Publications

4792

During a survey in 2003, safflower plants (Carthamus tinctorius) with phyllody symptoms were observed in production fields in several districts of Fars and Yazd provinces in Iran. Affected plants showed floral virescence, phyllody, proliferation of axillary buds, and little leaf symptoms. Incidence of the disease was less than 10%. Direct and nested PCR assays were used to verify association of phytoplasma with the disease. Total DNA was extracted from fresh, fine roots of eight phyllody-affected safflower plants and one symptomless plant. With phytoplasma universal primer pair P1/P7 (5′-AAGAGTTTGATCCTGGCTCAGGATT-3′/5′-CGTCCTTCATCGGCTCTT-3′), target DNA fragments of approximately 1.8 kb were amplified by direct PCR from phyllody-affected plants and Iranian cabbage yellows (ICY) phytoplasma used as a positive control. Reamplification of P1/P7 products with 16S rRNA gene primer pair R16F2n/R16R2 (5′-GAAACGACTGCTAAGACTGG-3′/5′-TGACGGGCGGTGTGTACAAACCCCG-3′) yielded fragments of the expected size (1.2 kb) from all eight diseased plants and the ICY-positive control. No products were amplified from the symptomless plant by either assay. R16F2n/R16R2 products were subjected to restriction fragment length polymorphism (RFLP) analysis by separate digestion with AluI, HaeIII, HhaI, HinfI, HpaII, MseI, RsaI, Sau3AI, or TaqI endonuclease. Comparison of resulting RFLP patterns with published patterns of other phytoplasmas (2) tentatively identified safflower phyllody (SP) phytoplasma as a member of clover proliferation group 16SrVI, subgroup C. HhaI digests also differentiated SP from ICY phytoplasma, a previously reported subgroup 16SrVI-A strain (3). After sequencing of the 16S rDNA fragment (GenBank Accession No. DQ88948), a BLAST search determined that SP phytoplasma shared closest homology with 16SrVI group members (‘Candidatus Phytoplasma trifolii’) and related strains (4). Furthermore, phylogenetic analysis of 16S rDNA sequences revealed SP phytoplasma to be most similar (99.7%) to brinjal little leaf (BLL) phytoplasma (GenBank Accession No. X83431). Analysis of putative restriction sites in 16S rRNA gene sequences revealed that SP and BLL shared identical restriction profiles and that both differed from the ‘Ca. Phytoplasma trifolii’ reference strain (GenBank Accession No. AY390261) because of the absence of a single HhaI site and the presence of an additional MseI site. Although safflower phyllody disease has been previously reported in Israel, the associated phytoplasma was classified as a strain of the aster yellows subgroup 16SrI-B (1). To our knowledge, this is the first report of safflower as a host of a ‘Ca. Phytoplasma trifolii’-related strain. References: (1) M. Klein. Plant Dis. Rep. 54:735, 1970. (2) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004. (3) M. Salehi et al. Plant Dis. 91:625, 2007. (4) K. Wang and C. Hiruki, Phytopathology 91:546, 2001.

ABSTRACTThe identity and ecophysiology of a group of uncultured protein-hydrolyzing epiphytic rods attached to filamentous bacteria in activated sludge from nutrient removal plants were investigated by using the full-cycle rRNA approach combined with microautoradiography and histochemical staining. The epiphytic group consists of three closely related clusters, each containing 11 to 16 clones. The closest related cultured isolate is the type strainHaliscomenobacter hydrossis(ATCC 27775) (<87% similarity) in the familySaprospiraceaeof the phylumBacteroidetes. Oligonucleotide probes at different hierarchical levels were designed for each cluster and used for ecophysiological studies. All three clusters behaved similarly in their physiology and were specialized in protein hydrolysis and used amino acids as energy and carbon sources. They were not involved in denitrification. No storage of polyphosphate and polyhydroxyalkanoates was found. They all colonized probe-defined filamentous bacteria belonging to the phylaChloroflexi,Proteobacteria, and candidate phylum TM7, with the exception of cluster 1, which did not colonize TM7 filaments. The three epiphytic clusters were all widespread in domestic and industrial wastewater treatment plants with or without biological phosphorus removal, constituting, in total, up to 9% of the bacterial biovolume. A new genus, “CandidatusEpiflobacter,” is proposed for this epiphytic group in activated-sludge treatment plants, where it presumably plays an important role in protein degradation.

