Investigation on Flavescence Dorée in North-Western Italy Identifies Map-M54 (16SrV-D/Map-FD2) as the Only Phytoplasma Genotype in Vitis vinifera L. and Reveals the Presence of New Putative Reservoir Plants.

Flavescence dorée (FD) is the most important phytoplasma-associated disease of the grapevine yellows complex in Europe. Recent studies highlighted a great genetic diversity within FD phytoplasma (FDp) strains and demonstrated that their diffusion is not related exclusively to the pathosystem including Vitis vinifera L. and Scaphoideus titanus but involves additional vectors and reservoir plants. This study aimed to investigate FD epidemiology in north-western Italy, with a particular focus on FDp hosts. During field surveys, leaf samples were collected from symptomatic grapevines and other symptomless plant species, and insects were collected within and around vineyards. Phytoplasmas belonging to the ribosomal group 16SrV were detected and typed using nested-PCR-based amplification and nucleotide sequence analyses of the map gene. All symptomatic grapevines were found to be infected by the FDp genotype M54, prevalent in S. titanus and also identified in other known and newly reported hosts. Interestingly, other FDp strains (M38, M50, M51, M121) and FDp-related strains (M39, M43, M48), never detected in grapevines, were largely identified in several known and newly reported host plants and insects including S. titanus. Such evidence confirmed the complexity of FD ecology, expanding the knowledge on the range of FDp host plants putatively involved in the disease spread.

Signaling Cross-Talk between Salicylic and Gentisic Acid in the 'Candidatus Phytoplasma Solani' Interaction with Sangiovese Vines.

"Bois noir" disease associated with 'Candidatus Phytoplasma solani' seriously compromises the production and survival of grapevines (Vitis vinifera L.) in Europe. Understanding the plant response to phytoplasmas should help to improve disease control strategies. Using a combined metabolomic and transcriptomic analysis, this work, therefore, investigated the phytoplasma-grapevine interaction in red cultivar Sangiovese in a vineyard over four seasonal growth stages (from late spring to late summer), comparing leaves from healthy and infected grapevines (symptomatic and symptomless). We found an accumulation of both conjugate and free salicylic acids (SAs) in the leaves of 'Ca. P. solani'-positive plants from early stages of infection, when plants are still asymptomatic. A strong accumulation of gentisic acid (GA) associated with symptoms progression was found for the first time. A detailed analysis of phenylpropanoids revealed a significant accumulation of hydroxycinnamic acids, flavonols, flavan 3-ols, and anthocyanin cyanidin 3-O-glucoside, which are extensively studied due to their involvement in the plant response to various pathogens. Metabolomic data corroborated by gene expression analysis indicated that phenylpropanoid biosynthetic and salicylic acid-responsive genes were upregulated in 'Ca. P. solani-positive plants compared to -negative ones during the observed period.

Influence of Nitrogen on Grapevine Susceptibility to Downy Mildew.

Downy mildew, caused by the obligate parasite Plasmopara viticola, is one of the most important threats to viticulture. The exploitation of resistant and susceptibility traits of grapevine is one of the most promising ways to increase the sustainability of disease management. Nitrogen (N) fertilization is known for influencing disease severity in the open field, but no information is available on its effect on plant-pathogen interaction. A previous RNAseq study showed that several genes of N metabolism are differentially regulated in grapevine upon P. viticola inoculation, and could be involved in susceptibility or resistance to the pathogen. The aim of this study was to evaluate if N fertilization influences: (i) the foliar leaf content and photosynthetic activity of the plant, (ii) P. viticola infectivity, and (iii) the expression of the candidate susceptibility/resistance genes. Results showed that N level positively correlated with P. viticola infectivity, confirming that particular attention should be taken in vineyard to the fertilization, but did not influence the expression of the candidate genes. Therefore, these genes are manipulated by the pathogen and can be exploited for developing new, environmentally friendly disease management tools, such as dsRNAs, to silence the susceptibility genes or breeding for resistance.

Disease Forecasting for the Rational Management of Grapevine Mildews in the Chianti Bio-District (Tuscany).

Downy and powdery mildews are major grapevine diseases. In organic viticulture, a few fungicides with protectant activities (copper and sulphur in particular) can be used, and their preventative application frequently leads to unneeded spraying. The adoption of an epidemiological disease forecasting model could optimise the timing of treatments and achieve a good level of disease protection. In this study, the effectiveness of the EPI (Etat Potentiel d'Infection) model in predicting infection risk for downy and powdery mildews was evaluated in nine organic vineyards located in Panzano in Chianti (FI), over a 2-year period (2020-2021). The reliability of the EPI model was investigated by comparing the disease intensities, the number of fungicide sprayings, the quantities of the fungicides (kg/ha), and the costs of the treatment achieved, with or without the use of the model, in a vineyard. The results obtained over two seasons indicated that, in most cases, the use of the EPI model accurately signalled the infection risk and allowed for a reduction in the frequency and cost of spraying, particularly for powdery mildew control (-40% sprayings, -20% costs compared to the farmer's schedule), without compromising crop protection. The use of the EPI model can, therefore, contribute to more-sustainable disease management in organic viticulture.

Biocontrol Potential of Endophytic Plant-Growth-Promoting Bacteria against Phytopathogenic Viruses: Molecular Interaction with the Host Plant and Comparison with Chitosan.

Endophytic plant-growth-promoting bacteria (ePGPB) are interesting tools for pest management strategies. However, the molecular interactions underlying specific biocontrol effects, particularly against phytopathogenic viruses, remain unexplored. Herein, we investigated the antiviral effects and triggers of induced systemic resistance mediated by four ePGPB (Paraburkholderia fungorum strain R8, Paenibacillus pasadenensis strain R16, Pantoea agglomerans strain 255-7, and Pseudomonas syringae strain 260-02) against four viruses (Cymbidium Ring Spot Virus-CymRSV; Cucumber Mosaic Virus-CMV; Potato Virus X-PVX; and Potato Virus Y-PVY) on Nicotiana benthamiana plants under controlled conditions and compared them with a chitosan-based resistance inducer product. Our studies indicated that ePGPB- and chitosan-treated plants presented well-defined biocontrol efficacy against CymRSV and CMV, unlike PVX and PVY. They exhibited significant reductions in symptom severity while promoting plant height compared to nontreated, virus-infected controls. However, these phenotypic traits showed no association with relative virus quantification. Moreover, the tested defense-related genes (Enhanced Disease Susceptibility-1 (EDS1), Non-expressor of Pathogenesis-related genes-1 (NPR1), and Pathogenesis-related protein-2B (PR2B)) implied the involvement of a salicylic-acid-related defense pathway triggered by EDS1 gene upregulation.

Real-Time On-Site Diagnosis of Quarantine Pathogens in Plant Tissues by Nanopore-Based Sequencing.

Rapid and sensitive assays for the identification of plant pathogens are necessary for the effective management of crop diseases. The main limitation of current diagnostic testing is the inability to combine broad and sensitive pathogen detection with the identification of key strains, pathovars, and subspecies. Such discrimination is necessary for quarantine pathogens, whose management is strictly dependent on genotype identification. To address these needs, we have established and evaluated a novel all-in-one diagnostic assay based on nanopore sequencing for the detection and simultaneous characterization of quarantine pathogens, using Xylella fastidiosa as a case study. The assay proved to be at least as sensitive as standard diagnostic tests and the quantitative results agreed closely with qPCR-based analysis. The same sequencing results also allowed discrimination between subspecies when present either individually or in combination. Pathogen detection and typing were achieved within 13 min of sequencing owing to the use of an internal control that allowed to stop sequencing when sufficient data had accumulated. These advantages, combined with the use of portable equipment, will facilitate the development of next-generation diagnostic assays for the efficient monitoring of other plant pathogens.

RNAi of a Putative Grapevine Susceptibility Gene as a Possible Downy Mildew Control Strategy.

