Scientific Reports

AbstractEleven haplotypes of the bacterium, ‘Candidatus Liberibacter solanacearum’, have been identified worldwide, several of which infect important agricultural crops. In the United States, haplotypes A and B are associated with yield and quality losses in potato, tomato, and other crops of the Solanaceae. Both haplotypes are vectored by potato psyllid, Bactericera cockerelli. Recently, a third haplotype, designated F, was identified in southern Oregon potato fields. To identify the vector of this haplotype, psyllids of multiple species were collected from yellow sticky cards placed near potato fields during two growing seasons. Over 2700 specimens were tested for ‘Ca. L. solanacearum’ by polymerase chain reaction. Forty-seven psyllids harbored the bacterium. The infected specimens comprised four psyllid species in two families, Aphalaridae and Triozidae (Hemiptera: Psylloidea). Nucleic acid and/or amino acid sequence analysis of the ‘Ca. L. solanacearum’ 16S ribosomal RNA, 50S ribosomal proteins L10/L12, and outer membrane protein identified three new haplotypes of the bacterium, designated as Aph1, Aph2 and Aph3, including two variants of Aph2 (Aph2a and Aph2b). The impact of these new haplotypes on solanaceous or other crops is not known. The vector of ‘Ca. L. solanacearum’ haplotype F was not detected in this study.

AbstractInterspecies hydrogen transfer (IHT) and direct interspecies electron transfer (DIET) are two syntrophy models for methanogenesis. Their relative importance in methanogenic environments is still unclear. Our recent discovery of a novel species Candidatus Geobacter eutrophica with the genetic potential of IHT and DIET may serve as a model species to address this knowledge gap. To experimentally demonstrate its DIET ability, we performed electrochemical enrichment of Ca. G. eutrophica-dominating communities under 0 and 0.4 V vs. Ag/AgCl based on the presumption that DIET and extracellular electron transfer (EET) share similar metabolic pathways. After three batches of enrichment, Geobacter OTU650, which was phylogenetically close to Ca. G. eutrophica, was outcompeted in the control but remained abundant and active under electrochemical stimulation, indicating Ca. G. eutrophica’s EET ability. The high-quality draft genome further showed high phylogenomic similarity with Ca. G. eutrophica, and the genes encoding outer membrane cytochromes and enzymes for hydrogen metabolism were actively expressed. A Bayesian network was trained with the genes encoding enzymes for alcohol metabolism, hydrogen metabolism, EET, and methanogenesis from dominant fermentative bacteria, Geobacter, and Methanobacterium. Methane production could not be accurately predicted when the genes for IHT were in silico knocked out, inferring its more important role in methanogenesis. The genomics-enabled machine learning modeling approach can provide predictive insights into the importance of IHT and DIET.

AbstractA recent survey in Germany revealed the wide presence of ‘Candidatus Phytoplasma ulmi’ in native elm stands. Accessions were studied for their genetic variability and phylogenetic relationship based on the conserved groEL and the variable imp gene. While the groEL sequences revealed a high intraspecific homology of more than 99%, the homology of the imp gene dropped to 71% between distantly related sequences. Twenty-nine groEL and 74 imp genotypes were distinguished based on polymorphic sites. Phylogenetic analysis of the groEL gene clustered all ‘Ca. P. ulmi’ strains and separated them from related phytoplasmas of the 16SrV group. The inferred phylogeny of the imp gene resulted in a different tree topology and separated the ‘Ca. P. ulmi’ genotypes into two clusters, one closely related to the flavescence dorée phytoplasma strain FD-D (16SrV-D), the other affiliated with the flavescence dorée phytoplasma strains FD-C and FD70 and the alder yellows phytoplasma (16SrV-C). In both phylograms, ‘Ca. P. ulmi’ genotypes from Scots elm trees formed a coherent cluster, while genotypes from European white elms and field elms grouped less strictly. The regional distribution pattern was congruent for some of the groEL and imp genotypes, but a strict linkage for all genotypes was not apparent.

