No description available.

No description available.

The aim of our work was to study the molecular mechanisms involved in symptoms appearance of plants inoculated either with a virus or with a virus-satellite complex. In the first case, we tried to set up a reliable method for an early identification of PVYNTN strains present in Italy and causing potato tuber necrosis. This, to prevent their spread in the field and to avoid severe yield losses, especially in seed potato production. We tried to localize the particular genomic region responsible for tuber necrosis. To this purpose, we carried out RT-PCR experiments using various primer combinations, covering PVY genomic regions larger than those previously used by other authors. As the previous researchers, though, we were not able to differentiate all NTN from others PVY strains. This probably because of the frequent virus variability, due to both genomic mutations and possible recombination events among different strains. In the second case, we studied the influence of Y-sat (CaRNA5 satellite) on symptoms of CMV (Cucumber mosaic virus) in Nicotiana benthamiana plants: strong yellowing appearance instead of simple mosaic. Wang et al (2004), inoculating the same infectious complex on tobacco plants transformed with a viral suppressor of plant silencing (HC-PRO), did not experience the occurrence of yellowing anymore and, therefore, hypotesized that changes in symptoms were due to plant post transcriptional gene silencing (PTGS) mechanism. In our case, inoculation of N. benthamiana plants transformed with another PTGS viral suppressor (p19), and other plants defective for RNA polymerase 6 (involved in systemic silencing), still resulted in yellowing appearance. This, to our opinion, suggests that in our system another possible mechanism is involved.

Beet soil-borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus (BNYVV) are members of Benyvirus genus. BSBMV has been reported only in the United States while BNYVV has a worldwide distribution. Both viruses are vectored by Polymyxa betae, possess similar host ranges, particles number and morphology. Both viruses are not serologically related but have similar genomic organizations. Field isolates consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs 1 and 2 are essential for infection and replication while RNAs 3 and 4 play important roles on plant and vector interactions, respectively. Nucleotide and amino acid analyses revealed BSBMV and BNYVV are different enough to be classified in two different species. Additionally in BNYVV/BSBMV mixed infections, a competition was previous described in sugar beet, where BNYVV infection reduces BSBMV accumulation in both susceptible and resistant cultivars. Considering all this observations we hypothesized that BNYVV and BSBMV crossed study, exploiting their similarities and divergences, can improve investigation of molecular interactions between sugar beets and Benyviruses. The main achievement of our research is the production of a cDNA biologically active clones collection of BNYVV and BSBMV RNAs, from which synthetic copies of both Benyviruses can be transcribed. Moreover, through recombination experiments we demonstrated, for the first time, the BNYVV RNA 1 and 2 capability to trans-replicate and encapsidate BSBMV RNA 3 and 4, either the BSBMV RNA 1 and 2 capability to replicate BNYVV RNA2 in planta. We also demonstrated that BSBMV RNA3 support long-distance movement of BNYVV RNA 1 and 2 in B. macrocarpa and that 85 foreign sequence as p29HA, GFP and RFP, are successfully expressed, in C. quinoa, by BSBMV RNA3 based replicon (RepIII) also produced by our research. These results confirm the close correlation among the two viruses. Interestingly, the symptoms induced by BSBMV RNA-3 on C. quinoa leaves are more similar to necrotic local lesions caused by BNYVV RNA-5 p26 than to strongly chlorotic local lesions or yellow spot induced by BNYVV RNA- 3 encoded p25. As previous reported BSBMV p29 share 23% of amino acid sequence identity with BNYVV p25 but identity increase to 43% when compared with sequence of BNYVV RNA-5 p26. Based on our results the essential sequence (Core region) for the longdistance movement of BSBMV and BNYVV in B. macrocarpa, is not only carried by RNA3s species but other regions, perhaps located on the RNA 1 and 2, could play a fundamental role in this matter. Finally a chimeric RNA, composed by the 5’ region of RNA4 and 3’ region of RNA3 of BSBMV, has been produced after 21 serial mechanically inoculation of wild type BSBMV on C. quinoa plants. Chimera seems unable to express any protein, but it is replicated and transcript in planta. It could represent an important tool to study the interactions between Benyvirus and plant host. In conclusion different tools, comprising a method to study synthetic viruses under natural conditions of inoculum through P. Betae, have been produced and new knowledge are been acquired that will allow to perform future investigation of the molecular interactions between sugar beets and Benyviruses.

