Publications

3512

Huanglongbing (HLB) is a devastating citrus disease caused by Candidatus Liberibacter asiaticus (CLas). Since its initial outbreak in Guangdong Province, China, it has spread to 10 provinces and caused significant economic losses. Hence, assessing CLas genetic diversity and demographic history is crucial for HLB epidemic prevention and control. In this study, we collected 500 leaf samples of CLas-infected plants from 10 provinces. We performed multi-loci sequence analysis on four gene fragments (omp, DnaA, GroEL, and SDE1) to explore the genetic differentiation and diversity of CLas in China. Our results indicated low nucleotide diversity (0.00005 ± 0.00001) in CLas, with the absence of significant systematic geographic structure in its distribution. Molecular variance analysis revealed predominant (81.7%) genetic variations within the population, with a minor variation (18.3%) occurring between populations as well as Yunnan provinces. In the Fujian population, significant gene exchange occurred with the other nine populations. Significant negative values in Tajima’s D and Fu’s FS neutrality tests indicated historical population expansions. The nucleotide mismatch distribution curve exhibits a single peak pattern, further supporting the expansion events. Our findings hold potential for advancing epidemiological research and providing suggestions for effective strategies to mitigate the spread of CLas and control HLB.

AbstractThe roles of Asgard archaea in eukaryogenesis and marine biogeochemical cycles are well studied, yet their contributions in soil ecosystems are unknown. Of particular interest are Asgard archaeal contributions to methane cycling in wetland soils. To investigate this, we reconstructed two complete genomes for soil-associated Atabeyarchaeia, a new Asgard lineage, and the first complete genome of Freyarchaeia, and defined their metabolismin situ. Metatranscriptomics highlights high expression of [NiFe]-hydrogenases, pyruvate oxidation and carbon fixation via the Wood-Ljungdahl pathway genes. Also highly expressed are genes encoding enzymes for amino acid metabolism, anaerobic aldehyde oxidation, hydrogen peroxide detoxification and glycerol and carbohydrate breakdown to acetate and formate. Overall, soil-associated Asgard archaea are predicted to be non-methanogenic acetogens, likely impacting reservoirs of substrates for methane production in terrestrial ecosystems.One-Sentence SummaryComplete genomes of Asgard archaea, coupled with metatranscriptomic data, indicate roles in production and consumption of carbon compounds that are known to serve as substrates for methane production in wetlands.

ABSTRACTMembers of the “CandidatusAccumulibacter” genus are widely studied as key polyphosphate-accumulating organisms (PAOs) in biological nutrient removal (BNR) facilities performing enhanced biological phosphorus removal (EBPR). This diverse lineage includes 18 “Ca. Accumulibacter” species, which have been proposed based on the phylogenetic divergence of the polyphosphate kinase 1 (ppk1) gene and genome-scale comparisons of metagenome-assembled genomes (MAGs). Phylogenetic classification based on the 16S rRNA genetic marker has been difficult to attain because most “Ca. Accumulibacter” MAGs are incomplete and often do not include the rRNA operon. Here, we investigate the “Ca. Accumulibacter” diversity in pilot-scale treatment trains performing BNR under low dissolved oxygen (DO) conditions using genome-resolved metagenomics. Using long-read sequencing, we recovered medium and high-quality MAGs for 5 of the 18 “Ca. Accumulibacter” species, all with rRNA operons assembled, which allowed a reassessment of the 16S rRNA-based phylogeny of this genus and an analysis of phylogeny based on the 23S rRNA gene. In addition, we recovered a cluster of MAGs that based on 16S rRNA, 23S rRNA,ppk1, and genome-scale phylogenetic analyses do not belong to any of the currently recognized “Ca. Accumulibacter” species for which we propose the new species designation “Ca. Accumulibacter jenkinsii” sp. nov. Relative abundance evaluations of the genus across all pilot plant operations revealed that regardless of the operational mode, “Ca. A. necessarius” and “Ca. A. propinquus” accounted for more than 40% of the “Ca. Accumulibacter” community, whereas the newly proposed “Ca. A. jenkinsii” accounted for about 5% of the “Ca. Accumulibacter” community.IMPORTANCEOne of the main drivers of energy use and operational costs in activated sludge processes is the amount of oxygen provided to enable biological phosphorus and nitrogen removal. Wastewater treatment facilities are increasingly considering reduced aeration to decrease energy consumption, and whereas successful BNR has been demonstrated in systems with minimal aeration, an adequate understanding of the microbial communities that facilitate nutrient removal under these conditions is still lacking. In this study, we used genome-resolved metagenomics to evaluate the diversity of the “CandidatusAccumulibacter” genus in pilot-scale plants operating with minimal aeration. We identified the “Ca.Accumulibacter” species enriched under these conditions, including one novel species for which we propose “Ca.Accumulibacter jenkinsii” sp. nov. as its designation. Furthermore, the MAGs obtained for 5 additional “Ca.Accumulibacter” species further refine the phylogeny of the “Ca.Accumulibacter” genus and provide new insight into its diversity within unconventional biological nutrient removal systems.

