Elisabeth Boudon-Padieu

Detection and differentiation of grapevine yellows phytoplasmas belonging to the elm yellows group and to the stolbur subgroup by PCR amplification of non-ribosomal DNA

Primer pairs were designed from a cloned DNA probe of a strain of flavescence dorée (FD) phytoplasma and from a cloned DNA probe of a strain of stolbur phytoplasma. Among an array of reference phytoplasma strains maintained in periwinkle, pair FD9f/r amplified a 1.3 kb DNA fragment only with phytoplasma strains of elm yellows (EY) group, i.e. two strains of FD and two strains of EY. Tru9I restriction analysis of the fragment amplified by FD9f/r revealed a diversity among EY-group phytoplasmas. The FD strains differed from the strains isolated from elm. The profile of the phytoplasmas infecting the grapevine samples from Catalonia and most of the samples from Northern Italy were identical to that of a FD strain. Three other profiles were detected in grapevine from Palatinate, in Germany.The two primer pairs derived from a stolbur strain, STOL4f/r and STOL11f2/r1, specifically amplified a 1.7 kb and a 0.9 kb DNA fragment, respectively, with all strains in the stolbur subgroup. However, the pair STOL4f/r did not recognise strain MOL. Both pairs allowed to detect phytoplasmas in diseased grapevines from France, Italy, Spain and Israel. Attempts to differentiate between phytoplasmas in the stolbur subgroup by restriction analyses failed. The pairs FD9f/r and STOL11f2/r1 could be used in the same reaction (multiplex PCR) to detect EY-group phytoplasmas, stolbur-subgroup phytoplasmas or both phytoplasmas simultaneously when template DNAs were mixed.

New mealybug species vectoring Grapevine leafroll-associated viruses-1 and -3 (GLRaV-1 and -3)

Many grape viruses, such as filamentous Grapevine leafroll-associated viruses in the Closteroviridae family, are spread primarily through infected propagating material. However, there is increasing evidence that leafroll disease are spread in the field by insect vectors, namely mealybugs and other scale insects. This study was carried out in the northern wine-growing regions of France where Grapevine leafroll-associated virus-1 and -3 (GLRaV-1 and -3) are the most widespread grape Ampelovirus species. The vineyards were inspected for presence of mealybug and scale insects and grapes infected by GLRaV-1 and -3. Mealybugs, Heliococcus bohemicus, Phenacoccus aceris (Pseudococcidae) and the soft scale insect Parthenolecanium corni (Coccidae), were capable of a transmission efficiency of 14%, 23% and 29% respectively. GLRaV-1 and -3 infections that resulted from virus transmission were confirmed with DAS-ELISA using polyclonal antibodies. This is the first report of GLRaV-1 and -3 transmission by mealybug and coccid species in France, and the first report of the ability of H. bohemicus and Phenacoccus aceris to transmit these viruses to grapevines. The relevance of these findings with regards to maintenance of virus-free grapevine stocks and to control leafroll spread in commercial vineyards is discussed.

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

ABSTRACT 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.

A New Natural Planthopper Vector of Stolbur Phytoplasma in the Genus Pentastiridius (Hemiptera: Cixiidae)

A new disease of sugar beet called Syndrome des Basses Richesses, which appeared in Burgundy and Franche-Comté, France, in 1991, is of uncertain aetiology. However, evidence for aerial transmission of the disease, symptom similarity with yellow wilt and preliminary results of phytoplasma detection, support the hypothesis of a phytoplasma being associated to the disease. A search for a natural phytoplasma vector, was conducted in Franche-Comté in 1997 and 1998, in an area where sugar beet crops had been affected since 1996. A cixiid, tentatively identified as Pentastiridius beieri, not described in the preceding years and not formerly reported as a phytoplasma vector, was present in sugar beet plots in high populations from June to August in 1997 and 1998. Individuals were captured and used for transmission experiments to periwinkle and sugar beet seedlings. They were further tested for the presence of a phytoplasma in their body, using PCR amplification of 16S rDNA of phytoplasmas. In 1997 and 1998, from 2% to 13.3% of the individuals carried a stolbur phytoplasma and insects which tested positive, appeared to have transmitted, through feeding, a stolbur phytoplasma to periwinkles and to sugar beets. This cixiid, whose vectoring capacity of stolbur phytoplasma to plants, is now clearly demonstrated, is available for experimental inoculations, in order to examine the role of phytoplasmas in the Syndrome des Basses Richesses, through the observation of symptom expression in phytoplasma-inoculated plants.

ELISA and dot-blot detection of flavescence dorée-MLO in individual leafhopper vectors during latency and inoculative state.

