Multiple lysine substitutions in the peptaibol trichogin GA IV enhance the antibiotic activity against plant pathogenic Pseudomonas syringae.

Plant diseases caused by Pseudomonas syringae are essentially controlled in the field with the use of copper-based products and antibiotics, raising environmental and safety concerns. Antimicrobial peptides (AMPs) derived from fungi may represent a sustainable alternative to those chemicals. Trichogin GA IV, a non-ribosomal, 11-residue long AMP naturally produced by the fungus Trichoderma longibrachiatum has the ability to insert into phospholipidic membranes and form water-filled pores, thereby perturbing membrane integrity and permeability. In previous studies, peptide analogs modified at the level of specific residues were designed to be water-soluble and active against plant pathogens. Here, we studied the role of glycine-to-lysine substitutions and of the presence of a C-terminal leucine amide on bioactivity against Pseudomonas syringae bacteria. P. syringae diseases affect a wide range of crops worldwide, including tomato and kiwifruit. Our results show that trichogin GA IV analogs containing two or three Gly-to-Lys substitutions are highly effective in vitro against P. syringae pv. tomato (Pst), displaying minimal inhibitory and minimal bactericidal concentrations in the low micromolar range. The same analogs are also able to inhibit in vitro the kiwifruit pathogen P. syringae pv. actinidiae (Psa) biovar 3. When sprayed on tomato plants 24 h before Pst inoculation, only tri-lysine containing analogs were able to significantly reduce bacterial titers and symptom development in infected plants. Our results point to a positive correlation between the number of lysine substitutions and the antibacterial activity. This correlation was supported by microscopy analyses performed with mono-, di- and tri-Lys containing analogs that showed a different degree of interaction with Pst cells and ultrastructural changes that culminated in cell lysis.

Glutamate dehydrogenase in "Liverworld"-A study in selected species to explore a key enzyme of plant primary metabolism in Marchantiophyta.

In plants, glutamate dehydrogenase (GDH) is an ubiquitous enzyme that catalyzes the reversible amination of 2-oxoglutarate in glutamate. It contributes to both the amino acid homeostasis and the management of intracellular ammonium, and it is regarded as a key player at the junction of carbon and nitrogen assimilation pathways. To date, information about the GDH of terrestrial plants refers to a very few species only. We focused on selected species belonging to the division Marchantiophyta, providing the first panoramic overview of biochemical and functional features of GDH in liverworts. Native electrophoretic analyses showed an isoenzymatic profile less complex than what was reported for Arabidposis thaliana and other angiosperms: the presence of a single isoform corresponding to an α-homohexamer, differently prone to thermal inactivation on a species- and organ-basis, was found. Sequence analysis conducted on amino acid sequences confirmed a high similarity of GDH in modern liverworts with the GDH2 protein of A. thaliana, strengthening the hypothesis that the duplication event that gave origin to GDH1-homolog gene from GDH2 occurred after the evolutionary bifurcation that separated bryophytes and tracheophytes. Experiments conducted on Marchantia polymorpha and Calypogeia fissa grown in vitro and compared to A. thaliana demonstrated through in gel activity detection and monodimensional Western Blot that the aminating activity of GDH resulted in strongly enhanced responses to ammonium excess in liverworts as well, even if at a different extent compared to Arabidopsis and other vascular species. The comparative analysis by bi-dimensional Western Blot suggested that the regulation of the enzyme could be, at least partially, untied from the protein post-translational pattern. Finally, immuno-electron microscopy revealed that the GDH enzyme localizes at the subcellular level in both mitochondria and chloroplasts of parenchyma and is specifically associated to the endomembrane system in liverworts.

The sieve-element endoplasmic reticulum: A focal point of phytoplasma-host plant interaction?

The rough endoplasmic reticulum (r-ER) is of paramount importance for adaptive responses to biotic stresses due to an increased demand for de novo synthesis of immunity-related proteins and signaling components. In nucleate cells, disturbance of r-ER integrity and functionality leads to the "unfolded protein response" (UPR), which is an important component of innate plant immune signalling. In contrast to an abundance of reports on r-ER responses to biotic challenges, sieve-element endoplasmic reticulum (SE-ER) responses to phytoplasma infection have not been investigated. We found that morphological SE-ER changes, associated with phytoplasma infection, are accompanied by differential expression of genes encoding proteins involved in shaping and anchoring the reticulum. Phytoplasma infection also triggers an increased release of bZIP signals from the (SE-ER)/r-ER and consequent differential expression of UPR-related genes. The modified expression patterns seem to reflect a trade-off between survival of host cells, needed for the phytoplasmic biotrophic lifestyle, and phytoplasmas. Specialized plasmodesmata between sieve element and companion cell may provide a corridor for transfer of phytoplasma effectors inducing UPR-related gene expression in companion cells.