AbstractTaxonomy is the science of defining and naming groups of biological organisms based on shared characteristics and, more recently, on evolutionary relationships. With the birth of novel genomics/bioinformatics techniques and the increasing interest in microbiome studies, a further advance of taxonomic discipline appears not only possible but highly desirable. The present work proposes a new approach to modern taxonomy, consisting in the inclusion of novel descriptors in the organism characterization: (1) the presence of associated microorganisms (e.g.: symbionts, microbiome), (2) the mitochondrial genome of the host, (3) the symbiont genome. This approach aims to provide a deeper comprehension of the evolutionary/ecological dimensions of organisms since their very first description. Particularly interesting, are those complexes formed by the host plus associated microorganisms, that in the present study we refer to as “holobionts”. We illustrate this approach through the description of the ciliateEuplotes vanleeuwenhoekisp. nov. and its bacterial endosymbiont “CandidatusPinguicoccus supinus” gen. nov., sp. nov. The endosymbiont possesses an extremely reduced genome (~ 163 kbp); intriguingly, this suggests a high integration between host and symbiont.

AbstractHuanglongbing (HLB), or Citrus Greening, is one of the most devastating diseases affecting agriculture today. Widespread throughout Citrus growing regions of the world, it has had severe economic consequences in all areas it has invaded. With no treatment available, management strategies focus on suppression and containment. Effective use of these costly control strategies relies on rapid and accurate identification of infected plants. Unfortunately, symptoms of the disease are slow to develop and indistinct from symptoms of other biotic/abiotic stressors. As a result, diagnosticians have focused on detecting the pathogen, Candidatus Liberibacter asiaticus, by DNA-based detection strategies utilizing leaf midribs for sampling. Recent work has shown that fibrous root decline occurs in HLB-affected trees before symptom development among leaves. Moreover, the pathogen, Ca. Liberibacter asiaticus, has been shown to be more evenly distributed within roots than within the canopy. Motivated by these observations, a longitudinal study of young asymptomatic trees was established to observe the spread of disease through time and test the relative effectiveness of leaf- and root-based detection strategies. Detection of the pathogen occurred earlier, more consistently, and more often in root samples than in leaf samples. Moreover, little influence of geography or host variety was found on the probability of detection.

AbstractThe phloem limited bacterium ‘Candidatus Liberibacter solanacearum’ (Lso) is associated with disease in Solanaceous and Apiaceous crops. This bacterium has previously been found in the UK in Trioza anthrisci, but its impact on UK crops is unknown. Psyllid and Lso diversity and distribution among fields across the major carrot growing areas of Scotland were assessed using real-time PCR and DNA barcoding techniques. Four Lso haplotypes were found: C, U, and two novel haplotypes. Lso haplotype C was also found in a small percentage of asymptomatic carrot plants (9.34%, n = 139) from a field in Milnathort where known vectors of this haplotype were not found. This is the first report of Lso in cultivated carrot growing in the UK and raises concern for the carrot and potato growing industry regarding the potential spread of new and existing Lso haplotypes into crops. Trioza anthrisci was found present only in sites in Elgin, Moray with 100% of individuals harbouring Lso haplotype C. Lso haplotype U was found at all sites infecting Trioza urticae and at some sites infecting Urtica dioica with 77.55% and 24.37% average infection, respectively. The two novel haplotypes were found in Craspedolepta nebulosa and Craspedolepta subpunctata and named Cras1 and Cras2. This is the first report of Lso in psyllids from the Aphalaridae. These new haplotypes were most closely related to Lso haplotype H recently found in carrot and parsnip. Lso was also detected in several weed plants surrounding carrot and parsnip fields. These included two Apiaceous species Aegropodium podagraria (hap undetermined) and Anthriscus sylvestris (hap C); one Gallium sp. (Rubiaceae) (hap undetermined); and Chenopodium album (Amaranthaceae) (hap undetermined).

