Pre-symptomatic modified phytohormone profile is associated with lower phytoplasma titres in an Arabidopsis seor1ko line.

The proteins AtSEOR1 and AtSEOR2 occur as conjugates in the form of filaments in sieve elements of Arabidopsis thaliana. A reduced phytoplasma titre found in infected defective-mutant Atseor1ko plants in previous work raised the speculation that non-conjugated SEOR2 is involved in the phytohormone-mediated suppression of Chrysanthemum Yellows (CY)-phytoplasma infection transmitted by Euscelidius variegatus (Ev). This early and long-lasting SEOR2 impact was revealed in Atseor1ko plants by the lack of detectable phytoplasmas at an early stage of infection (symptomless plants) and a lower phytoplasma titre at a later stage (fully symptomatic plants). The high insect survival rate on Atseor1ko line and the proof of phytoplasma infection at the end of the acquisition access period confirmed the high transmission efficiency of CY-phytoplasma by the vectors. Transmission electron microscopy analysis ruled out a direct role of SE filament proteins in physical phytoplasma containment. Time-correlated HPLC-MS/MS-based phytohormone analyses revealed increased jasmonate levels in midribs of Atseor1ko plants at an early stage of infection and appreciably enhanced levels of indole acetic acid and abscisic acid at the early and late stages. Effects of Ev-probing on phytohormone levels was not found. The results suggest that SEOR2 interferes with phytohormonal pathways in Arabidopsis midrib tissues in order to establish early defensive responses to phytoplasma infection.

Analysis of new grapevine Pinot gris virus (GPGV) isolates from Northeast Italy provides clues to track the evolution of a newly emerging clade.

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.

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.

Localization and subcellular association of Grapevine Pinot Gris Virus in grapevine leaf tissues.

Despite the increasing impact of Grapevine Pinot gris disease (GPG-disease) worldwide, etiology about this disorder is still uncertain. The presence of the putative causal agent, the Grapevine Pinot Gris Virus (GPGV), has been reported in symptomatic grapevines (presenting stunting, chlorotic mottling, and leaf deformation) as well as in symptom-free plants. Moreover, information on virus localization in grapevine tissues and virus-plant interactions at the cytological level is missing at all. Ultrastructural and cytochemical investigations were undertaken to detect virus particles and the associated cytopathic effects in field-grown grapevine showing different symptom severity. Asymptomatic greenhouse-grown grapevines, which tested negative for GPGV by real time RT-PCR, were sampled as controls. Multiplex real-time RT-PCR and ELISA tests excluded the presence of viruses included in the Italian certification program both in field-grown and greenhouse-grown grapevines. Conversely, evidence was found for ubiquitous presence of Grapevine Rupestris Stem Pitting-associated Virus (GRSPaV), Hop Stunt Viroid (HSVd), and Grapevine Yellow Speckle Viroid 1 (GYSVd-1) in both plant groups. Moreover, in every field-grown grapevine, GPGV was detected by real-time RT-PCR. Ultrastructural observations and immunogold labelling assays showed filamentous flexuous viruses in the bundle sheath cells, often located inside membrane-bound organelles. No cytological differences were observed among field-grown grapevine samples showing different symptom severity. GPGV localization and associated ultrastructural modifications are reported and discussed, in the perspective of assisting management and control of the disease.

Pea Broth Enhances the Biocontrol Efficacy of Lysobacter capsici AZ78 by Triggering Cell Motility Associated with Biogenesis of Type IV Pilus.

Bacterial cells can display different types of motility, due to the presence of external appendages such as flagella and type IV pili. To date, little information on the mechanisms involved in the motility of the Lysobacter species has been available. Recently, L. capsici AZ78, a biocontrol agent of phytopathogenic oomycetes, showed the ability to move on jellified pea broth. Pea broth medium improved also the biocontrol activity of L. capsici AZ78 against Plasmopara viticola under greenhouse conditions. Noteworthy, the quantity of pea residues remaining on grapevine leaves fostered cell motility in L. capsici AZ78. Based on these results, this unusual motility related to the composition of the growth medium was investigated in bacterial strains belonging to several Lysobacter species. The six L. capsici strains tested developed dendrite-like colonies when grown on jellified pea broth, while the development of dendrite-like colonies was not recorded in the media commonly used in motility assays. To determine the presence of genes responsible for biogenesis of the flagellum and type IV pili, the genome of L. capsici AZ78 was mined. Genes encoding structural components and regulatory factors of type IV pili were upregulated in L. capsici AZ78 cells grown on the above-mentioned medium, as compared with the other tested media. These results provide new insight into the motility mechanism of L. capsici members and the role of type IV pili and pea compounds on the epiphytic fitness and biocontrol features of L. capsici AZ78.

