Publications

4774

AbstractHuanglongbing (HLB) is a worldwide deadly citrus disease caused by the phloem-limited bacteria ‘Candidatus Liberibacter asiaticus’ (CLas) vectored by Asian citrus psyllids. In order to effectively manage this disease, it is crucial to understand the relationship among the bacterial isolates from different geographical locations. Whole genome sequencing approaches will provide more precise molecular characterization of the diversity among populations. Due to the lack of in vitro culture, obtaining the whole genome sequence of CLas is still a challenge, especially for medium to low titer samples. Hundreds of millions of sequencing reads are needed to get good coverage of CLas from an HLB positive citrus sample. In order to overcome this limitation, we present here a new method, Agilent SureSelect XT HS target enrichment, which can specifically enrich CLas from a metagenomic sample while greatly reducing cost and increasing whole genome coverage of the pathogen. In this study, the CLas genome was successfully sequenced with 99.3% genome coverage and over 72X sequencing coverage from low titer tissue samples (equivalent to 28.52 Cq using Li 16 S qPCR). More importantly, this method also effectively captures regions of diversity in the CLas genome, which provides precise molecular characterization of different strains.

ABSTRACTThe presences of ammonia tolerant ammonia-oxidizing archaea (AOA) in environments are always underestimated and their adaption to complex habitats has also rarely been reported. Here we present the physiological and genomic characteristics of an ammonia tolerant soil AOA strainCandidatusNitrosocosmicus agrestis. This strain was able to form aggregates and adhere on the surface of hydrophobic matrix. Ammonia-oxidizing activities were still observed at 200 mM NH4+(> 1500 μM of free ammonia) and 50 mM NO2-. Urea could be used as sole energy source but exogenous organics had no significant effect on the ammonia oxidation. Besides the genes involving in ammonia oxidation, carbon fixation and urea hydrolysis, the genome also encodes a full set of genes (GTs, GHs, CEs, MOP, LPSE, etc) that responsible for polysaccharide metabolism and secretion, suggesting the potential production of extracellular polymeric substances (EPS). Moreover, a pathway connecting urea cycle, polyamines synthesis and excretion was identified in the genome, which indicates the NH4+in cytoplasm could potentially be converted into polyamines and excreted out of cell, and then contributes to the high ammonia tolerance. Genes encoding the cytoplasmic carbonic anhydrase and putative polyamine exporter are unique inCa. Nitrosocosmicus agrestis or the genusCa. Nitrosocosmicus, suggesting the prevalence of ammonia tolerance in this clade. The proposed mechanism of ammonia tolerance via polyamines synthesis and export was verified by using transcriptional gene regulation and polyamines determination.IMPORTANCEAOA are ubiquitous in different environments and play a major role in nitrification. Though AOA have higher affinities for ammonia, their maximum specific cell activity and ammonia tolerance are usually much lower than AOB, resulting in low contribution to the global ammonia oxidation and N2O production. However, in some agricultural soils, the AOA activity would not be suppressed by the fertilization with high concentration of ammonium nitrogen, suggesting the presence of some ammonia tolerant species. This study provides some physiological and genomic characteristics for an ammonia tolerant soil AOA strainCa. Nitrosocosmicus agrestis and proposes some mechanisms of this AOA adapting to a variety of environments and tolerating to high ammonia. Ammonia tolerance of AOA was always underestimated in many previous studies, physiological and genomic analyses of this AOA clade are benefit to uncover the role of AOA playing in global environmental patterns.

Abstract Phagocytosis is a key eukaryotic feature, conserved from unicellular protists to animals, that enabled eukaryotes to feed on other organisms. It could also be a driving force behind endosymbiosis, a process by which α-proteobacteria and cyanobacteria evolved into mitochondria and plastids, respectively. Here we describe a planctomycete bacterium, ‘ Candidatus Uab amorphum’, which is able to engulf other bacteria and small eukaryotic cells through a phagocytosis-like mechanism. Observations via light and electron microscopy suggest that this bacterium digests prey cells in specific compartments. With the possible exception of a gene encoding an actin-like protein, analysis of the ‘ Ca . Uab amorphum’ genomic sequence does not reveal any genes homologous to eukaryotic phagocytosis genes, suggesting that cell engulfment in this microorganism is probably not homologous to eukaryotic phagocytosis. The discovery of this “phagotrophic” bacterium expands our understanding of the cellular complexity of prokaryotes, and may be relevant to the origin of eukaryotic cells.

