RESUMEN
Within the 16SrII phytoplasma group, subgroups A-X have been classified based on restriction fragment length polymorphism of their 16S rRNA gene, and two species have been described, namely 'Candidatus Phytoplasma aurantifolia' and 'Ca. Phytoplasma australasia'. Strains of 16SrII phytoplasmas are detected across a broad geographic range within Africa, Asia, Australia, Europe and North and South America. Historically, all members of the 16SrII group share ≥97.5â% nucleotide sequence identity of their 16S rRNA gene. In this study, we used whole genome sequences to identify the species boundaries within the 16SrII group. Whole genome analyses were done using 42 phytoplasma strains classified into seven 16SrII subgroups, five 16SrII taxa without official 16Sr subgroup classifications, and one 16SrXXV-A phytoplasma strain used as an outgroup taxon. Based on phylogenomic analyses as well as whole genome average nucleotide and average amino acid identity (ANI and AAI), eight distinct 16SrII taxa equivalent to species were identified, six of which are novel descriptions. Strains within the same species had ANI and AAI values of >97â%, and shared ≥80â% of their genomic segments based on the ANI analysis. Species also had distinct biological and/or ecological features. A 16SrII subgroup often represented a distinct species, e.g., the 16SrII-B subgroup members. Members classified within the 16SrII-A, 16SrII-D, and 16SrII-V subgroups as well as strains classified as sweet potato little leaf phytoplasmas fulfilled criteria to be included as members of a single species, but with subspecies-level relationships with each other. The 16SrXXV-A taxon was also described as a novel phytoplasma species and, based on criteria used for other bacterial families, provided evidence that it could be classified as a distinct genus from the 16SrII phytoplasmas. As more phytoplasma genome sequences become available, the classification system of these bacteria can be further refined at the genus, species, and subspecies taxonomic ranks.
Asunto(s)
Phytoplasma , Humanos , Phytoplasma/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Filogenia , ADN Bacteriano/genética , Composición de Base , Técnicas de Tipificación Bacteriana , Ácidos Grasos/químicaRESUMEN
Flavescence dorée (FD) is a European quarantine grapevine disease transmitted by the Deltocephalinae leafhopper Scaphoideus titanus. Whereas this vector had been introduced from North America, the possible European origin of FD phytoplasma needed to be challenged and correlated with ecological and genetic drivers of FD emergence. For that purpose, a survey of genetic diversity of these phytoplasmas in grapevines, S. titanus, black alders, alder leafhoppers and clematis were conducted in five European countries. Out of 132 map genotypes, only 11 were associated to FD outbreaks, three were detected in clematis, whereas 127 were detected in alder trees, alder leafhoppers or in grapevines out of FD outbreaks. Most of the alder trees were found infected, including 8% with FD genotypes M6, M38 and M50, also present in alders neighboring FD-free vineyards and vineyard-free areas. The Macropsinae Oncopsis alni could transmit genotypes unable to achieve transmission by S. titanus, while the Deltocephalinae Allygus spp. and Orientus ishidae transmitted M38 and M50 that proved to be compatible with S. titanus. Variability of vmpA and vmpB adhesin-like genes clearly discriminated 3 genetic clusters. Cluster Vmp-I grouped genotypes only transmitted by O. alni, while clusters Vmp-II and -III grouped genotypes transmitted by Deltocephalinae leafhoppers. Interestingly, adhesin repeated domains evolved independently in cluster Vmp-I, whereas in clusters Vmp-II and-III showed recent duplications. Latex beads coated with various ratio of VmpA of clusters II and I, showed that cluster II VmpA promoted enhanced adhesion to the Deltocephalinae Euscelidius variegatus epithelial cells and were better retained in both E. variegatus and S. titanus midguts. Our data demonstrate that most FD phytoplasmas are endemic to European alders. Their emergence as grapevine epidemic pathogens appeared restricted to some genetic variants pre-existing in alders, whose compatibility to S. titanus correlates with different vmp gene sequences and VmpA binding properties.
