Daffonchio, Daniele

ABSTRACTOne emerging disease of grapevine in Europe is Bois noir (BN), a phytoplasmosis caused by “CandidatusPhytoplasma solani” and spread in vineyards by the planthopperHyalesthes obsoletus(Hemiptera: Cixiidae). Here we present the first full characterization of the bacterial community of this important disease vector collected from BN-contaminated areas in Piedmont, Italy. Length heterogeneity PCR and denaturing gradient gel electrophoresis analysis targeting the 16S rRNA gene revealed the presence of a number of bacteria stably associated with the insect vector. In particular, symbiotic bacteria detected by PCR with high infection rates in adult individuals fell within the “CandidatusSulcia muelleri” cluster in theBacteroidetesand in the “CandidatusPurcelliella pentastirinorum” group in theGammaproteobacteria, both previously identified in different leafhoppers and planthoppers. A high infection rate (81%) was also shown for another symbiont belonging to theBetaproteobacteria, designated the HO1-V symbiont. Because of the low level of 16S rRNA gene identity (80%) with the closest relative, an uncharacterized symbiont of the tickHaemaphysalis longicornis, we propose the new name “CandidatusVidania fulgoroideae.” Other bacterial endosymbionts identified inH. obsoletuswere related to the intracellular bacteriaWolbachia pipientis,Rickettsiasp., and “CandidatusCardinium hertigii.” Fluorescentin situhybridization coupled with confocal laser scanning microscopy and transmission electron microscopy showed that these bacteria are localized in the gut, testicles, and oocytes. As “Ca.Sulcia” is usually reported in association with other symbiotic bacteria, we propose that inH. obsoletus, it may occur in a bipartite or even tripartite relationship between “Ca.Sulcia” and “Ca.Purcelliella,” “Ca.Vidania,” or both.

Summary ‘ Candidatus Liberibacter spp.’ cause serious plant diseases. ‘ Candidatus Liberibacter asiaticus’, ‘ Ca. L. americanus’ and ‘ Ca. L. africanus’ are the aetiological agents of citrus greening (Huanglongbing) in Asia, America and Africa. ‘ Candidatus Liberibacter solanacearum’ causes diseases in Solanaceae in America and New Zealand. All four species are vectored by psyllid insects of different genera. Here, we show that the pear psyllid pest Cacopsylla pyri (L.) hosts a novel liberibacter species that we named ‘ Ca. Liberibacter europaeus’. It can bloom to high titres in the psyllid host, with more than 10 9 16S rRNA gene copies per individual. Fluorescent in situ hybridization experiments showed that ‘ Ca. L. europaeus’ is present in the host midgut lumen, salivary glands and Malpighian tubules. ‘ Candidatus L. europaeus’ has a relatively high prevalence (> 51%) in C. pyri from different areas in the Piedmont and Valle d'Aosta regions in Italy and can be transmitted to pear plants in experimental transmission trials. However, even though high titres of the bacterium (more than 10 8 16S rRNA gene copies g −1 of pear plant tissue) could be detected, in the pear tissues no specific disease symptoms could be observed in the infected plants over a 6‐month period. Despite liberibacters representing potential quarantine organisms, ‘ Ca. L. europaeus’, first described in Italy and Europe, apparently behaves as an endophyte rather than a pathogen.

ABSTRACT Flavescence dorée (FD) is a grapevine disease that afflicts several wine production areas in Europe, from Portugal to Serbia. FD is caused by a bacterium, “ Candidatus Phytoplasma vitis,” which is spread throughout the vineyards by a leafhopper, Scaphoideus titanus (Cicadellidae). After collection of S. titanus specimens from FD-contaminated vineyards in three different areas in the Piedmont region of Italy, we performed a survey to characterize the bacterial microflora associated with this insect. Using length heterogeneity PCR with universal primers for bacteria we identified a major peak associated with almost all of the individuals examined (both males and females). Characterization by denaturing gradient gel electrophoresis confirmed the presence of a major band that, after sequencing, showed a 97 to 99% identity with Bacteroidetes symbionts of the “ Candidatus Cardinium hertigii” group. In addition, electron microscopy of tissues of S. titanus fed for 3 months on phytoplasma-infected grapevine plants showed bacterial cells with the typical morphology of “ Ca. Cardinium hertigii.” This endosymbiont, tentatively designated ST1-C, was found in the cytoplasm of previtellogenic and vitellogenic ovarian cells, in the follicle cells, and in the fat body and salivary glands. In addition, cell morphologies resembling those of “ Ca. Phytoplasma vitis” were detected in the midgut, and specific PCR assays indicated the presence of the phytoplasma in the gut, fat body and salivary glands. These results indicate that ST1-C and “ Ca. Phytoplasma vitis” have a complex life cycle in the body of S. titanus and are colocalized in different organs and tissues.