A novel Gram-stain-negative, aerobic, motile bacterial strain, D13-10-4-6T, was isolated from the bark sample of Populus × euramericana. The strain could grow at 15–35°C, at pH 6–10 and in 0–4% (w/v) NaCl, and the strain tested positive for oxidase and catalase activities. The main polar lipids were phosphatidylmonomethylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The main respiratory quinone was Q-10, and the predominant fatty acid was C18:1 ω7c. The phylogenetic analyses showed that the strain belonged to the genus Pseudogemmobacter of the family Rhodobacteraceae. The family Rhodobacteraceae is an ecologically diverse group that includes bacteria from aquatic to terrestrial ecosystems. As a consequence, the classification of the family Rhodobacteraceae is difficult, not least when the early taxonomy work relied heavily on 16S rRNA gene analysis. Recently, the taxonomic status of many members of the family has been revised based on the genome analysis; however, there are still some classification conflicts due to the lack of genome sequences and parallel publication time. In this study, phylogenetic trees based on 16S rRNA gene, gyrB gene, and 120 concatenated proteins, the average amino acid identity (AAI) and percentage of conserved proteins (POCP) have been used for the analysis of strain D13-10-4-6T and other members of 15 genera within the family to further clarify their taxonomic relationships. For the data of phylogeny, AAI, and POCP, the taxonomic proposals are (1) reclassification of Rhodobacter tardus as the type species of a novel genus, Stagnihabitans gen. nov., as Stagnihabitans tardus comb. nov.; (2) reclassification of Tabrizicola alkalilacus, Tabrizicola sediminis, Tabrizicola algicola into a novel genus, Pseudotabrizicola gen. nov., as Pseudotabrizicola alkalilacus comb. nov., Pseudotabrizicola sediminis comb. nov., Pseudotabrizicola algicola comb. nov.; (3) reclassification of Rhodobacter sediminicola into the genus Cereibacter as Cereibacter sediminicola comb. nov.; (4) reclassification of Rhodobacter flagellatus, Rhodobacter thermarum, and Xinfangfangia soli into the genus Tabrizicola as Tabrizicola flagellatus comb. nov., Tabrizicola thermarum comb. Nov., and Tabrizicola soli comb. nov.; (5) reclassification of Xinfangfangia humi into the genus Pseudogemmobacter as Pseudogemmobacter humicola comb. nov.; (6) classification of strain D13-10-4-6T as a novel species of the genus Pseudogemmobacter, for which the name P. hezensis sp. nov. is proposed, the type strain is D13-10-4-6T (= CFCC 12033T = KCTC 82215T).
Gnomoniopsis (Gnomoniaceae, Diaporthales) is a well-classified genus inhabiting leaves, branches and fruits of the hosts in three plant families, namely Fagaceae, Onagraceae and Rosaceae. In the present study, eighteen Gnomoniopsis isolates were obtained from diseased leaves of Fagaceae hosts collected from Fujian, Guangdong, Hainan, Henan, Jiangxi and Shaanxi provinces in China. Morphology from the cultures and phylogeny based on the 5.8S nuclear ribosomal DNA gene with the two flanking internally transcribed spacer (ITS) regions, the translation elongation factor 1-alpha (tef1) and the beta-tubulin (tub2) genes were employed to identify these isolates. As a result, seven species were revealed, viz. Gnomoniopsis castanopsidis, G. fagacearum, G. guangdongensis, G. hainanensis, G. rossmaniae and G. silvicola spp. nov, as well as a known species G. daii. In addition, G. daii was firstly reported on the host Quercus aliena.
The introduction of the pine wood nematode (PWN), Bursaphelenchus xylophilus, to new areas has impacted on the international economy. Therefore, accurate and reliable detection methods for PWN are essential for the control and management of this pest. A rapid and economic method for detecting PWN may be developed focusing on the PWN vector (Monochamus alternatus). This work standardized a loop-mediated isothermal amplification (LAMP) method using newly designed primer sequences based on the syg-2 gene, which encodes the synaptogenesis protein syg-2. Loopmediated isothermal amplification was conducted at 63°C for 60 min using six sets of primers. The result was confirmed by visual observation. A positive reaction was confirmed by SYBR Green I fluorescence dye under light thermal cycling. The lower limit of DNA detection was 51.4 pg/μl in both LAMP and 51.4 ng/μl in PCR. Therefore, the LAMP was 1,000 times more sensitive in DNA detection than PCR. The LAMP is a relatively new, highly accurate and rapid molecular technique that can rapidly detect infectious agents in the field without requiring sophisticated instruments, giving a visually readable result. The method greatly improves detection, without requiring professional knowledge and expensive, sophisticated equipment. Therefore, this system is suitable for quarantine and field detection. negative reaction (no target DNA template), the dye remained orange ( Figure 4). | DISCUSSIONBursaphelenchus xylophilus is an important pathogenic organism vectored between pine hosts by M. alternatus. Therefore, the number of M. alternatus is a crucial indicator of disease development. Unless B. xylophilus in M. alternatus is specifically treated in the primary phase of infection, PWD is fatal. The efficient, rapid detection of B. xylophilus from M. alternatus is necessary for monitoring and managing outbreaks, and in implementing specific therapeutics (Cardoso, Fonseca, & Abrantes, 2012; Wang et al., 2009). As the death of pines infected by PWD is very rapid, the novel PWD-detection technique must also be rapid.In the present work, we designed specific primers for rapid detection of B. xylophilus from M. alternatus using the LAMP. For this purpose, we targeted the conserved syg-2 gene reported by Gou (2014). The LAMP assay successfully detected all isolates of B. xylophilus, giving negative reactions in all other species tested. The high specificity of the LAMP is conferred by the six specially designed primers that recognize six regions on the syg-2 gene sequence, which are specific to B. xylophilus. The lower limit of DNA detection in the LAMP-based technique was 51.4 pg/μl of genomic DNA, compared with 51.4 ng/μl in conventional PCR. Therefore, the LAMP was 1,000-fold more sensitive than conventional PCR (Gou et al., 2015) and can rapidly detect B. xylophilus from M. alternatus.Given the high sensitivity of LAMP, post-amplification operations should be performed in a separate room, away from the reactions and reagents used in the PCR and LAMP, to reduce the chance of conta...
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