Asunto(s)
Hemípteros/microbiología , Insectos Vectores/microbiología , Phytoplasma/aislamiento & purificación , Enfermedades de las Plantas/microbiología , Vitis/microbiología , Animales , Bacterias , Proteínas Bacterianas/genética , Epidemias , Europa (Continente)/epidemiología , Variación Genética , Hemípteros/fisiología , Filogenia , Phytoplasma/clasificación , Phytoplasma/genética , Enfermedades de las Plantas/estadística & datos numéricosRESUMEN
To study the role of wild areas around the vineyards in the epidemiology of flavescence dorée (FD) and track the origin of new foci, two phytoplasma genetic markers, dnaK and malG, were developed for FD phytoplasma (FDp) characterization. The two genes and the vmpA locus were used to genetically characterize FDp populations at seven agroecosystems of a wine-growing Italian region. Vitis vinifera, "gone-wild" V. vinifera and rootstocks, Clematis spp., and Scaphoideus titanus adults were sampled within and outside the vineyards. A range of genotypes infecting the different hosts of the FDp epidemiological cycle was found. Type FD-C isolates were fairly homogeneous compared to type FD-D ones. Most of the FD-D variability was correlated with the malG sequence, and a duplication of this locus was demonstrated for this strain. Coinfection with FD-C and FD-D strains was rare, suggesting possible competition between the two. Similar levels of FDp genetic variation recorded for grapevines or leafhoppers of cultivated and wild areas and co-occurrence of many FDp genotypes inside and outside the vineyards supported the idea of the importance of wild or abandoned Vitis plants and associated S. titanus insects in the epidemiology of the disease. Genetic profiles of FDp found in Clematis were never found in the other hosts, indicating that this species does not take part in the disease cycle in the area. Due to the robustness of analyses using dnaK for discriminating between FD-C and FD-D strains and the high variability of malG sequences, these are efficient markers to study FDp populations and epidemiology at a small geographical scale.IMPORTANCE Flavescence dorée, a threatening disease of grapevine caused by FD phytoplasma (FDp), is distributed within the most important wine-producing areas of Europe and has severe effects on both vineyard productivity and landscape management. FDp is a quarantine pest in Europe, and despite the efforts to contain the pathogen, the disease is still spreading. In this work, new genetic markers for the fine genetic characterization of FDp at local scale are presented. Our findings improve the knowledge of FDp epidemiological cycle and offer the possibility of tracking the route of the FDp infection. In particular, due to its high genetic variability, one of the newly developed markers could be sufficient to track the origin of new infection foci, either from the wild areas or from nurseries.
Asunto(s)
Granjas , Variación Genética , Hemípteros/microbiología , Phytoplasma/genética , Enfermedades de las Plantas/microbiología , Animales , Clematis/microbiología , Italia , Phytoplasma/fisiología , Vitis/microbiologíaRESUMEN
Lavender decline compromises French lavender production, and preliminary data have suggested the involvement of "Candidatus Phytoplasma solani" in the etiology of the disease. In order to evaluate the epidemiological role of "Ca Phytoplasma solani," a 3-year survey was conducted in southeastern France. "Ca Phytoplasma solani" was detected in 19 to 56% of the declining plants, depending on seasons and cultivars, and its prevalence was correlated with symptom severity. Autumn was more favorable than spring for phytoplasma detection, and "Ca Phytoplasma solani" incidence was higher in Lavandula angustifolia than in Lavandula intermedia hybrids. Detection of the phytoplasma fluctuated over months, supporting the chronicity of infection. Three "Ca Phytoplasma solani" secY genotypes, S17, S16, and S14, were the most prevalent in lavender fields and were also detected in nurseries, whereas strains