Flavescence dorée, an MLO (mycoplasma-like organism) disease of grapevine, is vectored in the field by the leafhopperScaphoïdeus littoralis, and in the laboratory fromVicia faba toVicia faba by the leafhopperEuscelidius variegatus. Antibodies to partially purified MLOs from broadbeans orE. variegatus were raised in rabbits. ELISA allowed individual assay of leafhoppers almost without background to healthy leafhopper control. Positive ELISA reading was associated with biological transmission and demonstrated the infection in noninoculative leafhoppers, even during latency. Thus, ELISA detection was fast and more accurate than inoculative state. A first multiplication of antigen, leading to infectivity of leafhoppers, was followed by a fast and strong multiplication during the following weeks. Males were particularly affected. Differential acquisition between individuals was shown. Application toS. titanus naturally contaminated in the field allows survey of epidemic outbreak and search for other vector species.

Reduced fitness of the leafhopper vector Scaphoideus titanus exposed to Flavescence dorée phytoplasma

Scaphoideus titanus Ball (Homoptera: Cicadellidae), a specialist and univoltine leafhopper on grapevine (Vitis vinifera L.) (Vitaceae), is a vector of Flavescence dorée phytoplasma (FDP) in vineyards of European temperate areas. Males and females of the leafhopper were exposed to FDP by feeding on infected broad bean (Vicia faba L.) (Fabaceae). Detection of FDP by the amplification of phytoplasma DNA with polymerase chain reaction assays of individual insects revealed an acquisition rate of 91.4% (96/105) after an acquisition access period of 13 days. The adult life span of FD‐exposed males and females was much less than that of leafhoppers fed on healthy broad bean, as revealed by ANOVA on the quartiles of survival distribution and Weibull scale parameter. The progeny of exposed females (number of nymphs emerging from eggs deposited on woody cane segments) was significantly less than the progeny of unexposed females. Eggs produced by FD‐exposed females were slightly but significantly delayed in hatching. Reduced fecundity was confirmed by dissecting FD‐exposed and non‐exposed 42‐day‐old females and counting the number of fully sized eggs in each leafhopper. There was no evidence of transovarial passage of FDP in the offspring of infected females after 72 nymphs were reared on a healthy grapevine until the fifth instar or adult appearance and then confined on broad bean seedlings.

Scaphoideus titanus, a possible vector of grapevine yellows in New York.

Scaphoideus titanus, the natural vector of grapevine flavescence doree (FD), is widespread in New York. It was found closely associated with both wild Vitis riparia and cultivated V. vinifera, but it prefers V. riparia. Adult leafhoppers migrate from surrounding wild grapevines into the peripheral parts of vineyards. V. vinifera vines with symptoms of grapevine yellows (GY) disease were concentrated along the borders of vineyards. Even near GY-affected vineyards, V. riparia vines never exhibited symptoms of this disease. Symptoms of an MLO infection developed in 29% of Vicia faba plants fed on by field-collected S. titanus. However, potted V. vinifera «Chardonnay» on which the same leafhoppers were fed did not develop symptoms of GY within 1 yr (.)

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.

Generation and characterization of monoclonal antibodies to flavescence dorée phytoplasma: serological relationships and differences in electroblot immunoassay profiles of flavescence dorée and elm yellows phytoplasmas.

Eleven stable hybridoma cell lines secreting monoclonal antibodies specific for FD-phytoplasma, the pathogenic agent of grapevine Flavescence dorée, were produced by fusing a non-secreting myeloma cell line with spleen cells from Balb/c mice immunized with Flavescence dorée phytoplasma purified by immunoaffinity. These monoclonal antibodies were characterized for their recognition of phytoplasma proteins by western blot. Six of eleven reacted specifically in ELISA and immunoblotting with Elm-yellows phytoplasma. These antibodies did not react either in ELISA or in western blot with preparations from periwinkles infected with phytoplasmas that cause GYU (Grapevine Yellows from Udine), AP (Apple Proliferation), EAY (European Aster Yellows) and StolC (Stolbur from France). Two of these hybridoma lines were used routinely for the immunodiagnosis of Flavescence dorée phytoplasma in diseased grapevines.

Purification of grapevine flavescence dorée MLO (Mycoplasma-like organism) by immunoaffinity

To date MLO (Mycoplasma-like organism) remain non-culturable organisms and are difficult to extract in good conditions of purity and conservation from infected hosts (plants or leafhopper vectors). An immunoaffinity procedure that permits the purification of large quantities of Grapevine Flavescence dorée MLO (FD-MLO) is described, with covalently bound and oriented IgG molecules of a previously obtained anti-FD-MLO monoclonal antibody and elution of antigens in alkaline conditions. Evidence for purity and integrity of the eluted MLO is presented. The two main antigenic components detected by rabbit polyclonal antibodies to FD-MLO were shown to be different proteins and to contain different epitopes with the use of different monoclonal antibodies. DNA extracted from the purified FD-MLO fraction hybridized with an FD-MLO DNA-specific probe.