Jean-Luc Danet

The stolbur phytoplasma antigenic membrane protein gene stamp is submitted to diversifying positive selection.

Surface proteins play an important role in phytoplasma life cycle. The antigenic membrane protein (AMP) of Candidatus Phytoplasma asteris has been shown to interact with the insect microfilaments. Due to the synteny of the groL-amp-nadE genetic locus between phytoplasma genomes, the gene stamp that encodes the antigenic membrane protein of stolbur phytoplasma has been cloned and characterized. It encodes a 157 amino acid-long protein with a predicted signal peptide and a C-terminal hydrophobic alpha-helix. STAMP was 26-40% identical to AMP of Candidatus Phytoplasma asteris strains and 40% identical to AMP of Ca. P. japonicum. The expression of STAMP in Escherichia. coli produced a 16 kDa peptide recognized by an anti-stolbur monoclonal antibody. Stamp was more variable than the house-keeping gene secY and the ratio between non-synonymous over synonymous mutations (dN/dS ) was 2.78 for stamp as compared to 0.64 for secY. This indicates that stamp is submitted to a positive diversifying selection pressure.

Infection of the Circulifer haematoceps cell line Ciha-1 by Spiroplasma citri: the non-insect-transmissible strain 44 is impaired in invasion.

Successful transmission of Spiroplasma citri by its leafhopper vector requires a specific interaction between the spiroplasma surface and the insect cells. With the aim of studying these interactions at the cellular and molecular levels, a cell line, named Ciha-1, was established using embryonic tissues from the eggs of the S. citri natural vector Circulifer haematoceps. This is the first report, to our knowledge, of a cell line for this leafhopper species and of its successful infection by the insect-transmissible strain S. citri GII3. Adherence of the spiroplasmas to the cultured Ciha-1 cells was studied by c.f.u. counts and by electron microscopy. Entry of the spiroplasmas into the insect cells was analysed quantitatively by gentamicin protection assays and qualitatively by double immunofluorescence microscopy. Spiroplasmas were detected within the cell cytoplasm as early as 1 h after inoculation and survived at least 2 days inside the cells. Comparing the insect-transmissible GII3 and non-insect-transmissible 44 strains revealed that adherence to and entry into Ciha-1 cells of S. citri 44 were significantly less efficient than those of S. citri GII3.

Use the insiders: could insect facultative symbionts control vector-borne plant diseases?

Insect vector-borne plant diseases, particularly those whose causative agents are viral, or phloem- and xylem-restricted bacteria, greatly impact crop losses. Since plants are immobile, the epidemiology of vector-borne diseases greatly depends on insect vectors, which are the only means of dissemination for many pathogens. The effectiveness of a vector-borne pathogen relies upon the vectorial capacity, which is affected by vector density, feeding activity on hosts, longevity before and after pathogen ingestion, duration of the incubation period, and vector competence. During the last decade, research on human vector-borne epidemics has stimulated interest in novel control strategies targeting different parts of the vector cycle, and our purpose here is to draw parallels between this field of research and agronomy. We review the literature on insect vectors of crop diseases and their symbiotic microorganisms with the aim of suggesting future integrated management techniques based on current research on insect-vectored human diseases. Vector transmission is a complex process and different modes of transmission are encountered irrespective of the pathogen. Facultative symbionts have varied effects on life history traits that could be used for vector population control. Symbiont selection, transformation, and their manner of dissemination are important when developing an integrated vector management system based on symbiont manipulation. In the short term, progress on our knowledge of the microflora of insect vectors of plant diseases must be made. In the long term, symbiont manipulation, which has been successfully demonstrated against human insect-vectored diseases, could be adapted to insect-borne plant diseases to increase sustainable crop production.

Minor cultures as hosts for vectors of extensive crop diseases: Does Salvia sclarea act as a pathogen and vector reservoir for lavender decline?

Stolbur is a phytoplasma disease affecting crops worldwide. The planthopper Hyalesthes obsoletus is the main natural vector of ‘Candidatus Phytoplasma solani’ responsible of stolbur. In France, lavender (Lavandula angustifolia) and lavandin (Lavandulaxa0×xa0intermedia) are strongly affected by this phytoplasma. These plant species are both hosts for the phytoplasma and its insect vector. In 2011, catches of adults were exceptionally sizable on one of the clones of lavandin most tolerant to lavender decline. A high population level of ‘Ca. P. solani’ vector was also observed on the adjacent plot of clary sage, Salvia sclarea. In order to clarify the potential role of S. sclarea as a host plant for H. obsoletus and ‘Ca. P. solani,’ we conducted field surveys and laboratory experiments. The uprooting of clary sage and root examination showed the presence of nymphs during winter. Harvested nymphs have been reared on S. sclarea from seedlings in a greenhouse for many generations. By performing its whole lifecycle on clary sage, we demonstrated for the first time that S. sclarea is a host plant of H. obsoletus and could be a source of stolbur vector. Nevertheless, status of clary sage as host plant of phytoplasma in the field up to now is not so clear. On 42 Q-PCR runs done on S. sclarea, 41 were negative to the phytoplasma, and one positive. Experimental transmission with infected H. obsoletus sampled on infected lavender showed that clary sage plant could be infected, expressed symptoms and multiplied ‘Ca. P. solani.’

