Pseudomonas synxantha volatile organic compounds: efficacy against Cadophora luteo-olivacea and Botrytis cinerea of kiwifruit.

Volatile organic compounds (VOCs) are responsible for the antagonistic activity exerted by different biological control agents (BCAs). In this study, VOCs produced by Pseudomonas synxantha strain 117-2b were tested against two kiwifruit fungal postharvest pathogens: Cadophora luteo-olivacea and Botrytis cinerea, through in vitro and in vivo assays. In vitro results demonstrated that P. synxantha 117-2b VOCs inhibit mycelial growth of C. luteo-olivacea and B. cinerea by 56% and 42.8% after 14 and 5 days of exposition, respectively. In vivo assay demonstrated significant inhibitory effects. VOCs used as a biofumigant treatment reduced skin-pitting symptoms disease severity by 28.5% and gray mold incidence by 66.6%, with respect to the untreated control. BCA volatiles were analyzed by solid-phase microextraction coupled with gas chromatography-mass spectrometry (SPME-GC/MS), and among the detected compounds, 1-butanol, 3-methyl and 1-nonene resulted as the most produced. Their efficacy as pure synthetic compounds was assayed against mycelial growth of fungal pathogens by different concentrations (0.34, 0.56, and 1.12 µL mL-1 headspace). The effect of the application of VOCs as a biofumigant was also investigated as the expression level of seven defense-related genes of kiwifruit at different exposition times. The results indicated an enhancement of the expression of almost all the genes starting from 3 h of treatment. These results described P. synxantha VOCs characteristics and their potential as a promising method to adopt for protecting kiwifruit from postharvest diseases caused by C. luteo-olivacea and B. cinerea.

The Oxford Nanopore MinION as a Versatile Technology for the Diagnosis and Characterization of Emerging Plant Viruses.

The emergence of novel viral epidemics that could affect major crops represents a serious threat to global food security. The early and accurate identification of the causative viral agent is the most important step for a rapid and effective response to disease outbreaks. Over the last years, the Oxford Nanopore Technologies (ONT) MinION sequencer has been proposed as an effective diagnostic tool for the early detection and identification of emerging viruses in plants, providing many advantages compared with different high-throughput sequencing (HTS) technologies. Here, we provide a step-by-step protocol that we optimized to obtain the virome of "Lamon bean" plants (Phaseolus vulgaris L.), an agricultural product with Protected Geographical Indication (PGI) in North-East of Italy, which is frequently subjected to multiple infections caused by different RNA viruses. The conversion of viral RNA in ds-cDNA enabled the use of Genomic DNA Ligation Sequencing Kit and Native Barcoding DNA Kit, which have been originally developed for DNA sequencing. This allowed the simultaneous diagnosis of both DNA- and RNA-based pathogens, providing a more versatile alternative to the use of direct RNA and/or direct cDNA sequencing kits.

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.

Traditional Approaches and Emerging Biotechnologies in Grapevine Virology.

Environmental changes and global warming may promote the emergence of unknown viruses, whose spread is favored by the trade in plant products. Viruses represent a major threat to viticulture and the wine industry. Their management is challenging and mostly relies on prophylactic measures that are intended to prevent the introduction of viruses into vineyards. Besides the use of virus-free planting material, the employment of agrochemicals is a major strategy to prevent the spread of insect vectors in vineyards. According to the goal of the European Green Deal, a 50% decrease in the use of agrochemicals is expected before 2030. Thus, the development of alternative strategies that allow the sustainable control of viral diseases in vineyards is strongly needed. Here, we present a set of innovative biotechnological tools that have been developed to induce virus resistance in plants. From transgenesis to the still-debated genome editing technologies and RNAi-based strategies, this review discusses numerous illustrative studies that highlight the effectiveness of these promising tools for the management of viral infections in grapevine. Finally, the development of viral vectors from grapevine viruses is described, revealing their positive and unconventional roles, from targets to tools, in emerging biotechnologies.

Antifungal activity of chili pepper extract with potential for the control of some major pathogens in grapevine.