Peptide Analogs of a Trichoderma Peptaibol Effectively Control Downy Mildew in the Vineyard.

Plasmopara viticola, the agent of grapevine downy mildew, causes enormous economic damage, and its control is primarily based on the use of synthetic fungicides. The European Union policies promote reducing reliance on synthetic plant protection products. Biocontrol agents such as Trichoderma spp. constitute a resource for the development of biopesticides. Trichoderma spp. produce secondary metabolites such as peptaibols, but the poor water solubility of peptaibols limits their practical use as agrochemicals. To identify new potential bio-inspired molecules effective against P. viticola, various water-soluble peptide analogs of the peptaibol trichogin were synthesized. In grapevine leaf disk assays, the peptides analogs at a concentration of 50 µM completely prevented P. viticola infection after zoosporangia inoculation. Microscopic observations of one of the most effective peptides showed that it causes membrane lysis and cytoplasmic granulation in both zoosporangia and zoospores. Among the effective peptides, 4r was selected for a 2-year field trial experiment. In the vineyard, the peptide administered at 100 µM (equivalent to 129.3 g/ha) significantly reduced the disease incidence and severity on both leaves and bunches, with protection levels similar to those obtained using a cupric fungicide. In the second-year field trial, reduced dosages of the peptide were also tested, and even at the peptide concentration reduced by 50 or 75%, a significant decrease in the disease incidence and severity was obtained at the end of the trial. The peptide did not show any phytotoxic effect. Previously, peptide 4r had been demonstrated to be active against other fungal pathogens, including the grapevine fungus Botrytis cinerea. Thus, this peptide may be a candidate for a broad-spectrum fungicide whose biological properties deserve further investigation.

Effect of a triacontanol-rich biostimulant on the ripening dynamic and wine must technological parameters in Vitis vinifera cv. 'Ribolla Gialla'.

Biostimulants are organic compounds which can influence the biochemical activity of the whole plant. Lately, great attention has been focused on the possibility of using these stimulants in the viticulture sector. Due to this, the aim of this work was to investigate the foliar application of a biostimulant made by Fabaceae tissue, rich in amino acids and peptides along with the high presence of natural triacontanol (C30H62O) (>6 mg kg-1), previously reported in many crops as chemicals able to stimulate different yield components, the technological composition of musts still having an effect on some of the microbial population of different fruits/crops. Hence, this research was conducted during the growing seasons 2020 and 2021 in a commercial vineyard of the 'Ribolla Gialla' grapevine (Vitis vinifera, L.), in the Friuli Venezia Giulia Region (North-Eastern Italy), in order to understand the effect on this woody perennial crop not yet investigated. After a two-year-study, a physiological response occurred, as ripening and veraision were brought forward in the treated plants as well as the harvest time, having higher enological parameters (sugars, total titrable acidity and citric acid content) than the non-treated at every stage. Thus, grapes in the treated plants reached a full technological maturity earlier than the non-treated, in both study years. There was a positive effect on must microbial ecology important for winemaking, hence, the biostimulant have promoted the growth of the microbial community on berry skin translating into what found in the must.

Increased susceptibility to Chrysanthemum Yellows phytoplasma infection in Atcals7ko plants is accompanied by enhanced expression of carbohydrate transporters.

MAIN CONCLUSION: Loss of CALS7 appears to confer increased susceptibility to phytoplasma infection in Arabidopsis, altering expression of genes involved in sugar metabolism and membrane transport. Callose deposition around sieve pores, under control of callose synthase 7 (CALS7), has been interpreted as a mechanical response to limit pathogen spread in phytoplasma-infected plants. Wild-type and Atcals7ko mutants were, therefore, employed to unveil the mode of involvement of CALS7 in the plant's response to phytoplasma infection. The fresh weights of healthy and CY-(Chrysanthemum Yellows) phytoplasma-infected Arabidopsis wild type and mutant plants indicated two superimposed effects of the absence of CALS7: a partial impairment of photo-assimilate transport and a stimulated phytoplasma proliferation as illustrated by a significantly increased phytoplasma titre in Atcal7ko mutants. Further studies solely dealt with the effects of CALS7 absence on phytoplasma growth. Phytoplasma infection affected sieve-element substructure to a larger extent in mutants than in wild-type plants, which was also true for the levels of some free carbohydrates. Moreover, infection induced a similar upregulation of gene expression of enzymes involved in sucrose cleavage (AtSUS5, AtSUS6) and transmembrane transport (AtSWEET11) in mutants and wild-type plants, but an increased gene expression of carbohydrate transmembrane transporters (AtSWEET12, AtSTP13, AtSUC3) in infected mutants only. It remains still unclear how the absence of AtCALS7 leads to gene upregulation and how an increased intercellular mobility of carbohydrates and possibly effectors contributes to a higher susceptibility. It is also unclear if modified sieve-pore structures in mutants allow a better spread of phytoplasmas giving rise to higher titre.

