The biology of Cixius wagneri, the planthopper vector of ‘Candidatus Phlomobacter fragariae’ in strawberry production tunnels and its consequence for the epidemiology of strawberry marginal chlorosis

Abstract«Candidatus Phlomobacter fragariae» is the prevalent agent of strawberry marginal chlorosis (SMC) and is transmitted by the planthopper Cixius wagneri. Because the insect vector biology was unknown, a field experiment was set up to determine if it was able to reproduce on strawberry plants, to determine the number of insect generations per year and the ability of nymphs to transmit SMC. During spring 2004, 80 C. wagneri adults were delivered into 4 small insectproof tunnels containing 30 healthy plants. Fifteen percent of the delivered insect population were carrying the pathogen. In October 2004, only 3 young L1 instar nymphs were found in the first tunnel, demonstrating there were no new insect generations during the summer. In April 2005, 330 C. wagneri of early L1 to late L5 nymph instars were collected at the roots of the plants,clearly indicating that a single insect generation had overwintered as larvae andemerged at the following spring. All instars were shown to carry ‘Ca. P. fragariae’ (70 to 75 % of the larvae) and were able to transmit SMC as assessed by transmission assays. An insecticide treatment was applied in March 2005 in a third tunnel and a fourth tunnel was kept as a control. More than 120 C. wagneri adults were collected in the control tunnel 4 in June 2005 confirming that an insect generation arose in the tunnel, whereas no insects could be found in the treated tunnel 3. All plants were kept for two years, surveyed for symptom expression and tested for ‘Ca. P. fragariae’ infection by 16S-PCR. Results indicated a reduced mortality and SMC incidence in tunnel 3, and a higher mortality and SMC incidence in tunnel 2 than in tunnel 1, attesting that C. wagneri larvae had spread SMC and that an early insecticide treatment could control the disease.Keywords: Phloem-restricted bacteria, planthopper, insect vector, Fragaria x ananass

Detection of phloem restricted bacteria responsible for strawberry marginal chlorosis (SMC) by real-time PCR in a single assay

Two uncultured phloem restricted plant pathogens, the γ3 proteobacterium «Candidatus Phlomobacter fragariae » and the stolbur phytoplasma (group 16SrXII-A) are associated with strawberry marginal chlorosis (SMC) in France. As “Ca. P. fragariae” and stolbur phytoplasma induce identical symptoms, the only way to identify the pathogen infecting a given diseased plant is to perform conventional PCR assays. Because using two PCR techniques for detecting separately each of the two bacteria is time consuming and because specificity and sensitivity of the detection test needed to be improved, a new approach using triplex real time PCR was developed for the routine detection of “Ca. P. fragariae “ and stolbur phytoplasma. The real time PCR has the advantage of being faster reduces the risks of producing false positives. Furthermore, real-time PCR techniques provide the possibility of multiplexing by using probes with different compatible fluorescent dyes. Here, we present a new sensitive Taqman® method which permits the simultaneous amplification of three DNA targets in one test: the map gene of stolbur phytoplasma, the spoT gene of “Ca. P. fragariae” and the cox gene of strawberry chloroplast taken as an internal control. The specificity and the efficiency of this method were determined.Keywords: Strawberry Marginal Chlorosis, Triplex taqman® PCR ,Candidatus Phlomobacter fragariae, stolbur phytoplasma

PCR/RFLP-based method for molecular characterization of ‘Candidatus Phytoplasma prunorum’ strains using the aceF gene.

New molecular typing tools for phytoplasmas belonging to the 16SrX phytoplasma group have recently been developed based on the non-ribosomal genes aceF, pnp, imp, and SecY. In the present work we chose to perform a PCR-RFLP method based on the aceF gene. This genetic marker had previously shown high variability among strains of the 16SrX group, moreover, it had allowed for the differentiation of French hypovirulent ‘Candidatus Phytoplasma prunorum’ strains from virulent ones.Most of the stone fruit samples were collected in north-east Italy, although a few samples from Bosnia and Herzegovina, and Turkey were also included in the work to explore variability. French hypovirulent and virulent strains, one Azerbaijan strain and ‘Ca. P. prunorum’ strains maintained in periwinkles were used as reference strains. Some of the Italian samples were not collected in the field and they became infected by Cacopsylla pruni under controlled conditions.Sequencing of the aceF gene was performed on some of the samples tested and based on the alignment, a few restriction enzymes were selected for ‘Ca. P. prunorum’ strain differentiation. Nested PCR was performed using previously developed primers on all samples and RFLP analyses were carried out with BpiI, HaeIII and Tsp509I enzymes. BpiI and HaeIII enzymes generated two different profiles, one profile was undigested and the second one constituted by two different fragments. The Tsp509I enzyme enabled three different pattern types to be distinguished. Combining the results obtained with the three restriction enzymes, it was possible to distinguish between the ‘Ca. P. prunorum’ strains investigated in this study: 6 different RFLP subgroups AceF-A, -B, -C, -D, -E and –F. We confirmed that strains belonging to 4 subgroups, AceF-A, -B, -C and -E were present in north-east Italy, where a large number of the samples were processed. The strains of AceF-A and -E subgroups were the predominant ones (21.6% and 17.0%, respectively) and mixed infections of AceF-A+E subgroups (17.0%), and AceF-B+E (14.8%) subgroups were quite common. Keywords: phytoplasma, European stone fruit yellows, molecular differentiation, sequencin