Downy mildew, caused by the oomycete Plasmopara viticola, is one of the diseases causing the most severe economic losses to grapevine (Vitis vinifera) production. To date, the application of fungicides is the most efficient method to control the pathogen and the implementation of novel and sustainable disease control methods is a major challenge. RNA interference (RNAi) represents a novel biotechnological tool with a great potential for controlling fungal pathogens. Recently, a candidate susceptibility gene (VviLBDIf7) to downy mildew has been identified in V. vinifera. In this work, the efficacy of RNAi triggered by exogenous double-stranded RNA (dsRNA) in controlling P. viticola infections has been assessed in a highly susceptible grapevine cultivar (Pinot noir) by knocking down VviLBDIf7 gene. The effects of dsRNA treatment on this target gene were assessed by evaluating gene expression, disease severity, and development of vegetative and reproductive structures of P. viticola in the leaf tissues. Furthermore, the effects of dsRNA treatment on off-target (EF1α, GAPDH, PEPC, and PEPCK) and jasmonic acid metabolism (COI1) genes have been evaluated. Exogenous application of dsRNA led to significant reductions both in VviLBDIf7 gene expression, 5 days after the treatment, and in the disease severity when artificial inoculation was carried out 7 days after dsRNA treatments. The pathogen showed clear alterations to both vegetative (hyphae and haustoria) and reproductive structures (sporangiophores) that resulted in stunted growth and reduced sporulation. Treatment with dsRNA showed signatures of systemic activity and no deleterious off-target effects. These results demonstrated the potential of RNAi for silencing susceptibility factors in grapevine as a sustainable strategy for pathogen control, underlying the possibility to adopt this promising biotechnological tool in disease management strategies.

From plant resistance response to the discovery of antimicrobial compounds: The role of volatile organic compounds (VOCs) in grapevine downy mildew infection.

The discovery of new mechanisms of resistance and natural bioactive molecules could be two of the possible ways to reduce fungicide use in vineyard and assure an acceptable and sustainable protection against Plasmopara viticola, the grapevine downy mildew agent. Emission of volatile organic compounds (VOCs), such as terpenes, norisoprenoids, alcohols and aldehydes, is frequently induced in plants in response to attack by pathogens, such as P. viticola, that is known to cause a VOCs increment in cultivars harboring American resistance traits. In this study, the role of leaf VOCs in the resistance mechanism of two resistant cultivars (Mgaloblishvili, a pure Vitis vinifera cultivar, and Bianca, an interspecific hybrid) and the direct antimicrobial activity of four selected VOCs have been investigated. The leaf VOCs profiles, analyzed through solid-phase microextraction gas chromatography-mass spectrometry analysis, as well as the expression of six terpene synthases (TPSs), were determined upon pathogen inoculation. In both cultivars, the expression pattern of six TPSs increased soon after pathogen inoculation and an increment of nine VOCs has been detected. While in Mgaloblishvili VOCs were synthesized early after P. viticola inoculation, they constituted a late response to pathogen in Bianca. All the four terpenes (farnesene, nerolidol, ocimene and valencene), chosen according to the VOC profiles and gene expression analysis, caused a significant reduction (53-100%) in P. viticola sporulation. These results support the role of VOCs into defense mechanisms of both cultivars and suggest their potential role as a natural and eco-friendly solution to protect grapevine from P. viticola.

Rpv29, Rpv30 and Rpv31: Three Novel Genomic Loci Associated With Resistance to Plasmopara viticola in Vitis vinifera.

Plasmopara viticola (Berk. et Curt.) Berl. and de Toni, the agent of downy mildew, is one of the most important pathogens of European grapevine (Vitis vinifera L.). Extensive evaluation of cultivated grapevine germplasm has highlighted the existence of resistant phenotypes in the Georgian (Southern Caucasus) germplasm. Resistance is shown as a reduction in disease severity. Unraveling the genetic architecture of grapevine response to P. viticola infection is crucial to develop resistant varieties and reduce the impact of disease management. The aim of this work was to apply a genome-wide association (GWA) approach to a panel of Georgian-derived accessions phenotyped for P. viticola susceptibility and genotyped with Vitis18kSNP chip array. GWA identified three highly significant novel loci on chromosomes 14 (Rpv29), 3 (Rpv30) and 16 (Rpv31) associated with a low level of pathogen sporulation. Rpv29, Rpv30, and Rpv31 loci appeared to be associated with plant defense genes against biotic stresses, such as genes involved in pathogen recognition and signal transduction. This study provides the first evidence of resistant loci against P. viticola in V. vinifera germplasm, and identifies potential target genes for breeding P. viticola resistant grapevine cultivars.

