Publications

4372

AbstractA major bottleneck in identifying therapies to control citrus greening and other devastating plant diseases caused by fastidious pathogens is our inability to culture the pathogens in defined media or axenic cultures. As such, conventional approaches for antimicrobial evaluation (genetic or chemical) rely on time-consuming, low-throughput and inherently variable whole-plant assays. Here, we report that plant hairy roots support the growth of fastidious pathogens likeCandidatusLiberibacter spp., the presumptive causal agents of citrus greening, potato zebra chip and tomato vein greening diseases. Importantly, we leverage the microbial hairy roots for rapid, reproducible efficacy screening of multiple therapies. We identify six antimicrobial peptides, two plant immune regulators and eight chemicals which inhibitCandidatusLiberibacter spp. in plant tissues. The antimicrobials, either singly or in combination, can be used as near- and long-term therapies to control citrus greening, potato zebra chip and tomato vein greening diseases.

The recently described superphylum DPANN includes several phyla of uncultivated archaea with small cell sizes, reduced genomes, and limited metabolic capabilities. One of these phyla, “ Ca . Micrarchaeota,” comprises an enigmatic group of archaea found in acid mine drainage environments, the archaeal Richmond Mine acidophilic nanoorganisms (ARMAN) group. Analysis of their reduced genomes revealed the absence of key metabolic pathways consistent with their partner-associated lifestyle, and physical associations of ARMAN cells with their hosts were documented. However, “ Ca . Micrarchaeota” include several lineages besides the ARMAN group found in nonacidic environments, and none of them have been characterized. Here, we report a complete genome of “ Ca . Micrarchaeota” from a non-ARMAN lineage. Analysis of this genome revealed the presence of metabolic capacities lost in ARMAN genomes that could enable a free-living lifestyle. These results expand our understanding of genetic diversity, lifestyle, and evolution of “ Ca . Micrarchaeota.”

Bois noir is caused by ‘Candidatus Phytoplasma solani’, and it is one of the most important and widespread diseases in the Euro-Mediterranean region. There are complex interactions between phytoplasma and grapevines, weeds, and vectors. These ecological relationships can be tracked according to molecular epidemiology. The aims of the 2-year study (2014–2015) were to describe incidence and spatial distribution of Bois noir in a vineyard with three grapevine varieties in Sicily, and to identify the molecular types of the tuf and vmp1 genes in these naturally infected grapevines, according to the potential reservoir plants and vectors. Disease incidence in 2015 was significantly higher in ‘Chardonnay’ (up to 35%) than for ‘Nero d’Avola’ and ‘Pinot noir’ (<5%). All grapevine, weed, and insect samples were infected by ‘Ca. P. solani’ tuf-type b. Most of the collected insects were strictly related to Vitis spp. and belonged to Neoaliturus fenestratus, Empoasca spp., and Zygina rhamni. The characterization of the vmp1 gene revealed six different vmp types in grapevines (V1, V4, V9, V11, V12, V24), three in weeds (V4, V9, V11), and four in insects (V4, V9, V11, V24). Notably, V4, V9, appear both in hosts and vectors, with V9 predominant. Virtual restriction fragment length polymorphism (RFLP) analysis based on the nucleotide sequences supported the data of the conventional RFLP. Connections between the molecular data recorded in the vineyard ecosystems and the application of innovative tools based on the geostatistical analysis will contribute to further clarification of the specific ecological and epidemiological aspects of ‘Ca. P. solani’ in Sicily.

AbstractMembers of the genusDechloromonasare often abundant in enhanced biological phosphorus removal (EBPR) systems and are recognized putative polyphosphate accumulating organisms (PAOs), but their role in phosphate (P) removal is still unclear. Here, we used 16S rRNA gene sequencing and fluorescencein situhybridization (FISH) to investigate the abundance and distribution ofDechloromonasspp. in Danish wastewater treatment plants. Two species were abundant, novel, and uncultured, and could be targeted by existing FISH probes. Raman microspectroscopy of probe-defined organisms (FISH-Raman) revealed the levels and dynamics of important intracellular storage polymers in abundantDechloromonasspp. in the activated sludge from four full-scale EBPR plants and from a lab-scale sequencing batch reactor fed with different carbon sources (acetate, glucose, glycine, and glutamate). Moreover, 7 distinctDechloromonasspecies were determined from a set of 10 high-quality metagenome-assembled genomes (MAGs) from Danish EBPR plants, each encoding the potential for poly-P, glycogen, and polyhydroxyalkanoates (PHA) accumulation. The two most abundant species exhibited anin situphenotype in complete accordance with the metabolic information retrieved by the MAGs, with dynamic levels of poly-P, glycogen, and PHA during feast-famine anaerobic-aerobic cycling, legitimately placing these microorganisms among the important PAOs. As no isolates are available for the two species, we propose the namesCandidatusDechloromonas phosphatis andCandidatusDechloromonas phosphovora.

