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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.

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.

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).