Laser à semiconducteur à 852 nm bifrequence pompé optiquement pour les horloges atomiques CPT (poster)

National audienceNous présentons un laser à semiconducteur en cavité externe pompé optiquement, émettant sur deux fréquences optiques polarisées perpendiculairement, destiné au piégeage cohérent d'atomes (CPT) de Cs. L'émission est accordable autour de 852 nm. La différence de fréquence est ajustée grâce à une lame électro-optique autour de 9,2 GHz. La longueur d'onde du mode ordinaire est stabilisée sur la raie D2 du Cs et la différence de fréquence est asservie sur un signal de référence RF. En fonctionnement stabilisé, nous caractérisons les sources de bruits du laser afin d'évaluer les performances du laser en vue de son application dans une horloge atomique CPT

Dual-frequency VECSEL for atomic clocks using coherent population trapping

Workshop on Laser Diodes for Space Applications, Nov 2015, Palaiseau, FranceAtomic frequency references provide high-precision stable signals, which are crucial in the most demanding applications as high bitrate communication networks, high-end inertial navigation, or satellite positioning. One way to obtain those laser fields with low intensity-and frequency-noise is to use the dual-frequency and dual-polarization emission of an optically-pumped vertical external-cavity semiconductor laser (OP-VECSEL)

A robust and rapid xenograft model to assess efficacy of chemotherapeutic agents for human acute myeloid leukemia

International audienceRelevant preclinical mouse models are crucial to screen new therapeutic agents for acute myeloid leukemia (AML). Current in vivo models based on the use of patient samples are not easy to establish and manipulate in the laboratory. Our objective was to develop robust xenograft models of human AML using well-characterized cell lines as a more accessible and faster alternative to those incorporating the use of patient-derived AML cells. Five widely used AML cell lines representing various AML subtypes were transplanted and expanded into highly immunodeficient non-obese diabetic/LtSz-severe combined immunodeficiency IL2R gamma(null)(c) mice (for example, cell line-derived xenografts). We show here that bone marrow sublethal conditioning with busulfan or irradiation has equal efficiency for the xenotransplantation of AML cell lines. Although higher number of injected AML cells did not change tumor engraftment in bone marrow and spleen, it significantly reduced the overall survival in mice for all tested AML cell lines. On the basis of AML cell characteristics, these models also exhibited a broad range of overall mouse survival, engraftment, tissue infiltration and aggressiveness. Thus, we have established a robust, rapid and straightforward in vivo model based on engraftment behavior of AML cell lines, all vital prerequisites for testing new therapeutic agents in preclinical studies

A corpus-assisted study of the discourse marker well as an indicator of judges' institutional roles in court cases with litigants in person

In this paper, I concentrate on court cases with litigants in person (lay people who act on their own behalf in legal proceedings without a counsel or solicitor) and discuss the challenges of building a corpus of courtroom discourse where it is crucial to distinguish between speakers due to their distinct institutional roles. The corpus incorporates seven sub-corpora of verbatim transcripts from different court cases with litigants in person and comprises over eleven-million tokens. The focus of this paper is on the interplay between the legal and lay discourse types and how judges project their institutional roles through well-initiated turns directed at litigants in person and counsels. As a versatile discourse marker, well provides a good opportunity to explore how judges have to adapt their roles to ensure lay litigants in person receive the necessary support and that their lack of competence does not impede on the fairness of the proceedings. Given the breadth and importance of the topic of litigation in person, I discuss how the tools and approaches of corpus linguistics can be helpful in this multi-disciplinary area where multiple functions and uses of individual linguistic features need to be explored in depth

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