Brown rot caused by Monilinia laxa and Monilinia fructigena is considered one of the most important diseases affecting Prunus species. Although some losses can result from the rotten fruits in the orchard, most of the damage is caused to fruits during the post-harvest phase. Several studies reported that brown rot incidence during fruit development highly varies; it was found that at a period corresponding to the the pit hardening stage, fruit susceptibility drastically decreases, to be quickly restored afterwards. However the molecular basis of this phenomenon is still not well understood. Furthermore, no difference in the rot incidence was found between wound and un-wound fruits, suggesting that resistance associated more to a specifc biochemical response of the fruit, rather than to a higher mechanical resistance. So far, the interaction Monilinia-peach was analyzed through chemical approaches. In this study, a bio-molecular approach was undertaken in order to reveal alteration in gene expression associated to the variation of susceptibility. In this thesis three different methods for gene expression analysis were used to analyze the alterations in gene expression occurring in peach fruits during the pit hardening stage, in a period encompassing the temporary change in Monilinia susceptibility: real time PCR, microarray and cDNA AFLP techniques. In 2005, peach fruits (cv.K2) were weekly harvested during a 19-week long-period, starting from the fourth week after full bloom, until full maturity. At each sampling time, three replicates of 5 fruits each were dipped in the M.laxa conidial suspension or in distilled water, as negative control. The fruits were maintained at room temperature for 3 hours; afterwards, they were peeled with a scalpel; the peel was immediately frozen in liquid nitrogen and transferred to -80 °C until use. The degree of susceptibility of peach fruit to the pathogen was determined on 3 replicates of 20 fruits each, as percentage of infected fruits, after one week at 20 °C. Real time PCR analysis was performed to study the variation in expression of those genes encoding for the enzymes of the phenylpropanoid pathway (phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), cinnamate 4-hydroxylase (C4H), leucoanthocyanidine reductase (LAR), hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase (HQT) and of the jasmonate pathway, such as lipoxygenase (LOX), both involved in the production of important defense compounds. Alteration in gene expression was monitored on fruit samples of a period encompassing the pit hardening stage and the corresponding temporary resistance to M.laxa infections, weekly, from the 6thto the 12th week after full bloom (AFB) inoculated with M. laxa or mock-inoculated. The data suggest a critical change in the expression level of the phenylpropanoid pathway from the 7th to the 8th week AFB; such change could be directly physiologically associated to the peach growth and it could indirectly determine the decrease of susceptibility of peach fruit to Monilinia rot during the subsequent weeks. To investigate on the transcriptome variation underneath the temporary loss of susceptibility of peach fruits to Monilinia rot, the microarray and the cDNA AFLP techniques were used. The samples harvested on the 8th week AFB (named S, for susceptible ones) and on the 12th week AFB (named R, for resistant ones) were compared, both inoculated or mock-inoculated. The microarray experiments were carried out at the University of Padua (Dept. of Environmental Agronomy and Crop Science), using the μPEACH1.0 microarray together with the suited protocols. The analysis showed that 30 genes (corresponding to the 0.6% of the total sequences (4806) contained in the μPeach1.0 microarray) were found up-regulated and 31 ( 0.6%) down regulated in RH vs. SH fruits. On the other hand, 20 genes (0.4%) were shown to be up-regulated and 13 (0.3%) down-regulated in the RI vs. SI fruit. No genes were found differentially expressed in the mock-inoculated resistant fruits (RH) vs. the inoculated resistant ones (RI). Among the up-regulated genes an ATP sulfurylase, an heat shock protein 70, the major allergen Pru P1, an harpin inducing protein and S-adenosylmethionine decarboxylase were found, conversely among the down-regulated ones, cinnamyl alcohol dehydrogenase, an histidine- containing phosphotransfer protein and the ferritin were found. The microarray experimental results and the data indirectly derived, were tested by Real Time PCR analysis. cDNA AFLP analysis was also performed on the same samples. 