BACKGROUND: In recent years, biofungicides have drawn increasing interest in vineyards for a more sustainable integrated and copper-limited pest management. Among alternatives, botanicals could represent valuable tools, being rich sources of biologically active compounds. Conversely to the well-known antioxidant and biological properties in relation to health benefits, investigation on bioactivity of hot pungent Capsicum sp. products against fungal phytopathogens in vineyards is still scarce. Therefore, the present study aimed at exploring the biologically active compounds profile of a chili pepper (Capsicum chinense Jacq.) pod extract and its antimicrobial properties against some of the major fungal and Oomycetes pathogens of grapevine, including Botrytis cinerea Pers., Guignardia bidwellii (Ellis) Viala & Ravaz and Plasmopara viticola (Berk. & M.A. Curtis) Berl. & De Toni. RESULTS: The ethyl acetate-extracted oleoresin from the most pungent varieties was rich in capsaicinoids and polyphenols (371.09 and 268.5 µg mg-1 dry weight, respectively). Capsaicin and dihydrocapsaicin, hydroxycinnamic and hydroxybenzoic acids and quercetin derivatives were the most abundant, while carotenoids represented only a minor fraction. The oleoresin was efficient to inhibit all three pathogenic fungi and ED50 values were determined, evidencing that G. bidwellii was the more sensitive (0.233 ± 0.034 mg mL-1 ). CONCLUSION: The results suggested a potentiality of chili pepper extract for the control of some important grapevine pathogens, their possible application being helpful for the recommended limitation in extensive use of copper in vineyard. The complex mixture of high amounts of capsaicinoids, associated to specific phenolic acids and other minor bioactive components might contribute to the observed antimicrobial action of chili pepper extract. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

In vitro application of Eruca vesicaria subsp. sativa leaf extracts and associated metabolites reduces the growth of Oomycota species involved in Kiwifruit Vine Decline Syndrome.

This study aimed to determine whether leaf extracts from seven Eruca vesicaria subsp. sativa cultivars and their biochemically active compounds (glucosinolates and downstream-derived products) inhibit mycelia growth of three well-known pathogenic oomycetes, Phytopythium chamaehyphon, Phytopythium vexans and Phytophthora citrophthora; being the most significant in the development of Kiwifruit Vine Decline Syndrome (KVDS). Leaf extract quantity of 10, 20 and 30 mg were inoculated in Petri dish (90 mm Ø, each 22 mL of liquid medium - Potato Dextrose Agar), for in vitro bioassays. A pathogen plug was placed in the centre of each plate and the Oomycota colony perimeter was marked 5 days after inoculation. Radial colony growth was measured from 4 marks per plate 5, 10, and 15 days after inoculation, further elaborated with Image J software image analysis. Growth rates for all strains were inhibited by around 67% after 15 days. This was most pronounced when applying the highest concentration of leaf extract. By using Liquid Chromatography Mass Spectrometry (LC-MS) and Gas Chromatography Mass Spectrometry (GC-MS), fifteen glucosinolate compounds, of which glucosativin was found in the highest quantity, were identified. Concentrations of hydrolysis products produced by leaves (erucin and sativin) were also investigated, and were significantly associated with colony radial growth, especially towards Pp. chamaehyphon and Pp. vexans. Three downstream products of glucosinolates (two pure isothiocyanates, AITC and PEITC; and one indole I3C; all commonly present in Brassicaceae) were also tested, and a statistically significant inhibition of growth was observed at the highest concentration (0.6 µL).

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.

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.

Spatiotemporal and Quantitative Monitoring of the Fate of "Candidatus Phytoplasma Solani" in Tomato Plants Infected by Grafting.

