Publications

4364

A thermophilic, filamentous, heterotrophic bacterium, designated strain JAD2T, a member of an as-yet uncultivated lineage that is present and sometimes abundant in some hot springs worldwide, was isolated from sediment of Great Boiling Spring in Nevada, USA. Cells had a mean diameter of 0.3 µm and length of 4.0 µm, and formed filaments that typically ranged in length from 20 to 200 µm. Filaments were negative for the Gram stain reaction, spores were not formed and motility was not observed. The optimum temperature for growth was 72.5–75 °C, with a range of 67.5–75 °C, and the optimum pH for growth was 6.75, with a range of pH 6.5–7.75. Peptone, tryptone or yeast extract were able to support growth when supplemented with vitamins, but no growth was observed using a variety of defined organic substrates. Strain JAD2T was microaerophilic and facultatively anaerobic, with optimal growth at 1 % (v/v) O2 and an upper limit of 8 % O2. The major cellular fatty acids (>5 %) were C16 : 0, C19 : 0, C18 : 0, C20 : 0 and C19 : 1. The genomic DNA G+C content was 69.3 mol%. Phylogenetic and phylogenomic analyses using sequences of the 16S rRNA gene and other conserved genes placed JAD2T within the phylum Chloroflexi , but not within any existing class in this phylum. These results indicate that strain JAD2T is the first cultivated representative of a novel lineage within the phylum Chloroflexi , for which we propose the name Thermoflexus hugenholtzii gen. nov., sp. nov., within Thermoflexia classis nov., Thermoflexales ord. nov. and Thermoflexaceae fam. nov. The type strain of Thermoflexus hugenholtzii is JAD2T ( = JCM 19131T = CCTCC AB-2014030T).

In summer 2012, carrot (Daucus carota L.) plants displaying symptoms of leaf yellowing, stunting and proliferation of dwarfed shoots with bushy tops, and a dense hairy growth of secondary roots were observed. Symptomatic carrots were collected from three fields used for seed production and located in Region Centre of France near Orléans. The presence of psyllids (Psyllidae) in one of the fields was reported but they were not clearly identified. Fifty percent of the field was infected. Due to a large amount of plant debris, the harvested seeds were difficult to separate and the germination rate was low (from 10 to 77%), rendering them unmarketable. The symptoms observed were similar to those described for carrots infected by Aster yellows phytoplasma and ‘Candidatus Liberibacter solanacearum’ in Europe (3). Total DNA was extracted from petiole and root tissue of 16 symptomatic and 6 asymptomatic carrots (cv. Amsterdam, CAC3075), 2 samples of black nightshade leaves (Solanum nigrum) collected from the same fields, and 2 samples of carrot plants (cv Berlicum) grown in a high containment greenhouse, using a cetyl trimethyl ammonium bromide (CTAB) buffer extraction method. All DNA extracts were tested for phytoplasmas (1) and for ‘Ca. L. solanacearum’ by real-time PCR (2). DNA extracts were also tested for ‘Ca. L. solanacearum’ by PCR using primer pairs OA2/OI2c and CL514F/R to amplify a portion of 16S rDNA and rpIJ/rpIL ribosomal protein genes, respectively (4). DNA from greenhouse carrot plants yielded no amplicon with all PCR. Phytoplasma was not detected in any of the tested samples. However, amplification was observed with the real-time PCR assay for ‘Ca. L. solanacearum’ (2) for all DNA samples extracted from symptomatic and asymptomatic field carrots (cycle threshold [ct] values between 16.75 and 30.59), and from S. nigrum (ct between 31.62 and 33.25). For field carrot DNA, a 1,168-bp 16S rDNA fragment and a 669-bp rpIJ/rpIL fragment were amplified whereas DNA from S. nigrum yielded no amplicon. Four amplicons obtained from these PCR assays with both primer pairs from symptomatic carrot samples were sequenced directly (Beckmann Coulter Genomics, Grenoble, France). BLAST analysis of the 16S rDNA sequences (KF357911) showed 99% nucleotide identity to those of ‘Ca. L. solanacearum’ amplified from carrot in Finland (GU373049). The rpIJ/rpIL nucleotide sequences (KF357912) were 99% identical to sequences of the analogous rpIJ/rpIL ‘Ca. L. solanacearum’ ribosomal protein gene from carrot in Spain (JX308305). These results confirmed the presence of ‘Ca. L. solanacearum’ in all symptomatic and asymptomatic carrot sampled in Region Centre, France. To our knowledge, this is the first report of this pathogen in carrot in France. These results, in addition to those previously obtained (4), suggest a wider distribution of ‘Ca. L. solanacearum’ than previously reported in Europe and should lead plant health managers to consider this pathogen as an emerging threat. References: (1) N. M. Christensen et al. Mol. Plant Microbe Interact. 17:1175, 2004. (2) W. Li et al. J. Microbiol. Methods 78:59, 2009. (3) J. E. Munyaneza et al. Plant Dis. 94:639, 2010. (4) J. E. Munyaneza et al. Plant Dis. 96:453, 2012.