339 transcript derived fragments considered significant for Monilinia resistance, were selected, sequenced and classified. Genes potentially involved in cell rescue and defence were well represented (8%); several genes (12.1%) involved in the protein folding, post-transductional modification and genes (9.2%) involved in cellular transport were also found. A further 10.3% of genes were classified as involved in the metabolism of aminoacid, carbohydrate and fatty acid. On the other hand, genes involved in the protein synthesis (5.7%) and in signal transduction and communication (5.7%) were found. Among the most interesting genes found differentially expressed between susceptible and resistant fruits, genes encoding for pathogenesis related (PR) proteins were found. To investigate on the association of Monilinia resistance and PR biological function, the major allergen Pru P1 (GenBank accession AM493970) and its isoform (here named Pru P2), were expressed in heterologous system and in vitro assayed for their anti-microbial activity. The ribonuclease activity of the recombinant Pru P1 and Pru P2 proteins was assayed against peach total RNA. As the other PR10 proteins, they showed a ribonucleolytic activity, that could be important to contrast pathogen penetration. Moreover Pru P1 and Pru P2 recombinant proteins were checked for direct antimicrobial activity. No inhibitory effect of Pru P1 or Pru P2 was detected against the selected fungi.

Tuber borchii (Ascomycota, order Pezizales) is highly valued truffle sold in local markets in Italy. Despite its economic importance, knowledge on its distribution and population variation is scarce. The objective of this work was to investigate the evolutionary forces shaping the genetic structure of this fungus using coalescent and phylogenetic methods to reconstruct the evolutionary history of populations in Italy. To assess population structure, 61 specimens were collected from 11 different Provinces of Italy. Sampling was stratified across hosts and habitats to maximize coverage in native oak and pine stands and both mychorrizae and fruiting bodies were collected. Samples were identified considering anatomo-morphological characters. DNA was extracted and both multilocus (AFLP) and single-locus (18 loci from rDNA, nDNA, and mtDNA) approaches were used to look for polymorphisms. Screening AFLP profiles, both Jaccard and Dice coefficients of similarity were utilized to transform binary matrix into a distance matrix and then to desume Neighbour-Joining trees. Though these are only preliminary examinations, phylogenetic trees were totally concordant with those deriving from single locus analyses. Phylogenetic analyses of the nuclear loci were performed using maximum likelihood with PAUP and a combined phylogenetic inference, using Bayesian estimation with all nuclear gene regions, was carried out. To reconstruct the evolutionary history, we estimated recurrent migration, migration across the history of the sample, and estimated the mutation and approximate age of mutations in each tree using SNAP Workbench. The combined phylogenetic tree using Bayesian estimation suggests that there are two main haplotypes that are difficult to be differentiated on the basis of morphology, of ecological parameters and symbiontic tree. Between these two lineages, that occur in sympatry within T. borchii populations, there is no evidence of recurrent migration. However, migration over the history of the sample was asymmetrical suggesting that isolation was a result of interrupted gene flow followed by range expansion. Low levels of divergence between the haplotypes indicate that there are likely to be two cryptic species within the T. borchii population sampled. Our results suggest that isolation between populations of T. borchii could have led to reproductive isolation between two lineages. This isolation is likely due to sympatric speciation caused by a multiple colonization from different refugia or a recent isolation. In attempting to determinate whether these haplotypes represent separate species or a partition of the same species we applied Biological and Mechanistic species Concepts. Notwithstanding, further analyses are necessary to evaluate if selection favoured premating or post-mating isolation.