AbstractRickettsiales bacteria in arthropods play a significant role in both public health and arthropod ecology. However, the extensive genetic diversity of Rickettsiales endosymbionts of arthropods is still to be discovered. In 2016, 515 arthropods belonging to 9 species of four classes (Insecta, Chilopoda, Diplopoda and Arachnida) were collected in Serbia. The presence and genetic diversity of Rickettsiales bacteria were evaluated by characterizing the 16S rRNA (rrs), citrate synthase (gltA) and heat shock protein (groEL) genes. The presence of various Rickettsiales bacteria was identified in the majority of tested arthropod species. The results revealed co-circulation of five recognized Rickettsiales species including Rickettsia, Ehrlichia and Wolbachia, as well as four tentative novel species, including one tentative novel genus named Neowolbachia. These results suggest the remarkable genetic diversity of Rickettsiales bacteria in certain arthropod species in this region. Furthermore, the high prevalence of spotted fever group Rickettsia in Ixodes ricinus ticks highlights the potential public health risk of human Rickettsia infection.

Abstract Background ‘Candidatus Phytoplasma solani’ is endemic in Europe and infects a wide range of weeds and cultivated plants. Phytoplasmas are prokaryotic plant pathogens that colonize the sieve elements of their host plant, causing severe alterations in phloem function and impairment of assimilate translocation. Typical symptoms of infected plants include yellowing of leaves or shoots, leaf curling, and general stunting, but the molecular mechanisms underlying most of the reported changes remain largely enigmatic. To infer a possible involvement of Fe in the host-phytoplasma interaction, we investigated the effects of ‘Candidatus Phytoplasma solani’ infection on tomato plants (Solanum lycopersicum cv. Micro-Tom) grown under different Fe regimes. Results Both phytoplasma infection and Fe starvation led to the development of chlorotic leaves and altered thylakoid organization. In infected plants, Fe accumulated in phloem tissue, altering the local distribution of Fe. In infected plants, Fe starvation had additive effects on chlorophyll content and leaf chlorosis, suggesting that the two conditions affected the phenotypic readout via separate routes. To gain insights into the transcriptional response to phytoplasma infection, or Fe deficiency, transcriptome profiling was performed on midrib-enriched leaves. RNA-seq analysis revealed that both stress conditions altered the expression of a large (> 800) subset of common genes involved in photosynthetic light reactions, porphyrin / chlorophyll metabolism, and in flowering control. In Fe-deficient plants, phytoplasma infection perturbed the Fe deficiency response in roots, possibly by interference with the synthesis or transport of a promotive signal transmitted from the leaves to the roots. Conclusions ‘Candidatus Phytoplasma solani’ infection changes the Fe distribution in tomato leaves, affects the photosynthetic machinery and perturbs the orchestration of root-mediated transport processes by compromising shoot-to-root communication.

AbstractIn Argentina, amaranth is a promising crop due to high nutritional quality and ability to grow in a diversity of environments. In areas cultivated with amaranth, were observed plants exhibiting slow growth, deformed leaves, proliferation of shoots and malformed lateral panicles. Field survey revealed up to 96% disease incidence and 92% of the seeds collected from mother plants produced diseased seedlings. A phytoplasma was detected in association with seedlings and adult plants using nested PCR assays. Molecular identification by computer‐simulated RFLP and phylogenetic analysis evidenced the occurrence of a ‘Candidatus Phytoplasma hispanicum’‐related strain, affiliated with 16SrXIII‐A subgroup. The findings implicate amaranth as a new host for this subgroup and as a potential reservoir of the pathogen for other cultivated species. In addition, to the best of our knowledge, this study reports for the first time the presence of 16SrXIII‐A phytoplasma in Argentina and in South America. Furthermore, transmission assays pointed that naturally infected seed is an important vehicle of dissemination of the pathogen, threatening the expansion of the crop for new geographical areas.

Huanglongbing (HLB) or greening currently is the most devastating citrus disease worldwide. The fastidious phloem-colonizing bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) is the causal agent of citrus HLB in Florida. Bactericides containing the active ingredient oxytetracycline (OTC) have been used in foliar spray to control citrus HLB in Florida since 2016. However, the minimum concentration of OTC required to suppress CLas in planta remains unknown. We developed a new method for evaluating the effects of OTC treatment on CLas titers in infected plants and determined the relationship between OTC residue levels and control levels achieved for CLas using mathematical modeling in greenhouse and field experiments. In both greenhouse and field, OTC spray did not reduce the titers of CLas, and it produced undetectable or mild levels of OTC residue in leaves within 7 days post-application (DPA). In greenhouse, OTC injection at 0.05 g per tree decreased CLas titers to an undetectable level (cycle threshold value ≥ 36.0) from 7 to 30 DPA and produced a residue level of OTC at 0.68 to 0.73 µg/g of fresh tissue over this period. In the field, OTC injection at 0.50 g per tree resulted in the decline of CLas titers by 1.52 log reduction from 14 to 60 DPA, with residue levels of OTC at 0.27 to 0.33 µg/g of fresh tissue. In both trials, a first-order compart model of OTC residue dynamics in leaves of trunk-injected trees was specified for estimating the retention of effective concentrations. Furthermore, nonlinear modeling revealed significant positive correlations between OTC residue levels in leaves and the control levels for CLas achieved. The results suggested that the minimum concentrations of OTC required to suppress CLas populations in planta to below the detection limit are 0.68 and 0.86 µg/g and that the minimum concentrations of OTC required for initial inhibition of CLas growth in planta are ∼0.17 and ∼0.215 µg/g in leaf tissues under greenhouse and field conditions, respectively. This finding highlights that a minimum concentration of OTC should be guaranteed to be delivered to target CLas in infected plants for effective control of citrus HLB.

