Genetic diversity of Actinidia spp. shapes the oomycete pattern associated with Kiwifruit Vine Decline Syndrome (KVDS).

Kiwifruit Vine Decline Syndrome (KVDS) is an important soil-borne disease for the Italian kiwifruit industry, causing €300,000 in economic losses in 2020 alone. So far, the organisms recognized as involved in the aetiology of KVDS mainly belong to the Oomycota. As no effective management strategies exist, a promising approach to overcoming KVDS is the use of resistant species as rootstocks or for inclusion in breeding programs. Several Actinidia genotypes showing different level of resistance to KVDS were grown in disease-promoting soils. A metabarcoding approach was set up to identify KVDS-associated oomycetes and investigate whether the main species involved may vary according to plant genotype. Our results clearly showed significant differences between the genotypes in terms of oomycetes present in both plant rhizosphere and endosphere, which were strongly correlated with the symptoms displayed. We found out that the resistance of Actinidia macrosperma to KVDS is related to its ability to shape the pathobiome, particularly as far as the endosphere is concerned. In our conditions, Phytophthora sp. was predominantly found in sensitive genotypes, whilst Globisporangium intermedium was mainly detected in asymptomatic plants, suggesting that the latter species could compete with the recruitment of Phytophthora sp. in plants with different levels of resistance, consequently, explaining the onset of symptoms and the resistance condition.

The Virome of 'Lamon Bean': Application of MinION Sequencing to Investigate the Virus Population Associated with Symptomatic Beans in the Lamon Area, Italy.

'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.

Synergistic interaction between the type III secretion system of the endophytic bacterium Pantoea agglomerans DAPP-PG 734 and the virulence of the causal agent of olive knot Pseudomonas savastanoi pv. savastanoi DAPP-PG 722.

The endophytic bacterium Pantoea agglomerans DAPP-PG 734 was previously isolated from olive knots caused by infection with Pseudomonas savastanoi pv. savastanoi DAPP-PG 722. Whole-genome analysis of this P. agglomerans strain revealed the presence of a Hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS). To assess the role of the P. agglomerans T3SS in the interaction with P. savastanoi pv. savastanoi, we generated independent knockout mutants in three Hrp genes of the P. agglomerans DAPP-PG 734 T3SS (hrpJ, hrpN, and hrpY). In contrast to the wildtype control, all three mutants failed to cause a hypersensitive response when infiltrated in tobacco leaves, suggesting that P. agglomerans T3SS is functional and injects effector proteins in plant cells. In contrast to P. savastanoi pv. savastanoi DAPP-PG 722, the wildtype strain P. agglomerans DAPP-PG 734 and its Hrp T3SS mutants did not cause olive knot disease in 1-year-old olive plants. Coinoculation of P. savastanoi pv. savastanoi with P. agglomerans wildtype strains did not significantly change the knot size, while the DAPP-PG 734 hrpY mutant induced a significant decrease in knot size, which could be complemented by providing hrpY on a plasmid. By epifluorescence microscopy and confocal laser scanning microscopy, we found that the localization patterns in knots were nonoverlapping for P. savastanoi pv. savastanoi and P. agglomerans when coinoculated. Our results suggest that suppression of olive plant defences mediated by the Hrp T3SS of P. agglomerans DAPP-PG 734 positively impacts the virulence of P. savastanoi pv. savastanoi DAPP-PG 722.

Polymorphisms at the 3'end of the movement protein (MP) gene of grapevine Pinot gris virus (GPGV) affect virus titre and small interfering RNA accumulation in GLMD disease.

Grapevine Leaf Mottling and Deformation (GLMD) is a grapevine disease that has been associated with a trichovirus, the grapevine Pinot gris virus (GPGV). A wide diversity in the severity of GLMD disease symptoms has been recorded worldwide, but the relationship of this diversity to the sequence variation in the GPGV genome is still a matter of debate. Results from comparative analysis of GPGV genomic sequences have suggested an association of polymorphisms at the 3'-end of the movement protein (MP) with GLMD severity. Here, the 3'-terminus of the MP gene of a GPGV infectious clone derived from an isolate from grapevine showing severe symptoms (fvg-12), was substituted with a 356 bp synthetic DNA fragment having a sequence resembling that of another GPGV isolate (fvg-15), recovered from an asymptomatic grapevine. The clone containing this chimeric construct was root-inoculated in virus-free Kober rootstocks along with the clones containing the fvg-12 and fvg-15 full length sequence. Remarkable differences in virus titre, accumulation of GPGV-derived small interfering RNAs (siRNAs), alterations in the gene expression of boron transporters and, to a lesser extent, in symptom expression were recorded among plants infected with either one of the three GPGV derived clones. In particular, the chimeric clone behaviour was indistinguishable from that of the donor of the small 356 bp fragment and significantly different from the other. Thus, this work experimentally confirmed the critical role of the GPGV-MP C-terminus in determining the fate of the infection, as it had been previously hypothesized on the basis of comparative sequence analysis.