Lo studio “Lotta biologica a Fusarium solani f.sp. cucurbitae su zucchino” si colloca nell’ambito della difesa integrata delle colture orticole dalle fitopatie fungine, in particolare quelle causate da patogeni ad habitat terricolo nei confronti dei quali è sempre più frequente il ricorso a mezzi di lotta diversi dai prodotti chimici. Interessante e innovativa appare la prospettiva di utilizzare microrganismi adatti a svilupparsi nel suolo, competenti per la rizosfera delle piante e con spiccate caratteristiche d’antagonismo verso i patogeni tellurici e di stimolazione delle difese sistemiche della pianta. Il marciume del colletto delle cucurbitacee, causato da diversi patogeni tra cui Fusarium solani f.sp. cucurbitae, rappresenta la principale malattia fungina di tipo tellurica che colpisce lo zucchino ed il melone nella Pianura Padana e che può portare a consistenti perdite produttive. Indagini condotte dal 2004 da parte del Diproval nell’areale bolognese, hanno evidenziato un’elevata frequenza del patogeno su zucchino coltivato soprattutto in tunnel. Considerata la carenza di conoscenze locali di F. solani f.sp. cucurbitae e di mezzi chimici di lotta efficaci, la ricerca svolta ha inteso approfondire la diagnosi della malattia e le caratteristiche biologiche degli isolati locali, e valutare la possibilità di utilizzare metodi biologici di lotta contro questo patogeno, nonché di studiare alcune delle possibili modalità d’azione di microrganismi antagonisti. Sono state pertanto prelevate, da zone diverse del Bolognese, campioni di piante di zucchino che presentavano sintomi di marciume del colletto ed è stato isolato il patogeno, che in base alle caratteristiche morfologiche macro e microscopiche, alle prove di patogenicità su diversi ospiti e a saggi biomolecolari, è stato identificato come Fusarium solani f. sp. cucurbitae W.C. Snyder & H.N. Hansen razza 1. Dagli isolati di campo sono state realizzate un centinaio di colture monosporiche venti delle quali sono state utilizzate per la prosecuzione delle prove. I venti ceppi sono stati saggiati per la loro patogenicità inoculandoli in terriccio sterile e con trapianto di giovani piante di zucchino. E’ risultata un’elevata variabilità del livello di virulenza tra i ceppi, stimata da 39% a 83% riguardo la gravità della malattia e da 61 a 96% per la frequenza di malattia. Sono state condotte prove di accrescimento miceliare che hanno evidenziato differenze tra i ceppi e tra gli esperimenti condotti a tre diverse temperature (17, 23 e 28°C) alla luce ed al buio. La crescita maggiore complessivamente è stata ottenuta a 23°C. I venti ceppi hanno anche mostrato di produrre, in vitro, vari livelli di enzimi di patogenesi quali cellulasi, poligalatturonasi, pectin liasi e proteasi. E’ stata evidenziata unan correlazione significativa tra attività cellulasica e pectin liasica con frequenza e gravità della malattia dei venti ceppi del patogeno. Le prove relative al contenimento della malattia sono state condotte in cella climatica. Sono stati considerati prodotti commerciali (Remedier, Rootshield, Cedomon, Mycostop, Proradix, Clonotry) a base rispettivamente dei seguenti microrganismi: Trichoderma harzianum ICC012 + T. viride ICC080, T. harzianum T22, Pseudomonas chlororaphis MA342, Streptomyces griseoviridis K61, P. fluorescens proradix DSM13134 e T. harzianum + Clonostachys rosea). I prodotti sono stati somministrati sul seme, al terreno e su seme+terreno (esperimenti 1 e 2) e in vivaio, al trapianto e vivaio+trapianto (esperimenti 3 e 4), riproducendo situazioni di pratico impiego. L’inoculazione del patogeno (un ceppo ad elevata patogenicità, Fs7 ed uno a bassa patogenicità, Fs37) è stata effettuata nel terreno distribuendo uno sfarinato secco di semi di miglio e cereali colonizzati dal patogeno. La malattia è stata valutata come intensità e gravità. I prodotti sono stati impiegati in situazioni di particolare stress, avendo favorito particolarmente la crescita del patogeno. Complessivamente i risultati hanno evidenziato effetti di contenimento maggiore della malattia