ABSTRACT Microscopic examination of the hemolymph from diseased daphniids in 17 lakes in southwestern Michigan and five rock pools in southern Finland revealed the presence of tightly coiled bacteria that bore striking similarities to the drawings of a morphologically unique pathogen, “ Spirobacillus cienkowskii ,” first described by Elya Metchnikoff more than 100 years ago. The uncultivated microbe was identified as a deeply branching member of the Deltaproteobacteria through phylogenetic analyses of two conserved genes: the 16S rRNA-encoding gene ( rrs ) and the β-subunit of topoisomerase ( gyrB ). Fluorescence in situ hybridization confirmed that the rRNA gene sequence originated from bacteria with the tightly coiled morphology. Microscopy and PCR amplification with pathogen-specific primers confirmed infections by this bacterium in four species of Daphnia : Daphnia dentifera, D. magna, D. pulicaria , and D. retrocurva . Extensive field surveys reveal that this bacterium is widespread geographically and able to infect many different cladoceran species. In a survey of populations of D. dentifera in lakes in Michigan, we found the bacterium in 17 of 18 populations studied. In these populations, 0 to 12% of the individuals were infected, with an average of 3% during mid-summer and early autumn. Infections were less common in rock pool populations of D. magna in southern Finland, where the pathogen was found in 5 of 137 populations. The broad geographic distribution, wide host range, and high virulence of S. cienkowskii suggest it plays an important role in the ecology and evolution of daphniids.

Summary The widespread occurrence and diversity of ammonia oxidizing Archaea suggests their contribution to the nitrogen cycle is of global significance. Their distribution appeared limited to low‐ and moderate‐temperature environments until the recent finding of a diagnostic membrane lipid, crenarchaeol, in terrestrial hot springs. We report here the cultivation of a thermophilic nitrifier (‘ Candidatus Nitrosocaldus yellowstonii’), an autotrophic crenarchaeote growing up to 74°C by aerobic ammonia oxidation. The major core lipid of this archaeon growing at 72°C is crenarchaeol, providing the first direct evidence for its synthesis by a thermophile. These findings greatly extend the upper temperature limit of nitrification and document that the capacity for ammonia oxidation is broadly distributed among the Crenarchaeota .

Summary The facultative symbiont of Riftia pachyptila , named here Candidatus Endoriftia persephone , has evaded culture to date, but much has been learned regarding this symbiosis over the past three decades since its discovery. The symbiont population metagenome was sequenced in order to gain insight into its physiology. The population genome indicates that the symbionts use a partial Calvin–Benson Cycle for carbon fixation and the reverse TCA cycle (an alternative pathway for carbon fixation) that contains an unusual ATP citrate lyase. The presence of all genes necessary for heterotrophic metabolism, a phosphotransferase system, and dicarboxylate and ABC transporters indicate that the symbiont can live mixotrophically. The metagenome has a large suite of signal transduction, defence (both biological and environmental) and chemotaxis mechanisms. The physiology of Candidatus Endoriftia persephone is explored with respect to functionality while associated with a eukaryotic host, versus free‐living in the hydrothermal environment.

Atalantia buxifolia (Poir.) Oliv., synonym Severinia buxifolia (Poir.) Ten. as commonly found in literature, is a common landscape plant and a popular Chinese medicinal herb known as Jiubingle or Dongfengjie. It remains unclear if this rutaceous plant could host ‘Candidatus Liberibacter asiaticus’, the pathogen of citrus Huanglongbing (HLB) in Guangdong, P. R. China. This information is important for HLB control in citrus because infected A. buxifolia could serve as a source of inoculum. In August of 1994, three A. buxifolia plants adjacent to a citrus experimental orchard of the South China Agricultural University at Guangzhou were found showing leaf mottle/yellowing symptoms. Two buds from each plant were grafted onto three mandarin trees (Citrus reticulata cv. Pongan) in a psyllid-proof screenhouse for indexing. By October of 1995, typical leaf mottle symptoms were observed in all three grafted trees compared with a healthy control. In March of 1996, one of the A. buxifolia plants was transferred to a screenhouse and has been maintained there. The leaf mottle/yellowing symptoms persisted but did not significantly affect plant growth. DNA was extracted from leaf samples in October 2006 by using the CTAB (cetyltrimethylammoniumbromide) method and assayed by nested-PCR using the general bacterial 16S rDNA primer set fDl/rD1 as the first round of amplification and primer set OI1/OI2c as second round amplification (1,3). After agarose gel electrophoresis and staining with ethidium bromide, an approximate 1.1-kb DNA band was detected in symptomatic samples but not healthy leaf samples of A. buxifolia and C. reticulata. XbaI digestion of the amplicons yielded approximate 500- and 600-bp fragments, characteristic of ‘Ca. L. asiaticus’. Similarly, a standard PCR with primer set A5/J2 (3) yielded an approximate 700-bp DNA band characteristic of ‘Ca. L. asiaticus’ from symptomatic samples only. To our knowledge, this is the first report of graft transmission and PCR detection of ‘Ca. L. asiaticus’ from A. buxifolia in Guangdong, P. R. China. This work also confirms the findings from Taiwan (2) that A. buxifolia could serve as a source of ‘Ca. L. asiaticus’. References: (1) X. Deng et al. Online publication. doi:10.1094/PHP-2007-0419-01-BR. Plant Health Progress, 2007. (2) T.-H. Hung et al. Eur. J. Plant Pathol. 107:183, 2001. (3) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996.