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.

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.

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.

Genome Sequence and Antifungal Activity of Two Niche-Sharing Pseudomonas protegens Related Strains Isolated from Hydroponics.

This work reports the comparison of the genome sequence and the ability to inhibit fungal growth of two Pseudomonas protegens related strains that were isolated from the same hydroponic culture of lamb's lettuce. The two strains were very similar in their core genome but one strain, Pf4, contained three gene clusters for the production of secondary metabolites, i.e., pyoluteorin (plt), pyrrolnitrin (prn), and rhizoxin (rzx), that were missing in the other strain, Pf11. The difference between the two strains was not due to simple insertion events, but to a relatively complex differentiation focused on the accessory genomes. In dual culture assays, both strains inhibited nearly all tested fungal strains, yet Pf4 exerted a significantly stronger fungal growth inhibition than Pf11. In addition to the differences in the secondary metabolite production associated genes abundance, the genome of Pf4 was more stable, smaller in size and with a lower number of transposons. The preservation of a dynamic equilibrium within natural populations of different strains comprised in the same species but differing in their secondary metabolite repertoire and in their genome stability may be functional to the adaptation to environmental changes.

Assembly of Phytoplasma Genome Drafts from Illumina Reads Using Phytoassembly.

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.

An Effective Pipeline Based on Relative Coverage for the Genome Assembly of Phytoplasmas and Other Fastidious Prokaryotes.

BACKGROUND: For the plant pathogenic phytoplasmas, as well as for several fastidious prokaryotes, axenic cultivation is extremely difficult or not possible yet; therefore, even with second generation sequencing methods, obtaining the sequence of their genomes is challenging due to host sequence contamination. OBJECTIVE: With the Phytoassembly pipeline here presented, we aim to provide a method to obtain high quality genome drafts for the phytoplasmas and other uncultivable plant pathogens, by exploiting the coverage differential in the ILLUMINA sequences from the pathogen and the host, and using the sequencing of a healthy, isogenic plant as a filter. VALIDATION: The pipeline has been benchmarked using simulated and real ILLUMINA runs from phytoplasmas whose genome is known, and it was then used to obtain high quality drafts for three new phytoplasma genomes. CONCLUSION: For phytoplasma infected samples containing >2-4% of pathogen DNA and an isogenic reference healthy sample, the resulting assemblies can be next to complete. The Phytoassembly source code is available on GitHub at https://github.com/cpolano/phytoassembly.

Genomic Structural Variations Affecting Virulence During Clonal Expansion of Pseudomonas syringae pv. actinidiae Biovar 3 in Europe.

Pseudomonas syringae pv. actinidiae (Psa) biovar 3 caused pandemic bacterial canker of Actinidia chinensis and Actinidia deliciosa since 2008. In Europe, the disease spread rapidly in the kiwifruit cultivation areas from a single introduction. In this study, we investigated the genomic diversity of Psa biovar 3 strains during the primary clonal expansion in Europe using single molecule real-time (SMRT), Illumina and Sanger sequencing technologies. We recorded evidences of frequent mobilization and loss of transposon Tn6212, large chromosome inversions, and ectopic integration of IS sequences (remarkably ISPsy31, ISPsy36, and ISPsy37). While no phenotype change associated with Tn6212 mobilization could be detected, strains CRAFRU 12.29 and CRAFRU 12.50 did not elicit the hypersensitivity response (HR) on tobacco and eggplant leaves and were limited in their growth in kiwifruit leaves due to insertion of ISPsy31 and ISPsy36 in the hrpS and hrpR genes, respectively, interrupting the hrp cluster. Both strains had been isolated from symptomatic plants, suggesting coexistence of variant strains with reduced virulence together with virulent strains in mixed populations. The structural differences caused by rearrangements of self-genetic elements within European and New Zealand strains were comparable in number and type to those occurring among the European strains, in contrast with the significant difference in terms of nucleotide polymorphisms. We hypothesize a relaxation, during clonal expansion, of the selection limiting the accumulation of deleterious mutations associated with genome structural variation due to transposition of mobile elements. This consideration may be relevant when evaluating strategies to be adopted for epidemics management.

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.

Draft genome of a Xanthomonas perforans strain associated with pith necrosis.

Xanthomonas perforans causes bacterial spot of tomato and pepper. A genome draft of an unusual isolate (strain 4P1S2), differing in that it was associated with stem pith necrosis, was assembled from Illumina MiSeq sequencing data using the draft of X. perforans strain 91-118 as a reference. The resulting draft (accession number JRWW00000000) largely overlapped with the reference draft. In addition, the reads not mapping on the reference assembly were selected and used for a further assembly, that revealed a large putative plasmid. The analysis of the predicted proteins showed only few gene features that could be potentially implicated in the switch of a phytopathological behavior.

