Publications

4474

A novel Gram-stain-positive, rod shaped and anaerobic bacterium, designated as W6T, was isolated from Shengli oilfield in China. Strain W6T was observed to grow from 20 to 45 °C with pH 6.5–9.0 (optimally at 40 °C and pH of 7.5) and without addition of NaCl. The major cellular fatty acids were iso-C15 : 0 (29.1%), C14 : 0 (27.0%) and C16 : 0 (12.2%), and the main polar lipids were lipids (L) and aminolipids (AL). The DNA G+C content is 42.9 mol%. Based on 16S rRNA gene sequence analysis, strain W6T showed highest similarities to Tissierella creatinini DSM 9508T (94.9%) and Soehngenia saccharolytica DSM 12858T (94.1%). The morphological, physiological, biochemical, phylogenetic and phylogenomic analyses demonstrated strain W6T (CCAM 534T=DSM 28124T=CGMCC 1.5291T) represents a novel species in a new genus, for which the name Gudongella oleilytica gen. nov. sp. nov. is proposed. The family Tissierellaceae is proposed as a new family containing the genera Anaerosalibacter , Gudongella, Keratinibaculum , Soehngenia , Sporanaerobacter , Tepidimicrobium , Tissierella , Urmitella and species Clostridium ultunense based on the phylogenetic and phylogenomic analyses.

‘Candidatus Liberibacter asiaticus’ (CLas) is an unculturable α-proteobacterium associated with citrus Huanglongbing (HLB; yellow shoot disease). PCR procedures that accurately confirm or exclude CLas infection in citrus tissue/Asian citrus psyllid (ACP) samples are critical for HLB management. When CLas was described in 1994, a 23-bp signature oligonucleotide sequence (OI1) in the 16S rRNA gene (rrs, three genomic copies) was identified based on Sanger sequencing. OI1 contains single nucleotide polymorphisms (SNPs) distinguishing CLas from non-CLas species. The SNPs were used to design the primer HLBas, a key primer for a commonly used TaqMan PCR system (HLBas-PCR) for CLas detection. Recent developments in next-generation sequencing technology have led to the identification of 15 CLas whole genome sequence strains (WGSs). Analyses of CLas WGSs have generated a significant amount of biological information that could help to improve CLas detection. Utilizing the WGS information, this study re-evaluated the sequence integrity of OI1/HLBas and identified and/or confirmed a missing nucleotide G in the two primers. Replacement primers for OI1 and HLBas are proposed. At low cycle threshold (Ct) values (e.g., <30), HLBas-PCR remained reliable in CLas determination. At high Ct values (e.g., >30), HLBas-PCR alone was unreliable in differentiating whether samples contain low CLas titers or whether CLas is not present. The availability of ribonucleotide reductase (RNR)-PCR derived from the five-copy nrdB gene helped to resolve this problem. To further enhance low CLas titer detection, a 4CP-PCR system, based on a four-copy genomic locus, was developed. Evaluation of 107 HLB samples (94 citrus and 13 ACP) showed that 4CP-PCR was more sensitive than HLBas-PCR and shared similar sensitivity with RNR-PCR.