Bacterial microbiota associated with insect vectors of grapevine Bois noir disease in relation to phytoplasma infection.

Bois noir is a grapevine disease causing severe yield loss in vineyards worldwide. It is associated with 'Candidatus Phytoplasma solani', a phloem-limited bacterium transmitted by polyphagous insects. Due to its complex epidemiology, it is difficult to organize effective containment measures. This study aimed to describe the bacterial microbiota associated with 'Candidatus Phytoplasma solani' infected and non-infected insect hosts and vectors to investigate if phytoplasma presence can shape the microbiota. Alpha-diversity analysis showed a low microbiota diversity in these insects, in which few genera were highly abundant. Beta-diversity analysis revealed that the xylem- and phloem-feeding behavior influences the microbiota structure. Moreover, it highlighted that phytoplasma infection is associated with a restructuring of microbiota exclusively in Deltocephalinae insect vectors. Obtained data showed that 'Candidatus Phytoplasma solani' may have adverse effects on the endosymbionts Sulcia and Wolbachia, suggesting a possible fitness modification in the insects. The phytoplasma-antagonistic Dyella was not found in any of the examined insect species. The results indicate an interesting perspective regarding the microbial signatures associated with xylem- and phloem-feeding insects, and determinants that could be relevant to establish whether an insect species can be a vector or not, opening up new avenues for developing microbial resource management-based approaches.

Proposal of A New Bois Noir Epidemiological Pattern Related to 'Candidatus Phytoplasma Solani' Strains Characterized by A Possible Moderate Virulence in Tuscany.

Bois noir (BN), associated with 'Candidatus Phytoplasma solani' (CaPsol), is the most widespread disease of the grapevine yellows complex worldwide. In this work, BN epidemiology was investigated in a case study vineyard where an unusual CaPsol strain, previously detected only in other host plants, was found to be prevalent in grapevine. Experimental activities included: symptom observation; sampling of symptomatic vines, Auchenorrhyncha specimens, and weeds; molecular detection and typing of CaPsol strains; statistical analyses for determining possible relationships between CaPsol relative concentration, strain type, and symptom severity. Among insects, Reptalus quinquecostatus was the most abundant and was found to be highly infected by CaPsol, while Hyalesthes obsoletus, the main CaPsol vector, was not caught. Moreover, R. quinquecostatus harbored CaPsol strains carrying uniquely the stamp sequence variant St10, also identified as prevalent in vines and in the majority of weeds, and all the secY variants identified in the vineyard. Statistical analyses revealed that CaPsol strains carrying the St10 variant are not associated with severe symptoms, suggesting their possible moderate virulence. Based on such evidence, a new BN epidemiological pattern related to these CaPsol strains and involving grapevine, R. quinquecostatus, and/or weeds is proposed. Furthermore, the possible presence of other players (vectors and weeds) involved in CaPsol transmission to grapevines was highlighted.

Novel Aspects on The Interaction Between Grapevine and Plasmopara viticola: Dual-RNA-Seq Analysis Highlights Gene Expression Dynamics in The Pathogen and The Plant During The Battle For Infection.

Mgaloblishvili, a Vitis vinifera cultivar, exhibits unique resistance traits against Plasmopara viticola, the downy mildew agent. This offers the unique opportunity of exploring the molecular responses in compatible and incompatible plant-pathogen interaction. In this study, whole transcriptomes of Mgaloblishvili, Pinot noir (a V. vinifera susceptible cultivar), and Bianca (a resistant hybrid) leaves, inoculated and non-inoculated with the pathogen, were used to identify P. viticola effector-encoding genes and plant susceptibility/resistance genes. Multiple effector-encoding genes were identified in P. viticola transcriptome, with remarkable expression differences in relation to the inoculated grapevine cultivar. Intriguingly, five apoplastic effectors specifically associated with resistance in V. vinifera. Gene coexpression network analysis identified specific modules and metabolic changes occurring during infection in the three grapevine cultivars. Analysis of these data allowed, for the first time, the detection in V. vinifera of a putative P. viticola susceptibility gene, encoding a LOB domain-containing protein. Finally, the de novo assembly of Mgaloblishvili, Pinot noir, and Bianca transcriptomes and their comparison highlighted novel candidate genes that might be at the basis of the resistant phenotype. These results open the way to functional analysis studies and to new perspectives in molecular breeding of grapevine for resistance to P. viticola.

