Metodi molecolari nello studio della variabilità genetica di Tuber borchii

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.

Risposte di resistenza a batteri fitopatogeni di importanti specie coltivate indotte da molecole segnale di diversa natura

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.

Identification and genetic diversity in phytoplasmas associated with diseases of cassava and other agronomic relevant crops in south-east Asia and Latin America

Identification and genetic diversity of phytoplasmas infecting tropical plant species, selected among those most agronomically relevant in South-east Asia and Latin America were studied. Correlation between evolutionary divergence of relevant phytoplasma strains and their geographic distribution by comparison on homologous genes of phytoplasma strains detected in the same or related plant species in other geographical areas worldwide was achieved. Molecular diversity was studied on genes coding ribosomal proteins, groEL, tuf and amp besides phytoplasma 16S rRNA. Selected samples infected by phytoplasmas belonging to diverse ribosomal groups were also studied by in silico RFLP followed by phylogenetic analyses. Moreover a partial genome annotation of a ‘Ca. P. brasiliense’ strain was done towards future application for epidemiological studies. Phytoplasma presence in cassava showing frog skin (CFSD) and witches’ broom (CWB) diseases in Costa Rica - Paraguay and in Vietnam – Thailand, respectively, was evaluated. In both cases, the diseases were associated with phytoplasmas related to aster yellows, apple proliferation and “stolbur” groups, while only phytoplasma related to X-disease group in CFSD, and to hibiscus witches’ broom, elm yellows and clover proliferation groups in CWB. Variability was found among strains belonging to the same ribosomal group but having different geographic origin and associated with different disease. Additionally, a dodder transmission assay to elucidate the role of phytoplasmas in CWB disease was carried out, and resulted in typical phytoplasma symptoms in periwinkle plants associated with the presence of aster yellows-related strains. Lethal wilt disease, a severe disease of oil palm in Colombia that is spreading throughout South America was also studied. Phytoplasmas were detected in symptomatic oil palm and identified as ‘Ca. P. asteris’, ribosomal subgroup 16SrI-B, and were distinguished from other aster yellows phytoplasmas used as reference strains; in particular, from an aster yellows strain infecting corn in the same country.