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3843

Bacteria-like endosymbionts of females of the plant-parasitic nematodes Globodera rostochiensis and Heterodera goettingiana and juveniles of Heterodera glycines were first observed during transmission electron microscopy (TEM) studies conducted in the 1970s. These organisms were characterized as being rod-shaped, ranging in size from 0.3 to 0.5 μm in diameter and 1.8 to 3 μm in length and containing structures labelled as striated inclusion bodies or tubular structures. A population of H. glycines was obtained from the soybean field where infected nematodes were first discovered in order to conduct TEM studies of females and males and to determine the phylogenetic position of the H. glycines endosymbiont among bacteria by studying the 16S rRNA and gyrB gene sequences. The bacterium was observed in the pseudocoelom and intestine of juveniles, females and males, in hypodermal chords of juveniles and males, in ovary walls and in oocytes and spermatozoa. The bacterium was polymorphic, measuring 0.4–0.8×2.5–4.5 μm, and many specimens contained an array of microfilament-like structures similar to those observed in ‘Candidatus Cardinium hertigii’, the endosymbiont of Encarsia spp. wasps. Phylogenetic analysis of the 16S rRNA and gyrB genes of the H. glycines-infecting bacterium revealed 93 % and 81 % sequence identity, respectively, to the homologous genes in ‘Candidatus C. hertigii’. Thus, the name ‘Candidatus Paenicardinium endonii’ is proposed for the bacterial endosymbiont of the plant-parasitic nematode H. glycines.

Symbiotic interactions with bacteria are essential for the survival and reproduction of many insects. The European beewolf (Philanthus triangulum, Hymenoptera, Crabronidae) engages in a highly specific association with bacteria of the genus Streptomyces that appears to protect beewolf offspring against infection by pathogens. Using transmission and scanning electron microscopy, the bacteria were located in the antennal glands of female wasps, where they form dense cell clusters. Using genetic methods, closely related streptomycetes were found in the antennae of 27 Philanthus species (including two subspecies of P. triangulum from distant localities). In contrast, no endosymbionts could be detected in the antennae of other genera within the subfamily Philanthinae (Aphilanthops, Clypeadon and Cerceris). On the basis of morphological, genetic and ecological data, ‘Candidatus Streptomyces philanthi’ is proposed. 16S rRNA gene sequence data are provided for 28 ecotypes of ‘Candidatus Streptomyces philanthi’ that reside in different host species and subspecies of the genus Philanthus. Primers for the selective amplification of ‘Candidatus Streptomyces philanthi’ and an oligonucleotide probe for specific detection by fluorescence in situ hybridization (FISH) are described.

“Candidatus phytoplasma asteris” and related strains (i.e., aster yellows group 16SrI) have been associated with diseases of numerous plant species worldwide. Symptoms of aster yellows (AY) have been reported on rapeseed/canola (Brassica napus and B. rapa) crops in Saskatchewan (SK) and Manitoba, Canada since 1953 (2). Symptoms generally include stunting, virescence, leaf yellowing or purpling, phyllody, and formation of bladder-like siliques. A total of 120 mature B. rapa cv. AC Sunbeam plants exhibiting AY symptoms were collected in commercial fields near Medstead, SK during 2003 and 2004 (one field per year). As described previously (4), total genomic DNA was extracted from leaf, stem, roots, and seeds collected from the 120 plants, from seeds from the seed lots sown in 2003 and 2004, and from leaf and stem tissue of 20 greenhouse-grown plants from each seed lot. The latter DNA samples were assayed for phytoplasma DNA by a nested polymerase chain reaction (PCR) assay incorporating phytoplasma universal 16S rRNA primer pairs P1/P6 (1) followed by R16R2/R16F2 (4). Seed samples analyzed from the 2003 and 2004 seed lots and tissues of the 40 greenhouse-grown plants all tested negative for phytoplasma DNA using this assay. Leaf, stem, and/or root tissues of all plants collected in the field in 2003 (60 plants) and 2004 (60 plants) and 71.1% (315 of 443) of seed samples (five seeds per sample) tested positive for the presence of phytoplasma DNA, as evidenced by the presence of an expected band of 1.2 kb on the gels after the second amplification with primers R16R2/R16F2. Nested PCR products from plant samples collected in 2003 were cloned, sequenced, and compared with phytoplasma sequences archived in the GenBank nucleotide database. On this basis, phytoplasmas detected in plants or their seeds collected in 2003 were found to be most similar (98.8%) to CHRY (Accession No. AY180956), a 16SrI-A subgroup strain, or were most similar (98.9%) to isolate 99UW89 (Accession no. AF268407), a known 16SrI-B subgroup strain. Sequences of phytoplasmas detected in plants or their seeds in 2004 were obtained by direct sequencing of rRNA products amplified from samples using PCR incorporating primer pairs P1/P6 and P4/P7 (3). Analysis of sequence data revealed that phytoplasmas in these plants were all most similar (99.5%) to AY-WB (Accession no. AY389828), a 16SrI-A subgroup member. The nucleotide sequences have been deposited with GenBank under Accession nos. DQ404346, DQ404347, and DQ411470. To our knowledge, this is the first report of 16SrI-A and 16SrI-B subgroup phytoplasmas infecting plants and seed of B. rapa in Saskatchewan. References: (1) I.-M. Lee et al. Phytopathology, 83:834, 1993. (2) W. E. Sackston. Can. Plant Dis. Surv. 33:41, 1953. (3) L. B. Sharmila et al. J. Plant Biochem. Biotech. 13:1, 2004. (4) E. Tanne et al. Phytopathology, 91:741, 2001.