Genome wide sequence analysis grants unbiased definition of species boundaries in "Candidatus Phytoplasma".

The phytoplasmas are currently named using the Candidatus category, as the inability to grow them in vitro prevented (i) the performance of tests, such as DNA-DNA hybridization, that are regarded as necessary to establish species boundaries, and (ii) the deposition of type strains in culture collections. The recent accession to complete or nearly complete genome sequence information disclosed the opportunity to apply to the uncultivable phytoplasmas the same taxonomic approaches used for other bacteria. In this work, the genomes of 14 strains, belonging to the 16SrI, 16SrIII, 16SrV and 16SrX groups, including the species "Ca. P. asteris", "Ca. P. mali", "Ca. P. pyri", "Ca. P. pruni", and "Ca. P. australiense" were analyzed along with Acholeplasma laidlawi, to determine their taxonomic relatedness. Average nucleotide index (ANIm), tetranucleotide signature frequency correlation index (Tetra), and multilocus sequence analysis of 107 shared genes using both phylogenetic inference of concatenated (DNA and amino acid) sequences and consensus networks, were carried out. The results were in large agreement with the previously established 16S rDNA based classification schemes. Moreover, the taxonomic relationships within the 16SrI, 16SrIII and 16SrX groups, that represent clusters of strains whose relatedness could not be determined by 16SrDNA analysis, could be comparatively evaluated with non-subjective criteria. "Ca. P. mali" and "Ca. P. pyri" were found to meet the genome characteristics for the retention into two different, yet strictly related species; representatives of subgroups 16SrI-A and 16SrI-B were also found to meet the standards used in other bacteria to distinguish separate species; the genomes of the strains belonging to 16SrIII were found more closely related, suggesting that their subdivision into Candidatus species should be approached with caution.

Genome drafts of four phytoplasma strains of the ribosomal group 16SrIII.

By applying a coverage-based read selection and filtration through a healthy plant dataset, and a post-assembly contig selection based on homology and linkage, genome sequence drafts were obtained for four phytoplasma strains belonging to the 16SrIII group (X disease clade), namely Vaccinium Witches' Broom phytoplasma (647 754 nt in 272 contigs), Italian Clover Phyllody phytoplasma strain MA (597 245 nt in 197 contigs), Poinsettia branch-inducing phytoplasma strain JR1 (631 440 nt in 185 contigs) and Milkweed Yellows phytoplasma (583 806 nt in 158 contigs). Despite assignment to different 16SrIII subgroups, the genomes of the four strains were similar, comprising a highly conserved core (92-98 % similar in their nucleotide sequence among each other over alignments about 500 kb in length) and a minor strain-specific component. As far as their protein complement was concerned, they did not differ significantly in their basic metabolism potential from the genomes of other wide-host-range phytoplasmas sequenced previously, but were distinct from strains of other species, as well as among each other, in genes encoding functions conceivably related to interactions with the host, such as membrane trafficking components, proteases, DNA methylases, effectors and several hypothetical proteins of unknown function, some of which are likely secreted through the Sec-dependent secretion system. The four genomes displayed a group of genes encoding hypothetical proteins with high similarity to a central domain of IcmE/DotG, a core component of the type IVB secretion system of Gram-negative Legionella spp. Conversely, genes encoding functional GroES/GroEL chaperones were not detected in any of the four drafts. The results also indicated the significant role of horizontal gene transfer among different 'Candidatus Phytoplasma' species in shaping phytoplasma genomes and promoting their diversity.

Application of immunoelectron microscopy techniques in the diagnosis of phytoplasma diseases.

An immunoelectron microscopy technique was applied to label Chrysanthemum leuchanthemum phytoplasma in infected leaf tissues of Chrysanthemum leuchanthemum L. and Catharanthus roseus L. plants. Specific monoclonal antibodies at different dilutions and secondary antimouse antibody conjugated with colloidal gold particles of different sizes were used. The monoclonal antibodies demonstrated their specificity against the antigen; immunocytological methods permitted the precise localization and identification of phytoplasmas in thin sections from infected tissues.