The genus Synechococcus represents an enigmatic group of cyanobacteria with very simple unicellular morphology and polyphyletic evolutionary origin. Here, we describe a new genus based on strain of Synechococcus-like cyanobacterium. The strain was isolated from the peat bog Klin (Slovakia), where it occupies different niches such as hyaline cells of Sphagnum, sheaths of cyanobacteria, dead cells of desmids, carapaces of dead crustaceans, and solitary in detritus. We describe this new genus using a combination of molecular, morphological and ecological features. A phylogeny of the 16S rRNA gene, 16S-23S ITS and rbcL loci showed a separate position of the investigated strain and its close proximity to filamentous cyanobacteria. Therefore, it is a novel lineage of Synechococcus-like cyanobacteria illustrating the polyphyletic nature of the genus Synechococcus. Moreover, the strain exhibits unique morphological and ecological features, which allow us to erect the new monospecific genus Neosynechococcus.

DNA of several spotted fever group rickettsiae was found in ticks in Israel. The findings include evidence for the existence of Rickettsia africae and Candidatus Rickettsia barbariae in ticks in Israel. The DNA of R. africae was detected in a Hyalomma detritum tick from a wild boar and DNA of C. Rickettsia barbariae was detected in Rhipicephalus turanicus and Rhipicephalus sanguineus collected from vegetation. The DNA of Rickettsia massiliae was found in Rh. sanguineus and Haemaphysalis erinacei, whereas DNA of Rickettsia sibirica mongolitimonae was detected in a Rhipicephalus (Boophilus) annulatus. Clinicians should be aware that diseases caused by a variety of rickettsiae previously thought to be present only in other countries outside of the Middle East may infect residents of Israel who have not necessarily traveled overseas. Furthermore, this study reveals again that the epidemiology of the spotted fever group rickettsiae may not only involve Rickettsia conorii but may include other rickettsiae.

ABSTRACT “ Candidatus Synechococcus spongiarum” is a cyanobacterial symbiont widely distributed in sponges, but its functions at the genome level remain unknown. Here, we obtained the draft genome (1.66 Mbp, 90% estimated genome recovery) of “ Ca. Synechococcus spongiarum” strain SH4 inhabiting the Red Sea sponge Carteriospongia foliascens . Phylogenomic analysis revealed a high dissimilarity between SH4 and free-living cyanobacterial strains. Essential functions, such as photosynthesis, the citric acid cycle, and DNA replication, were detected in SH4. Eukaryoticlike domains that play important roles in sponge-symbiont interactions were identified exclusively in the symbiont. However, SH4 could not biosynthesize methionine and polyamines and had lost partial genes encoding low-molecular-weight peptides of the photosynthesis complex, antioxidant enzymes, DNA repair enzymes, and proteins involved in resistance to environmental toxins and in biosynthesis of capsular and extracellular polysaccharides. These genetic modifications imply that “ Ca. Synechococcus spongiarum” SH4 represents a low-light-adapted cyanobacterial symbiont and has undergone genome streamlining to adapt to the sponge’s mild intercellular environment. IMPORTANCE Although the diversity of sponge-associated microbes has been widely studied, genome-level research on sponge symbionts and their symbiotic mechanisms is rare because they are unculturable. “ Candidatus Synechococcus spongiarum” is a widely distributed uncultivated cyanobacterial sponge symbiont. The genome of this symbiont will help to characterize its evolutionary relationship and functional dissimilarity to closely related free-living cyanobacterial strains. Knowledge of its adaptive mechanism to the sponge host also depends on the genome-level research. The data presented here provided an alternative strategy to obtain the draft genome of “ Ca. Synechococcus spongiarum” strain SH4 and provide insight into its evolutionary and functional features.

