Publications

4493

‘Candidatus Phytoplasma prunorum,’ which causes European stone fruit yellows (ESFY), is the prevalent phytoplasma affecting Prunus spp. in Europe. It is closely related to ‘Ca. P. pyri,’ which causes pear decline (PD) in pear trees. Both phytoplasma belong to the ribosomal group 16Sr-X and are naturally transmitted by different species of Cacopsylla spp. (4). In North America, ‘Ca. P. pyri’ is responsible for peach yellow leaf roll (PYLR), transmitted by Cacopsylla pyricola from pear to peach trees (1). In Spain, ‘Ca. P. prunorum’ is widespread on Prunus spp., but its occurrence on Prunus persicae is very low and ‘Ca. P. pyri’ is present in every pear orchard (3). During 2012, a previously unreported syndrome including early reddening, leaf curling, decline, abnormal fruits, and in some cases chlorosis and death of peach trees was reported on peach in Lleida, northern Spain. Symptoms were different to ESFY and PYLR, in that flowering disorders such as ESFY or yellows were not apparent, and reddening and decline were the most common symptoms. The disease was present in a wide range of varieties and rootstocks, suggesting insect transmission in an area where C. pruni, vector of ‘Ca. P. prunorum,’ was not previously reported, but C. pyri was abundant in pear orchards. Shoot samples from 20 symptomatic peach trees were collected in seven orchards within a 2 km2 area with an estimated incidence of 40%, which was higher in the borders. DNA was extracted from 1 g of leaf midribs and phloem tissue and amplified with ribosomal universal primers P1/P7 followed by nested PCR with R16F2n/R16R2 and specific primers fO1/rO1 that target the 16Sr-X group (3). The final PCR products were digested with RsaI enzyme. Amplifications with non-ribosomal specific primers, Imp ESFY, Imp PD A and Imp PD B that amplify sequences of gene Imp, that encode a phytoplasma membrane protein, were also carried out (2). Tissue samples with ESFY and PD and peach seedlings were used as positive and negative controls, respectively. Amplified PCR products were sequenced and compared to sequences deposited in GenBank. Phytoplasmas were detected in 18 of the 20 samples analyzed. No phytoplasmas were detected in negative peach controls. All digestions of fO1/rO1 PCR products from peach samples showed a PD profile, while no ESFY profile was detected. All samples were positive with specific primers Imp PD A and B. None of the peach samples were positive with the specific Imp-ESFY primers. Sequencing of R16 and Imp PDA and B amplicons revealed the presence of a stable isolate. The sequences were submitted to the European nucleotide archive (ENA) with the accession nos. HG737345 and HG737344. Based on the 16S rDNA sequence, this strain is 100% homologous to the reference strain PD1 (GenBank Accession No. AJ542543) and 99.55% homologous to strain PD 33 Lib (GenBank FN600725) based on the Imp gene sequence. This is the first report of PD phytoplasma in peach trees in Spain, and the first report in Europe of PD phytoplasma causing economically important outbreaks in peach orchards, following a pattern that could be similar to PYLR in North America. This strain is genetically closer to some European or Middle Eastern PDs than to North American PYLR. References: (1) C. L. Blomquist et al. Plant Dis. 86:759, 2002. (2) J. L. Danet et al. Microbiology 157:438, 2011. (3) M. Garcia-Chapa et al. J. Phytopathol. 151:584, 2003. (4) E. Seemüller et al. Int. J. Syst. Evol. Microbiol. 54:1217, 2004.

