Publications

3807

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 Background ‘Candidatus Phytoplasma ulmi’ is the agent associated with elm yellows and has been categorised in the European Union as a quarantine pathogen. For central and northern European countries, information on the occurrence and distribution of the pathogen and its impact on elms is scarce, so a survey of native elm trees has been conducted in Germany. Results About 6500 samples from Ulmus minor, Ulmus laevis and Ulmus glabra, were collected nationwide. Phytoplasma detection was performed by applying a universal 16Sr DNA-based quantitative PCR (qPCR) assay and a novel ‘Ca. P. ulmi’ specific qPCR assay targeting the 16S–23S spacer region. Both assays revealed that 28% of the samples were infected by ‘Ca. P. ulmi’, but infection rates of the elm species and regional incidences differed. The phytoplasma presence in the trees was not correlated to disease-specific symptoms. The survey identified a regional disparity of infection which was high in east, south and central Germany, whereas only a few infected sites were found in the western and northern parts of the country. Monitoring the seasonal titre of ‘Ca. P. ulmi’ in an infected tree by qPCR revealed a high colonisation in all parts of the tree throughout the year. Conclusions ‘Ca. P. ulmi’ is widely present in elms in Germany. The rare occurrence of symptoms indicates either a high degree of tolerance in elm populations or a low virulence of pathogen strains enabling high infection rates in a long-living host.

Abstract BACKGROUND Rickettsia spp. are human pathogens that cause a number of diseases and are transmitted by arthropods, including ixodid ticks. Estonia contributes a region, where the distribution area of two exophilic tick species of known medical importance, Ixodes persulcatus and I. ricinus, overlap. The presence of the nidicolous rodent-associated I. trianguliceps has recently been shown for Estonia. Although there is no Estonian data available on human disease caused by tick-borne Rickettsia spp., the presence of three Rickettsia species in non-nidicolous ticks, albiet at very dissimilar rates, was also previously reported. The aim of this studywas to screen, identify and characterize Rickettsia species in nidicolous and non-nidicolous ticks attached to rodents. RESULTS Nymphs and larvae of I. ricinus ( n = 1004), I . persulcatus ( n = 75) and I. trianguliceps ( n = 117) attached to rodents and shrews caught in different parts of Estonia were studied for the presence of Rickettsia spp. by nested PCR. Ticks were removed from 314 small animals of 5 species (bank voles Myodes glareolus , yellow necked mice Apodemus flavicollis , striped field mice A. agrarius, pine voles M. subterranius and common shrews S. araneus) . Rickettsial DNA was detected in 8,7% (103/1186) studied ticks. In addition to R. helvetica, previously found in questing ticks, this study reports the first identification of the recently described I. trianguliceps- associated Candidatus R. uralica in west of the Ural.

Abstract Background: 'Candidatus Liberibacter asiaticus' (Las) is the pathogenic bacterium that causes Huanglongbing in citrus plants, as well as in several types of experimental plants. Las releases a set of effectors to modulate host responses. One of these critical effectors is Sec-delivered effector 1 (SDE1), which induces chlorosis in Nicotiana benthamiana. Results: Four SDE1-interacting proteins were identified from N. benthamiana, including DEAD-box RNA helicase DDX3, 26S proteasome non-ATPase regulatory subunit PSMD14, an ARM repeat protein, and a hypothetical protein. Gene silencing revealed that knockdown of the NbDDX3 gene led to chlorosis in N. benthamiana leaves. Fluorescent signal detection revealed that SDE1 was localized to the cell membrane, cytoplasm, and nucleus. Simultaneously, NbDD3 was expressed in cytoplasmic vesicles, as well as in the cell membrane. The interactions between SDE1 and NbDDX3 were shown to be localized on cell membrane using co-localization and bimolecular fluorescence complementation analysis. Moreover, the transcription of NbDDX3 gene was substantially suppressed in N. benthamiana plants that expressed SDE1. Conclusion: Las effector SDE1 interacts with NbDDX3 at the cell membrane. Most importantly, the transient expression of SDE1 exerts a suppression effect on the transcription of NbDDX3 gene. The silencing of NbDDX3 leads to leaf chlorosis in N. benthamiana. This provided evidence to understand the molecular events in association with chlorosis induced by SDE1.

Abstract‘Candidatus Liberibacter asiaticus’ (CLas) is an unculturable, Gram-negative, phloem restricted plant pathogenic bacterium associated with a very serious disease of citrus worldwide known as Citrus Huanglongbing (HLB). CLas is widely spread in the Americas. In Cuba, CLas has been associated with HLB symptoms and has seriously affected the Cuban citrus industry. In this short communication we discuss the identification of CLas-infected sour orange in urban areas of Mayabeque Province in Cuba, an area previously unexplored for the presence of HLB, and a host widely cultivated in gardens and yards along Cuba. We used for the first time the bacteria molecular barcode chaperonin-60 universal target (cpn60 UT) to identify and to detect CLas in HLB-symptomatic host plants.