Infection by phloem-limited phytoplasma affects mineral nutrient homeostasis in tomato leaf tissues.

Phytoplasmas are sieve-elements restricted wall-less, pleomorphic pathogenic microorganisms causing devastating damage to over 700 plant species worldwide. The invasion of sieve elements by phytoplasmas has several consequences on nutrient transport and metabolism, anyway studies about changes of the mineral-nutrient profile following phytoplasma infections are scarce and offer contrasting results. Here, we examined changes in macro- and micronutrient concentration in tomato plant upon 'Candidatus Phytoplasma solani' infection. To investigate possible effects of 'Ca. P. solani' infection on mineral element allocation, the mineral elements were separately analysed in leaf midrib, leaf lamina and root. Moreover, we focused our analysis on the transcriptional regulation of genes encoding trans-membrane transporters of mineral nutrients. To this aim, a manually curated inventory of differentially expressed genes encoding transporters in tomato leaf midribs was mined from the transcriptional profile of healthy and infected tomato leaf midribs. Results highlighted changes in ion homeostasis in the host plant, and significant modulations at transcriptional level of genes encoding ion transporters and channels.

Transcriptomic and Ultrastructural Analyses of Pyricularia Oryzae Treated With Fungicidal Peptaibol Analogs of Trichoderma Trichogin.

Eco-friendly analogs of Trichogin GA IV, a short peptaibol produced by Trichoderma longibrachiatum, were assayed against Pyricularia oryzae, the causal agent of rice blast disease. In vitro and in vivo screenings allowed us to identify six peptides able to reduce by about 70% rice blast symptoms. One of the most active peptides was selected for further studies. Microscopy analyses highlighted that the treated fungal spores could not germinate and the fluorescein-labeled peptide localized on the spore cell wall and in the agglutinated cytoplasm. Transcriptomic analysis was carried out on P. oryzae mycelium 3 h after the peptide treatment. We identified 1,410 differentially expressed genes, two-thirds of which upregulated. Among these, we found genes involved in oxidative stress response, detoxification, autophagic cell death, cell wall biogenesis, degradation and remodeling, melanin and fatty acid biosynthesis, and ion efflux transporters. Molecular data suggest that the trichogin analogs cause cell wall and membrane damages and induce autophagic cell death. Ultrastructure observations on treated conidia and hyphae confirmed the molecular data. In conclusion, these selected peptides seem to be promising alternative molecules for developing effective bio-pesticides able to control rice blast disease.

Gimme shelter: three-dimensional architecture of the endoplasmic reticulum, the replication site of grapevine Pinot gris virus.

Grapevine leaf mottling and deformation is a novel grapevine disease that has been associated with grapevine Pinot gris virus (GPGV). The virus was observed exclusively inside membrane-bound structures in the bundle sheath cells of the infected grapevines. As reported widely in the literature, many positive-sense single-stranded RNA viruses modify host-cell membranes to form a variety of deformed organelles, which shelter viral genome replication from host antiviral compounds. Morphologically, the GPGV-associated membranous structures resemble the deformed endoplasmic reticulum described in other virus-host interactions. In this study we investigated the GPGV-induced membranous structures observed in the bundle sheath cells of infected plants. The upregulation of different ER stress-related genes was evidenced by RT-qPCR assays, further confirming the involvement of the ER in grapevine/GPGV interaction. Specific labelling of the membranous structures with an antibody against luminal-binding protein identified them as ER. Double-stranded RNA molecules, which are considered intermediates of viral replication, were localised exclusively in the ER-derived structures and indicated that GPGV exploited this organelle to replicate itself in a shelter niche. Novel analyses using focussed ion-beam scanning electron microscopy (FIB-SEM) were performed in grapevine leaf tissues to detail the three-dimensional organisation of the ER-derived structures and their remodelling due to virus replication.

Trigger and Suppression of Antiviral Defenses by Grapevine Pinot Gris Virus (GPGV): Novel Insights into Virus-Host Interaction.