We investigated the bacterial community compositions and phosphorus removal performance under sludge bulking and non-bulking conditions in two biological wastewater treatment systems (conventional A2/O (anaerobic/anoxic/aerobic) and inverted A2/O (anoxic/anaerobic/aerobic) processes) receiving the same raw wastewater. Sludge bulking resulted in significant shift in bacterial compositions from Proteobacteria dominance to Actinobacteria dominance, characterized by the significant presence of filamentous ‘Candidatus Microthrix parvicella’. Quantitative real-time polymerase chain reaction (PCR) analysis revealed that the relative abundance of ‘Candidatus Accumulibacter phosphatis’, a key polyphosphate-accumulating organism responsible for phosphorus removal, with respect to 16s rRNA genes of total bacteria was 0.8 and 0.7%, respectively, for the conventional and inverted A2/O systems when sludge bulking occurred, which increased to 8.2 and 12.3% during the non-bulking period. However, the total phosphorus removal performance during the bulking period (2-week average: 97 ± 1 and 96 ± 1%, respectively) was not adversely affected comparable to that during the non-bulking period (2-week average: 96 ± 1 and 96 ± 1%, respectively). Neisser staining revealed the presence of large polyphosphate granules in ‘Candidatus Microthrix parvicella’, suggesting that this microbial group might have been responsible for phosphorus removal during the sludge bulking period when ‘Candidatus Accumulibacter phosphatis’ was excluded from the systems.

Corn reddening (CR) or maize redness is a severe disease of corn (Zea mays L.) associated with ‘Candidatus Phytoplasma solani’ or stolbur phytoplasma (16SrXII-A). In Serbia, CR is continually present at a low frequency, while two outbreaks occurred in the late 1950s and 1990s. Its etiology was molecularly determined in 2006 (1). The first severe outbreak in Bulgaria was observed in Kneja in 1992, and in 2010 typical CR symptoms (leaf reddening, premature drying, and shriveled grains) were observed from Byala Slatina to Pleven. Although the number of CR affected plants was highly variable in different fields, the disease incidence in most cases was 30 to 50%, with an estimated yield reduction of about 20%. Leaf samples from four symptomatic corn plants were collected from Kneja, northwestern Bulgaria, in mid-August 2013. Extraction of DNA was performed from the main leaf midrib tissues using the CTAB method. Separate PCRs were carried out for amplification of the phytoplasma 16S rDNA and tuf genes using the phytoplasma specific primers P1/P7 and TufAyf/r, respectively. DNA from asymptomatic corn plants and reactions without template DNA were employed as negative controls, while DNA from periwinkle tissue infected with ‘Ca. P. asteris’ was used as a positive control. Amplicons of the expected sizes (1.7 and 0.9 kbp, respectively) were produced with DNA from three out of four symptomatic corn samples, while no amplification was observed with DNA from one symptomatic corn sample (probably because samples were collected during a drought period) nor the DNA from asymptomatic plants and negative control. RFLP analyses performed on 16S rDNA and tuf gene amplicons using Tru1I and HpaII restriction enzymes, respectively, revealed the presence of ‘Ca. P. solani’ (16SrXII-A, tuf type b) in all three positive samples (3). Both amplicons of a selected representative sample 241/13 were directly sequenced by a commercial service and the obtained sequences were deposited in NCBI GenBank under the accession number KF907506 for the16S rDNA (1,684 bp) and KF907507 for the tuf gene (896 bp). The 16S rDNA sequence of phytoplasma detected in Bulgarian corn shared a complete sequence identity with ‘Ca. P. solani’ strain from Serbian corn (JQ730750.1) and >99.7% sequence identity with the reference strain STOL (AF248959), while the tuf gene nucleotide sequence shared complete sequence identity with several ‘Ca. P. solani’ (e.g., Serbian strain 284/09, FO393427), thus confirming, with both genes, the affiliation of phytoplasmas in Bulgarian corn to ‘Ca. P. solani’. This study adds new information on CR prevalence, previously reported in neighboring countries. Further studies will investigate the roles of Hyalesthes obsoletus and Reptalus panzeri, both polyphagous Cixiidae reported as CR vectors (2,4) in disease transmission in Bulgaria. References: (1) B. Duduk and A. Bertaccini. Plant Dis. 90:1313, 2006. (2) J. Jović et al. Eur. J. Plant Pathol. 118:85, 2007. (3) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (4) N. Mori et al. Bull. Insectol. 66:245, 2013.