AbstractIn this study, newly identified small molecules were examined for efficacy against ‘Candidatus Liberibacter asiaticus’ in commercial groves of sweet orange (Citrus sinensis) and white grapefruit (Citrus paradisi) trees. We used benzbromarone and/or tolfenamic acid delivered by trunk injection. We evaluated safety and efficacy parameters by performing RNAseq of the citrus host responses, 16S rRNA gene sequencing to characterize citrus-associated microbial communities during treatment, and qRT-PCR as an indirect determination of ‘Ca. L. asiaticus’ viability. Analyses of the C. sinensis transcriptome indicated that each treatment consistently induced genes associated with normal metabolism and growth, without compromising tree viability or negatively affecting the indigenous citrus-associated microbiota. It was found that treatment-associated reduction in ‘Ca. L. asiaticus’ was positively correlated with the proliferation of several core taxa related with citrus health. No symptoms of phytotoxicity were observed in any of the treated trees. Trials were also performed in commercial groves to examine the effect of each treatment on fruit productivity, juice quality and efficacy against ‘Ca. L. asiaticus’. Increased fruit production (15%) was observed in C. paradisi following twelve months of treatment with benzbromarone and tolfenamic acid. These results were positively correlated with decreased ‘Ca. L. asiaticus’ transcriptional activity in root samples.

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.

ABSTRACTCold seeps and hydrothermal vents deliver large amounts of methane and other gaseous alkanes into marine surface sediments. Consortia of archaea and partner bacteria thrive on the oxidation of these alkanes and its coupling to sulfate reduction. The inherently slow growth of the involved organisms and the lack of pure cultures have impeded the understanding of the molecular mechanisms of archaeal alkane degradation. Here, using hydrothermal sediments of the Guaymas Basin (Gulf of California) and ethane as substrate we cultured microbial consortia of a novel anaerobic ethane oxidizer Candidatus Ethanoperedens thermophilum (GoM-Arc1 clade) and its partner bacterium Candidatus Desulfofervidus auxilii previously known from methane-oxidizing consortia. The sulfate reduction activity of the culture doubled within one week, indicating a much faster growth than in any other alkane-oxidizing archaea described before. The dominance of a single archaeal phylotype in this culture allowed retrieving a closed genome of Ca. Ethanoperedens, a sister genus of the recently reported ethane oxidizer Candidatus Argoarchaeum. The metagenome-assembled genome of Ca. Ethanoperedens encoded for a complete methanogenesis pathway including a methyl-coenzyme M reductase (MCR) that is highly divergent from those of methanogens and methanotrophs. Combined substrate and metabolite analysis showed ethane as sole growth substrate and production of ethyl-coenzyme M as activation product. Stable isotope probing showed that the enzymatic mechanisms of ethane oxidation in Ca. Ethanoperedens is fully reversible, thus its enzymatic machinery has potential for the biotechnological development of microbial ethane production from carbon dioxide.IMPORTANCEIn the seabed gaseous alkanes are oxidized by syntrophic microbial consortia that thereby reduce fluxes of these compounds into the water column. Because of the immense quantities of seabed alkane fluxes, these consortia are key catalysts of the global carbon cycle. Due to their obligate syntrophic lifestyle, the physiology of alkane-degrading archaea remains poorly understood. We have now cultivated a thermophilic, relatively fast-growing ethane oxidizer in partnership with a sulfate-reducing bacterium known to aid in methane oxidation, and have retrieved the first complete genome of a short-chain alkane-degrading archaeon. This will greatly enhance the understanding of non-methane alkane activation by non-canonical methyl-coenzyme M reductase enzymes, and provide insights into additional metabolic steps and the mechanisms underlying syntrophic partnerships. Ultimately, this knowledge could lead to the biotechnological development of alkanogenic microorganisms to support the carbon neutrality of industrial processes.EtymologyEthanoperedens. ethano, (new Latin): pertaining to ethane; peredens (Latin): consuming, devouring; thermophilum. (Greek): heat-loving. The name implies an organism capable of ethane oxidation at elevated temperatures.LocalityEnriched from hydrothermally heated, hydrocarbon-rich marine sediment of the Guaymas Basin at 2000 m water depth, Gulf of California, Mexico.DiagnosisAnaerobic, ethane-oxidizing archaeon, mostly coccoid, about 0.7 μm in diameter, forms large irregular cluster in large dual-species consortia with the sulfate-reducing partner bacterium ‘Candidatus Desulfofervidus auxilii’.