detected in surrounding bindweed and wild carrots were mostly of the S1 and S4 genotypes. This suggests that lavender is the main pathogen reservoir of the epidemic. Adults and nymphs of the planthopper vector Hyalesthes obsoletus were commonly captured in lavender fields and were shown to harbor mainly the prevalent phytoplasma genotypes detected in lavenders. The "Ca Phytoplasma solani" genotype S17 was transmitted to Catharanthus roseus periwinkle by naturally infected H. obsoletus Finally, the inventory of the bacterial community of declining lavenders that tested negative for "Ca Phytoplasma solani" by 16S rRNA deep sequencing ruled out the involvement of other phloem-limited bacterial pathogens.IMPORTANCE The etiology and main pathways for the spread of lavender decline, an infectious disease affecting French lavender production since the 1960s, have remained unclear, hampering the development of efficient control strategies. An extensive survey of lavender fields led to the conclusion that "Candidatus Phytoplasma solani" was chronically infecting declining lavenders and was associated with large infectious populations of Hyalesthes obsoletus planthoppers living on the crop itself. Lavender appeared to be the main reservoir host for lavender-specific phytoplasma strains, an unusual feature for this phytoplasma, which usually propagates from reservoir weeds to various economically important crops. These results point out the necessity to protect young lavender fields from the initial phytoplasma inoculum coming from surrounding lavender fields or from infected nurseries and to promote agricultural practices that reduce the development of H. obsoletus vector populations.
Asunto(s)
Lavandula/microbiología , Phytoplasma/clasificación , Phytoplasma/patogenicidad , Enfermedades de las Plantas/microbiología , Animales , Francia , Genotipo , Técnicas de Genotipaje , Hemípteros/microbiología , Epidemiología Molecular , Filogenia , Phytoplasma/genética , Phytoplasma/aislamiento & purificación , Prevalencia , ARN Ribosómico 16S/genética , Vinca/microbiologíaRESUMEN
Flavescence dorée (FD) phytoplasma from 16SrV-C and -D subgroups cause severe damage to grapevines throughout Europe. This phytoplasma is transmitted from grapevine to grapevine by the sap-sucking leafhopper Scaphoideus titanus. European black alder and clematis serve as perennial plant reservoirs for 16SrV-C phytoplasma strains, and their host range has recently been extended to hazelnuts. In Slovenia, hazelnut orchards are declining due to 16SrV phytoplasma infections, where large populations of the non-autochthonous leafhopper Orientus ishidae have been observed. To better characterise the phytoplasma-induced decline of hazelnut and possible transmission fluxes between these orchards and grapevine, genetic diversity of 16SrV phytoplasmas in grapevine, hazelnut and leafhoppers was monitored from 2017 to 2022. The nucleotide sequence analysis was based on the map gene. The most prevalent map genotype in grapevine in all wine-growing regions of Slovenia was M54, which accounted for 84% of the 176 grapevines tested. Besides M54, other epidemic genotypes with lower frequency were M38 (6%), M51 (3%), M50 (2%) and M122 (1%). M38, M50 and M122 were also detected in infected cultivated hazelnuts and in specimens of O. ishidae leafhopper caught in declining hazelnut orchards. It suggests that this polyphagous vector could be responsible for phytoplasma infection in hazelnut orchards and possibly for some phytoplasma exchanges between hazelnuts and grapevine. We hereby describe new genotypes: M158 in grapevine as well as four never reported genotypes M159 to M162 in hazelnut. Of these four genotypes in hazelnut, one (M160) was also detected in O. ishidae. Analysis of additional genes of the new genotypes allowed us to assign them to the VmpA-III cluster, which corresponds to the 16SrV-C strains previously shown to be compatible with S. titanus transmission.
RESUMEN
[This corrects the article DOI: 10.3389/fpls.2023.1217425.].
