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

Triplex real-time PCR assay for sensitive and simultaneous detection of grapevine phytoplasmas of the 16SrV and 16SrXII-A groups with an endogenous analytical control

Flavescence dorée (FD) and Bois noir (BN) are the two main yellows of grapevine in Europe and are caused by phytoplasmas of the 16SrV and 16SrXII-A groups respectively. A new triplex real-time PCR assay was developed in order to detect simultaneously the FD and BN phytoplasmas as well as grapevine chloroplastic DNA with TaqMan minor groove binder probes. Each set of designed primers and probes specifically detected the map gene of the FD and BN phytoplasmas, respectively and did not detect phytoplasmas from other phylogenetic groups. PCR efficiencies varied from 90 to 110 %. The PCR assay showed good intra-test and inter-test reproducibility. Triplex real-time PCR was compared to the conventional biplex nested-PCR method. The sensitivity of the real-time PCR, tested on several infected periwinkle and grapevine samples, was up to 5 and 100 times higher for the BN-P and the FD-P targets, respectively. Out of 109 grapevine samples analysed 10, which were negative with the nested PCR, turned to be FD-P positive with the real-time PCR. A decision scheme was set up according to the Ct values of the FD-P, BN-P and grapevine targets in order to assess the routine detection results

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

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

Evidence for Transcript Networks Composed of Chimeric RNAs in Human Cells

The classic organization of a gene structure has followed the Jacob and Monod bacterial gene model proposed more than 50 years ago. Since then, empirical determinations of the complexity of the transcriptomes found in yeast to human has blurred the definition and physical boundaries of genes. Using multiple analysis approaches we have characterized individual gene boundaries mapping on human chromosomes 21 and 22. Analyses of the locations of the 5′ and 3′ transcriptional termini of 492 protein coding genes revealed that for 85% of these genes the boundaries extend beyond the current annotated termini, most often connecting with exons of transcripts from other well annotated genes. The biological and evolutionary importance of these chimeric transcripts is underscored by (1) the non-random interconnections of genes involved, (2) the greater phylogenetic depth of the genes involved in many chimeric interactions, (3) the coordination of the expression of connected genes and (4) the close in vivo and three dimensional proximity of the genomic regions being transcribed and contributing to parts of the chimeric RNAs. The non-random nature of the connection of the genes involved suggest that chimeric transcripts should not be studied in isolation, but together, as an RNA network

The Major Antigenic Membrane Protein of “Candidatus Phytoplasma asteris” Selectively Interacts with ATP Synthase and Actin of Leafhopper Vectors

Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of “Candidatus Phytoplasma asteris”, the chrysanthemum yellows phytoplasmas (CYP) strain, and three others as non-vectors. Interactions between a labelled (recombinant) CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity