http://http://dspace.unitus.it:80 The DSpace digital repository system captures, stores, indexes, preserves, and distributes digital research material. Mon, 20 May 2013 13:44:45 GMT 2013-05-20T13:44:45Z http://hdl.handle.net/2067/97 Title: Analisi genetica e molecolare dei batteri implicati nella "moria" del nocciolo Authors: Proietti Zolla, Cristina Abstract: La dott. Proietti Zolla, con la tesi del suo dottorato “Analisi genetica e molecolare dei batteri implicati nella moria del nocciolo” ha condotto una ricerca finalizzata allo studio dei batteri coinvolti nella “moria” del nocciolo. Gli studi sono iniziati con l’identificazione dei batteri isolati da piante di nocciolo sintomatiche e ritrovati all’interno e all’esterno dell’insetto Anisandrus dispar attraverso metodi morfologici e molecolari basati sulla analisi delle sequenze del gene 16S rRNA. Quindi la ricerca è proseguita nello studio delle interazioni genetiche, per comprendere le relazioni esistenti tra questi batteri e quelli considerati coinvolti nella “moria” del nocciolo. L’allineamento delle sequenze del gene 16S ottenute con i programmi CLUSTALW e PHYLIPH ha permesso di ottenere un diagramma che mostra la relazione tra i ceppi. Gli stessi batteri analizzati mediante i primer 16S sono stati sottoposti ad analisi con AFLP. Le bande ottenute utilizzando tutte le combinazioni di primer sono state utilizzate per computare un’unica matrice di similarità utilizzando il coefficiente di Jaccard e per costruire un albero filogenetico. Sia l’analisi 16S che AFLP hanno consentito di evidenziare tra i batteri isolati diversità genetiche intraspecifiche e intrageneriche. La tecnica AFLP rispetto al 16S ha messo meglio in evidenza la presenza di polimorfismi tra i batteri appartenenti alla stessa specie, in particolare tra i batteri identificati come Pseudomonas spp. isolati nell’Anisandro e nella pianta, nonché tra ceppi di P. avellanae isolati nella provincia di Viterbo e in Grecia. L’individuazione dei polimorfismi genetici è stata utilizzata per la messa a punto di marcatori SCAR specifici per l’identificazione di P: avellanae. I primer SCAR sono stati ottenuti tagliando i frammenti AFLP polimorfici dal gel, clonandoli in cellule di Escherichia coli competenti, amplificandoli con gli stessi primer utilizzati per ottenere gli AFLP e allineando le sequenze ottenute in EMBL. I primer SCAR disegnati a partire dagli AFLP si sono rivelati specifici solo per i ceppi di P. avellanae e non hanno dato alcun amplificato né con i ceppi di P. syringae né con le Brennerie, né con l’E. amylovora. Inoltre gli stessi primer testati sui batteri isolati dall’insetto non hanno dato alcun amplificato escludendo così la possibilità che l’insetto sia vettore per questo batterio all’interno della pianta.; Pseudomonas avellanae, the main cause of hazelnut decline, causes severe economic losses to hazelnut (Corylus avellana L.) production in central Italy. In the same years in which Pseudomonas avellanae it was identified like the pathogenic responsible of the cancer of the hazelnut in the north of Greece, a similar disease was identified in the province of Viterbo. On the basis of morphological, biochemical, physiological and pathogenicity tests, the etiological agent has been identified as a bacterium belonging to 'true erwiniae group' and very closed to Erwinia amylovora and E. rubrifaciens. It was believed therefore that the agent of the “moria” could be a strain of E. amylovora highly specialized on the hazelnut. Other authors think that the causal agent is P. avellanae. Current assays used to identify virulent strains of P. avellanae, by plating on culture media and the use of biochemical, physiological, nutritional and pathogenicity tests, are too labour-intensive for routine use. Analysis of 16S rRNA sequences is used in order to fastly identify the bacteria isolates from hazelnut orchards in the Viterbo province, and from internal/esxternal of the insect Anisandrus dispar, supposed carrier of the pathogenic. The analysis has concurred of giving one first indication of which bacteria was probable pathogenic and inducing the "moria". Therefore they have been studied the intrageneric and intergeneric relations, confronting the molecular markers obtained from two different method: analysis of the mutations of 16S rRNA and AFLP technique. Both 16S analysis and AFLP have concurred to evidence between the bacteria the intrageneric diversities, but AFLP technique highlighted better the presence of polymorphism between the bacteria pertaining to the same species, in particular between the bacteria identifies like Pseudomonas spp. isolates in Anisandrus dispar and in plant, let alone between P. avellanae strains isolated in the province of Viterbo and Greece. The detect of the genetic polimorphism is used for design specific markers SCAR for the identification of P. avellanae. Primer pairs were designed on the basis of the sequence data obtained from the excised AFLP fragments. All P. avellanae strains, either from Greece or from Italy, produced an amplicon. No products were produced from the water controls, nor from other plant-pathogenic or hazelnut-associated bacteria. In particular, no amplicons were observed neither from P. s. pv. syringae , neither from Brennerie or Erwinia amylovora. Moreover the same primers tested on the bacteria isolated from the insect, have not given some amplified concurring to exclude, with a high reliability, the possibility that the insect vehicles P. avellanae in the plant. Description: Dottorato di ricerca in Protezione della piante Sun, 09 Apr 2006 22:00:00 GMT http://hdl.handle.net/2067/97 2006-04-09T22:00:00Z http://hdl.handle.net/2067/1190 Title: Bio-ethology of Anisandrus dispar F. and Its Possible Involvement in Dieback (Moria) Diseases of Hazelnut (Corylus avellana L.) Plants in Central Italy Authors: Balestra, Giorgio M.; Bucini, Danilo; Paparatti, Bruno; Speranza, Stefano; Proietti Zolla, Cristina; Pucci, Claudio; Varvaro, Leonardo Abstract: Hazelnut is one of the most important orchards in central Italy (Viterbo province). More than eighty phytophagous insect pests adversely affect hazelnut orchards, but only a few of these, such as Anisandrus dispar F. (Coleoptera, Scolytidae), induce severe damage. A bacterial disease (called moria) constitutes one of the main phytopathological problems of hazelnut plants in central Italy. Two years ago, the Lazio Regional Government and local hazelnut cooperatives supported a research into the bio-ethology of A. dispar and its possible association with moria disease on hazelnut plants in Viterbo. In 2003 and 2004 two experimental hazelnut areas were selected in the Capranica and Caprarola districts (Viterbo), where eighteen chemio-chromotrophic traps were installed to study the dynamic population of A. dispar and to catch live Scolytidae females. Representative samples of live A. dispar females were used to isolate and identify the bacterial populations present both out- side and inside the insects. After two years 5,726 A. dispar females had been caught. Of more than 1,400 live A. dispar females, 10% were submitted to microbiological ana- lyses by morphological, physiological, biochemical and molecular techniques. The populations of the main bacteria (by outside and inside) associated with the phyto- phagous were identified as Erwinia billingae, Brenneria quercina, Pantoea cedenensis and Pseudomonas spp. Studies are currently in progress to: i) clarify the biological cycle of A. dispar; ii) identify the role (direct and/or indirect) of the insect respect to the epidemiology of moria disease; iii) carry on pathogenicity tests on bacterial isolates to prove their involvement in bacteriosis; iv) develop specific primers to identify the presence of these bacteria when associated with the insect and with asymptomatic hazelnut plants; v) verify the influence of environmental parameters on the biology of both the insect and the disease. Fri, 31 Dec 2004 23:00:00 GMT http://hdl.handle.net/2067/1190 2004-12-31T23:00:00Z