AbstractBackgroundThe olive fly,Bactrocera oleae,is the most important insect pest in olive production, causing economic damage to olive crops worldwide. In addition to extensive research onB. oleaecontrol methods, scientists have devoted much effort in the last century to understanding olive fly endosymbiosis with a bacterium eventually identified asCandidatusErwinia dacicola. This bacterium plays a relevant role in olive fly fitness. It is vertically transmitted, and it benefits both larvae and adults in wild populations; however, the endosymbiont is not present in lab colonies, probably due to the antibiotics and preservatives required for the preparation of artificial diets. Endosymbiont transfer from wildB. oleaepopulations to laboratory-reared ones allows olive fly mass-rearing, thus producing more competitive flies for future Sterile Insect Technique (SIT) applications.ResultsWe tested the hypothesis thatCa. E. dacicola might be transmitted from wild, naturally symbiotic adults to laboratory-reared flies. Several trials have been performed with different contamination sources ofCa. E. dacicola, such as ripe olives and gelled water contaminated by wild flies, wax domes containing eggs laid by wild females, cages dirtied by faeces dropped by wild flies and matings between lab and wild adults. PCR-DGGE, performed with the primer set 63F-GC/518R, demonstrated that the transfer of the endosymbiont from wild flies to lab-reared ones occurred only in the case of cohabitation.ConclusionsCohabitation of symbiotic wild flies and non-symbiotic lab flies allows the transfer ofCa. E. dacicola through adults. Moreover, PCR-DGGE performed with the primer set 63F-GC/518R was shown to be a consistent method for screeningCa. E. dacicola, also showing the potential to distinguish between the two haplotypes (htA and htB). This study represents the first successful attempt at horizontal transfer ofCa. E. dacicola and the first step in acquiring a better understanding of the endosymbiont physiology and its relationship with the olive fly. Our research also represents a starting point for the development of a laboratory symbiotic olive fly colony, improving perspectives for future applications of the Sterile Insect Technique.

Abstract Background Microbial communities recurrently establish metabolic associations resulting in increased fitness and ability to perform complex tasks, such as xenobiotic degradation. In a previous study, we have described a sulfonamide-degrading consortium consisting of a novel low-abundant actinobacterium, named strain GP, and Achromobacter denitrificans PR1. However, we found that strain GP was unable to grow independently and could not be further purified. Results Previous studies suggested that strain GP might represent a new putative species within the Leucobacter genus (16S rRNA gene similarity < 97%). In this study, we found that average nucleotide identity (ANI) with other Leucobacter spp. ranged between 76.8 and 82.1%, further corroborating the affiliation of strain GP to a new provisional species. The average amino acid identity (AAI) and percentage of conserved genes (POCP) values were near the lower edge of the genus delimitation thresholds (65 and 55%, respectively). Phylogenetic analysis of core genes between strain GP and Leucobacter spp. corroborated these findings. Comparative genomic analysis indicates that strain GP may have lost genes related to tetrapyrrole biosynthesis and thiol transporters, both crucial for the correct assembly of cytochromes and aerobic growth. However, supplying exogenous heme and catalase was insufficient to abolish the dependent phenotype. The actinobacterium harbors at least two copies of a novel genetic element containing a sulfonamide monooxygenase (sadA) flanked by a single IS1380 family transposase. Additionally, two homologs of sadB (4-aminophenol monooxygenase) were identified in the metagenome-assembled draft genome of strain GP, but these were not located in the vicinity of sadA nor of mobile or integrative elements. Conclusions Comparative genomics of the genus Leucobacter suggested the absence of some genes encoding for important metabolic traits in strain GP. Nevertheless, although media and culture conditions were tailored to supply its potential metabolic needs, these conditions were insufficient to isolate the PR1-dependent actinobacterium further. This study gives important insights regarding strain GP metabolism; however, gene expression and functional studies are necessary to characterize and further isolate strain GP. Based on our data, we propose to classify strain GP in a provisional new species within the genus Leucobacter, ‘Candidatus Leucobacter sulfamidivorax‘.