Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale.

To study the role of wild areas around the vineyards in the epidemiology of flavescence dorée (FD) and track the origin of new foci, two phytoplasma genetic markers, dnaK and malG, were developed for FD phytoplasma (FDp) characterization. The two genes and the vmpA locus were used to genetically characterize FDp populations at seven agroecosystems of a wine-growing Italian region. Vitis vinifera, "gone-wild" V. vinifera and rootstocks, Clematis spp., and Scaphoideus titanus adults were sampled within and outside the vineyards. A range of genotypes infecting the different hosts of the FDp epidemiological cycle was found. Type FD-C isolates were fairly homogeneous compared to type FD-D ones. Most of the FD-D variability was correlated with the malG sequence, and a duplication of this locus was demonstrated for this strain. Coinfection with FD-C and FD-D strains was rare, suggesting possible competition between the two. Similar levels of FDp genetic variation recorded for grapevines or leafhoppers of cultivated and wild areas and co-occurrence of many FDp genotypes inside and outside the vineyards supported the idea of the importance of wild or abandoned Vitis plants and associated S. titanus insects in the epidemiology of the disease. Genetic profiles of FDp found in Clematis were never found in the other hosts, indicating that this species does not take part in the disease cycle in the area. Due to the robustness of analyses using dnaK for discriminating between FD-C and FD-D strains and the high variability of malG sequences, these are efficient markers to study FDp populations and epidemiology at a small geographical scale.IMPORTANCE Flavescence dorée, a threatening disease of grapevine caused by FD phytoplasma (FDp), is distributed within the most important wine-producing areas of Europe and has severe effects on both vineyard productivity and landscape management. FDp is a quarantine pest in Europe, and despite the efforts to contain the pathogen, the disease is still spreading. In this work, new genetic markers for the fine genetic characterization of FDp at local scale are presented. Our findings improve the knowledge of FDp epidemiological cycle and offer the possibility of tracking the route of the FDp infection. In particular, due to its high genetic variability, one of the newly developed markers could be sufficient to track the origin of new infection foci, either from the wild areas or from nurseries.

Nectar yeasts enhance the interaction between Clematis akebioides and its bumblebee pollinator.

It has been hypothesised that intense metabolism of nectar-inhabiting yeasts (NIY) may change nectar chemistry, including volatile profile, which may affect pollinator foraging behaviours and consequently plant fitness. However, empirical evidence for the plant-microbe-pollinator interactions remains little known. To test this hypothesis, we use a bumblebee-pollinated vine Clematis akebioides endemic to southwest China as an experimental model plant. To quantify the incidence and density of Metschnikowia reukaufii, a cosmopolitan NIY in floral nectar, a combination of yeast cultivation and microscopic cell-counting method was used. To examine the effects of NIY on plant-pollinator interactions, we used real flowers filled with artificial nectar with or without yeast cells. Then the volatile metabolites produced in the yeast-inoculated nectar were analysed with coupled gas chromatography and mass spectrometry (GC-MS). On average 79.3% of the C. akebioides flowers harboured M. reukaufii, and cell density of NIY was high to 7.4 × 104 cells mm-3 . In the field population, the presence of NIY in flowers of C. akebioides increased bumblebee (Bombus friseanus) pollinator visitation rate and consequently seed set per flower. A variety of fatty acid derivatives produced by M. reukaufii may be responsible for the above beneficial interactions. The volatiles produced by the metabolism of M. reukaufii may serve as an honest signal to attract bumblebee pollinators and indirectly promote the female reproductive fitness of C. akebioides, forming a potentially tripartite plant-microbe-pollinator mutualism.

Genetic diversity of European phytoplasmas of the 16SrV taxonomic group and proposal of 'Candidatus Phytoplasma rubi'.

In addition to the grapevine flavescence dorée phytoplasmas, other members of taxonomic group 16SrV phytoplasmas infect grapevines, alders and species of the genera Clematis and Rubus in Europe. In order to investigate which phytoplasmas constitute discrete, species-level taxa, several strains were analysed by comparing their 16S rRNA gene sequences and a set of five housekeeping genes. Whereas 16S rRNA gene sequence similarity values were >97.5 %, the proposed threshold to distinguish two 'Candidatus Phytoplasma' taxa, phylogenetic analysis of the combined sequences of the tuf, rplV-rpsC, rplF-rplR, map and uvrB-degV genetic loci showed that two discrete phylogenetic clusters could be clearly distinguished. The first cluster grouped flavescence dorée (FD) phytoplasmas, alder yellows (AldY) phytoplasmas, Clematis (CL) phytoplasmas and the Palatinate grapevine yellows (PGY) phytoplasmas. The second cluster comprised Rubus stunt (RS) phytoplasmas. In addition to the specificity of the insect vector, the Rubus stunt phytoplasma contained specific sequences in the 16S rRNA gene. Hence, the Rubus stunt phytoplasma 16S rRNA gene was sufficiently differentiated to represent a novel putative taxon: 'Candidatus Phytoplasma rubi'.