Abstract“CandidatusLiberibacter asiaticus” (CLas) andSpiroplasma citriare phloem-limited bacteria that infect citrus and are transmitted by insect vectors.S. citricauses citrus stubborn disease (CSD) and is vectored by the beet leafhopper in California.CLas is associated with the devastating citrus disease, Huanglongbing (HLB), and is vectored by the Asian citrus psyllid.CLas is a regulatory pathogen spreading in citrus on residential properties in southern California and is an imminent threat to spread to commercial citrus plantings. CSD is endemic in California and has symptoms in citrus that can be easily confused with HLB. Consequently, a multiplex qPCR and duplex droplet digital PCR (ddPCR) were developed for simultaneous detection ofCLas andS. citri. The multiplex qPCR assay was designed to detect multicopy genes ofCLas - RNR (5 copies) andS. citri– SPV1 ORF1 (13 copies), respectively, and citrus cytochrome oxidase (COX) as internal positive control. Absolute quantitation of these pathogens was achieved by duplex ddPCR as a supplement for marginal qPCR results. Duplex ddPCR allowed higher sensitivity than qPCR for detection ofCLas andS. citri. ddPCR showed higher resilience to inhibitors and yielded highly reproducible results. The multiplex qPCR assay has the benefit of testing both pathogens at reduced cost and can serve to augment the official regulatory protocol forCLas detection in California. Moreover, the ddPCR provided unambiguous absolute detection ofCLas andS. citriat very low concentrations without any standards for pathogen titer.

AbstractNonulosonic acids (NulOs) are a family of acidic carbohydrates with a nine-carbon backbone, which include different related structures, such as sialic acids. They have mainly been studied for their relevance in animal cells and pathogenic bacteria. Recently, sialic acids have been discovered as important compound in the extracellular matrix of virtually all microbial life and in “CandidatusAccumulibacter phosphatis”, a well-studied polyphosphate-accumulating organism, in particular. Here, bioaggregates highly enriched with these bacteria (approx. 95% based on proteomic data) were used to study the production of NulOs in an enrichment of this microorganism. Fluorescence lectin-binding analysis, enzymatic quantification, and mass spectrometry were used to analyze the different NulOs present, showing a wide distribution and variety of these carbohydrates, such as sialic acids and bacterial NulOs, in the bioaggregates. Phylogenetic analysis confirmed the potential of “Ca. Accumulibacter” to produce different types of NulOs. Proteomic analysis showed the ability of “Ca. Accumulibacter” to reutilize and reincorporate these carbohydrates. This investigation points out the importance of diverse NulOs in non-pathogenic bacteria, which are normally overlooked. Sialic acids and other NulOs should be further investigated for their role in the ecology of “Ca. Accumulibacter” in particular, and biofilms in general.Key Points“Ca.Accumulibacter” has the potential to produce a range of nonulosonic acids.Mass spectrometry and lectin binding can reveal the presence and location of nonulosonic acids.Role of nonulosonic acid in non-pathogenic bacteria needs to be studied in detail.

The classDeltaproteobacteriacomprises an ecologically and metabolically diverse group of bacteria best known for dissimilatory sulphate reduction and predatory behaviour. Although this lineage is the fourth described class of the phylumProteobacteria, it rarely affiliates with other proteobacterial classes and is frequently not recovered as a monophyletic unit in phylogenetic analyses. Indeed, one branch of the classDeltaproteobacteriaencompassingBdellovibrio-like predators was recently reclassified into a separate proteobacterial class, theOligoflexia. Here we systematically explore the phylogeny of taxa currently assigned to these classes using 120 conserved single-copy marker genes as well as rRNA genes. The overwhelming majority of markers reject the inclusion of the classesDeltaproteobacteriaandOligoflexiain the phylumProteobacteria. Instead, the great majority of currently recognized members of the classDeltaproteobacteriaare better classified into four novel phylum-level lineages. We propose the namesDesulfobacterotaphyl. nov. andMyxococcotaphyl. nov. for two of these phyla, based on the oldest validly published names in each lineage, and retain the placeholder name SAR324 for the third phylum pending formal description of type material. Members of the classOligoflexiarepresent a separate phylum for which we propose the nameBdellovibrionotaphyl. nov. based on priority in the literature and general recognition of the genusBdellovibrio. Desulfobacterotaphyl. nov. includes the taxa previously classified in the phylumThermodesulfobacteria, and these reclassifications imply that the ability of sulphate reduction was vertically inherited in theThermodesulfobacteriarather than laterally acquired as previously inferred. Our analysis also indicates the independent acquisition of predatory behaviour in the phylaMyxococcotaandBdellovibrionota, which is consistent with their distinct modes of action. This work represents a stable reclassification of one of the most taxonomically challenging areas of the bacterial tree and provides a robust framework for future ecological and systematic studies.