Giuseppe Firrao

Extreme environments are a largely unexplored reservoir of microbial diversity, with a remarkable potential to be exploited in agriculture. One hundred and seventeen yeast isolates, derived from different ecosystems in Italy, Sweden, Algeria, and France, were molecularly identified, and the most represented genus was Aureobasidium (57%). A phylogenetic analysis based on a multi-locus sequence typing (ITS, ELO, EF-1alpha) was conducted to characterize the black yeasts’ population. To investigate A. pullulans extremophilic and extremotolerant behaviour, different temperatures and pH, together with the enzymatic production, were evaluated. The strains were tested by in vitro and in vivo assays against the postharvest fungal pathogen Monilinia fructicola as potential biocontrol agents (BCAs). Results displayed a great ecological variability concerning strains’ growth and cell production depending on different culture conditions. However, a remarkable thermotolerance aptitude was detected in almost all the strains. In particular, the strains belonging to Group 2 (Algerian Desert) and 3 (Alto Adige Region) showed, respectively, higher thermotolerance and biocontrol ability. These findings showed how some extreme environments could represent a promising source for new potential BCAs. However, further studies are needed to investigate the mechanisms of action of these putative BCAs for application during the postharvest phase.

Xylophilus ampelinus is a xylematic bacterium causing bacterial blight of grapevine, a disease regarded as a potential threat for viticulture in several countries. Currently, PCR detection is pivotal in diagnostic protocols due to the bacterium's infrequent occurrence in the field and the technical advantages of PCR. Recent metagenomic studies have unveiled diversity in its taxonomic domain, unknown when the most widely used assays for the detection of X. ampelinus infections were developed. In particular, PCR assays relying on highly conserved sequence regions, such as those surrounding ribosomal RNA genes, may be substituted with more specific PCR assays. In this study, we first investigated the diversity of detectable grapevine endophytes related to but different from X. ampelinus and delineated the genotaxonomic boundaries of the species in relation to the known (meta)genomes of closely related bacteria. Then, by exploiting the wealth of genomes now available for bacteria classified in the Burkholderiales , we devised several sets of primers targeting only X. ampelinus and its closest relatives. These primers were employed to (i) genotaxonomically circumscribe the grapevine endophyte species related to but distinguishable from X. ampelinus and (ii) develop a robust multiplex PCR assay expected to be specific for the species X. ampelinus based on in vitro and in silico evidence. The adoption of the multiplex PCR assay presented here is expected to reduce the risk of false positives in the diagnosis of bacterial blight of grapevine.

Since 2012, kiwifruit vine decline syndrome (KVDS) has severely compromised all major kiwifruit-growing areas in Italy. Thus far, etiological studies were mainly focused on waterlogging effects or on the isolation of microorganisms from diseased plants; therefore, an all-encompassing picture of KVDS microbiota is still missing. This work aims to describe oomycete and fungal communities associated with KVDS and to identify key taxa potentially involved in the disease through a metabarcoding approach on root endosphere and rhizosphere samples. Two nearby fields with similar pedoclimatic conditions were identified based on KVDS spreading during a 4-year survey (2016 to 2019). In total, four sampling areas were selected, one from the control field with no sign of KVDS (asymptomatic site) and three from the KVDS-affected field (diseased site): (i) asymptomatic until the sampling date in 2018, (ii) symptomatic since 2018, and (iii) symptomatic since 2017. Total genomic DNA samples were subjected to a nested PCR approach separately targeting the internal transcribes spacer 2 regions of fungal and oomycete communities. The communities were compared in terms of α- and β-diversities, and key taxa were identified using univariate differential abundance tests. Major differences in taxa distribution were observed between samples from the different sites (asymptomatic and diseased) and were mostly linked to the oomycete community. Phytophthora sojae was the main taxa characterizing the diseased site and supposed to be involved in the disease and Phytopythium spp. were found related to the different plant health status. Finally, Dactylonectria macrodidyma, Phytopythium citrinum, and Thielaviopsis basicola were also proposed as new KVDS-related pathogens.

