Publications

4374

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.

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.

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).