Identification and ecology of alternative insect vectors of 'Candidatus Phytoplasma solani' to grapevine.

Bois noir, a disease of the grapevine yellows complex, is associated with 'Candidatus Phytoplasma solani' and transmitted to grapevines in open fields by the cixiids Hyalesthes obsoletus and Reptalus panzeri. In vine-growing areas where the population density of these vectors is low within the vineyard, the occurrence of bois noir implies the existence of alternative vectors. The aim of this study was to identify alternative vectors through screening of the Auchenorrhyncha community, phytoplasma typing by stamp gene sequence analyses, and transmission trials. During field activities, conducted in Northern Italy in a vineyard where the bois noir incidence was extremely high, nine potential alternative insect vectors were identified according to high abundance in the vineyard agro-ecosystem, high infection rate, and harbouring phytoplasma strains characterized by stamp gene sequence variants found also in symptomatic grapevines. Transmission trials coupled with molecular analyses showed that at least eight species (Aphrodes makarovi, Dicranotropis hamata, Dictyophara europaea, Euscelis incisus, Euscelidius variegatus, Laodelphax striatella, Philaenus spumarius, and Psammotettix alienus/confinis) are alternative vectors of 'Candidatus Phytoplasma solani' to grapevines. These novel findings highlight that bois noir epidemiology in vineyard agro-ecosystems is more complex than previously known, opening up new perspectives in the disease management.

Unique resistance traits against downy mildew from the center of origin of grapevine (Vitis vinifera).

The Eurasian grapevine (Vitis vinifera), an Old World species now cultivated worldwide for high-quality wine production, is extremely susceptible to the agent of downy mildew, Plasmopara viticola. The cultivation of resistant V. vinifera varieties would be a sustainable way to reduce the damage caused by the pathogen and the impact of disease management, which involves the economic, health and environmental costs of frequent fungicide application. We report the finding of unique downy mildew resistance traits in a winemaking cultivar from the domestication center of V. vinifera, and characterize the expression of a range of genes associated with the resistance mechanism. Based on comparative experimental inoculations, confocal microscopy and transcriptomics analyses, our study shows that V. vinifera cv. Mgaloblishvili, native to Georgia (South Caucasus), exhibits unique resistance traits against P. viticola. Its defense response, leading to a limitation of P. viticola growth and sporulation, is determined by the overexpression of genes related to pathogen recognition, the ethylene signaling pathway, synthesis of antimicrobial compounds and enzymes, and the development of structural barriers. The unique resistant traits found in Mgaloblishvili highlight the presence of a rare defense system in V. vinifera against P. viticola which promises fresh opportunities for grapevine genetic improvement.

Competition assays revealed Paenibacillus pasadenensis strain R16 as a novel antifungal agent.

