Publications

4797

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.

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 .

ABSTRACT Soil nitrification plays an important role in the reduction of soil fertility and in nitrate enrichment of groundwater. Various ammonia-oxidizing archaea (AOA) are considered to be members of the pool of ammonia-oxidizing microorganisms in soil. This study reports the discovery of a chemolithoautotrophic ammonia oxidizer that belongs to a distinct clade of nonmarine thaumarchaeal group I.1a, which is widespread in terrestrial environments. The archaeal strain MY2 was cultivated from a deep oligotrophic soil horizon. The similarity of the 16S rRNA gene sequence of strain MY2 to those of other cultivated group I.1a thaumarchaeota members, i.e., Nitrosopumilus maritimus and “ Candidatus Nitrosoarchaeum koreensis,” is 92.9% for both species. Extensive growth assays showed that strain MY2 is chemolithoautotrophic, mesophilic (optimum temperature, 30°C), and neutrophilic (optimum pH, 7 to 7.5). The accumulation of nitrite above 1 mM inhibited ammonia oxidation, while ammonia oxidation itself was not inhibited in the presence of up to 5 mM ammonia. The genome size of strain MY2 was 1.76 Mb, similar to those of N. maritimus and “ Ca . Nitrosoarchaeum koreensis,” and the repertoire of genes required for ammonia oxidation and carbon fixation in thaumarchaeal group I.1a was conserved. A high level of representation of conserved orthologous genes for signal transduction and motility in the noncore genome might be implicated in niche adaptation by strain MY2. On the basis of phenotypic, phylogenetic, and genomic characteristics, we propose the name “ Candidatus Nitrosotenuis chungbukensis” for the ammonia-oxidizing archaeal strain MY2.

In this study, the taxonomic position and group classification of the phytoplasma associated with a lethal yellowing-type disease (LYD) of coconut (Cocos nucifera L.) in Mozambique were addressed. Pairwise similarity values based on alignment of nearly full-length 16S rRNA gene sequences (1530 bp) revealed that the Mozambique coconut phytoplasma (LYDM) shared 100 % identity with a comparable sequence derived from a phytoplasma strain (LDN) responsible for Awka wilt disease of coconut in Nigeria, and shared 99.0–99.6 % identity with 16S rRNA gene sequences from strains associated with Cape St Paul wilt (CSPW) disease of coconut in Ghana and Côte d’Ivoire. Similarity scores further determined that the 16S rRNA gene of the LYDM phytoplasma shared <97.5 % sequence identity with all previously described members of ‘Candidatus Phytoplasma ’. The presence of unique regions in the 16S rRNA gene sequence distinguished the LYDM phytoplasma from all currently described members of ‘Candidatus Phytoplasma ’, justifying its recognition as the reference strain of a novel taxon, ‘Candidatus Phytoplasma palmicola’. Virtual RFLP profiles of the F2n/R2 portion (1251 bp) of the 16S rRNA gene and pattern similarity coefficients delineated coconut LYDM phytoplasma strains from Mozambique as novel members of established group 16SrXXII, subgroup A (16SrXXII-A). Similarity coefficients of 0.97 were obtained for comparisons between subgroup 16SrXXII-A strains and CSPW phytoplasmas from Ghana and Côte d’Ivoire. On this basis, the CSPW phytoplasma strains were designated members of a novel subgroup, 16SrXXII-B.

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.