Wild indigo (Tephrosia purpurea (L.) Pers.) grows as a common weed throughout the Indian subcontinent. The plant has pinnate leaves, white or purplish flowers, and flat hairy pods, and is cultivated as a green manure crop. The plant extracts contain compounds such as tephrosin, an aromatic ester, prenylated flavonoid, and sesquiterpene (2) that have medicinal properties. The newly recognized disease, Tephrosia purpurea witches' broom (TPWB), was characterized by chlorosis, stunting, and proliferative branching, which were suggestive of phytoplasma infection during a field survey conducted in November 2013. To determine the presence of phytoplasma, 2 g of compound leaves from three symptomatic and asymptomatic plants were used for total DNA extraction using the CTAB method. The phytoplasma 16S rRNA gene was detected in all three symptomatic plants using nested PCR with universal phytoplasma primer pairs, P1/P7 followed by R16F2n/R16R2 (4). No amplification was observed in DNA isolated from asymptomatic plants. PCR fragments (1,246 bp in length) generated from symptomatic T. purpurea plants were sequenced directly using five different primers viz. 343R, 536F, 704F, 907R, and 1103F. TPWB phytoplasma 16S rRNA gene sequence (GenBank Accession No. HG792252) showed 99.12% homology with a ‘Candidatus Phytoplasma aurantifolia’ strain WBDL (U15442) when compared using the EzTaxon 16S rRNA database (3). Virtual restriction fragment length polymorphism (RFLP) analysis was carried out on the obtained sequence using iPhyClassifier (5). The virtual RFLP pattern derived from the HG792252 sequence was different to the reference patterns of previously established 16Sr groups and subgroups. The reference pattern of the 16Sr group II, subgroup C (AJ293216) was most similar with a similarity coefficient of 0.92, which placed it in a new subgroup, 16Sr II-M (1). Furthermore, virtual RFLP results were confirmed by digesting R16F2n/R16R2 amplicon with BstUI, DraI, HinfI, HpaI, and MseI restriction enzymes according to manufacturer's instructions. To our knowledge, this is the first report of a ‘Ca. P. aurantifolia’-related strain associated with witches'-broom disease of T. purpurea in India. References: (1) H. Cai et al. Int. J. Syst. Evol. Microbiol. 58:1448, 2008. (2) A. K. Khalafalah et al. Pharmacognosy Res. 2:72, 2010. (3) O.-S. Kim et al. Int. J. Syst. Evol. Microbiol. 62:716, 2012. (4) C. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996. (5) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

Citrus huanglongbing (HLB) is a century-old destructive disease which presents an unprecedented challenge to citrus industries worldwide. In Florida, HLB is associated with the phloem-limited bacterium ‘Candidatus Liberibacter asiaticus’ and is mainly transmitted by Asian citrus psyllid (Diaphorina citri). Quantification of the pathogen population in a host aids in investigation of virulence mechanisms and disease management. Recently a procedure was developed to detect live bacterial populations using a novel DNA-binding dye, propidium monoazide, in conjunction with real-time polymerase chain reaction (PMA-qPCR). Chinese box orange (Severinia buxifolia) is a common ornamental present in Florida which could host D. citri and ‘Ca. L. asiaticus’. For 20 months, the change of the live ‘Ca. L. asiaticus’ populations in graft- and psyllid-transmitted Valencia sweet orange (Citrus sinensis ‘Valencia’) and S. buxifolia plants was monitored by PMA-qPCR. Our results showed that the live ‘Ca. L. asiaticus’ population was significantly lower in the months of December, January, and February than the rest of the year in both hosts. No statistically significant pattern in the total bacterial population was observed in either host. This pattern may indicate a seasonal growth of ‘Ca. L. asiaticus’ along with the growth of both plants. These new findings should provide useful information on HLB management.

ABSTRACT Members of the Cronobacter genus are associated with serious infections in neonates. This is the first report of the draft genome sequence for the newly proposed species Cronobacter colletis .

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.