Scientific Reports

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 Galium 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 ofAnaplasmaspp. andEhrlichiaspp. 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 therrsanddsbgenes ofAnaplasmaspp. andEhrlichiaspp., respectively. The assays were positive in 27.57% (91/330) of theAnaplasmaspp. and 24.54% (81/330) of theEhrlichiaspp. Of the 91 positiveAnaplasmaspp. samples, 56.04% were positive in a conventional PCR assay targeting the 23S–5S intergenic region. Phylogenetic and distance analyses based on therrsgene allocatedAnaplasmasequences from sloths captured in Rondônia and Pará states in a single clade, which was closely related to theA. marginale,A. ovis,andA. capraclades. The sequences detected in southern anteaters from São Paulo were allocated in a clade closely related to sequences ofAnaplasmaspp. detected inNasua nasua,Leopardus pardalis, andCerdocyon thousin Brazil. These sequences were positioned close toA. odocoileisequences. Genotype analysis corroborated previous findings and demonstrated the circulation of two distinctAnaplasmagenotypes in animals from north and southeast Brazil. The first genotype was new. The second was previously detected inN. nasuain Mato Grosso do Sul state. The intergenic region analyses also demonstrated two distinct genotypes ofAnaplasma. The sequences detected in Xenarthra from Pará and Rondônia states were closely related to those inA. marginale,A. ovis,andA. capra.Anaplasmaspp. sequences detected in Xenarthra from São Paulo and were allocated close to those inA. phagocytophilum. The analyses based on thedsbgene grouped theEhrlichiaspp. sequences with sequences ofE. canis(São Paulo, Mato Grosso do Sul, and Pará) andE. minasensis(Rondônia and Pará). The data indicate the occurrence ofE. canisandE. minasensisand two possible newCandidatusspecies ofAnaplasmaspp. 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.

AbstractInferring the dispersal processes of vector-borne plant pathogens is a great challenge because the plausible epidemiological scenarios often involve complex spread patterns at multiple scales. The spatial genetic structure of ‘CandidatusPhytoplasma prunorum’, responsible for European stone fruit yellows disease, was investigated by the application of a combination of statistical approaches to genotype data of the pathogen sampled from cultivated and wild compartments in three FrenchPrunus-growing regions. This work revealed that the prevalence of the different genotypes is highly uneven both between regions and compartments. In addition, we identified a significant clustering of similar genotypes within a radius of 50 km or less, but not between nearby wild and cultivatedPrunus. We also provide evidence that infected plants are transferred between production areas, and that both species of theCacopsylla prunicomplex can spread the pathogen. Altogether, this work supports a main epidemiological scenario where ‘Ca. P. prunorum’ is endemic in — and generally acquired from — wildPrunusby its immature psyllid vectors. The latter then migrate to shelter plants that epidemiologically connect sites less than 50 km apart by later providing infectious mature psyllids to their “migration basins”. Such multi-scale studies could be useful for other pathosystems.

AbstractHuanglongbing (HLB) is a serious disease of Citrus sp. worldwide. In Africa and the Mascarene Islands, a similar disease is known as African citrus greening (ACG) and is associated with the bacterium Candidatus Liberibacter africanus (Laf). In recent years, Candidatus Liberibacter asiaticus (Las) associated with the severe HLB has been reported in Ethiopia. Thus, we aimed to identify the Liberibacter species affecting citrus, the associated vectors in Eastern Africa and their ecological distribution. We assessed the presence of generic Liberibacter in symptomatic leaf samples by quantitative PCR. Subsequently, we sequenced the 50 S ribosomal protein L10 (rplJ) gene region in samples positive for Liberibacters and identified the species by comparison with public sequence data using phylogenetic reconstruction and genetic distances. We detected generic Liberibacter in 26%, 21% and 66% of plants tested from Uganda, Ethiopia and Kenya, respectively. The rplJ sequences revealed the most prevalent Liberibacters in Uganda and Ethiopia were LafCl (22%) and Las (17%), respectively. We detected Las in Kenya for the first time from three sites in the coastal region. Finally, we modelled the potential habitat suitability of Las in Eastern Africa using MaxEnt. The projection showed large areas of suitability for the pathogen in the three countries surveyed. Moreover, the potential distribution in Eastern Africa covered important citrus-producing parts of Ethiopia, Kenya, Uganda and Tanzania, and included regions where the disease has not been reported. These findings will guide in the development of an integrated pest management strategy to ACG/HLB management in Africa.

AbstractBois noir, a disease of the grapevine yellows complex, is associated with ‘CandidatusPhytoplasma solani’ and transmitted to grapevines in open fields by the cixiidsHyalesthes obsoletusandReptalus panzeri. In vine-growing areas where the population density of these vectors is low within the vineyard, the occurrence of bois noir implies the existence of alternative vectors. The aim of this study was to identify alternative vectors through screening of the Auchenorrhyncha community, phytoplasma typing bystampgene sequence analyses, and transmission trials. During field activities, conducted in Northern Italy in a vineyard where the bois noir incidence was extremely high, nine potential alternative insect vectors were identified according to high abundance in the vineyard agro-ecosystem, high infection rate, and harbouring phytoplasma strains characterized bystampgene sequence variants found also in symptomatic grapevines. Transmission trials coupled with molecular analyses showed that at least eight species (Aphrodes makarovi,Dicranotropis hamata,Dictyophara europaea,Euscelis incisus,Euscelidius variegatus,Laodelphax striatella,Philaenus spumarius, andPsammotettix alienus/confinis) are alternative vectors of ‘CandidatusPhytoplasma solani’ to grapevines. These novel findings highlight that bois noir epidemiology in vineyard agro-ecosystems is more complex than previously known, opening up new perspectives in the disease management.