Abstract‘Candidatus Liberibacter solanacearum’ (Lso) is a pathogen of solanaceous crops. Two haplotypes of Lso (LsoA and LsoB) are present in North America; both are transmitted by the tomato psyllid, Bactericera cockerelli (Šulc), in a circulative and propagative manner and cause damaging plant diseases (e.g. Zebra chip in potatoes). In this study, we investigated the acquisition and transmission of LsoA or LsoB by the tomato psyllid. We quantified the titer of Lso haplotype A and B in adult psyllid guts after several acquisition access periods (AAPs). We also performed sequential inoculation of tomato plants by adult psyllids following a 7-day AAP and compared the transmission of each Lso haplotype. The results indicated that LsoB population increased faster in the psyllid gut than LsoA. Further, LsoB population plateaued after 12 days, while LsoA population increased slowly during the 16 day-period evaluated. Additionally, LsoB had a shorter latent period and higher transmission rate than LsoA following a 7 day-AAP: LsoB was first transmitted by the adult psyllids between 17 and 21 days following the beginning of the AAP, while LsoA was first transmitted between 21 and 25 days after the beginning of the AAP. Overall, our data suggest that the two Lso haplotypes have distinct acquisition and transmission rates. The information provided in this study will improve our understanding of the biology of Lso acquisition and transmission as well as its relationship with the tomato psyllid at the gut interface.

AbstractAnaplasmataceae agents are obligatory intracellular Gram-negative α-proteobacteria that are transmitted mostly by arthropod vectors. Although mammals of the Superorder Xenarthra (sloths, anteaters, and armadillos) have been implicated as reservoirs for several zoonotic agents, only few studies have sought to detect Anaplasmataceae agents in this group of mammals. This study aimed to investigate the occurrence and genetic diversity of Anaplasma spp. and Ehrlichia spp. in blood and spleen samples of free-living Xenarthra from four different states in Brazil (São Paulo, Mato Grosso do Sul, Rondônia, and Pará). Nested and conventional PCR screening assays were performed to detect the rrs and dsb genes of Anaplasma spp. and Ehrlichia spp., respectively. The assays were positive in 27.57% (91/330) of the Anaplasma spp. and 24.54% (81/330) of the Ehrlichia spp. Of the 91 positive Anaplasma spp. samples, 56.04% were positive in a conventional PCR assay targeting the 23S–5S intergenic region. Phylogenetic and distance analyses based on the rrs gene allocated Anaplasma sequences from sloths captured in Rondônia and Pará states in a single clade, which was closely related to the A. marginale, A. ovis, and A. capra clades. The sequences detected in southern anteaters from São Paulo were allocated in a clade closely related to sequences of Anaplasma spp. detected in Nasua nasua, Leopardus pardalis, and Cerdocyon thous in Brazil. These sequences were positioned close to A. odocoilei sequences. Genotype analysis corroborated previous findings and demonstrated the circulation of two distinct Anaplasma genotypes in animals from north and southeast Brazil. The first genotype was new. The second was previously detected in N. nasua in Mato Grosso do Sul state. The intergenic region analyses also demonstrated two distinct genotypes of Anaplasma. The sequences detected in Xenarthra from Pará and Rondônia states were closely related to those in A. marginale, A. ovis, and A. capra. Anaplasma spp. sequences detected in Xenarthra from São Paulo and were allocated close to those in A. phagocytophilum. The analyses based on the dsb gene grouped the Ehrlichia spp. sequences with sequences of E. canis (São Paulo, Mato Grosso do Sul, and Pará) and E. minasensis (Rondônia and Pará). The data indicate the occurrence of E. canis and E. minasensis and two possible new Candidatus species of Anaplasma spp. in free-living mammals of the Superorder Xenarthra in Brazil.

AbstractThis study reveals that the abundance of the filament Kouleothrix (Eikelboom type 1851) correlated positively with poor settleability of activated sludge biomass in a Japanese full-scale nutrient removal wastewater treatment plant sampled over a one-year period. 16S rRNA amplicon sequence data confirmed that Kouleothrix was the dominant filament in the plant, with a relative abundance of 3.06% positively correlated with sludge volume index (SVI) (R = 0.691). Moreover, Kouleothrix (type 1851) appeared to form interfloc bridges, typical of bulking sludge, regardless of season. Together with earlier studies that indicated the responsibility of Kouleothrix (type 1851) on bulking events, these data suggest that their high relative abundances alone may be responsible for sludge bulking. 16S rRNA qPCR data for this filament showed changes in its relative abundance correlated with changes in several operational parameters, including mixed liquor temperature, sludge retention time, and suspended solids concentration, and it may be that manipulating these may help control Kouleothrix bulking.

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.