General Medicine

Huanglongbing (HLB) is a devastating citrus disease that has caused massive economic losses to the citrus industry worldwide. The disease is endemic in most citrus-producing areas of southern China, especially in the sweet orange orchards where soil acidification has intensified. In this work, we used lime as soil pH amendment to optimize soil pH and enhance the endurance capacity of citrus against Candidatus Liberibacter asiaticus (CLas). The results showed that regulation of soil acidity is effective to reduce the occurrence of new infections and mitigate disease severity in the presence of HLB disease. We also studied the associated molecular mechanism and found that acid soil improvement can (i) increase the root metabolic activity and up-regulate the expression of ion transporter-related genes in HLB-infected roots, (ii) alleviate the physiological disorders of sieve tube blockage of HLB-infected leaves, (iii) strengthen the citrus immune response by increasing the expression of genes involved in SAR and activating the salicylic acid signal pathway, (iv) up-regulate 55 proteins related to stress/defence response and secondary metabolism. This study contributes to a better understanding of the correlation between environment factors and HLB disease outbreaks and also suggests that acid soil improvement is of potential value for the management of HLB disease in southern China.

A novel endophytic actinobacterium, designated strain EGI 650086T, was isolated from the roots of Anabasis elatior (C.A.Mey.) Schischk. collected in Xinjiang, north-west China. The taxonomic position of the strain was investigated using a polyphasic taxonomic approach. Growth occurred at 15–40 °C, pH 6.0–8.0 and in the presence of 0–6 % NaCl (w/v). Phylogenetic analysis based on 16S rRNA gene sequence and concatenation of 22 protein marker genes revealed that strain EGI 650086T formed a monophyletic clade within the genus Amycolatopsis and shared the highest sequence similarities with Amycolatopsis nigrescens JCM 14717T (97.1 %) and Amycolatopsis sacchari DSM 44468T (97.0 %). Sequence similarities with type strains of other species of the genus Amycolatopsis were less than 97.0 %. The average nucleotide identity and DNA–DNA hybridization values between strain EGI 650086T and the reference strains were 78.1–79.8 % and 22.1–23.0 %, respectively. The genome of strain EGI 650086T was 10.9 Mb, with a DNA G+C content of 70.1 mol%. The diagnostic diamino acid in the peptidoglycan was meso-diaminopimelic acid. The major whole-cell sugars contained arabinose, galactose, glucose and ribose. The predominant menaquinones were MK-9 (H4) and MK-9 (H2). Major fatty acids were iso-C16 : 0 and summed feature 4 (iso-C17 : 1 I and/or anteiso-C17 : 1 B). The polar lipid profile of strain EGI 650086T included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides, two unknown phospholipids, an unknown glycolipid and an unknown lipid. Polyphasic taxonomic characteristics indicated that strain EGI 650086T represents a novel species of the genus Amycolatopsis , for which the name Amycolatopsis anabasis sp. nov. is proposed. The type strain is EGI 650086T (=KCTC 49044T=CGMCC 4.7188T).

AbstractPalaeontologically, eubacteria are > 3× older than neomura (eukaryotes, archaebacteria). Cell biology contrasts ancestral eubacterial murein peptidoglycan walls and derived neomuran N-linked glycoprotein coats/walls. Misinterpreting long stems connecting clade neomura to eubacteria on ribosomal sequence trees (plus misinterpreted protein paralogue trees) obscured this historical pattern. Universal multiprotein ribosomal protein (RP) trees, more accurate than rRNA trees, are taxonomically undersampled. To reduce contradictions with genically richer eukaryote trees and improve eubacterial phylogeny, we constructed site-heterogeneous and maximum-likelihood universal three-domain, two-domain, and single-domain trees for 143 eukaryotes (branching now congruent with 187-protein trees), 60 archaebacteria, and 151 taxonomically representative eubacteria, using 51 and 26 RPs. Site-heterogeneous trees greatly improve eubacterial phylogeny and higher classification, e.g. showing gracilicute monophyly, that many ‘rDNA-phyla’ belong in Proteobacteria, and reveal robust new phyla Synthermota and Aquithermota. Monoderm Posibacteria and Mollicutes (two separate wall losses) are both polyphyletic: multiple outer membrane losses in Endobacteria occurred separately from Actinobacteria; neither phylum is related to Chloroflexi, the most divergent prokaryotes, which originated photosynthesis (new model proposed). RP trees support an eozoan root for eukaryotes and are consistent with archaebacteria being their sisters and rooted between Filarchaeota (=Proteoarchaeota, including ‘Asgardia’) and Euryarchaeota sensu-lato (including ultrasimplified ‘DPANN’ whose long branches often distort trees). Two-domain trees group eukaryotes within Planctobacteria, and archaebacteria with Planctobacteria/Sphingobacteria. Integrated molecular/palaeontological evidence favours negibacterial ancestors for neomura and all life. Unique presence of key pre-neomuran characters favours Planctobacteria only as ancestral to neomura, which apparently arose by coevolutionary repercussions (explained here in detail, including RP replacement) of simultaneous outer membrane and murein loss. Planctobacterial C-1 methanotrophic enzymes are likely ancestral to archaebacterial methanogenesis and β-propeller-α-solenoid proteins to eukaryotic vesicle coats, nuclear-pore-complexes, and intraciliary transport. Planctobacterial chaperone-independent 4/5-protofilament microtubules and MamK actin-ancestors prepared for eukaryote intracellular motility, mitosis, cytokinesis, and phagocytosis. We refute numerous wrong ideas about the universal tree.

