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The planthopper Pentastiridius leporinus (L.) (Hemiptera: Cixiidae) has been identified as the main vector of 'Candidatus Arsenophonus phytopathogenicus', a plant pathogenic bacterium associated to a sugar beet disease in eastern France called syndrome 'basses richesses'. In a 2-yr survey (2006-07), we quantified the abundance of P. leporinus populations migrating into 29 sugar beet fields in eastern France. Sticky traps posted in these fields were monitored on a twice-weekly (2006) or weekly (2007) basis. Subsets of the captured planthoppers were tested for the presence of Ca. A. phytopathogenicus through polymerase chain reaction (PCR). Our results showed that planthoppers colonized sugar beet fields in June and July of each year, following temporal patterns of migration that were fitted to logistic functions. The number of planthoppers migrating into sugar beet fields greatly varied among the fields and the years surveyed, averaging from a few (2-10) to over 400 planthoppers per trap. Interestingly, the prevalence of planthoppers infected by Ca. A. phytopathogenicus increased nonlinearly with the abundance of planthoppers captured on the traps. The proportion of infection for Ca. A. phytopathogenicus ranged from ≍0.07-1 (total infection) in small (2-10 planthoppers per trap) and large (400 planthoppers per trap) populations, respectively. We hypothesize that the outbreaks of P. leporinus in sugar beet fields, and the consequent increased rates of planthoppers infected by the Ca. A. phytopathogenicus, are key factors leading to the emergence of the sugar beet disease in eastern France.

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Pentastiridius leporinus is an important vector of sugar beet pathogens in eastern France. An electron microscope survey on the insect-associated microflora revealed the occurrence of intranuclear prokaryotic cells in every internal organ analysed. These bacteria, which could also be found in the cytoplasm surrounding the nucleus, had a homogeneous coccoid (ca. 0.45 microm) or rod (0.45-1 microm) shape. The presence of three membrane layers was observed, the outermost forming a kind of vacuole containing generally a single microorganism. No cytopathological abnormalities were detected in the infected cells. To our knowledge, this is the first report of a hemipteran species infected by intranuclear bacteria. The possible identity of this microorganism is discussed.

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ABSTRACT Elicitins, small proteins secreted by Phytophthora and Pythium spp., display the ability to induce plant resistance toward pathogens. Ultrastructural investigations of cryptogein-treated tobacco plants evidenced host defense responses such as (i) formation of a calcium pectate gel in intercellular spaces of parenchymas, (ii) impregnation of pectin by phenolic compounds in intercellular spaces of phloem bundles, and (iii) accumulation of phloem proteins (P proteins) in the lumen of leaf sieve elements. These cytological modifications lead to the enhancement of physical barriers that prevent pathogen ingress and restrict host tissue colonization when cryptogein-treated tobacco plants were challenged with the pathogen Phytophthora parasitica. Wall appositions also were observed at most sites of penetration of hyphae. Moreover, growing hyphae exhibited severe morphological damages, suggesting a modified toxic environment. The same induction of P proteins in mature sieve tubes of tobacco leaves was obtained with oligandrin treatment, another elicitin. Cryptogein or oligandrin treatment prevented symptom expression in phytoplasma-infected tobacco plants in contrast with nontreated tobacco plants. Moreover, P protein plugs and occlusion of pore sites by callose were evidenced in sieve elements of treated plants. Both these phloem modifications might prevent the in planta movement of phloem-restricted microorganisms.

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ABSTRACT The syndrome "basses richesses" of sugar beet (SBR) was first observed in 1991 in Burgundy, France. A cixiid planthopper, Pentastiridius beieri, has been proved to be involved in the transmission to sugar beet of a stolbur phytoplasma, which could be detected in some affected plants. In 2000, periwinkle and sugar beet exposed to field-collected cixiids developed symptoms similar to SBR on sugar beet. Use of 4'-6-diamidino-2-phenylindole (DAPI) staining and transmission electron microscopy confirmed the presence of phytoplasma in some of the plants, which were also positive for this pathogen in a polymerase chain reaction (PCR) analysis. A phloem-restricted gram-negative bacteria was seen in all other plants with symptoms but PCR-negative for phytoplasma. Three primer pairs reported as diagnostic for phloem-limited bacteria were tested but only primers specific for 'Candidatus Phlomobacter fragariae' gave a positive signal, which related to the presence of DAPI-stained bacteria-like objects in diseased plants. Although phytoplasma and bacterium-like organisms were associated with the same macroscopic symptoms on sugar beet, histochemical analysis of phloem cells showed that phytoplasma were associated with cell necrosis and cell wall lignification, while bacteria were associated with these same abnormalities as well as deposit of phenolic compounds in the lumen of phloem cells.