Since its discovery in 2012, the role of the trichovirus named grapevine Pinot gris virus (GPGV) as the causative agent of grapevine leaf mottling and deformation (GMLD) syndrome, a grapevine disease for which symptoms had been observed in north Italy and western Slovenia a few years earlier, has been a matter of discussion. In the following 10 years of investigations, evidence has emerged of a role for the genetic diversity of the virus and for the varietal diversity of the host, as well as their interaction during antiviral defence (i.e. RNA silencing) and virus‐mediated defence suppression. In a context of wider research efforts, several relevant aspects of GPGV evolution and GMLD epidemiology were also clarified. Nevertheless, a complete understanding of the molecular events and the environmental conditions that could affect viral infection severity and symptom display remains a challenging puzzle. In the meantime, we are witnessing a large spread of the virus, which has almost completely permeated the regions where it first appeared, while spreading through most areas of grapevine cultivation. A deeper knowledge of the molecular context of the disease is key to establishing whether the impressive potential of GPGV to spread makes it a candidate as the next emerging threat for the grapevine industry.

‘Lamon bean’ is a protected geographical indication (PGI) for a product of four varieties of bean (Phaseolus vulgaris L.) grown in a specific area of production, which is located in the Belluno district, Veneto region (N.E. of Italy). In the last decade, the ‘Lamon bean’ has been threatened by severe virus epidemics that have compromised its profitability. In this work, the full virome of seven bean samples showing different foliar symptoms was obtained by MinION sequencing. Evidence that emerged from sequencing was validated through RT-PCR and ELISA in a large number of plants, including different ecotypes of Lamon bean and wild herbaceous hosts that may represent a virus reservoir in the field. Results revealed the presence of bean common mosaic virus (BCMV), cucumber mosaic virus (CMV), peanut stunt virus (PSV), and bean yellow mosaic virus (BYMV), which often occurred as mixed infections. Moreover, both CMV and PSV were reported in association with strain-specific satellite RNAs (satRNAs). In conclusion, this work sheds light on the cause of the severe diseases affecting the ‘Lamon bean’ by exploitation of MinION sequencing.

Grapevine Pinot gris virus (GPGV) is an emerging trichovirus that has been putatively associated with a novel grapevine disease known as grapevine leaf mottling and deformation (GLMD). Yet the role of GPGV in GLMD disease is poorly understood, since it has been detected both in symptomatic and symptomless grapevines. We exploited a recently constructed GPGV infectious clone (pRI::GPGV-vir) to induce an antiviral response in Nicotiana benthamiana plants. In silico prediction of virus-derived small interfering RNAs and gene expression analyses revealed the involvement of DCL4, AGO5, and RDR6 genes during GPGV infection, suggesting the activation of the posttranscriptional gene-silencing (PTGS) pathway as a plant antiviral defense. PTGS suppression assays in transgenic N. benthamiana 16c plants revealed the ability of the GPGV coat protein to suppress RNA silencing. This work provides novel insights on the interaction between GPGV and its host, revealing the ability of the virus to trigger and suppress antiviral RNA silencing.[ ] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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.

Grapevine Pinot gris disease (GPGD) has been associated with a trichovirus, namely grapevine Pinot gris virus (GPGV), although the virus has been reported in both symptomatic and asymptomatic plants. Despite the puzzling aetiology of the disease and potentially important role of GPGV, the number of fully sequenced isolates is still rather limited. With the aim of increasing the knowledge on intraspecific diversity and evolution, nine GPGV isolates were collected from different vineyards in the Friuli Venezia Giulia region (Northeast Italy), cloned, sequenced, and subjected to robust phylogenetic and other analyses. The results provided hints on the evolutionary history of the virus, the occurrence of recombination, and the presence of clade-specific SNPs in sites of putative protein modifications with potential impact on the interaction with the host.

Genome drafts for the phytoplasmas may be rapidly and efficiently assembled from NGS sequence data alone exploiting the proper bioinformatic tools and starting from properly collected samples. Here, we describe the use of the Phytoassembly pipeline ( https://github.com/cpolano/phytoassembly ), a fully automated tool that accepts as input row Illumina data from two samples (a phytoplasma infected sample and a healthy reference sample) to produce a phytoplasma genome draft, using the healthy plant host genome as a filter and profiting from the difference in reads coverage between the genome of the pathogen and that of the host. For phytoplasma infected samples containing >2% of pathogen DNA and an isogenic healthy reference sequence the resulting assemblies span the almost entire genomes.