The development of new sustainable containment strategies of plant diseases is very important to guarantee food security while reducing the environmental impact of agriculture. Research of new biocontrol agents is a long and difficult process that involves several steps that start from the identification of possible candidates which, for example, show antibiotic activities, and ends with in field, large scale trials. In this study, the plant growth promoting potential and antifungal effect exerted by a novel, putative candidate biocontrol agent, strain R16, identified as Paenibacillus pasadenensis by sequence analysis of 16S rRNA and rpoB genes, against three important plant pathogenic fungi (Botrytis cinerea, Fusarium verticillioides, and Phomopsis viticola), were assessed. Biochemical assays to determine plant growth promoting potential gave negative results for siderophore production and phosphate solubilization, and positive results for ACC-deamination and IAA production. Further biochemical assays for endophytic lifestyle and antifungal activity gave positive results for catalase and chitinase activity, respectively. In vitro antagonism assays showed that strain R16 is effective against B. cinerea, reducing mycelial growth both in dual-culture and through volatile substances, characterized to be mostly composed by farnesol, and inhibiting conidia germination. Good antagonistic potential was also observed in vitro towards P. viticola, but not towards F. verticillioides. Moreover, in vivo assays confirmed the strain R16 activity reduced the infection rate on B. cinerea-inoculated berries. The obtained results firstly proved that P. pasadenesis strain R16 is a putative plant growth promoter and effective against phytopathogenic fungi. Further studies will be needed to investigate the possible application of this strain as a biocontrol agent.

Identification and Characterization of New 'Candidatus Phytoplasma solani' Strains Associated with Bois Noir Disease in Vitis vinifera L. Cultivars Showing a Range of Symptom Severity in Georgia, the Caucasus Region.

Evidence from a preliminary survey highlighted that 'Candidatus Phytoplasma solani', the etiological agent of bois noir (BN) disease of grapevine, infects grapevine varieties in Georgia, a country of the South Caucasus. In this study, field surveys were carried out to investigate the BN symptom severity in international and Georgian native varieties. 'Ca. P. solani' was detected and identified by polymerase chain reaction-based amplification and restriction fragment length polymorphism analysis of 16S ribosomal DNA, and further characterized by multiple gene typing analysis (vmp1 and stamp genes). Obtained data highlighted that the majority of Georgian grapevine varieties showed moderate and mild symptoms, whereas international cultivars exhibited severe symptoms. Molecular characterization of 'Ca. P. solani' from grapevine revealed the presence of 11 distinct phytoplasma types. Only one type (VmGe12/StGe7) was identical to a strain previously reported in periwinkle from Lebanon; the other 'Ca. P. solani' types are described here for the first time. Phylogenetic analyses of vmp1 and stamp gene concatenated nucleotide sequences showed that 'Ca. P. solani' strains in Georgia are associated mainly with the bindweed-related BN host system. Moreover, the fact that 'Ca. P. solani' strains are distributed in grapevine cultivars showing a range of symptom intensity suggests a different susceptibility of such local cultivars to BN.

'Candidatus Phytoplasma phoenicium' associated with almond witches'-broom disease: from draft genome to genetic diversity among strain populations.

BACKGROUND: Almond witches'-broom (AlmWB), a devastating disease of almond, peach and nectarine in Lebanon, is associated with 'Candidatus Phytoplasma phoenicium'. In the present study, we generated a draft genome sequence of 'Ca. P. phoenicium' strain SA213, representative of phytoplasma strain populations from different host plants, and determined the genetic diversity among phytoplasma strain populations by phylogenetic analyses of 16S rRNA, groEL, tufB and inmp gene sequences. RESULTS: Sequence-based typing and phylogenetic analysis of the gene inmp, coding an integral membrane protein, distinguished AlmWB-associated phytoplasma strains originating from diverse host plants, whereas their 16S rRNA, tufB and groEL genes shared 100 % sequence identity. Moreover, dN/dS analysis indicated positive selection acting on inmp gene. Additionally, the analysis of 'Ca. P. phoenicium' draft genome revealed the presence of integral membrane proteins and effector-like proteins and potential candidates for interaction with hosts. One of the integral membrane proteins was predicted as BI-1, an inhibitor of apoptosis-promoting Bax factor. Bioinformatics analyses revealed the presence of putative BI-1 in draft and complete genomes of other 'Ca. Phytoplasma' species. CONCLUSION: The genetic diversity within 'Ca. P. phoenicium' strain populations in Lebanon suggested that AlmWB disease could be associated with phytoplasma strains derived from the adaptation of an original strain to diverse hosts. Moreover, the identification of a putative inhibitor of apoptosis-promoting Bax factor (BI-1) in 'Ca. P. phoenicium' draft genome and within genomes of other 'Ca. Phytoplasma' species suggested its potential role as a phytoplasma fitness-increasing factor by modification of the host-defense response.