AbstractCandidatusAltiarchaea are widespread across aquatic subsurface ecosystems and possess a highly conserved core genome, yet adaptations of this core genome to different biotic and abiotic factors based on gene expression remain unknown. Here, we investigated the metatranscriptome of twoCa. Altiarchaeum populations that thrive in two substantially different subsurface ecosystems. In Crystal Geyser, a high-CO2groundwater system in the USA,Ca. Altiarchaeum crystalense co-occurs with the symbiontCa. Huberiarchaeum crystalense, while in the Muehlbacher sulfidic spring in Germany, an artesian spring high in sulfide concentration,Ca. A. hamiconexum is heavily infected with viruses. We here mapped metatranscriptome reads against their genomes to analyze thein situexpression profile of their core genomes. Out of 537 shared gene clusters, 331 were functionally annotated and 130 differed significantly in expression between the two sites. Main differences were related to genes involved in cell defense like CRISPR-Cas, virus defense, replication, and transcription as well as energy and carbon metabolism. Our results demonstrate that altiarchaeal populations in the subsurface are likely adapted to their environment while influenced by other biological entities that tamper with their core metabolism. We consequently posit that viruses and symbiotic interactions can be major energy sinks for organisms in the deep biosphere.(Originality-Significance StatementOrganisms of the uncultivated phylumCa. Altiarchaeota are globally widespread and fulfill essential roles in carbon cycling,e.g., carbon fixation in the continental subsurface. Here, we show that the transcriptional activity of organisms in the continental subsurface differ significantly depending on the geological and microbial setting of the ecosystem explaining many of the previously observed physiological traits of this organism group.)

The methanogenic strain Mx-05T was isolated from the human fecal microbiome. A phylogenetic analysis based on the 16S rRNA gene and protein marker genes indicated that the strain is affiliated with the order Methanomassiliicoccales. It shares 86.9% 16S rRNA gene sequence identity with Methanomassiliicoccus luminyensis, the only member of this order previously isolated. The cells of Mx-05T were non-motile cocci, with a diameter range of 0.4–0.7 μm. They grew anaerobically and reduced methanol, monomethylamine, dimethylamine, and trimethylamine into methane, using H2 as an electron donor. H2/CO2, formate, ethanol, and acetate were not used as energy sources. The growth of Mx-05T required an unknown medium factor(s) provided by Eggerthella lenta and present in rumen fluid. Mx-05T grew between 30 °C and 40 °C (optimum 37 °C), over a pH range of 6.9–8.3 (optimum pH 7.5), and between 0.02 and 0.34 mol.L−1 NaCl (optimum 0.12 mol.L−1 NaCl). The genome is 1.67 Mbp with a G+C content of 55.5 mol%. Genome sequence annotation confirmed the absence of the methyl branch of the H4MPT Wood–Ljungdahl pathway, as described for other Methanomassiliicoccales members. Based on an average nucleotide identity analysis, we propose strain Mx-05T as being a novel representative of the order Methanomassiliicoccales, within the novel family Methanomethylophilaceae, for which the name Methanomethylophilus alvi gen. nov, sp. nov. is proposed. The type strain is Mx-05T (JCM 31474T).