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.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

NoPv1: a synthetic antimicrobial peptide aptamer targeting the causal agents of grapevine downy mildew and potato late blight.

Grapevine (Vitis vinifera L.) is a crop of major economic importance. However, grapevine yield is guaranteed by the massive use of pesticides to counteract pathogen infections. Under temperate-humid climate conditions, downy mildew is a primary threat for viticulture. Downy mildew is caused by the biotrophic oomycete Plasmopara viticola Berl. & de Toni, which can attack grapevine green tissues. In lack of treatments and with favourable weather conditions, downy mildew can devastate up to 75% of grape cultivation in one season and weaken newly born shoots, causing serious economic losses. Nevertheless, the repeated and massive use of some fungicides can lead to environmental pollution, negative impact on non-targeted organisms, development of resistance, residual toxicity and can foster human health concerns. In this manuscript, we provide an innovative approach to obtain specific pathogen protection for plants. By using the yeast two-hybrid approach and the P. viticola cellulose synthase 2 (PvCesA2), as target enzyme, we screened a combinatorial 8 amino acid peptide library with the aim to identify interacting peptides, potentially able to inhibit PvCesa2. Here, we demonstrate that the NoPv1 peptide aptamer prevents P. viticola germ tube formation and grapevine leaf infection without affecting the growth of non-target organisms and without being toxic for human cells. Furthermore, NoPv1 is also able to counteract Phytophthora infestans growth, the causal agent of late blight in potato and tomato, possibly as a consequence of the high amino acid sequence similarity between P. viticola and P. infestans cellulose synthase enzymes.

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.

With or Without You: Altered Plant Response to Boron-Deficiency in Hydroponically Grown Grapevines Infected by Grapevine Pinot Gris Virus Suggests a Relation Between Grapevine Leaf Mottling and Deformation Symptom Occurrence and Boron Plant Availability.

Despite the increasing spread of Grapevine Leaf Mottling and Deformation (GLMD) worldwide, little is known about its etiology. After identification of grapevine Pinot gris virus (GPGV) as the presumptive causal agent of the disease in 2015, various publications have evaluated GPGV involvement in GLMD. Nevertheless, there are only partial clues to explain the presence of GPGV in both symptomatic and asymptomatic grapevines and the mechanisms that trigger symptom development, and so a consideration of new factors is required. Given the similarities between GLMD and boron (B)-deficiency symptoms in grapevine plants, we posited that GPGV interferes in B homeostasis. By using a hydroponic system to control B availability, we investigated the effects of different B supplies on grapevine phenotype and those of GPGV infection on B acquisition and translocation machinery, by means of microscopy, ionomic and gene expression analyses in both roots and leaves. The transcription of the genes regulating B homeostasis was unaffected by the presence of GPGV alone, but was severely altered in plants exposed to both GPGV infection and B-deficiency, allowing us to speculate that the capricious and patchy occurrence of GLMD symptoms in the field may not be related solely to GPGV, but to GPGV interference in plant responses to different B availabilities. This hypothesis found preliminary positive confirmations in analyses on field-grown plants.

The rhizosphere signature on the cell motility, biofilm formation and secondary metabolite production of a plant-associated Lysobacter strain.

Lysobacter spp. are common bacterial inhabitants of the rhizosphere of diverse plant species. However, the impact of the rhizosphere conditions on their physiology is still relatively understudied. To provide clues on the behaviour of Lysobacter spp. in this ecological niche, we investigated the physiology of L. capsici AZ78 (AZ78), a biocontrol strain isolated from tobacco rhizosphere, on a common synthetic growth medium (LBA) and on a growth medium containing components of the plant rhizosphere (RMA). The presence of a halo surrounding the AZ78 colony on RMA was a first visible effect related to differences in growth medium composition and it corresponded to the formation of a large outer ring. The lower quantity of nutrients available in RMA as compared with LBA was associated to a higher expression of a gene encoding cAMP-receptor-like protein (Clp), responsible for cell motility and biofilm formation regulation. AZ78 cells on RMA were motile, equipped with cell surface appendages and organised in small groups embedded in a dense layer of fibrils. Metabolic profiling by mass spectrometry imaging revealed increased diversity of analytes produced by AZ78 on RMA as compared with LBA. In particular, putative cyclic lipodepsipeptides, polycyclic tetramate macrolactams, cyclic macrolactams and other putative secondary metabolites with antibiotic activity were identified. Overall, the results obtained in this study shed a light on AZ78 potential to thrive in the rhizosphere by its ability to move, form biofilm and release secondary metabolites.

