When a Palearctic bacterium meets a Nearctic insect vector: Genetic and ecological insights into the emergence of the grapevine Flavescence dorée epidemics in Europe.

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.

Lavender Decline in France Is Associated with Chronic Infection by Lavender-Specific Strains of "Candidatus Phytoplasma solani".

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.

Variable Membrane Protein A of Flavescence Dorée Phytoplasma Binds the Midgut Perimicrovillar Membrane of Euscelidius variegatus and Promotes Adhesion to Its Epithelial Cells.

Phytoplasmas are uncultivated plant pathogens and cell wall-less bacteria and are transmitted from plant to plant by hemipteran insects. The phytoplasma's circulative propagative cycle in insects requires the crossing of the midgut and salivary glands, and primary adhesion to cells is an initial step toward the invasion process. The flavescence dorée (FD) phytoplasma possesses a set of variable membrane proteins (Vmps) exposed on its surface, and this pathogen is suspected to interact with insect cells. The results showed that VmpA is expressed by the flavescence dorée phytoplasma present in the midgut and salivary glands. Phytoplasmas cannot be cultivated at present, and no mutant can be produced to investigate the putative role of Vmps in the adhesion of phytoplasma to insect cells. To overcome this difficulty, we engineered the Spiroplasma citri mutant G/6, which lacks the ScARP adhesins, for VmpA expression and used VmpA-coated fluorescent beads to determine if VmpA acts as an adhesin in ex vivo adhesion assays and in vivo ingestion assays. VmpA specifically interacted with Euscelidiusvariegatus insect cells in culture and promoted the retention of VmpA-coated beads to the midgut of E. variegatus In this latest case, VmpA-coated fluorescent beads were localized and embedded in the perimicrovillar membrane of the insect midgut. Thus, VmpA functions as an adhesin that could be essential in the colonization of the insect by the FD phytoplasmas.IMPORTANCE Phytoplasmas infect a wide variety of plants, ranging from wild plants to cultivated species, and are transmitted by different leafhoppers, planthoppers, and psyllids. The specificity of the phytoplasma-insect vector interaction has a major impact on the phytoplasma plant host range. As entry into insect cells is an obligate process for phytoplasma transmission, the bacterial adhesion to insect cells is a key step. Thus, studying surface-exposed proteins of phytoplasma will help to identify the adhesins implicated in the specific recognition of insect vectors. In this study, it is shown that the membrane protein VmpA of the flavescence dorée (FD) phytoplasma acts as an adhesin that is able to interact with cells of Euscelidiusvariegatus, the experimental vector of the FD phytoplasma.

Adaptation of lettuce mosaic virus to Catharanthus roseus involves mutations in the central domain of the VPg.

An isolate of Lettuce mosaic virus (LMV, a Potyvirus) infecting Madagascar periwinckle (Catharanthus roseus) was identified and characterized by Illumina deep sequencing. LMV-Cr has no close affinities to previously sequenced LMV isolates and represents a novel, divergent LMV clade. Inoculation experiments with other representative LMV isolates showed that they are unable to infect C. roseus, which was not known to be a host for LMV. However, three C. roseus variants of one of these isolates, LMV-AF199, could be selected and partially or completely sequenced. These variants are characterized by the accumulation of mutations affecting the C-terminal part of the cylindrical inclusion (CI) helicase and the central part of the VPg. In particular, a serine to proline mutation at amino acid 143 of the VPg was observed in all three independently selected variants and is also present in the LMV-Cr isolate, making it a prime candidate as a host-range determinant. Other mutations at VPg positions 65 and 144 could also contribute to the ability to infect C. roseus. Inoculation experiments involving a recombinant LMV expressing a permissive lettuce eukaryotic translation initiation factor 4E (eIF4E) suggest that eIF4E does not contribute to the interaction of most LMV isolates with C. roseus.

Multilocus sequence analysis reveals the genetic diversity of European fruit tree phytoplasmas and supports the existence of inter-species recombination.