One thermophilic (strain IMO-1T) and two mesophilic (strains KIBI-1T and YMTK-2T) non-spore-forming, non-motile, Gram-negative, multicellular filamentous micro-organisms, which were previously isolated as members of the tentatively named class ‘Anaerolineae’ of the phylum Chloroflexi, were characterized. All isolates were strictly anaerobic micro-organisms. The length of the three filamentous isolates was greater than 100 μm and the width was 0.3–0.4 μm for strain IMO-1T, 0.4–0.5 μm for strain KIBI-1T and thinner than 0.2 μm for strain YMTK-2T. Strain IMO-1T could grow at pH 6.0–7.5 (optimum growth at pH 7.0). The optimal temperature for growth of strain IMO-1T was around 50 °C (growth occurred between 42 and 55 °C). Growth of the mesophilic strains KIBI-1T and YMTK-2T occurred at pH 6.0–7.2 with optimal growth at pH 7.0. Both of the mesophilic strains were able to grow in a temperature range of 25–50 °C with optimal growth at around 37 °C. Yeast extract was required for growth of all three strains. All the strains could grow with a number of carbohydrates in the presence of yeast extract. The G+C contents of the DNA of strains IMO-1T, KIBI-1T and YMTK-2T were respectively 53.3, 59.5 and 48.2 mol%. Major fatty acids for thermophilic strain IMO-1T were anteiso-C17 : 0, iso-C15 : 0, C16 : 0 and anteiso-C15 : 0, whereas those for mesophilic strains KIBI-1T and YMTK-2T were branched C14 : 0, iso-C15 : 0, C16 : 0 and branched C17 : 0, and branched C17 : 0, C16 : 0, C14 : 0 and C17 : 0, respectively. Detailed phylogenetic analyses based on their 16S rRNA gene sequences indicated that the isolates belong to the class-level taxon ‘Anaerolineae’ of the bacterial phylum Chloroflexi, which for a long time had been considered as a typical uncultured clone cluster. Their morphological, physiological, chemotaxonomic and genetic traits strongly support the conclusion that these strains should be described as three novel independent taxa in the phylum Chloroflexi. Here, Anaerolinea thermolimosa sp. nov. (type strain IMO-1T=JCM 12577T=DSM 16554T), Levilinea saccharolytica gen. nov., sp. nov. (type strain KIBI-1T=JCM 12578T=DSM 16555T) and Leptolinea tardivitalis gen. nov., sp. nov. (type strain YMTK-2T=JCM 12579T=DSM 16556T) are proposed. In addition, we formally propose to subdivide the tentative class-level taxon ‘Anaerolineae’ into Anaerolineae classis nov. and Caldilineae classis nov. We also propose the subordinate taxa Anaerolineales ord. nov., Caldilineales ord. nov., Anaerolineaceae fam. nov. and Caldilineaceae fam. nov.

ABSTRACT Members of the genus Arsenophonus comprise a large group of bacterial endosymbionts that are widely distributed in arthropods of medical, veterinary, and agricultural importance. At present, little is known about the role of these bacteria in arthropods, because few representatives have been isolated and cultured in the laboratory. In the current study, we describe the isolation and pure culture of an Arsenophonus endosymbiont from the hippoboscid louse fly Pseudolynchia canariensis . We propose provisional nomenclature for this bacterium in the genus Arsenophonus as “ Candidatus Arsenophonus arthropodicus.” Phylogenetic analyses indicate that “ Candidatus Arsenophonus arthropodicus” is closely related to the Arsenophonus endosymbionts found in psyllids, whiteflies, aphids, and mealybugs. The pure culture of this endosymbiont offers new opportunities to examine the role of Arsenophonus in insects. To this end, we describe methods for the culture of “ Candidatus Arsenophonus arthropodicus” in an insect cell line and the transformation of this bacterium with a broad-host-range plasmid.