Community structure of arbuscular mycorrhizal fungi in a primary successional volcanic desert on the southeast slope of Mount Fuji.

Community structure of arbuscular mycorrhizal fungi (AMF), evaluated as spore samples and mycorrhizal roots of four herbaceous plant species, was investigated at different altitudes in a primary successional volcanic desert on Mount Fuji using molecular methods (fragment and sequence analysis of the large ribosomal subunit RNA gene). In total, 17 different AMF clades were identified, and most were members of the Glomaceae, Acaulosporaceae, and Gigasporaceae. The AMF community structures detected by spore sampling were inconsistent with those from plant roots. Of all AMF clades, six (35.3%) were detected only on the basis of spores, six (35.3%) only in roots, and five corresponded to both spores and roots (29.4%). Although an Acaulospora species was the most dominant among spores (67.1%), it accounted for only 6.8% in root samples. A species analysis of AMF communities at different altitudes demonstrated that AMF species diversity increased as altitude decreased and that the species enrichment at lower altitudes resulted from the addition of new species rather than species replacement. The inconsistencies in the species composition of spore communities with those in roots and the change in species diversity with altitude are discussed.

[Keratomycosis due to Cylindrocarpon lichenicola]. / Keratomykose durch Cylindrocarpon lichenicola.

Two independent males acquired keratomycosis at a few months interval. The culturally proven pathogen was Cylindrocarpon lichenicola (C. Massal) D. Hawksworth. For one patient Clematis plants were suspected of source of infection. Both patients did not remember traumata; they had no contact with each other, and their residences were far apart. One patient was treated with voriconazole locally and systemically for 12 weeks, the other patient with amphotericin B locally for 6 weeks. Both treatment regimens were successful. Cylindrocarpon lichenicola is characterised by keratinolytic properties. Following molecular-genetic studies the pathogen was recently reclassified as Fusarium lichenicola.

Arbuscular mycorrhizal colonization of the dominant plant species in primary successional volcanic deserts on the Southeast slope of Mount Fuji.

Arbuscular mycorrhizal (AM) colonization was observed on four plant species in primary successional volcanic deserts on the Southeast slope of Mount Fuji. The AM colonization of the dominant species, Polygonum cuspidatum, contradicts the conclusion that Polygonaceae are often regarded as being non-mycorrhizal species. The secondary dominant species, Polygonum weyrichii var. alpinum, formed no mycorrhizas. The roots of Cirsium purpuratum, Clematis stans and Campanula punctata ssp. hondoensis, showed a higher percentage of AM colonization than P. cuspidatum. AM colonization and spore density in the rhizosphere soil of P. cuspidatum significantly decreased as elevation increased. AM colonization in roots of Cirsium purpuratum and Clematis stans also tended to decrease with increased altitudes. Cirsium purpuratum and Campanula punctata ssp. hondoensis formed single structural types of Arum- and Paris-type, respectively, whereas P. cuspidatum and Clematis stans formed both Arum- and Paris-type morphologies.

Okibacterium fritillariae gen. nov., sp. nov., a novel genus of the family Microbacteriaceae.

Okibacterium fritillariae gen. nov., sp. nov. (type strain VKM Ac-2059T = IFO 16404T) is proposed for aerobic, oxidase- and catalase-positive, coryneform bacteria isolated from seeds of Fritillaria ruthenica Wikstr. and Clematis recta L. Strains of the new genus form a distinct branch within the phylogenetic cluster composed of members of the family Microbacteriaceae and are characterized by B-type peptidoglycan containing amino acids glycine, glutamate, homoserine, alanine and lysine, the glycolyl type of muramic acid, the major menaquinones MK-10 and MK-11, the principal phospholipids phosphatidylglycerol and diphosphatidylglycerol, and a DNA G+C content of approximately 67 mol %.