'Candidatus Phytoplasma solani' interferes with the distribution and uptake of iron in tomato.

BACKGROUND: 'Candidatus Phytoplasma solani' is endemic in Europe and infects a wide range of weeds and cultivated plants. Phytoplasmas are prokaryotic plant pathogens that colonize the sieve elements of their host plant, causing severe alterations in phloem function and impairment of assimilate translocation. Typical symptoms of infected plants include yellowing of leaves or shoots, leaf curling, and general stunting, but the molecular mechanisms underlying most of the reported changes remain largely enigmatic. To infer a possible involvement of Fe in the host-phytoplasma interaction, we investigated the effects of 'Candidatus Phytoplasma solani' infection on tomato plants (Solanum lycopersicum cv. Micro-Tom) grown under different Fe regimes. RESULTS: Both phytoplasma infection and Fe starvation led to the development of chlorotic leaves and altered thylakoid organization. In infected plants, Fe accumulated in phloem tissue, altering the local distribution of Fe. In infected plants, Fe starvation had additive effects on chlorophyll content and leaf chlorosis, suggesting that the two conditions affected the phenotypic readout via separate routes. To gain insights into the transcriptional response to phytoplasma infection, or Fe deficiency, transcriptome profiling was performed on midrib-enriched leaves. RNA-seq analysis revealed that both stress conditions altered the expression of a large (> 800) subset of common genes involved in photosynthetic light reactions, porphyrin / chlorophyll metabolism, and in flowering control. In Fe-deficient plants, phytoplasma infection perturbed the Fe deficiency response in roots, possibly by interference with the synthesis or transport of a promotive signal transmitted from the leaves to the roots. CONCLUSIONS: 'Candidatus Phytoplasma solani' infection changes the Fe distribution in tomato leaves, affects the photosynthetic machinery and perturbs the orchestration of root-mediated transport processes by compromising shoot-to-root communication.

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.

Sieve element biology provides leads for research on phytoplasma lifestyle in plant hosts.

Phytoplasmas reside exclusively in sieve tubes, tubular arrays of sieve element-companion cell complexes. Hence, the cell biology of sieve elements may reveal (ultra)structural and functional conditions that are of significance for survival, propagation, colonization, and effector spread of phytoplasmas. Electron microscopic images suggest that sieve elements offer facilities for mobile and stationary stages in phytoplasma movement. Stationary stages may enable phytoplasmas to interact closely with diverse sieve element compartments. The unique, reduced sieve element outfit requires permanent support by companion cells. This notion implies a future focus on the molecular biology of companion cells to understand the sieve element-phytoplasma inter-relationship. Supply of macromolecules by companion cells is channelled via specialized symplasmic connections. Ca2+-mediated gating of symplasmic corridors is decisive for the communication within and beyond the sieve element-companion cell complex and for the dissemination of phytoplasma effectors. Thus, Ca2+ homeostasis, which affects sieve element Ca2+ signatures and induces a range of modifications, is a key issue during phytoplasma infection. The exceptional physical and chemical environment in sieve elements seems an essential, though not the only factor for phytoplasma survival.

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.

DAPI and Confocal Laser-Scanning Microscopy for In Vivo Imaging of Phytoplasmas.

As phytoplasmas are located inside the phloem tissue, always surrounded by numerous layers of other cells, they can result difficult candidates for microscopical investigations. Moreover, the necessity to kill the plant tissues for microscopy observations causes instantaneous and irreversible modifications in the sieve elements, leading to misleading information and erroneous interpretations. Phytoplasmas were here investigated in intact Vicia faba host plants using DAPI as fluorescent probe and confocal laser scanning microscopy. The described nondestructive technique may be applied for the imaging of phytoplasmas and of different pathogen-related responses in planta.

Correction to: Jasmonate-mediated defence responses, unlike salicylate-mediated responses, are involved in the recovery of grapevine from bois noir disease.

CORRECTION: Following publication of the original article [1], it came to the attention of the authors that they had omitted to acknowledge the University of Parma. The Acknowledgement section should read as follows: "The authors kindly acknowledge the University of Parma (Department of Chemistry, Life Sciences and Environmental Sustainability; formerly Department of Life Sciences/Evolutionary and Functional Biology) for the transfer of funds obtained from the Ager project: GIALLUMI DELLA VITE: TECNOLOGIE INNOVATIVE PER LA DIAGNOSI E LO STUDIO DELLE INTERAZIONI PIANTA/PATOGENO, BANDO AGER VITICOLTURA DA VINO".