Orthology-Based Estimate of the Contribution of Horizontal Gene Transfer from Distantly Related Bacteria to the Intraspecific Diversity and Differentiation of Xylella fastidiosa

Xylella fastidiosa is a xylem-limited bacterium phylogenetically related to the xanthomonads, with an unusually large and diversified range of plant hosts. To ascertain the origin of its peculiarities, its pan-genome was scanned to identify the genes that are not coherent with its phylogenetic position within the order Xanthomonadales. The results of the analysis revealed that a large fraction of the genes of the Xylella pan-genome have no ortholog or close paralog in the order Xanthomonadales. For a significant part of the genes, the closest homologue was found in bacteria belonging to distantly related taxonomic groups, most frequently in the Betaproteobacteria. Other species, such as Xanthomonas vasicola and Xanthomonas albilineans which were investigated for comparison, did not show a similar genetic contribution from distant branches of the prokaryotic tree of life. This finding indicates that the process of acquisition of DNA from the environment is still a relevant component of Xylella fastidiosa evolution. Although the ability of Xylella fastidiosa strains to recombine among themselves is well known, the results of the pan-genome analyses stressed the additional relevance of environmental DNA in shaping their genomes, with potential consequences on their phytopathological features

Quantitative real-time PCR and high-resolution melting (HRM) analysis for strain-specific monitoring of fluorescent pseudomonads used as biocontrol agents against soil-borne pathogens of food crops

Fluorescent pseudomonads colonizing roots of crop plants and producing antifungal metabolites are regarded as a reliable alternative to chemical fungicides against soil-borne phytopathogens. Key factors in successful pathogen control are presence and activity at the appropriate concentration, time, and place of biocontrol agents. Thus, quantification methods to monitor population dynamics are pivotal to the development of reliable application protocols. Real-time PCR is nowadays the most widespread cultureindependent technique for the detection and enumeration of different target sequences. Here, its implementation with high resolution melting analysis as a powerful tool to accurately discriminate microbial inoculants is discussed

Micotossine: prevenire è molto, molto meglio che curare

Food contamination with fungal secondary metabolites has been a major limit for population wellness and development in the past. Although today social protection against this plague has been established in most countries, there are still many areas in the world where mycotoxin contamination causes illness and even death, due to the lack of healthy conditions for the storage of commodities. Information and knowledge are the main weapons available to contrast the danger of food contamination by mycotoxins. In several instances, information about the severity of the issue alone would have been effective in preventing cases of acute intoxications. Prevention is the winning strategy not only at the level of the end consumer, but also as far as the production is concerned. Thanks to the advances in the understanding of fungal epidemiology and mycotoxin synthesis, strategies for the prevention or at least for the limitation of mycotoxin contaminations have been developed in recent years. As far as cereal production in Italy is concerned, proper storage conditions are now widely adopted, and protocols for good practice in cultivation are being followed by an increasing number of producers. In addition, rapid methods for the early detection of contaminants and quality assessment have been developed and, when introduced in the routine field operation, may lead to substantial improvements of product quality

Brittle Materials and Stress Concentrations: are they able to withstand?

AbstractThe combination of brittleness in materials and stress concentrations may lead to premature fracture of structural components. To improve the modelling capability more and more sophisticated methods have to be employed. To the purpose a Finite Fracture Mechanics (FFM) criterion based on the contemporaneous fulfilment of a stress requirement and the energy balance has been proposed in the literature. This coupled approach is here refined and applied to investigate brittle fracture in rounded V-notched samples under mode I loading. The failure FFM condition is expressed by a system of two equations in two unknowns: the critical crack advance and the apparent generalized fracture toughness (i.e., the failure load). The refined criterion is validated by its implementation with various experimental data, available in the literature, related to ceramic, metallic, and plastic brittle materials