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A polymerase chain reaction (PCR)-based method was developed for the detection of phytoplasma in insect feeding medium (sucrose). A correlation was established between the transmissibility of Flavescence dorée phytoplasma in the experimental leafhopper vector Euscelidius variegatus and its detection by PCR in the insect feeding medium. However, phytoplasma were detected in the insects' bodies 3 weeks before they began to transmit. Hence, PCR assays of the sucrose medium reflected phytoplasma vectoring ability probably by detecting it in the insect saliva, whereas detection of phytoplasma in the insect's body did not identify it as a vector. The assay was applied to two field-collected leafhoppers suspected of being phytoplasma vectors in Israel (Orosius albicinctus and Anaceratagallia laevis). The presence of phytoplasma in the body of specimens of the latter species was assayed by PCR in 1999. Phytoplasmas were detected in insects' bodies throughout the year, with no specific seasonal pattern. In the saliva, however, no phytoplasma could be detected in the autumn. This seasonal pattern supported the validity of the feeding-medium tests and their correlation to the insect's ability to transmit phytoplasma. Transmission assays indicated, to our knowledge for the first time, that O. albicinctus and A. laevis are vectors of phytoplasma in Israel. A simple PCR-based assay is thus provided, circumventing the need for tedious biological assays and enabling epidemiological studies of phytoplasma transmissibility on a large scale.

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Phytoplasmas are plant-pathogenic mollicutes restricted to phloem. They belong to several groups in a unique phylogenetic clade. Non-related phytoplasmas may infect the same plant species, often with similar symptoms. Hence methods are needed to specifically localize phytoplasmas and to study their multiplication and movement in their hosts. Conditions for post-embedding in situ hybridization (ISH) with transmission electron microscopy using oligodeoxynucleotides as probes for labelling of phytoplasmas in plant tissues have been searched. Sections of acrylic resin-embedded tissues of phytoplasma-infected periwinkle were submitted to ISH using digoxigenin or biotin-labelled oligoprobes (22 mers). These probes were the complementary sequences of primers used in group-specific polymerase chain reaction (PCR) amplification of 16S rDNA of stolbur and elm yellows phytoplasma, respectively. Together with preliminary digestion with pepsin, different in situ denaturation conditions and formamide concentrations were tested. The grids were incubated in the hybridization mixture at 37 degreesC overnight. Detection of hybridized material was performed with gold immunocytochemistry. Specificity of labelling was checked with appropriate controls. Stringency conditions could be found to ensure specific hybridization with such short probes. A specific labelling was obtained for stolbur phytoplasma on groups of mature as well as senescent phytoplasma cells. The results show that oligonucleotides may be used as probes for phytoplasma identification in post-embedding ISH with electron microscopy.

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Restriction fragment length polymorphism (RFLP) analyses were performed on polymerase chain reaction (PCR) amplimers of phytoplasmal DNA from eight samples obtained from Ulmus spp. (elms) affected by elm yellows (EY) in Italy and the United States, from Catharanthus roseus infected with strain EY1, and from five other plant species infected with phytoplasmas of the EY group sensu lato (group 16SrV). RFLP profiles obtained with restriction enzyme TaqI from ribosomal DNA amplified with primer pair P1/P7 differentiated elm-associated phytoplasmas from strains originally detected in Apocynum cannabinum, Prunus spp., Rubus fruticosus, Vitis vinifera, and Ziziphus jujuba. RFLP profiles obtained similarly with BfaI differentiated strains from A. cannabinum and V. vinifera from other phytoplasmas of group 16SrV. Elm-associated strains from within the United States had two RFLP patterns in ribosomal DNA based on presence or absence of an RsaI site in the 16S-23S spacer. Elm-associated phytoplasma strains from Italy were distinguished from those of American origin by RFLPs obtained with MseI in the same fragment of non-ribosomal DNA. Strain HD1, which was discovered in A. cannabinum associated with EY-diseased elms in New York State, was unique among the strains studied.

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Flavescence dorée (FD), a grapevine yellows disease, is caused by a mycoplasma-like organism (MLO). A colloidal gold indirect immunolabeling technique identified MLO in salivary glands of a vector leafhopper, Euscelidius variegatus. After aldehyde fixation, tissue samples were prepared by cryoultramicrotomy or embedding in acrylic resins. Double fixation with aldehydes and osmium retroxide, followed by embedding in epon, was also performed. Thin or semi-thin serial sections were treated with polyclonal anti-FD-MLO rabbit antibodies, then with gold-conjugated anti-rabbit IgG. Labeling was revealed using the silver enhancement technique for light microscopy. MLO in frozen thin sections of glands were efficiently labeled. Optimal results were obtained with 4% paraformaldehyde-0.1% glutaraldehyde fixation and low-temperature embedding in LR White resin. Both scattered MLO and unusual dense forms of MLO were easily detected with the electron-dense gold probe. This method distinguished MLO from other membrane-limited bodies and provided a good tool for studying infection in large regions of FD-infected tissues by light microscopy.

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Three hybridomas producing monoclonal antibodies specific for the mycoplasma-like organism (MLO), pathogenic agent of grapevine flavescence dorée, were obtained after fusion between spleen cells from a mouse immunized against flavescence dorée MLO and Sp2/O-Ag-14 mouse myeloma cells. These hybridomas were selected in an indirect sandwich ELISA in which antibodies from two different animal species were used (rabbit and mouse). This assay is convenient for anti-MLO monoclonal antibody screening, because of its sensitivity, specificity and good preservation of antigens. The three monoclonal antibodies were examined for reactivity towards the MLO causal agents of 15 plant yellows. None of the three were implicated in a serological relationship between the MLO of these plant yellows and flavescence dorée-MLO. Reactivity of the three monoclonal antibodies was checked towards two other isolates of flavescence dorée. One of the three antibodies detected the wild isolates.

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