Publications

4373

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.

One thermophilic (strain IMO-1T) and two mesophilic (strains KIBI-1Tand 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 phylumChloroflexi, 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-1Tand thinner than 0.2 μm for strain YMTK-2T. Strain IMO-1Tcould grow at pH 6.0–7.5 (optimum growth at pH 7.0). The optimal temperature for growth of strain IMO-1Twas around 50 °C (growth occurred between 42 and 55 °C). Growth of the mesophilic strains KIBI-1Tand YMTK-2Toccurred 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-1Tand YMTK-2Twere respectively 53.3, 59.5 and 48.2 mol%. Major fatty acids for thermophilic strain IMO-1Twere anteiso-C17 : 0, iso-C15 : 0, C16 : 0and anteiso-C15 : 0, whereas those for mesophilic strains KIBI-1Tand YMTK-2Twere branched C14 : 0, iso-C15 : 0, C16 : 0and branched C17 : 0, and branched C17 : 0, C16 : 0, C14 : 0and 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 phylumChloroflexi, 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 phylumChloroflexi. Here,Anaerolinea thermolimosasp. nov. (type strain IMO-1T=JCM 12577T=DSM 16554T),Levilinea saccharolyticagen. nov., sp. nov. (type strain KIBI-1T=JCM 12578T=DSM 16555T) andLeptolinea tardivitalisgen. 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’ intoAnaerolineaeclassis nov. andCaldilineaeclassis nov. We also propose the subordinate taxaAnaerolinealesord. nov.,Caldilinealesord. nov.,Anaerolineaceaefam. nov. andCaldilineaceaefam. nov.

ABSTRACT A novel bacterial strain that was isolated from an Italian soil and was designated SOSP1-21 T forms branched mycelia in solid and liquid media and has a filamentous morphology similar to that of some genera belonging to the Actinobacteria . Electron microscopy showed that this organism has a grape-like appearance, resulting from interlacing of spores originating from sporophoric hyphae. Ten strains that are morphologically related to SOSP1-21 T were recovered from soil. Phylogenetic analyses of 16S rRNA gene segments confirmed the relatedness of these strains to SOSP1-21 T and indicated that the newly isolated strains form separate clades in a deeply branching lineage. The closest matches for the 16S rRNA sequences of all the strains (around 79% identity) were matches with representatives of the Chloroflexi , although the affiliation with this division was not supported by high bootstrap values. The strains are mesophilic aerobic heterotrophs and are also capable of growing under microaerophilic conditions. They all stain gram positive. Strain SOSP1-21 T contains ornithine, alanine, glutamic acid, serine, and glycine as the peptidoglycan amino acids. In addition, an unusual level of C16:1 2OH (30%) was found in the cellular fatty acids. The G+C content of SOSP1-21 T genomic DNA is 53.9%, and MK-9(H 2 ) was the only menaquinone detected. All these data suggest that SOSP1-21 T and the related strains may constitute a new division of filamentous, spore-forming, gram-positive bacteria. We propose the name Ktedobacter racemifer gen. nov., sp. nov. for strain SOSP1-21 T .

“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.

Although no loss of crown shape or unusual growth were evident on two mature Chinaberry trees (Melia azedarach L.) located near the citadel in central Hué city, Vietnam, leaves on both trees displayed distinctive interveinal yellowing during September 2003. This symptom was reminiscent in appearance to foliar discoloration previously observed on mature Chinaberry trees in El Torno, Santa Cruz, Bolivia that was subsequently attributed to phytoplasma infection of these trees (2). Eight samples of yellowed leaves were collected from affected trees and preserved by pressing and drying for later analysis. Total nucleic acids were extracted from 0.5 g of each leaf sample and assayed for phytoplasma DNA using a polymerase chain reaction (PCR) assay with phytoplasma universal rRNA primer pair P1 and P7 (4). No visible product was generated from any Chinaberry sample while a product of expected size (1.8 kb) was obtained from DNA of a periwinkle plant (Catharanthus roseus (L.) G. Don) infected with “Candidatus Phytoplasma asteris”-related strain eastern aster yellows (EAY) and included as a known positive control in the assay. After P1/P7-primed products were reamplified by PCR with nested phytoplasma universal 16S rRNA primer pair R16mF2/R16mR1 (1), a 1.4-kb product of predicted size was obtained from the eight samples and EAY positive control, whereas no product was obtained from DNA of seed-grown healthy periwinkle included as a negative control. Digests of nested PCR products (1.4 kb) with HaeIII or MseI endonuclease, and electrophoresis of digests through 8% polyacrylamide gels, revealed no apparent differences in restriction fragment patterns among products from Chinaberry samples. However, HaeIII and MseI patterns differed from those obtained by digestion of nested PCR products from EAY, a known 16SrI-A subgroup phytoplasma (3), with these enzymes. Chinaberry phytoplasmas were definitively identified as group 16SrI strains after reevaluation of samples by a PCR incorporating ribosomal protein (rp) gene primer pair rpF1/rpR1 and reamplification of resulting products with nested 16SrI group-specific primer pair rp(I)F1A/rp(I)R1A (3). A 1.2-kb product of expected size was obtained from all Chinaberry samples and EAY positive control only. Restriction fragment length polymorphism patterns produced by DraI or SspI endonuclease digests of nested PCR products (1.2 kb) revealed no differences among Chinaberry samples, although patterns associated with each enzyme differed from those observed for the EAY positive control. Sequence comparison and phylogenetic analysis of Chinaberry yellows phytoplasma (CbY-V) 16Sr DNA (GenBank Accession No. AY863003) determined this strain to be most closely related (99.65%) to Epilobium phyllody phytoplasma, a 16SrI-B subgroup strain (3). However, based on analysis of rp gene sequences (GenBank Accession No. DQ321823), strain CbY-V was judged most similar (99.59%) to cabbage proliferation, a well characterized 16SrI-B subgroup, rpI-B subgroup phytoplasma (3). To our knowledge, this is the first record of phytoplasma infection of Chinaberry, a common urban shade tree in Vietnam. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) N. A. Harrison et al. Plant Pathol. 52:147, 2003. (3) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004 (4) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.