nel caso del ceppo Fs37, meno virulento. La malattia è stata ridotta quasi sempre da tutti i formulati e quello che l’ha ridotta maggiormente è stato Cedomon. Il contenimento della malattia somministrando i prodotti solo nel terreno di semina o di trapianto è stato in generale quello più basso. Il contenimento più elevato è stato ottenuto con la combinazione di due tipologie di trattamento, seme+terreno e vivaio+trapianto. Le differenze tra i prodotti sono risultate più evidenti nel caso del ceppo Fs7. Per quanto riguarda lo studio di alcune delle modalità d’azione dei microrganismi contenuti nei formulati più efficaci, è stato verificato che tutti sono stati in grado di inibire, se pur in vario modo, la crescita del patogeno in vitro. Gli antagonisti più efficaci sono stati S. griseoviridis K61 (Mycostop) e P. fluorescens proradix DSM13134). I ceppi di Trichoderma, ed in particolare T.harzianum T22 (Rootshield), sono risultati i più attivi colonizzatori del substrato. Riguardo il fenomeno dell’antibiosi, il batterio P. fluorescens proradix DSM13134 ha mostrato di produrre i metaboliti non volatili più efficaci nel ridurre lo sviluppo del patogeno. Nelle condizioni sperimentali adottate anche i due ceppi di T. viride ICC080 e T. harzianum ICC012 hanno dimostrato di produrre metaboliti efficaci. Tali risultati, anche se relativi a prove in vitro, possono contribuire alla comprensione dei meccanismi dei microrganismi sul contenimento dell’infezione relativamente al rapporto diretto sul patogeno. E’ stato inoltre verificato che tutti i microrganismi saggiati sono dotati di competenza rizosferica e solo i batteri di endofitismo. Si conclude che, nonostante l’elevata pressione infettiva del patogeno che ha certamente influito negativamente sull’efficacia dei microrganismi studiati, i microrganismi antagonisti possono avere un ruolo nel ridurre l’infezione di F. solani f.sp. cucurbitae razza 1.

Blue mould caused by Penicillium expansum Link is one of the most destructive rot of pome fruit in all growing areas (Snowdon, 1990; Jones and Aldwinckle, 1991; Tonini,1996) In the past, Penicillium rot has been controlled by fungicide postharvest treatment mainly by thiabendazole (TBZ) and benomyl (Hardenburg and Spalding, 1972), but their intense use produced the appearance of resistant strains with a great reduction of their activity The aims of the present study were to characterize the isolates of Pencillium sp causing blue mold on pear in Italy by physiological and biochemical parameters. In particular differencing also the behavior of isolates to relationship with sensitivity or resistance to TBZ treatments. We have examined the early stage of infection in relation to enzyme activity, local modulation of pH, production of organic acids, and to secondary metabolism of pathogen. The results described here confirm that the majority of P. expansum isolates from pears packing houses are resistant to TBZ, Among the TBZ-resistant isolates scored in this work, different isolates (RR) showed higher percentage of conidial germination on TBZ-amended medium compared to non amended medium. This may indicate a stimulatory effect of TBZ on conidial germination. Therefore TBZ treatments are not only ineffective for controlling P. expansum, but they may also increase the severity of blue mould on fruits. In the absence of fungicide, isolates showed a significant difference for infection severity, R and RR isolates are characterized by higher pathogenic fitness on fruits, producing larger lesions than S isolates. These data are supported by the study with laboratory-induced resistant isolates, which shows the lack of correlation between TBZ resistance and osmotic sensitivity, and highlights the association between TBZ resistance and infection severity (Baraldi et al 2003). Enzymatic screening gave a positive reaction to esterase, urease, pectinase activity, in addition, the pathogen is able to synthesize a complex enzyme act to degrade the main components of the cell wall especially pectin and cellulose. Isolated sensitive and resistant