RESUMEN
In addition to the grapevine flavescence dorée phytoplasmas, other members of taxonomic group 16SrV phytoplasmas infect grapevines, alders and species of the genera Clematis and Rubus in Europe. In order to investigate which phytoplasmas constitute discrete, species-level taxa, several strains were analysed by comparing their 16S rRNA gene sequences and a set of five housekeeping genes. Whereas 16S rRNA gene sequence similarity values were >97.5â%, the proposed threshold to distinguish two 'Candidatus Phytoplasma' taxa, phylogenetic analysis of the combined sequences of the tuf, rplV-rpsC, rplF-rplR, map and uvrB-degV genetic loci showed that two discrete phylogenetic clusters could be clearly distinguished. The first cluster grouped flavescence dorée (FD) phytoplasmas, alder yellows (AldY) phytoplasmas, Clematis (CL) phytoplasmas and the Palatinate grapevine yellows (PGY) phytoplasmas. The second cluster comprised Rubus stunt (RS) phytoplasmas. In addition to the specificity of the insect vector, the Rubus stunt phytoplasma contained specific sequences in the 16S rRNA gene. Hence, the Rubus stunt phytoplasma 16S rRNA gene was sufficiently differentiated to represent a novel putative taxon: 'Candidatus Phytoplasma rubi'.
Asunto(s)
Variación Genética , Phytoplasma/clasificación , Phytoplasma/genética , Alnus/microbiología , Proteínas Bacterianas/genética , Clematis/microbiología , Análisis por Conglomerados , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Europa (Continente) , Datos de Secuencia Molecular , Filogenia , ARN Ribosómico 16S/genética , Rosaceae/microbiología , Análisis de Secuencia de ADN , Vitis/microbiologíaRESUMEN
The assembly of 20,000 sequencing reads obtained from shotgun and chromosome-specific libraries of the Spiroplasma citri genome yielded 77 chromosomal contigs totaling 1,674 kbp (92%) of the 1,820-kbp chromosome. The largest chromosomal contigs were positioned on the physical and genetic maps constructed from pulsed-field gel electrophoresis and Southern blot hybridizations. Thirty-eight contigs were annotated, resulting in 1,908 predicted coding sequences (CDS) representing an overall coding density of only 74%. Cellular processes, cell metabolism, and structural-element CDS account for 29% of the coding capacity, CDS of external origin such as viruses and mobile elements account for 24% of the coding capacity, and CDS of unknown function account for 47% of the coding capacity. Among these, 21% of the CDS group into 63 paralog families. The organization of these paralogs into conserved blocks suggests that they represent potential mobile units. Phage-related sequences were particularly abundant and include plectrovirus SpV1 and SVGII3 and lambda-like SpV2 sequences. Sixty-nine copies of transposases belonging to four insertion sequence (IS) families (IS30, IS481, IS3, and ISNCY) were detected. Similarity analyses showed that 21% of chromosomal CDS were truncated compared to their bacterial orthologs. Transmembrane domains, including signal peptides, were predicted for 599 CDS, of which 58 were putative lipoproteins. S. citri has a Sec-dependent protein export pathway. Eighty-four CDS were assigned to transport, such as phosphoenolpyruvate phosphotransferase systems (PTS), the ATP binding cassette (ABC), and other transporters. Besides glycolytic and ATP synthesis pathways, it is noteworthy that S. citri possesses a nearly complete pathway for the biosynthesis of a terpenoid.
Asunto(s)
Bacteriófagos/genética , Cromosomas Bacterianos/genética , Cromosomas Bacterianos/virología , Evolución Molecular , Recombinación Genética , Spiroplasma citri/genética , Spiroplasma citri/virología , Proteínas Bacterianas/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Genoma Bacteriano , Secuencias Repetitivas Esparcidas , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Análisis de Secuencia de ADN , Eliminación de Secuencia , Transposasas/genéticaRESUMEN
Studies of phytoplasma-insect vector interactions and epidemiological surveys of plant yellows associated with the stolbur phytoplasma (StolP) require the identification of relevant candidate genes and typing markers. A recent StolP genome survey identified a partial coding sequence, SR01H10, having no homologue in the "Candidatus Phytoplasma asteris" genome but sharing low similarity with a variable surface protein of animal mycoplasmas. The complete coding sequence and its genetic environment have been fully characterized by chromosome walking. The vmp1 gene encodes a protein of 557 amino acids predicted to possess a putative signal peptide and a potential C-terminal transmembrane domain. The mature 57.8-kDa VMP1 protein is likely to be anchored in the phytoplasma membrane with a large N-terminal hydrophilic part exposed to the phytoplasma cell surface. Southern blotting experiments detected multiple sequences homologous to vmp1 in the genomes of nine StolP isolates. vmp1 is variable in size, and eight different vmp1 RsaI restriction fragment length polymorphism types could be distinguished among 12 StolP isolates. Comparison of vmp1 sequences revealed that insertions in largest forms of the gene encode an additional copy of a repeated domain of 81 amino acids, while variations in 11-bp repeats led to gene disruption in two StolP isolates. vmp1 appeared to be much more variable than three housekeeping genes involved in protein translation, maturation, and secretion and may therefore be involved in phytoplasma-host interactions.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de la Membrana/genética , Phytoplasma/genética , Polimorfismo Genético , Proteínas Bacterianas/química , ADN Bacteriano/química , ADN Bacteriano/genética , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Filogenia , Señales de Clasificación de Proteína , Análisis de Secuencia de ADN , Homología de SecuenciaRESUMEN
Flavescence dorée (FD) is a quarantine disease of grapevine, involving interactions between the plants, leafhopper vectors, and FD phytoplasma. Characterizing the susceptibility of vine varieties could limit disease propagation. After extensive surveys in vineyards, we showed that Cabernet Sauvignon (CS) is highly susceptible, with a high proportion of symptomatic branches and phytoplasma titers, in contrast to Merlot (M). Localized insect transmissions and grafting showed that phytoplasma circulate in the whole plant in the CS cultivar, but in M they are restricted to the transmission point. Insect-mediated transmission under high confinement mimicking natural conditions confirmed these phenotypes and allowed the classification of 28 Vitis accessions into three distinct categories, according to the percentage of infected plants and their phytoplasma titers. Reduced symptoms, low phytoplasma titers, and low percentages of infected plants were found to be associated in the Vitis vinifera cultivars tested. Interestingly, the low susceptibility of M was observed for one of its parents, i.e., Magdeleine Noire des Charentes. Rootstocks and their Vitis parents, although having high percentages of infected plants and intermediate to high phytoplasma titers, shared a symptomless response. This is troubling, because rootstocks can constitute a silent reservoir of contamination in mother plants or when they grow wild nearby vineyards. Altogether, data suggest distribution of genetic traits within the Vitis genus involved in insect-mediated phytoplasma transmission, multiplication, circulation, and symptom development.
RESUMEN
ABSTRACT Marginal chlorosis has affected strawberry production in France for about 15 years. A phloem-restricted uncultured bacterium, "Candidatus Phlomobacter fragariae," is associated with the disease. A large-scale survey for marginal chlorosis in French strawberry production fields and nurseries by polymerase chain reaction amplification of "Ca. P. fragariae" 16S rDNA revealed that symptoms of marginal chlorosis were not always induced by "Ca. P. fragariae" and that the stolbur phytoplasma could induce identical symptoms. "Ca. P. fragariae" was found to be predominant in strawberry production fields, whereas the stolbur phytoplasma was predominantly detected in nurseries. Two transmission periods of the disease, one in spring and the other from late summer to early fall, were evident. Cixius wagneri planthoppers captured on infected strawberry plants were demonstrated to be efficient vectors of "Ca. P. fragariae." The involvement in natural disease spread of the whitefly Trialeurodes vaporariorum, previously shown to acquire and multiply "Ca. P. fragariae" under greenhouse conditions, remains uncertain.