The genetic diversity of three temperate fruit tree phytoplasmas 'Candidatus Phytoplasma prunorum', 'Ca. P. mali' and 'Ca. P. pyri' has been established by multilocus sequence analysis. Among the four genetic loci used, the genes imp and aceF distinguished 30 and 24 genotypes, respectively, and showed the highest variability. Percentage of substitution for imp ranged from 50 to 68 % according to species. Percentage of substitution varied between 9 and 12 % for aceF, whereas it was between 5 and 6 % for pnp and secY. In the case of 'Ca P. prunorum' the three most prevalent aceF genotypes were detected in both plants and insect vectors, confirming that the prevalent isolates are propagated by insects. The four isolates known to be hypo-virulent had the same aceF sequence, indicating a possible monophyletic origin. Haplotype network reconstructed by eBURST revealed that among the 34 haplotypes of 'Ca. P. prunorum', the four hypo-virulent isolates also grouped together in the same clade. Genotyping of some Spanish and Azerbaijanese 'Ca. P. pyri' isolates showed that they shared some alleles with 'Ca. P. prunorum', supporting for the first time to our knowledge, the existence of inter-species recombination between these two species.

Infection of the Circulifer haematoceps cell line Ciha-1 by Spiroplasma citri: the non-insect-transmissible strain 44 is impaired in invasion.

Successful transmission of Spiroplasma citri by its leafhopper vector requires a specific interaction between the spiroplasma surface and the insect cells. With the aim of studying these interactions at the cellular and molecular levels, a cell line, named Ciha-1, was established using embryonic tissues from the eggs of the S. citri natural vector Circulifer haematoceps. This is the first report, to our knowledge, of a cell line for this leafhopper species and of its successful infection by the insect-transmissible strain S. citri GII3. Adherence of the spiroplasmas to the cultured Ciha-1 cells was studied by c.f.u. counts and by electron microscopy. Entry of the spiroplasmas into the insect cells was analysed quantitatively by gentamicin protection assays and qualitatively by double immunofluorescence microscopy. Spiroplasmas were detected within the cell cytoplasm as early as 1 h after inoculation and survived at least 2 days inside the cells. Comparing the insect-transmissible GII3 and non-insect-transmissible 44 strains revealed that adherence to and entry into Ciha-1 cells of S. citri 44 were significantly less efficient than those of S. citri GII3.

Sequences essential for transmission of Spiroplasma citri by its leafhopper vector, Circulifer haematoceps, revealed by plasmid curing and replacement based on incompatibility.

Spiroplasma citri GII3 contains highly related low-copy-number plasmids pSci1 to -6. Despite the strong similarities between their replication regions, these plasmids coexist in the spiroplasma cells, indicating that they are mutually compatible. The pSci1 to -6 plasmids encode the membrane proteins known as S. citri adhesion-related proteins (ScARPs) (pSci1 to -5) and the hydrophilic protein P32 (pSci6), which had been tentatively associated with insect transmission, as they were not detected in non-insect-transmissible strains. With the aim of further investigating the role of plasmid-encoded determinants in insect transmission, we have constructed S. citri mutant strains that differ in their plasmid contents by developing a plasmid curing/replacement strategy based on the incompatibility of plasmids having identical replication regions. Experimental transmission of these S. citri plasmid mutants through injection into the leafhopper vector Circulifer haematoceps revealed that pSci6, more precisely, the pSci6_06 coding sequence, encoding a protein of unknown function, was essential for transmission. In contrast, ScARPs and P32 were dispensable for both acquisition and transmission of the spiroplasmas by the leafhopper vector, even though S. citri mutants lacking pSci1 to -5 (encoding ScARPs) were acquired and transmitted at lower efficiencies than the wild-type strain GII3.

"Candidatus Phlomobacter fragariae" Is the Prevalent Agent of Marginal Chlorosis of Strawberry in French Production Fields and Is Transmitted by the Planthopper Cixius wagneri (China).

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.

Tuf and secY PCR amplification and genotyping of phytoplasmas.

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.