A novel obligately anaerobic, thermophilic and formate-utilizing bacterium K32T was isolated from Shengli oilfield of China. Cells were straight rods (0.4–0.8 µm × 2.5–8.0 µm), Gram-stain-positive, non-spore-forming and slightly motile. Optimum growth occurred with pH of 7 and 0.5 g l–1 NaCl under temperature of 55–60 °C. Nitrate could be reduced into nitrite, syntrophic formate oxidation to methane and carbon dioxide occurred when co-culturing strain K32T and Methanothermobacter thermautotrophicus ΔH. The main cellular fatty acids were iso-C15 : 0 (24.0 %), anteiso-C15 : 0 (21.7 %), C16 : 0 (12.7 %) and C14 : 0 (10.8 %), and the main polar lipid was phosphatidylglycerol. The G+C content of the genomic DNA was 46.3 mol%. The 16S rRNA gene sequence of K32T shared ≤90.4 % of sequence similarity to closest type strains of Desulfitibacter alkalitolerans , Calderihabitans maritimus and members of the genus Moorella . Based on the phenotypic, biochemical and genotypic characterization, Zhaonella formicivorans gen. nov., sp. nov. is proposed with K32T (=CCAM 584T =DSM 107278T=CGMCC1.5297T) as the type strain, which is the first representative of Zhaonellaceae fam. nov. In addition, the order Thermoanaerobacterales and family Peptococcaceae were reclassified, and three novel families in the novel order of Moorellales ord. nov. were also proposed.

Most of the microorganisms responsible for vector-borne diseases (VBD) have hematophagous arthropods as vector/reservoir. Recently, many new species of microorganisms phylogenetically related to agents of VBD were found in a variety of aquatic eukaryotic hosts; in particular, numerous new bacterial species related to the genus Rickettsia (Alphaproteobacteria, Rickettsiales) were discovered in protist ciliates and other unicellular eukaryotes. Although their pathogenicity for humans and terrestrial animals is not known, several indirect indications exist that these bacteria might act as etiological agents of possible VBD of aquatic organisms, with protists as vectors. In the present study, a novel strain of the Rickettsia-Like Organism (RLO) endosymbiont “Candidatus (Ca.) Trichorickettsia mobilis” was identified in the macronucleus of the ciliate Paramecium multimicronucleatum. We performed transfection experiments of this RLO to planarians (Dugesia japonica) per os. Indeed, the latter is a widely used model system for studying bacteria pathogenic to humans and other Metazoa. In transfection experiments, homogenized paramecia were added to food of antibiotic-treated planarians. Treated and non-treated (i.e. control) planarians were investigated at day 1, 3, and 7 after feeding for endosymbiont presence by means of PCR and ultrastructural analyses. Obtained results were fully concordant and suggest that this RLO endosymbiont can be transiently transferred from ciliates to metazoans, being detected up to day 7 in treated planarians’ enterocytes. Our findings might offer insights into the potential role of ciliates or other protists as putative vectors for diseases caused by Rickettsiales or other RLOs and occurring in fish farms or in the wild.

In prokaryotic taxonomy, a set of criteria is commonly used to delineate species. These criteria are generally based on cohesion at the phylogenetic, phenotypic and genomic levels. One such criterion shown to have promise in the genomic era is average nucleotide identity (ANI), which provides an average measure of similarity across homologous regions shared by a pair of genomes. However, despite the popularity and relative ease of using this metric, ANI has undergone numerous refinements, with variations in genome fragmentation, homologue detection parameters and search algorithms. To test the robustness of a 95–96 % species cut-off range across all the commonly used ANI approaches, seven different methods were used to calculate ANI values for intra- and interspecies datasets representing three classes in the Proteobacteria . As a reference point, these methods were all compared to the widely used blast-based ANI (i.e. ANIb as implemented in JSpecies), and regression analyses were performed to investigate the correlation of these methods to ANIb with more than 130000 individual data points. From these analyses, it was clear that ANI methods did not provide consistent results regarding the conspecificity of isolates. Most of the methods investigated did not correlate perfectly with ANIb, particularly between 90 and 100% identity, which includes the proposed species boundary. There was also a difference in the correlation of methods for the different taxon sets. Our study thus suggests that the specific approach employed needs to be considered when ANI is used to delineate prokaryotic species. We furthermore suggest that one would first need to determine an appropriate cut-off value for a specific taxon set, based on the intraspecific diversity of that group, before conclusions on conspecificity of isolates can be made, and that the resulting species hypotheses be confirmed with analyses based on evolutionary history as part of the polyphasic approach to taxonomy.

Abstract The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is an important insect pest of the citrus crop worldwide. It vectors the pathogen ‘Candidatus Liberibacter asiaticus’ (CLas) that causes a serious disease known as citrus greening. Here, we tested the infection frequency of Wolbachia and CLas from 100 D. citri individuals collected from two host plants belonging to families Rutaceae (Citrus reticulata Blanco) and Boraginaceae (Cordia myxa L.) using molecular methods. The following trend of endosymbionts infection in adult D. citri was found; 85.4% (35/41) by Wolbachia, and 19.5% (8/41) by CLas collected from C. reticulata plants and 65.4% (17/26) by Wolbachia, and 15.4% (4/26) by CLas in case of C. myxa plant. However, 61.5% (8/13) nymphs collected from C. reticulata and 20.0% (4/20) collected from C. myxa plants were infected by Wolbachia, while no nymph was infected by CLas collected from either host plants. Findings from this work represent the first report of CLas presence in D. citri feeding on C. myxa plants. By studying the presence of CLas with other endosymbiotic bacteria, future basic and applied research to develop control strategies can be prioritized.