'Candidatus Phytoplasma solani', a novel taxon associated with stolbur- and bois noir-related diseases of plants.

Phytoplasmas classified in group 16SrXII infect a wide range of plants and are transmitted by polyphagous planthoppers of the family Cixiidae. Based on 16S rRNA gene sequence identity and biological properties, group 16SrXII encompasses several species, including 'Candidatus Phytoplasma australiense', 'Candidatus Phytoplasma japonicum' and 'Candidatus Phytoplasma fragariae'. Other group 16SrXII phytoplasma strains are associated with stolbur disease in wild and cultivated herbaceous and woody plants and with bois noir disease in grapevines (Vitis vinifera L.). Such latter strains have been informally proposed to represent a separate species, 'Candidatus Phytoplasma solani', but a formal description of this taxon has not previously been published. In the present work, stolbur disease strain STOL11 (STOL) was distinguished from reference strains of previously described species of the 'Candidatus Phytoplasma' genus based on 16S rRNA gene sequence similarity and a unique signature sequence in the 16S rRNA gene. Other stolbur- and bois noir-associated ('Ca. Phytoplasma solani') strains shared >99 % 16S rRNA gene sequence similarity with strain STOL11 and contained the signature sequence. 'Ca. Phytoplasma solani' is the only phytoplasma known to be transmitted by Hyalesthes obsoletus. Insect vectorship and molecular characteristics are consistent with the concept that diverse 'Ca. Phytoplasma solani' strains share common properties and represent an ecologically distinct gene pool. Phylogenetic analyses of 16S rRNA, tuf, secY and rplV-rpsC gene sequences supported this view and yielded congruent trees in which 'Ca. Phytoplasma solani' strains formed, within the group 16SrXII clade, a monophyletic subclade that was most closely related to, but distinct from, that of 'Ca. Phytoplasma australiense'-related strains. Based on distinct molecular and biological properties, stolbur- and bois noir-associated strains are proposed to represent a novel species level taxon, 'Ca. Phytoplasma solani'; STOL11 is designated the reference strain.

Restructuring of endophytic bacterial communities in grapevine yellows-diseased and recovered Vitis vinifera L. plants.

Length heterogeneity-PCR assays, combined with statistical analyses, highlighted that the endophytic bacterial community associated with healthy grapevines was characterized by a greater diversity than that present in diseased and recovered plants. The findings suggest that phytoplasmas can restructure the bacterial community by selecting endophytic strains that could elicit a plant defense response.

'Candidatus Liberibacter europaeus' sp. nov. that is associated with and transmitted by the psyllid Cacopsylla pyri apparently behaves as an endophyte rather than a pathogen.

'Candidatus Liberibacter spp.' cause serious plant diseases. 'Candidatus Liberibacter asiaticus', 'Ca. L. americanus' and 'Ca. L. africanus' are the aetiological agents of citrus greening (Huanglongbing) in Asia, America and Africa. 'Candidatus Liberibacter solanacearum' causes diseases in Solanaceae in America and New Zealand. All four species are vectored by psyllid insects of different genera. Here, we show that the pear psyllid pest Cacopsylla pyri (L.) hosts a novel liberibacter species that we named 'Ca. Liberibacter europaeus'. It can bloom to high titres in the psyllid host, with more than 10(9) 16S rRNA gene copies per individual. Fluorescent in situ hybridization experiments showed that 'Ca. L. europaeus' is present in the host midgut lumen, salivary glands and Malpighian tubules. 'Candidatus L. europaeus' has a relatively high prevalence (> 51%) in C. pyri from different areas in the Piedmont and Valle d'Aosta regions in Italy and can be transmitted to pear plants in experimental transmission trials. However, even though high titres of the bacterium (more than 10(8) 16S rRNA gene copies g(-1) of pear plant tissue) could be detected, in the pear tissues no specific disease symptoms could be observed in the infected plants over a 6-month period. Despite liberibacters representing potential quarantine organisms, 'Ca. L. europaeus', first described in Italy and Europe, apparently behaves as an endophyte rather than a pathogen.