p23 genes, single strand conformation polymorphism and partial sequencing) showed that the obtained CTV isolates had a high homology (99%) with mild CTV strains (T 30 and T 385). Moreover, survey on the genetic flow and analysis of synonymous/nonsynonymous, by MEGA 5.0 and DNAsp 5.0 programs, demonstrated the provenience of the new “tristeza” foci near Palermo from the first one reported in 2006, probably due to aphid diffusion. Regarding the effect of artificial mycorrhization on fitness of citrange seedlings and micro-propagated plants, first findings suggest an improvement of some vegetative parameters. In addition, a collection of colonies of genera Fusarium and Trichoderma from roots and rhizosphere of citranges, using suitable selective substrates, was obtained. In vitro antagonism tests allowed to select a Trichoderma isolate, called 1TC, able to compete with the most of Fusaria. Appropriate mycorrhizal inocula and selected antagonists towards soil-borne pathogens, improving the vegetative performance of plants, both in the nursery and in the field, could improve the host tolerance to CTV.

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

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 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. Key words: Vitis vinifera L., DNA extraction, PCR, fungi, bacteria.

In the last five years, we have observed a new decline of Olea europaea in north-western and southern Sicily (insular Italy). The syndrome consists in more or less extensive leaf chlorosis, sometimes with irregular necrotic marginal or apical spots. Apical defoliation of twigs showing cortical necrosis and withering has often been observed. Isolations, carried out in 2007/08, yilded a complex of fungal genera associated with the syndrome. Among these, Stemphylium sp., a well-known foliar pathogen of other plants, was constantly isolated from all the alterations observed. In December 2010, single spore colonies of this fungus were used for artificial inoculations on healthy 3-year-old olive plants (cv. Biancolilla). Necrotic foliar spots, extending from the inoculum site to the apex leaf, and twig cortical necrosis with darkening of internal tissues were, observed from 15/40 and 90/120 days, respectively, after inoculations. Colonies of Stemphylium sp. were always re-isolated from all symptomatic organs, thus fulfilling Koch’s postulates. To our knowledge, this is the first report of Stemphylium sp. as a pathogen to olive trees in Sicily. Further morphological and molecular studies are in progress to achieve specific identification.

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 that bacteria were located in different layers of the leaf tissue. At the same time, one cleared disk of each foliar sample was crushed and plated on Plate Count Agar to isolate cultivable endophytic bacteria. Some of these microorganisms were presumptively identified, by partial 16S rRNA gene sequencing, as Burkholderia phytofirmans, thus suggesting their potential role as plant growth promoters and sources of resistance against pathogenic agents, such as fungi.

STUDY ON THE CONTROL OF BACTROCERA OLEAE AND FUNGI INDUCING EARLY LEAF AND FRUIT DROP IN OLIVE GROWING FOR TABLE OLIVES AND OIL PRODUCTION IN SICILY. Susceptibility of olive cultivars to olive fruit fly, damage thresholds, sampling methods, effectiveness of products deterring olive fruit fly oviposition, foliar chlorosis and withering of branches have been investigated. Riassunto Tra le cultivar saggiate (16 siciliane e 6 estere) esiste un ampio gradiente nella suscettibilità agli attacchi di B. oleae, andando dalle più suscettibili Nocellara del Belice, Giarraffa, Cerasuola alle meno preferite; tra queste ultime vi sono cultivar con caratteristiche peculiari diversificate: Nocellara messinese (tardiva a drupa grande), Minuta (precoce a drupa piccola), Moresca (precoce a drupa media), Picholine (tardiva a drupa media). Il colore verde è chiaramente preferito dalla mosca delle olive, mentre altre caratteristiche fisiche e chimiche indagate quali dimensione della drupa, coefficiente di sfericità, contenuto in acqua, olio e oleuropeina non hanno evidenziato correlazioni statisticamente significative con il livello d’infestazione. La analisi effettuate sulla qualità degli oli evidenziano un’assenza di correlazione statisticamente significativa tra questa ed il livello d’infestazione raggiunto alla raccolta, sia essa totale o dannosa, mentre tale correlazione è stata registrata nei confronti dell’infestazione dannosa media registrata nei due mesi precedenti la raccolta. Per le olive da mensa la convinzione diffusa che le semplici punture di ovideposizione compromettano l’estetica delle olive trasformate viene smentita dai risultati delle indagini svolte; i segni di tali punture dopo la trasformazione sono poco o per nulla visibili ad occhio nudo. Difatti la soglia di dannosità adottata dagli operatori della trasformazione non tiene conto delle punture, ma delle larve di III età, delle loro gallerie, e ancora di più dei fori d’uscita. I due metodi semplificati di misurazione dell’infestazione della mosca delle olive provati appaiono entrambi idonei all’individuazione delle soglie d’intervento e facilmente applicabili dagli olivicoltori, anche se il metodo che ricorre all’uso della lente contafili e del coltello da innesto fornisce valori più precisi e quasi identici al campionamento che necessita dell’analisi al microscopio stereoscopico. Tra i prodotti repellenti e antiovideponenti saggiati, il caolino e l’idrossido di rame si sono dimostrati così efficaci, e privi di effetti negativi su olio e olive da mensa, da poter suggerire un loro utilizzo sia nell’olivicoltura biologica che in quella convenzionale. I risultati dello studio sui funghi agenti di filloptosi e carpoptosi anticipata inducono a ipotizzare una scarsa interazione tra i microrganismi fungini isolati (Alternaria, Cladosporium, Phoma, Rhizoctonia) e le alterazioni osservate.