are characterized by a good activity of pectinase, especially from poligactoronase, which, as already reported by several studies (D'hallewin et al, 2004; Prusky et al, 2004), are the basis of degradative process of cell wall. Also, although the measure was minor also highlighted some activities of cellulase, but even note in the production of this kind of cellulase and hemicellulase P. Expansum were not targeted, studies have found no other source of information in this regard. Twenty isolates of Penicillium expansum, were tested in vitro ad in vivo for acid production ability and pH drop. We have found that modulation of pH and the organic acids extrusion were influence to various parameter:  Initial pH: in general, the greatest reduction of pH was observed in isolates grown at pH 7, except for four isolates that maintained the pH of the medium close to 7, the others significantly decreased the pH, ranging from 5.5 to 4.1.. In extreme acid condition (pH 3,0) growth and modulation of pH is most lower respect optimal condition (pH 5,0). Also isolates R and RR have showed a greater adaptation to environmental condition more than isolates S.  Time: although the acidification continues for some days, PH modulation is strongest in early hours (48-72 hours)of inoculation process. Time also affects the quality of organic acids, for example in vitro results showed an initial abundant production of succinc acid, followed to important production of galacturoinc acid.  Substrates: there are many differences for the type of acids produced in vitro and in vivo. Results showed in vivo an abundant production of galacturonic, malic, and citric acids and some unknown organic acids in smaller concentrations. Secondary metabolite analysis revealed intra-specific differences, and patulin was found in all isolates, but most significant reduction was observed between in vitro and in vivo samples. There was no correlation between the concentration of patulin, and the percentage of infected fruits, but sample with a lower infection severity of rotten area than the others, showed a significantly lower mycotoxin concentration than samples with a higher lesion diameter of rotten area. Beyond of patulin was detected the presence of another secondary metabolite, penitrem A.

A modern management of crop protection should be based on integrated control programmes, including the use of environmentally safe products. Antagonistic/beneficial bacteria and resistance inducers may have a great potential in the prophylaxis of diseases caused by common and quarantine pathogens. This work was carried out to confirm the ability of the known strain IPV-BO G19 (Pseudomonas fluorescens) against fire blight (Erwinia amylovora), as well as to evaluate their efficacy against southern bacterial wilt of tomato (Ralstonia solanacearum) and grapevine crown gall (Agrobacterium vitis). A virulent strain of R. solanacearum race 3 was inhibited by the antagonist on plate. When the pathogen was inoculated 48 h after their application to the root apparatus of tomato plants grown in a climatic chamber, bacterial wilt progression rate was clearly reduced. Moreover the defence response evoked by IPV-BO G19 was studied in tomato plants by monitoring the transcription of genes codifying for three PRs as PR-1a, PR-4, PR-5 and for an intracellular chitinase using multiplex RT-PCR and Real Time RT-PCR. In two field trials during 2005 and 2006, the strain IPV-BO G19 was compared with biofungicides and some abiotic elicitors to protect actively growing shoots of pear scions against fire blight. In both trials, IPV-BO G19 plus Na-alginate gave a high level of protection, three weeks after wound inoculation with E. amylovora. In pear leaf tissues treated with the antagonistic strain IPV-BO G19, catalase, superoxyde dismutase and peroxidise activity was evaluated as markers of induced resistance. The IPV-BO G19 strain was compared with other bioagents and resistance inducers to prevent grapevine crown gall under glasshouse and vineyard conditions.

No description available.