Understanding how phytoplasmas move and multiply within the host plant is fundamental for plant-pathogen interaction studies. In recent years, the tomato has been used as a model plant to study this type of interaction. In the present work, we investigated the distribution and multiplication dynamics of one strain of "Candidatus Phytoplasma (Ca. P.) solani", (16SrXII-A) in tomato (Solanum lycopersicum L., cv. Micro-Tom) plants. We obtained infected plants by grafting, a fast and effective method to maintain phytoplasma infection. In planta spread and multiplication of "Ca. P. solani" was monitored over time using qualitative and quantitative qPCR. Root, apical shoot, lower leaves, and upper leaves were sampled at each sampling time. We hypothesized that "Ca. P. solani" from the grafting site reached firstly the highest leaf, the apex and the roots; subsequently, the phytoplasmas spread to the rest of the upper leaves and then progressively to the lower leaves. Significant differences were found in "Ca. P. solani" titer among different plant tissues. In particular, the concentration of phytoplasma in the roots was significantly higher than that in the other plant compartments in almost all the sampling dates. Since the roots show rapid colonization and the highest concentration of phytoplasmas, they represent the ideal tissue to sample for an early, sensitive and robust diagnosis.

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.

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.

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.

Symptoms of Phytoplasma Diseases.

Phytoplasmas are associated with diseases in several hundreds of cultivated herbaceous and woody plants. Their impact in agriculture and the periodical outbreak of worrying epidemics make very important, besides precise laboratory-based diagnosis, the direct in-field recognition of phytoplasma disease symptoms. Even if some symptoms are typical of this kind of pathogens, in-field diagnosis requires the knowledge of the host plant, strong field experience, and awareness of the symptom variability of the various organs of the plant during different seasons and under various environmental conditions. It is therefore very important to be familiar with factors like environmental conditions, agronomical features, and disease progression that influence symptom expression. Therefore, a satisfactory diagnosis should be based on repeated and complete observations scored over the entire plant and across different times of the year. A more suitable diagnosis is possible if the observer is able to recognize and distinguish the symptoms of other biotic or abiotic diseases. A general rule is to observe three different symptoms, at least, and to seek input from the grower about the initial development, frequency, diffusion, and particular characteristics of the disease.After a short introduction the following symptoms are presented: the most common and representative symptoms caused by phytoplasmas; the most common symptoms of phytoplasma diseases occurring in particular plant organs, with some references to specific diseases; phytoplasma symptoms on the model plant periwinkle (Vinca rosea or Catharanthus roseus); the main factors influencing phytoplasma symptoms expression; and several practical procedures that should be followed for suitable diagnosis. A series of original photos have been included to illustrate typical symptoms.

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.

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.

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.

Involvement of plasma membrane peroxidases and oxylipin pathway in the recovery from phytoplasma disease in apple (Malus domestica).

Apple trees (Malus domestica Borkh.) may be affected by apple proliferation (AP), caused by 'Candidatus Phytoplasma mali'. Some plants can spontaneously recover from the disease, which implies the disappearance of symptoms through a phenomenon known as recovery. In this article it is shown that NAD(P)H peroxidases of leaf plasma membrane-enriched fractions exhibited a higher activity in samples from both AP-diseased and recovered plants. In addition, an increase in endogenous SA was characteristic of the symptomatic plants, since its content increased in samples obtained from diseased apple trees. In agreement, phenylalanine ammonia lyase (PAL) activity, a key enzyme of the phenylpropanoid pathway, was increased too. Jasmonic acid (JA) increased only during recovery, in a phase subsequent to the pathological state, and in concomitance to a decline of salicylic acid (SA). Oxylipin pathway, responsible for JA synthesis, was not induced during the development of AP-disease, but it appeared to be stimulated when the recovery occurred. Accordingly, lipoxygenase (LOX) activity, detected in plasma membrane-enriched fractions, showed an increase in apple leaves obtained from recovered plants. This enhancement was paralleled by an increase of hydroperoxide lyase (HPL) activity, detected in leaf microsomes, albeit the latter enzyme was activated in either the disease or recovery conditions. Hence, a reciprocal antagonism between SA- and JA-pathways could be suggested as an effective mechanism by which apple plants react to phytoplasma invasions, thereby providing a suitable defense response leading to the establishment of the recovery phenomenon.