Maternally inherited obligate endosymbionts codiverge with their invertebrate hosts and reflect their host’s evolutionary history. Whiteflies (Hemiptera: Aleyrodidae) harbor one obligate endosymbiont, Candidatus Portiera aleyrodidarum (hereafter Portiera). Portiera was anciently acquired by whitefly and has been coevolving with its host ever since. Uncovering the divergence of endosymbionts provides a fundamental basis for inspecting the coevolutionary processes between the bacteria and their hosts. To illustrate the divergence of Portiera lineages across different whitefly species, we sequenced the Portiera genome from Aleyrodes shizuokensis and conducted a comparative analysis on the basic features and gene evolution with bacterial genomes from five whitefly genera, namely Aleurodicus, Aleyrodes, Bemisia, Pealius, and Trialeurodes. The results indicated that Portiera from Bemisia possessed significantly larger genomes, fewer coding sequences (CDSs), and a lower coding density. Their gene arrangement differed notably from those of other genera. The phylogeny of the nine Portiera lineages resembled that of their hosts. Moreover, the lineages were classified into three distinct genetic groups based on the genetic distance, one from Aleurodicus (Aleurodicinae), one from Bemisia (Aleyrodinae), and another from Aleyrodes, Pealius, and Trialeurrodes (Aleyrodinae). Synonymous and nonsynonymous rate analyses, parity rule 2 plot analyses, neutrality plot analyses, and effective number of codons analyses supported the distinction of the three genetic groups. Our results indicated that Portiera from distant hosts exhibit distinct genomic contents, implying codivergence between hosts and their endosymbionts. This work will enhance our understanding of coevolution between hosts and their endosymbionts.

Candidatus Liberibacter spp is the most prevalent microorganism in the citrus plant, associated with Citrus Huanglongbing (HLB), which is transmitted by the psyllid vector. In Colombia, the vector Diaphorina citri Kugayama has been reported in different regions, but “Ca. Liberibacter asiaticus” (CLas) has only been detected in insect vectors, not in citrus host plants. To identify the presence and quantify the pathogen in citrus tissues, we employed a combined strategy that involved three techniques based on polymerase chain reaction (PCR). First, we used endpoint PCR with specific primers for CLas (OI1-OI2c) to confirm the infection. Second, we used qPCR with specific primers CIT295a – CIT298 designed on 16S rDNA gene regions to quantify the pathogen load. Finally, we employed droplet digital PCR (ddPCR) to determine the copy number of the pathogen in citrus tissues using the β-subunit of ribonucleotide reductase (RNR) gene (nrdB) that is specific to CLas. We identified the presence of CLas in citrus plants for the first time in Colombia and quantified its titer in the plant tissue. We employed ddPCR and qPCR to provide crucial information for the country's disease management, control strategies, and general crop health.

Methane emission by terrestrial invertebrates is restricted to millipedes, termites, cockroaches, and scarab beetles. The arthropod-associated archaea known to date belong to the orders Methanobacteriales, Methanomassiliicoccales, Methanomicrobiales, and Methanosarcinales, and in a few cases also to non-methanogenic Nitrososphaerales and Bathyarchaeales. However, all major host groups are severely undersampled, and the taxonomy of existing lineages is not well developed. Full-length 16S rRNA gene sequences and genomes of arthropod-associated archaea are scarce, reference databases lack resolution, and the names of many taxa are either not validly published or under-classified and require revision. Here, we investigated the diversity of archaea in a wide range of methane-emitting arthropods, combining phylogenomic analysis of isolates and metagenome-assembled genomes (MAGs) with amplicon sequencing of full-length 16S rRNA genes. Our results allowed us to describe numerous new species in hitherto undescribed taxa among the orders Methanobacteriales (Methanacia, Methanarmilla, Methanobaculum, Methanobinarius, Methanocatella, Methanoflexus, Methanorudis, and Methanovirga, all gen. nova), Methanomicrobiales (Methanofilum and Methanorbis, both gen. nova), Methanosarcinales (Methanofrustulum and Methanolapillus, both gen. nova), Methanomassiliicoccales (Methanomethylophilaceae fam. nov., Methanarcanum, Methanogranum, Methanomethylophilus, Methanomicula, Methanoplasma, Methanoprimaticola, all gen. nova), and the new family Bathycorpusculaceae (Bathycorpusculum gen. nov.). Reclassification of amplicon libraries from this and previous studies using this new taxonomic framework revealed that arthropods harbor only CO2 and methyl-reducing hydrogenotrophic methanogens. Numerous genus-level lineages appear to be present exclusively in arthropods, suggesting long evolutionary trajectories with their termite, cockroach, and millipede hosts, and a radiation into various microhabitats and ecological niches provided by their digestive tracts (e.g., hindgut compartments, gut wall, or anaerobic protists). The distribution patterns among the different host groups are often complex, indicating a mixed mode of transmission and a parallel evolution of invertebrate and vertebrate-associated lineages.