An endopolygalacturonase gene of Diaporthe helianthi

In an attempt to define virulence determinants of Diaporthe helianthi, the causative agent of sunflower stem canker, the gene named Dhpg, coding for an endopolygalacturonase, was investigated in the highly virulent strain 8/96 isolated from a diseased plant in France. The 1130 bp coding sequence, in addition to 1473 bp from the upstream region, were cloned, sequenced and analyzed. Dhpg was most closely related to polygalacturonase genes from phytopathogenic fungi, such as Ophiostoma ulmi, O. novo-ulmi, and Gibberella zeae. A Southern blot RFLP analysis proved that Dhpg was represented in single-copy in the genome of the isolate 8/96. Analyses carried out both in vitro on liquid cultures and in vivo on host infected tissues provided evidence of the constitutive expression of the Dhpg transcript under all tested conditions. Moreover, an European collection of D. helianthi isolates was screened for the presence of Dhpg homologues by PCR, revealing the same single band in all French and Yugoslavian isolates, while one Romanian and all Italian isolates displayed a variable pattern. This genetic variability related to the different geographic origin of D. helianthi is consistent with data previously reported for different loci on the same set of isolates. This is the first report of a polygalacturonase gene in D. helianthi

Polyketide synthases of Diaporthe helianthi and involvement of DhPKS1 in virulence on sunflower

Background The early phases of Diaporthe helianthi pathogenesis on sunflower are characterized by the production of phytotoxins that may play a role in host colonisation. In previous studies, phytotoxins of a polyketidic nature were isolated and purified from culture filtrates of virulent strains of D. helianthi isolated from sunflower. A highly aggressive isolate (7/96) from France contained a gene fragment of a putative nonaketide synthase (lovB) which was conserved in a virulent D. helianthi population. Results In order to investigate the role of polyketide synthases in D. helianthi 7/96, a draft genome of this isolate was examined. We were able to find and phylogenetically analyse 40 genes putatively coding for polyketide synthases (PKSs). Analysis of their domains revealed that most PKS genes of D. helianthi are reducing PKSs, whereas only eight lacked reducing domains. Most of the identified PKSs have orthologs shown to be virulence factors or genetic determinants for toxin production in other pathogenic fungi. One of the genes (DhPKS1) corresponded to the previously cloned D. helianthi lovB gene fragment and clustered with a nonribosomal peptide synthetase (NRPS) -PKS hybrid/lovastatin nonaketide like A. nidulans LovB. We used DhPKS1 as a case study and carried out its disruption through Agrobacterium-mediated transformation in the isolate 7/96. D. helianthi DhPKS1 deleted mutants were less virulent to sunflower compared to the wild type, indicating a role for this gene in the pathogenesis of the fungus. Conclusion The PKS sequences analysed and reported here constitute a new genomic resource that will be useful for further research on the biology, ecology and evolution of D. helianthi and generally of fungal plant pathogens

Agroinoculation of Grapevine Pinot Gris Virus in tobacco and grapevine provides insights on viral pathogenesis

The Grapevine Pinot Gris disease (GPG-d) is a novel disease characterized by symptoms such as leaf mottling and deformation, which has been recently reported in grapevines, and mostly in Pinot gris. Plants show obvious symptoms at the beginning of the growing season, while during summer symptom recovery frequently occurs, manifesting as symptomless leaves. A new Trichovirus, named Grapevine Pinot gris virus (GPGV), which belongs to the family Betaflexiviridae was found in association with infected plants. The detection of the virus in asymptomatic grapevines raised doubts about disease aetiology. Therefore, the primary target of this work was to set up a reliable system for the study of the disease in controlled conditions, avoiding interfering factor(s) that could affect symptom development. To this end, two clones of the virus, pRI::GPGV-vir and pRI::GPGV-lat, were generated from total RNA collected from one symptomatic and one asymptomatic Pinot gris grapevine, respectively. The clones, which encompassed the entire genome of the virus, were used in Agrobacterium-mediated inoculation of Vitis vinifera and Nicotiana benthamiana plants. All inoculated plants developed symptoms regardless of their inoculum source, demonstrating a correlation between the presence of GPGV and symptomatic manifestations. Four months post inoculum, the grapevines inoculated with the pRI::GPGV-lat clone developed asymptomatic leaves that were still positive to GPGV detection. Three to four weeks later (i.e. ca. 5 months post inoculum), the same phenomenon was observed in the grapevines inoculated with pRI::GPGV-vir. This observation perfectly matches symptom progression in infected field-grown grapevines, suggesting a possible role for plant antiviral mechanisms, such as RNA silencing, in the recovery process.</div