Previous investigation on five cultivars of healthy Sicilian grapevine allowed the isolation of endophytic bacteria belonging to Bacillus genus from different organs (bud, leaf, stalk and shoot). The aim of this work was to use fluorescence in situ hybridization (FISH) experiments in healthy and damaged leaf tissues of Vitis vinifera to visualize and localize bacteria associated with plant materials. The leaves were cleared to minimize the autofluorescence of the plant fragments. The use of fluorescently labelled bacterial probe Eub338 in FISH experiments on discoloured grapevine leaf disks allowed the estimation of the spatial distribution of different bacterial colonies. At the same time, one cleared disk each foliar sample was minced on Plate Count Agar to detect cultivable endophytic bacteria. The combined microscopic approach showed a differential location of microbial colonies within the leaf tissues examined. Particularly, the bacterial colonies were found in the veins, cells, hairs, intercellular spaces and more in the cut edges of leaf disk. Several bacterial species were isolated from leaf tissue. The high presence of microbial colonies in leaf tissue suggests a potential use of these endophytic bacteria as plant growth promoting and sources of resistance against pathogenic agents, such as fungi. Further studies are required to understand the role of bacterial endophytes in plant-microbe inter- actions. Besides, the DGGE (Denaturing Gradient Gel Electrophoresis) analysis from foliar disks will be carried out to detect uncultivable endophytic bacterial populations.

1 Giovanni Fatta, Tiziana Campisi, Mario Li Castri, Stefano Lo Piccolo e Calogero Vinci Università degli Studi di Palermo, Dipartimento di Progetto e Costruzione Edilizia(DPCE) LA TRADIZIONE COSTRUTTIVA NELL'AREA DELLE MADONIE Sommario Nonostante sia sempre stata terra di passaggio e di conquista, in stretta relazione con altre culture tecniche, la Sicilia esprime anche nell’edilizia di base la propria insularità, non di rado manifestata in maniera originale, strettamente legata all’ambiente in cui si sviluppa. Le forti modificazioni e gli stravolgimenti prodotti nell’ultimo dopoguerra dalle architetture di importazione imposte ai delicati tessuti storici, dall’edilizia di quantità e dall’abusivismo, hanno ferito ma non nascondono quei segni unitari che caratterizzano vaste aree, così da far riconoscere l’esistenza di una vera e propria architettura regionale caratterizzata dall’uso di alcuni modelli formali, costruttivi e tipologici di base adeguati all’ambiente e al contesto insediativo. Coscienti dell’estremo degrado fisico e sociale dovuto alla sub-urbanizzazione e alla globalizzazione dell’edilizia di molte delle aree cittadine e periferiche, volta alla uniformazione basata su presunti criteri applicati a prescindere dal tempo e dal luogo, in questo studio volto alla valorizzazione e la tutela della varietà e della differenza si analizza il vasto territorio montano delle Madonie che comprende, in un Parco Regionale, quindici centri urbani densi di storia e cultura. La distanza dalle città e dagli agglomerati industriali, insieme alle difficili collegamenti stradali, hanno consentito la permanenza non solo episodica dei valori materici, tecnologici e formali proposti dalla Architettura Regionale, fondata su una radicata tradizione locale e su caratteri architettonici consolidati dalle “regole dell’arte”. L’analisi e la conoscenza delle antiche tecniche e dei materiali tradizionali e del significato funzionale e formale degli elementi architettonici che costituiscono le “parole” dell’ambiente costruito, fornisce i riferimenti necessari per recuperare, nello spirito evolutivo dell’attività costruttiva, il significato costruttivo, funzionale ed estetico degli organismi edilizi diffusi nell’entroterra siciliano e della loro capacità di costituire parte integrante del sistema ambientale. Attraverso un repertorio di casi concreti, ritenuti sufficientemente rappresentativi della complessa realtà locale, è stato inizialmente possibile analizzare la tipologia aggregativa e funzionale del tessuto storico montano nelle varianti condizionate da giaciture, allineamenti, localizzazioni. Si è parallelamente affiancato l’approfondimento sui materiali e le tecniche costruttive che connotano gli elementi costitutivi dell’edilizia storica locale, quali murature, solai, volte, coperture, infissi, aggetti, pavimentazioni interne ed esterne, rivestimenti, intonaci, etc. Le fasi di conoscenza e di analisi, consolidate nelle relazioni tra i diversi aspetti ed elementi, costituiscono un valido supporto richiesto dalle comunità locali da tradurre in regole costruttive in grado di valorizzare i livelli qualitativi dell’architettura tradizionale.