Areca catechu palm is an important cash plant in Hainan Island of China and even tropical regions worldwide. Areca catechu palm yellow leaf (AcYL) disease caused by the phytoplasmas is a devastating disease for the plant production. In the study, the phytoplasmas associated with the AcYL diseases were identified and characterized based on the conserved genes of the phytoplasmas, and genetic variation and phylogenetic relationship of the phytoplasma strains in the 16SrXXXII group was demonstrated. The results indicated that Areca catechu palm showing yellow leaf symptoms were single infected by ‘Candidatus Phytoplasma malaysianum’-related strains belonging to 16SrXXXII-D subgroup. BLAST and multiple sequence alignment analysis based on 16S rRNA and secA genes showed that the AcYL phytoplasmas shared 100% sequence identity and 100% homology with the ‘Ca. Phytoplasma malaysianum’-related strains. Phylogenetic analysis indicated that the AcYL phytoplasmas and ‘Ca. Phytoplasma malaysianum’-related strains belonging to 16SrXXXII group were clustered into one clade with a 100% bootstrap value. Based on computer-simulated digestions, 6 kinds of RFLP patterns within 16SrXXXII group were obtained and a novel subgroup in the 16Sr group was recommended to propose to describe the relevant strains in this 16Sr subgroup. To our knowledge, this is the first report that Areca catechu palm showing yellow leaf symptoms infected by ‘Ca. Phytoplasma malaysianum’-related strains belonging to 16SrXXXII group. And a novel 16Sr subgroup 16SrXXXII-F was proposed based on the systematical analysis of genetic variation of all the phytoplasmas within 16SrXXXII group. The findings of this study would support references for monitoring the epidemiology and developing effective prevention strategies of the AcYL diseases.

Citrus greening disease was first reported in Saudi Arabia during the 1970’s when characteristic foliar and fruit symptoms were observed in commercial citrus groves, however, “Candidatus Liberibacter asiaticus” (CLas) was not detected in symptomatic trees until 1981-1984 when CLas-like cells were observed by transmission electron microscopy in leaves collected from symptomatic citrus groves in southwestern Saudi Arabia. Despite the anticipated establishment of the CLas-Asian citrus psyllid (ACP) (Diaphorina citri Kuwayama) pathosystem, CLas presence has not been verified in suspect trees nor have ACP infestations been documented. Given the recent expansion of citrus production in Saudi Arabia, a systematic country-wide survey was carried out to determine the potential CLas distribution in the thirteen citrus-growing regions of the country. Citrus trees were surveyed for presence of CLas-psyllid vector(s) and characteristic disease symptoms in commercial and urban citrus trees. Adult psyllids collected from infested citrus trees were identified as ACP based on morphological characteristics. Real-time, quantitative PCR amplification (qPCR) of the CLas β-subunit of the ribonucleotide reductase (RNR) gene from citrus leaf and fruit samples and/or ACP adults, revealed trees were positive for CLas detection in ten of the 13 survey regions, however, CLas was undetectable in ACP adults. Phylogenetic and SNPs analyses of a PCR-amplified, cloned fragment of the CLas 16S rRNA gene (~1.1 kbp) indicated Saudi Arabian isolates were most closely related to Florida, USA isolates. Analysis of climate variables indicated that the distribution of the ACP-CLas pathosystem observed in Saudi Arabia was consistent with published predictions of terrains most likely to support establishment.