Huanglongbing is an unculturable vascular citrus pathogen transmitted from infected to healthy plants through grafting or by citrus psyllids, Diaphorina citri mainly in Asia and America and Trioza erytreae in Africa. This phloem limited gram-negative bacterium causes dramatic yield losses and is classified into three species based on 16S rDNA sequence analysis (2): (i) ‘Candidatus Liberibacter asiaticus’ (Las), the most epidemiologically active, widespread and heat tolerant species; (ii) ‘Ca. L. africanus’ (Laf), only found in Africa; and (iii) the newly described ‘Ca. L. americanus’ (Lam), which appeared in 2005 in Brazil (5). Considered as a quarantine organism in America and Europe, Las is actively affecting North America and Asia, and research is leading toward psyllid management and resistance breeding. Despite the fact that Reunion Island has successfully controlled Las by introducing a psyllid parasitoid, Tamarixia radiata (1), this strategy was less effective or reproducible within other territories. D. citri was first detected in Guadeloupe in 1998, where the control of the the psyllid population has been effective with T. radiata (3); and was first detected in Martinique in 2012. Following the outbreak in the United States and the Caribbean, and also supported by reports of symptoms in citrus orchards, local National Plant Protection Organizations (NPPO) organized a detection survey across both islands to verify the occurrence of Huanglongbing. Since 2012, 450 sites were prospected each year in Martinique and Guadeloupe, where 20 leaves from 10 to 30 trees were analyzed. DNA extraction was performed (DNeasy Plant Mini Kit, Qiagen) on fresh or dried leaf midribs, along with negative control midribs (Citrus paradisi ‘Star Rubis’) and PCR amplification was done with the species-specific primers A2/J5 (4) and GB1/GB3 (5). Only Las-specific 703-bp amplicons were obtained (n = 43) and 20 were sequenced (Beckman Coulter Genomics, United Kingdom; sequences available through GenBank Accession Nos. KF699074 to KF699093) and blasted against the National Center for Biotechnology Information non-redondant database (NCBI-nr). BLAST analysis revealed 100% identity with the 50S ribosomal protein subunit L1 (rplA) and L10 (rplJ) of ‘Ca. L. asiaticus’ (all strains), and no significant homology to other organisms. Additionally, sequence assembly on a reference genome (NC_012985) showed 100% homology. Huanglongbing was detected in Guadeloupe on March 2012 at Le Moule (East coast) in a Tahiti lime orchard (C. latifolia) and crossed the island in 6 months. Las was detected in Martinique on May 2013 on Tahiti lime (C. latifolia) at Bellefontaine (Northwest) in a private garden and at Le Lorrain (Northeast) in an orchard. Other species from the Rutaceae family were affected by HLB (C. reticulat and C. sinensis) on both islands; however, few of the positive samples showed HLB symptoms (blotchy mottle patterns and green islands on leaves), but presented symptoms similar to nutrient deficiencies. Despite the former presence of T. radiata in Guadeloupe and its detection in Martinique a few weeks after the detection of D. citri, where it had a mean parasitism rate of 70%, an outbreak of HLB spread across both islands. These analyses confirm the presence of HLB in Martinique and Guadeloupe and to our knowledge represent the first report of Las in the French West Indies. Introduction events remain unclear, but this report raises the importance of plant certification, psyllid population control, and surveillance of territories close to the French West Indies, with regards to the risk that HLB presents to citrus production worldwide. References: (1) B. Aubert et al. Fruits. 38, 1983. (2) J. M. Bové. J. Plant Pathol. 88:1, 2006. (3) J. Etienne et al. Fruits. 56:05, 2001. (4) A. Hocquellet et al. Mol. Cell. Probes 13:5, 1999. (5) D. C. Teixeira et al. Mol. Cell. Probes 19:3, 2005.