Important genetic diversity of ‘Candidatus Phytoplasma solani’ related strains associated with bois noir grapevine yellows and planthoppers in Azerbaijan

Bois noir (BN) is an important grapevine yellows endemic to the Euro-Mediterranean basin caused by ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’), a non culturable plant pathogenic Mollicute. Bois noir symptoms could be associated with ‘Ca. P. solani’ in two Azerbaijanian vineyards where disease incidence and severity were recorded for five local Vitis vinifera cultivars. In order to gain insight into the epidemiology of Bois noir in Azerbaijan, ‘Ca. P. solani’ isolates infecting plants were characterized by multi-locus sequence analysis and their secY and stamp gene sequences compared to that of the strains detected in other plants and in local Cixiidae planthoppers. Genotypes were determined for two non-ribosomal house-keeping genes, namely tuf and secY, as well as two variable markers namely Stamp and mleP1 genes, that respectively encode the antigenic membrane protein AMP and a 2-Hydroxycarboxylate transporter. The Azerbaijanian BN phytoplasma isolates corresponded to three tufB and secY genotypes. A finer differentiation of Azerbaijanian ‘Ca. P. solani’ isolates was obtained with mleP1 as five different mleP1 genetic variants were found. Finally, Stamp gene allowed differentiating four new genotypes in grapevine among the 10 new Stamp genotypes detected in various plants in Azerbaijan. The preliminary survey for infected insects conducted in northern Azerbaijan, led to the identification of Hyalesthes obsoletus and Reptalus noahi as potential vectors for two ‘Ca. P. solani’ new genotypes phylogenetically distant from the known genetic clusters. Altogether these results indicate an important genetic diversity of BN phytoplasmas in Azerbaijan that certainly result from spread through local insect vectors.

Variable membrane protein A of flavescence dorée phytoplasma binds the midgut perimicrovillar membrane of Euscelidius variegatus and promotes adhesion to its epithelial cells

ABSTRACT Phytoplasmas are uncultivated plant pathogens and cell wall-less bacteria and are transmitted from plant to plant by hemipteran insects. The phytoplasmas circulative propagative cycle in insects requires the crossing of the midgut and salivary glands, and primary adhesion to cells is an initial step toward the invasion process. The flavescence dorée (FD) phytoplasma possesses a set of variable membrane proteins (Vmps) exposed on its surface, and this pathogen is suspected to interact with insect cells. The results showed that VmpA is expressed by the flavescence dorée phytoplasma present in the midgut and salivary glands. Phytoplasmas cannot be cultivated at present, and no mutant can be produced to investigate the putative role of Vmps in the adhesion of phytoplasma to insect cells. To overcome this difficulty, we engineered the Spiroplasma citri mutant G/6, which lacks the ScARP adhesins, for VmpA expression and used VmpA-coated fluorescent beads to determine if VmpA acts as an adhesin in ex vivo adhesion assays and in vivo ingestion assays. VmpA specifically interacted with Euscelidius variegatus insect cells in culture and promoted the retention of VmpA-coated beads to the midgut of E. variegatus. In this latest case, VmpA-coated fluorescent beads were localized and embedded in the perimicrovillar membrane of the insect midgut. Thus, VmpA functions as an adhesin that could be essential in the colonization of the insect by the FD phytoplasmas. IMPORTANCE Phytoplasmas infect a wide variety of plants, ranging from wild plants to cultivated species, and are transmitted by different leafhoppers, planthoppers, and psyllids. The specificity of the phytoplasma-insect vector interaction has a major impact on the phytoplasma plant host range. As entry into insect cells is an obligate process for phytoplasma transmission, the bacterial adhesion to insect cells is a key step. Thus, studying surface-exposed proteins of phytoplasma will help to identify the adhesins implicated in the specific recognition of insect vectors. In this study, it is shown that the membrane protein VmpA of the flavescence dorée (FD) phytoplasma acts as an adhesin that is able to interact with cells of Euscelidius variegatus, the experimental vector of the FD phytoplasma.

Tuf and secY PCR amplification and genotyping of phytoplasmas.

Tuf and secY genotyping techniques have been developed to distinguish phytoplasma strains. Tuf polymerase chain reaction sequence analyses are available for phytoplasma taxonomic groups 16SrI, 16SrV, 16SrXII-A, and XII-B. In addition to their use to confirm the taxonomic status of phytoplasma strains, they allow the spread of phytoplasma strains in host plants and insect vectors to be traced. SecY is more variable than tuf and is therefore more discriminatory than tuf, but secY and tuf phylogenies show congruence.

A ‘Candidatus Phytoplasma omanense’-related strain detected in yellowing grapevine, stunted bindweed and Cixiidae planthoppers in Lebanon

Grapevine yellows (GY) affecting Lebanese vineyards are reported to be associated with ‘Candidatus Phytoplasma solani’, the bois noir (BN) etiological agent. However, during a field survey in June 2014 for BN in Mansoura municipality of West Bekaa, Lebanon, ‘Candidatus Phytoplasma omanense’ was detected in a grapevine sample, cultivar Syrah, exhibiting leaf scorch and discoloration, using a phytoplasma universal nested-PCR and sequencing of the 1.2 kbp 16SrDNA amplicon. The same 1.2 kbp 16SrDNA sequence could be amplified from Hyalesthes obsoletus and Reptalus sp. Cixiidae planthoppers collected on Convolvulus arvensis (bindweed) plants in June 2014 in Aammiq municipality of West Bekaa. Yellowing and stunted bindweed plants collected in 2015 in Kefraya and Aamiq municipalities of West Bekaa were also found infected with the same phytoplasma strain. A 16S rDNA RFLP assay was designed to differentiate this phytoplasma from ‘Ca. P. solani’ and ‘Ca. P. phoenicium’, which are endemic to the Bekaa region. ‘Ca. P. omanense’ is reported for the first time in grapevine and in Cixiidae planthoppers already known to respectively host and vector ‘Ca. P. solani’ strains associated with BN disease in grapevine. This result highlights the need for a precise survey of phytoplasmas associated to grapevine yellows in the Eastern Mediterranean basin, and for investigating the possible role of ‘Ca. P. omanense’ as a new threat to Euro-Mediterranean viticulture.