The stolbur phytoplasma antigenic membrane protein gene stamp is submitted to diversifying positive selection.

Surface proteins play an important role in phytoplasma life cycle. The antigenic membrane protein (AMP) of 'Candidatus Phytoplasma asteris' has been shown to interact with the insect microfilaments. Due to the synteny of the groL-amp-nadE genetic locus between phytoplasma genomes, the gene stamp that encodes the antigenic membrane protein of stolbur phytoplasma has been cloned and characterized. It encodes a 157 amino acid-long protein with a predicted signal peptide and a C-terminal hydrophobic alpha-helix. STAMP was 26-40% identical to AMP of 'Candidatus Phytoplasma asteris' strains and 40% identical to AMP of 'Ca. P. japonicum'. The expression of STAMP in Escherichia. coli produced a 16 kDa peptide recognized by an anti-stolbur monoclonal antibody. Stamp was more variable than the house-keeping gene secY and the ratio between non-synonymous over synonymous mutations (dN/dS ) was 2.78 for stamp as compared to 0.64 for secY. This indicates that stamp is submitted to a positive diversifying selection pressure.

Plasmid pSci6 from Spiroplasma citri GII-3 confers insect transmissibility to the non-transmissible strain S. citri 44.

The insect-transmissible strain GII-3 of Spiroplasma citri contains plasmids pSci1-6, five of which (pSci1-5) encode adhesin-like proteins and one (pSci6) encodes protein P32, which has been associated with insect transmissibility. In contrast, S. citri strains ASP-1 and 44, which cannot be transmitted via injection into the leafhopper vector Circulifer haematoceps, lack these proteins and also do not carry plasmids pSci1-6. To further study the apparent relationship between the presence of plasmids and insect transmissibility, plasmids from S. citri GII-3 were introduced into the insect-non-transmissible S. citri strain 44 by electrotransformation using the tetM gene as the selection marker. Tetracycline-resistant transformants were shown to carry one, two or three distinct plasmids. Plasmids pSci1-6 were all detected in the transformants, pSci1 being the most frequently found, alone or together with other plasmids. Selected S. citri 44 transformants having distinct plasmid contents were submitted, separately or in combination, to experimental transmission to periwinkle (Catharanthus roseus) plants via injection into the leafhopper vector. The occurrence of symptomatic plants indicated that, in contrast to S. citri 44, spiroplasmal transformants were transmitted to the host plant, in which they multiplied. Spiroplasma cultures isolated from these infected plants all contained pSci6, leading to the conclusion that, under the experimental conditions used, transformation by pSci6 conferred insect transmissibility to S. citri strain 44. This is believed to be the first report of a phenotypic change associated with transformation of S. citri by natural plasmids.

'Candidatus Liberibacter americanus', associated with citrus huanglongbing (greening disease) in São Paulo State, Brazil.