RESUMEN
Tuf and secY genotyping techniques have been developed to distinguish phytoplasma strains. Tuf polymerase chain reaction sequence analyses are available for phytoplasma taxonomic groups 16SrI, 16SrV, 16SrXII-A, and XII-B. In addition to their use to confirm the taxonomic status of phytoplasma strains, they allow the spread of phytoplasma strains in host plants and insect vectors to be traced. SecY is more variable than tuf and is therefore more discriminatory than tuf, but secY and tuf phylogenies show congruence.
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Proteínas Bacterianas/genética , Genotipo , Factor Tu de Elongación Peptídica/genética , Phytoplasma/genética , Reacción en Cadena de la Polimerasa , Filogenia , Phytoplasma/clasificación , Polimorfismo de Longitud del Fragmento de Restricción , ARN Ribosómico 16S/genéticaRESUMEN
Vineyards of southern France and northern Italy are affected by the flavescence dorée (FD) phytoplasma, a quarantine pathogen transmitted by the leafhopper of Nearctic origin Scaphoideus titanus. To better trace propagation of FD strains and identify possible passage between the vineyard and wild plant compartments, molecular typing of phytoplasma strains was applied. The sequences of the two genetic loci map and uvrB-degV, along with the sequence of the secY gene, were determined among a collection of FD and FD-related phytoplasmas infecting grapevine, alder, elm, blackberry, and Spanish broom in Europe. Sequence comparisons and phylogenetic analyses consistently indicated the existence of three FD phytoplasma strain clusters. Strain cluster FD1 (comprising isolate FD70) displayed low variability and represented 17% of the disease cases in the French vineyard, with a higher incidence of the cases in southwestern France. Strain cluster FD2 (comprising isolates FD92 and FD-D) displayed no variability and was detected both in France (83% of the cases) and in Italy, whereas the more-variable strain cluster FD3 (comprising isolate FD-C) was detected only in Italy. The clonal property of FD2 and its wide distribution are consistent with diffusion through propagation of infected-plant material. German Palatinate grapevine yellows phytoplasmas (PGY) appeared variable and were often related to some of the alder phytoplasmas (AldY) detected in Italy and France. Finally, phylogenetic analyses concluded that FD, PGY, and AldY were members of the same phylogenetic subclade, which may have originated in Europe.
Asunto(s)
Alnus/microbiología , Genes Bacterianos/genética , Filogenia , Phytoplasma/genética , Enfermedades de las Plantas/microbiología , Vitis/microbiología , Secuencia de Bases , Cartilla de ADN/genética , Europa (Continente) , Componentes del Gen , Datos de Secuencia Molecular , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Secuencia de ADNRESUMEN
Almonds (Prunus amygdalus) represent an important crop in most Mediterranean countries. A new and devastating disease of almond trees in Lebanon was recently reported, characterized by the development of severe witches'-brooms on which no flowers or fruits developed, and leading to tree death within a few years. A phytoplasma was detected in diseased trees by PCR amplification of rRNA operon sequences, and RFLP patterns of amplified DNA indicated that the phytoplasma belonged to the pigeon pea witches'-broom (PPWB) group. In the present work, the presence of a phytoplasma in symptomatic plants was confirmed by electron microscopy; this phytoplasma was graft-transmissible to almond, plum and peach seedlings. The phytoplasma was characterized by sequence analysis of rRNA genes and was shown to be different from the phytoplasmas previously described in the PPWB group. A 16S rDNA phylogenetic tree identified the almond tree phytoplasma as a member of a distinct subclade of the class Mollicutes. Oligonucleotides have been defined for specific detection of the new phytoplasma. The almond phytoplasma from Lebanon was shown to be identical to a phytoplasma that induces a disease called 'almond brooming' in Iran, but different from another PPWB-group phytoplasma that infects herbaceous annual plants in Lebanon. Based on its unique properties, the name 'Candidatus Phytoplasma phoenicium' is proposed for the phytoplasma associated with almond witches'-broom in Lebanon and Iran.