Vector transmission of a plant-pathogenic bacterium in the Arsenophonus clade sharing ecological traits with facultative insect endosymbionts.

The planthopper Pentastiridius leporinus (Hemiptera: Cixiidae) is the major vector of a nonculturable plant-pathogenic gamma-3 proteobacterium associated with a disease of sugar beet called syndrome "basses richesses" (SBR). The bacterium, here called SBR bacterium, belongs to the Arsenophonous clade, which includes mostly insect-associated facultative symbionts. Assays using field-collected planthopper nymphs and adults were carried out to investigate the interaction of SBR bacterium with the insect vector and its transmission to sugar beet. Field-collected planthoppers showed a percentage of infection that averaged from 57% for early instar nymphs to near 100% for late instar nymphs and emerging adults. SBR bacterium was persistently transmitted by emerging adults. Root-feeding nymphs were able to inoculate SBR bacterium to sugar beet. The bacterium was transmitted vertically from infected parental females to their respective offspring with an average frequency of 30%. Real-time polymerase chain reaction assays on dissected planthopper internal organs revealed a high concentration of the bacterium within male and female reproductive organs and within female salivary glands. SBR-like bacteria were observed through transmission electron microscopy in the cytoplasm of different insect organs including ovaries, salivary glands, and guts with no evidence for cytological disorders. SBR bacterium seems to share common ecological traits of insect-transmitted plant pathogens and facultative insect endosymbionts suggesting it may have evolved primarily as an insect-associated bacterium.

Characterization and evolution of two bacteriome-inhabiting symbionts in cixiid planthoppers (Hemiptera: Fulgoromorpha: Pentastirini).

Like other plant sap-sucking insects, planthoppers within the family Cixiidae (Insecta: Hemiptera: Fulgoromorpha) host a diversified microbiota. We report the identification and first molecular characterization of symbiotic bacteria in cixiid planthoppers (tribe: Pentastirini). Using universal eubacterial primers we first screened the eubacterial 16S rRNA sequences in Pentastiridius leporinus (Linnaeus) with PCR amplification, cloning, and restriction fragment analysis. We identified three main 16S rRNA sequences that corresponded to a Wolbachia bacterium, a plant pathogenic bacterium, and a novel gammaproteobacterial symbiont. A fourth bacterial species affiliated with 'Candidatus Sulcia muelleri' was detected in PCR assays using primers specific for the Bacteroidetes. Within females of two selected cixiid planthoppers, P. leporinus and Oliarus filicicola, fluorescence In situ hybridization analysis and transmission electron microscopy observations showed that 'Ca. Sulcia muelleri' and the novel gammaproteobacterial symbiont were housed in separate bacteriomes. Phylogenetic analysis revealed that both of these symbionts occurred in at least four insect genera within the tribe Pentastirini. 'Candidatus Purcelliella pentastirinorum' was proposed as the novel gammaproteobacterial symbiont.

A chromosome map of the Flavescence doree phytoplasma.

The Flavescence dorée phytoplasma (FD-P), a non-cultivable, plant-pathogenic bacterium of the class Mollicutes, is the causal agent of a quarantine disease affecting vineyards of southern Europe, mainly in southern France and northern Italy. To investigate FD-P diversity and phytoplasma genetic determinants governing the FD-P life cycle, a genome project has been initiated. A physical map of the chromosome of FD-P strain FD92, purified from infected broad beans, was constructed by performing restriction digests of the chromosome and resolving the fragments by PFGE. Single and double digestions of the chromosome with the enzymes SalI, BssHII, MluI and EagI were performed and used to map 13 restriction sites on the FD-P chromosome. The size of the chromosome was calculated to be 671 kbp. Southern blot analyses using cloned phytoplasma probes were carried out to assist in the arrangement of contiguous restriction fragments and to map eight genetic loci, including the two rRNA operons, the tuf, uvrB-degV and secY-map (FD9) genes, the FD2 marker and two orphan sequences (FDDH29 and FDSH05) isolated through subtractive suppression hybridization.

Insect Vectors (Hemiptera: Cixiidae) and Pathogens Associated with the Disease Syndrome "Basses Richesses" of Sugar Beet in France.

The syndrome "basses richesses" (SBR) is a disease of sugar beet in eastern France associated with two phloem-restricted, nonculturable plant pathogens: a stolbur phytoplasma and a γ-3 proteobacterium, here called SBR bacterium. Three planthopper (Hemiptera: Cixiidae) species were found to live near and within sugar beet fields in eastern France: Cixius wagneri, Hyalesthes obsoletus, and Pentastiridius leporinus. The role of these planthoppers in spreading the two pathogens to sugar beet was studied. Based on its abundance and high frequency of infection with the SBR bacterium, P. leporinus was considered to be the economic vector of SBR disease. C. wagneri, the primary vector of 'Candidatus Phlomobacter fragariae' to strawberry in western France, also was found to be infected by the SBR bacterium and to transmit the pathogen to sugar beet. Neither C. wagneri nor P. leporinus were infected by stolbur phytoplasma. Populations of H. obsoletus living on bindweed (Convolvulus arvensis) and nettle (Urtica dioica) collected near sugar beet fields did not carry the SBR bacterium, but were highly infected with two restriction fragment length polymorphism-differentiable stolbur phytoplasma isolates. In transmission assays, only the bindweed phytoplasma isolate was transmissible to and pathogenic on sugar beet. When compared with controls, the bindweed stolbur phytoplasma and SBR bacterium similarly reduced the biomass of sugar beet plants, but the phytoplasma caused greater reductions in taproot biomass and sugar content than the SBR bacterium.