Symptoms of huanglongbing (HLB) were reported in São Paulo State (SPS), Brazil, in March 2004. In Asia, HLB is caused by 'Candidatus Liberibacter asiaticus' and in Africa by 'Candidatus Liberibacter africanus'. Detection of the liberibacters is based on PCR amplification of their 16S rRNA gene with specific primers. Leaves with blotchy mottle symptoms characteristic of HLB were sampled in several farms of SPS and tested for the presence of liberibacters. 'Ca. L. asiaticus' was detected in a small number of samples but most samples gave negative PCR results. Therefore, a new HLB pathogen was suspected. Evidence for an SPS-HLB bacterium in symptomatic leaves was obtained by PCR amplification with universal primers for prokaryotic 16S rRNA gene sequences. The amplified 16S rRNA gene was cloned and sequenced. Sequence analysis and phylogeny studies showed that the 16S rRNA gene possessed the oligonucleotide signatures and the secondary loop structure characteristic of the alpha-Proteobacteria, including the liberibacters. The 16S rRNA gene sequence phylogenetic tree showed that the SPS-HLB bacterium clustered within the alpha-Proteobacteria, the liberibacters being its closest relatives. For these reasons, the SPS-HLB bacterium is considered a member of the genus 'Ca. Liberibacter'. However, while the 16S rRNA gene sequences of 'Ca. L. asiaticus' and 'Ca. L. africanus' had 98.4% similarity, the 16S rRNA gene sequence of the SPS-HLB liberibacter had only 96.0% similarity with the 16S rRNA gene sequences of 'Ca. L. asiaticus' or 'Ca. L. africanus'. This lower similarity was reflected in the phylogenetic tree, where the SPS-HLB liberibacter did not cluster within the 'Ca. L asiaticus'/'Ca. L. africanus group', but as a separate branch. Within the genus 'Candidatus Liberibacter' and for a given species, the 16S/23S intergenic region does not vary greatly. The intergenic regions of three strains of 'Ca. L. asiaticus', from India, the People's Republic of China and Japan, were found to have identical or almost identical sequences. In contrast, the intergenic regions of the SPS-HLB liberibacter, 'Ca. L. asiaticus' and 'Ca. L. africanus' had quite different sequences, with similarity between 66.0 and 79.5%. These results confirm that the SPS-HLB liberibacter is a novel species for which the name 'Candidatus Liberibacter americanus' is proposed. Like the African and the Asian liberibacters, the 'American' liberibacter is restricted to the sieve tubes of the citrus host. The liberibacter could also be detected by PCR amplification of the 16S rRNA gene in Diaphorina citri, the psyllid vector of 'Ca. L. asiaticus', suggesting that this psyllid is also a vector of 'Ca. L. americanus' in SPS. 'Ca. L. americanus' was detected in 216 of 218 symptomatic leaf samples from 47 farms in 35 municipalities, while 'Ca. L. asiaticus' was detected in only 4 of the 218 samples, indicating that 'Ca. L. americanus' is the major cause of HLB in SPS.

Spiralin is not essential for helicity, motility, or pathogenicity but is required for efficient transmission of Spiroplasma citri by its leafhopper vector Circulifer haematoceps.

Spiralin is the most abundant protein at the surface of the plant pathogenic mollicute Spiroplasma citri and hence might play a role in the interactions of the spiroplasma with its host plant and/or its insect vector. To study spiralin function, mutants were produced by inactivating the spiralin gene through homologous recombination. A spiralin-green fluorescent protein (GFP) translational fusion was engineered and introduced into S. citri by using an oriC-based targeting vector. According to the strategy used, integration of the plasmid by a single-crossover recombination at the spiralin gene resulted in the expression of the spiralin-GFP fusion protein. Two distinct mutants were isolated. Western and colony immunoblot analyses showed that one mutant (GII3-9a5) did produce the spiralin-GFP fusion protein, which was found not to fluoresce, whereas the other (GII3-9a2) produced neither the fusion protein nor the wild-type spiralin. Both mutants displayed helical morphology and motility, similarly to the wild-type strain GII-3. Genomic DNA analyses revealed that GII3-9a5 was unstable and that GII3-9a2 was probably derived from GII3-9a5 by a double-crossover recombination between plasmid sequences integrated into the GII3-9a5 chromosome and free plasmid. When injected into the leafhopper vector Circulifer haematoceps, the spiralinless mutant GII3-9a2 multiplied to high titers in the insects (1.1 x 10(6) to 2.8 x 10(6) CFU/insect) but was transmitted to the host plant 100 times less efficiently than the wild-type strain. As a result, not all plants were infected, and symptom production in these plants was delayed for 2 to 4 weeks compared to that in the wild-type strain. In the infected plants however, the mutant multiplied to high titers (1.2 x 10(6) to 1.4 x 10(7) CFU/g of midribs) and produced the typical symptoms of the disease. These results indicate that spiralin is not essential for pathogenicity but is required for efficient transmission of S. citri by its insect vector.

'Candidatus phytoplasma phoenicium' sp. nov., a novel phytoplasma associated with an emerging lethal disease of almond trees in Lebanon and Iran.

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.