Plasmon resonance tuning using DNA origami actuation.

A strategy for an innovative, continuous and reversible LSPR tuning using DNA origami actuation to modulate the nanometric separation of two gold nanoparticles has been developed. The actuation mechanism is based on DNA hybridization, in particular three different DNA sequences were shown to induce resonance shift of up to 6 nm.

Draft Genome Sequence of Erwinia oleae, a Bacterium Associated with Olive Knots Caused by Pseudomonas savastanoi pv. savastanoi.

Erwinia oleae is a nonpathogenic bacterial species isolated from olive knots caused by Pseudomonas savastanoi pv. savastanoi. Since the presence of E. oleae in the knots increases disease severity, interspecies interactions with the pathogen are hypothesized. Here, we report the first draft genome sequence of the E. oleae type strain.

Draft Genome Sequence of Pseudomonas savastanoi pv. savastanoi Strain DAPP-PG 722, Isolated in Italy from an Olive Plant Affected by Knot Disease.

Olive knot disease, caused by the bacterium Pseudomonas savastanoi pv. savastanoi, seriously affects olive trees in the Mediterranean basin. Here, we report the draft genome sequence of P. savastanoi pv. savastanoi DAPP-PG 722, a strain isolated in Italy from an olive plant affected by knot disease.

Draft Genome Sequence of a Hypersensitive Reaction-Inducing Pantoea agglomerans Strain Isolated from Olive Knots Caused by Pseudomonas savastanoi pv. savastanoi.

Pantoea agglomerans strains inducing a hypersensitive reaction in tobacco leaves are frequently isolated inside olive knots caused by Pseudomonas savastanoi pv. savastanoi. Here, we report the draft genome sequence of the Italian P. agglomerans strain, which is able to increase olive knot disease severity when coinoculated with P. savastanoi pv. savastanoi.

A DNA origami nanorobot controlled by nucleic acid hybridization.

A prototype for a DNA origami nanorobot is designed, produced, and tested. The cylindrical nanorobot (diameter of 14 nm and length of 48 nm) with a switchable flap, is able to respond to an external stimulus and reacts by a physical switch from a disarmed to an armed configuration able to deliver a cellular compatible message. In the tested design the robot weapon is a nucleic acid fully contained in the inner of the tube and linked to a single point of the internal face of the flap. Upon actuation the nanorobot moves the flap extracting the nucleic acid that assembles into a hemin/G-quadruplex horseradish peroxidase mimicking DNAzyme catalyzing a colorimetric reaction or chemiluminescence generation. The actuation switch is triggered by an external nucleic acid (target) that interacts with a complementary nucleic acid that is beard externally by the nanorobot (probe). Hybridization of probe and target produces a localized structural change that results in flap opening. The flap movement is studied on a two-dimensional prototype origami using Förster resonance energy transfer and is shown to be triggered by a variety of targets, including natural RNAs. The nanorobot has potential for in vivo biosensing and intelligent delivery of biological activators.

A Genomic redefinition of Pseudomonas avellanae species.

The circumscription of bacterial species is a complex task. So far, DNA-DNA hybridization (DDH), 16S rRNA gene sequencing, and multiocus sequence typing analysis (MLSA) are currently the preferred techniques for their genetic determination. However, the average nucleotide identity (ANI) analysis of conserved and shared genes between two bacterial strains based on the pair-wise genome comparisons, with support of the tetranucleotide frequency correlation coefficients (TETRA) value, has recently been proposed as a reliable substitute for DDH. The species demarcation boundary has been set to a value of 95-96% of the ANI identity, with further confirmation through the assessment of the corresponding TETRA value. In this study, we performed a genome-wide MLSA of 14 phytopathogenic pseudomonads genomes, and assessed the ANI and TETRA values of 27 genomes, representing seven out of the nine genomospecies of Pseudomonas spp. sensu Gardan et alii, and their phylogenetic relationships using maximum likelihood and Bayesian approaches. The results demonstrate the existence of a well demarcated genomic cluster that includes strains classified as P. avellanae, P. syringae pv. theae, P. s. pv. actinidiae and one P. s. pv. morsprunorum strain all belonging to the single species P. avellanae. In addition, when compared with P. avellanae, five strains of P. s. pv. tomato, including the model strain DC3000, and one P. s. pv. lachrymans strain, appear as very closely related to P. avellanae, with ANI values of nearly 96% as confirmed by the TETRA analysis. Conversely, one representative strain, previously classified as P. avellanae and isolated in central Italy, is a genuine member of the P. syringae species complex and can be defined as P. s. pv. avellanae. Currently. The core and pan genomes of P. avellanae species consist of 3,995 and 5,410 putative protein-coding genes, respectively.