Multilocus sequence typing confirms the close genetic interrelatedness of three distinct flavescence dorée phytoplasma strain clusters and group 16SrV phytoplasmas infecting grapevine and alder in Europe.

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.

Characterization of a gamma-3 Proteobacteria Responsible for the Syndrome "Basses Richesses" of Sugar Beet Transmitted by Pentastiridius sp. (Hemiptera, Cixiidae).

ABSTRACT The disease syndrome "basses richesses" (SBR) has affected sugar beet crops in Burgundy (France) since 1991. It mainly is associated with an uncultivable phloem-restricted bacterium-like organism (BLO) called SBR BLO. Transmission tests showed that field-collected Pentastiridius sp. (Hemiptera, Cixiidae) were able to transmit the SBR BLO to sugar beet. In the present work, sequences of a 1,507-bp 16S ribosomal (r)DNA fragment of SBR BLO were amplified from DNA extracts of SBR-affected field sugar beet plants, of field-collected Pentastiridius sp. plant-hoppers, and of Pentastiridiussp.-exposed sugar beet seedlings that expressed SBR symptoms. The sequences showed total identity, confirming the role of SBR BLO in the etiology of SBR and the vector role of Pentastiridius sp. Our surveys on SBR-affected sugar beet plants and Pentastiridius sp. planthoppers collected in different fields and different years suggest that a unique BLO was involved in SBR. Furthermore, comparison of 16S rDNA sequences permitted the identification of the SBR BLO as a new plant-pathogenic gamma-3 proteobacteria different from 'Candidatus Phlomobacter fragariae,' another BLO responsible for marginal chlorosis disease of strawberry in France. Phylogenetic analysis revealed a close relationship between the SBR bacterium and several bacteria described as endosymbionts of hemipteran insects.

Insect injection and artificial feeding bioassays to test the vector specificity of flavescence dorée phytoplasma.

ABSTRACT The specificity of vector transmission of Flavescence dorée phytoplasma (FDP) was tested by injecting FDP, extracted from laboratory-reared infective Euscelidius variegatus, into specimens of 15 other hemipteran insect species collected in European vineyards. Concentrations of viable phytoplasma extracts and latency in vectors were monitored by injection of healthy-reared E. variegatus leafhoppers. Based on these preliminary results, insects were injected by using phytoplasma extracts that ensured the highest rate of FDP acquisition and transmission by E. variegatus. Transmission into an artificial diet through a Parafilm membrane about 3 weeks after insect injection was attempted. FDP-injected insects that belonged to 15 hemipteran species were confined in cages and fed through the membrane for a 4- to 5-day inoculation access period. FDP DNA was detected by polymerase chain reaction (PCR) in the feeding buffer fed upon by Anoplotettix fuscovenosus, Aphrodes makarovi,E. variegatus, and Euscelis incisus. PCR amplification with specific primers detected FDP DNA in injected insects of all test insect species. Band intensity was positively correlated with the transmissibility of FDP. Transmission of FDP to plants by feeding was confirmed for Anoplotettix fuscovenosus, E. variegatus, and Euscelis incisus, but not for Aphrodes makarovi. Our results suggest that vector competency of FDP is restricted to specimens belonging to the family Cicadellidae, subfamily Deltocephalinae.

Effect of two strains of Flavescence dorée phytoplasma on the survival and fecundity of the experimental leafhopper vector Euscelidius variegatus Kirschbaum.

We have investigated the influence on longevity and fecundity of Flavescence dorée phytoplasma (FDP), the agent of a grapevine yellows disease, in the experimental vector Euscelidius variegatus Kirschbaum. Late instar nymphs were exposed to one or the other of two strains of FDP (FD92 and FD2000) by feeding on infected broad bean (Vicia faba L.) or on healthy broad bean or maize (Zea mays L.) for an acquisition access period of 13 days. Detection of FDP in individual insects was done with PCR assays and revealed that almost all exposed leafhoppers had acquired FDP, for both FD92 and FD2000 strains. FDP infection significantly reduced the life span of males and females (ANOVA of the quartiles of survival distribution and Weibull scale parameter). FDP-exposed females produced significantly fewer nymphs. The two FDP strains had similar effects on reduction of survival and fecundity of leafhoppers. There was no significant differences in longevity of E. variegatus males exposed to FD broad bean than held on healthy broad bean or maize, but female survival and fecundity were reduced when they fed on maize versus healthy broad bean.