Genomic and phenotypic signatures of bacteriophage coevolution with the phytopathogen Pseudomonas syringae.

The rate and trajectory of evolution in an obligate parasite is critically dependent on those of its host(s). Adaptation to a genetically homogeneous host population should theoretically result in specialization, while adaptation to an evolving host population (i.e., coevolution) can result in various outcomes including diversification, range expansion, and/or local adaptation. For viruses of bacteria (bacteriophages, or phages), our understanding of how evolutionary history of the bacterial host(s) impacts viral genotypic and phenotypic evolution is currently limited. In this study, we used whole genome sequencing and two different metrics of phage impacts to compare the genotypes and phenotypes of lytic phages that had either coevolved with or were repeatedly passaged on an unchanging (ancestral) strain of the phytopathogen Pseudomonas syringae. Genomes of coevolved phages had more mutations than those of phages passaged on a constant host, and most mutations were in genes encoding phage tail-associated proteins. Phages from both passaging treatments shared some phenotypic outcomes, including range expansion and divergence across replicate populations, but coevolved phages were more efficient at reducing population growth (particularly of sympatric coevolved hosts). Genotypic similarity correlated with infectivity profile similarity in coevolved phages, but not in phages passaged on the ancestral host. Overall, while adaptation to either host type (coevolving or ancestral) led to divergence in phage tail proteins and infectivity patterns, coevolution led to more rapid molecular changes that increased bacterial killing efficiency and had more predictable effects on infectivity range. Together, these results underscore the important role of hosts in driving viral evolution and in shaping the genotype-phenotype relationship.

Understanding the Impacts of Bacteriophage Viruses: From Laboratory Evolution to Natural Ecosystems.

Viruses of bacteria (bacteriophages or phage) have broad effects on bacterial ecology and evolution in nature that mediate microbial interactions, shape bacterial diversity, and influence nutrient cycling and ecosystem function. The unrelenting impact of phages within the microbial realm is the result, in large part, of their ability to rapidly evolve in response to bacterial host dynamics. The knowledge gained from laboratory systems, typically using pairwise interactions between single-host and single-phage systems, has made clear that phages coevolve with their bacterial hosts rapidly, somewhat predictably, and primarily by counteradapting to host resistance. Recent advancement in metagenomics approaches, as well as a shifting focus toward natural microbial communities and host-associated microbiomes, is beginning to uncover the full picture of phage evolution and ecology within more complex settings. As these data reach their full potential, it will be critical to ask when and how insights gained from studies of phage evolution in vitro can be meaningfully applied to understanding bacteria-phage interactions in nature. In this review, we explore the myriad ways that phagesshape and are themselves shaped by bacterial host populations and communities, with a particular focus on observed and predicted differences between the laboratory and complex microbial communities.

Water stress and disruption of mycorrhizas induce parallel shifts in phyllosphere microbiome composition.

Water and nutrient acquisition are key drivers of plant health and ecosystem function. These factors impact plant physiology directly as well as indirectly through soil- and root-associated microbial responses, but how they in turn affect aboveground plant-microbe interactions are not known. Through experimental manipulations in the field and growth chamber, we examine the interacting effects of water stress, soil fertility, and arbuscular mycorrhizal fungi on bacterial and fungal communities of the tomato (Solanum lycopersicum) phyllosphere. Both water stress and mycorrhizal disruption reduced leaf bacterial richness, homogenized bacterial community composition among plants, and reduced the relative abundance of dominant fungal taxa. We observed striking parallelism in the individual microbial taxa in the phyllosphere affected by irrigation and mycorrhizal associations. Our results show that soil conditions and belowground interactions can shape aboveground microbial communities, with important potential implications for plant health and sustainable agriculture.

Bacteriophage uptake by mammalian cell layers represents a potential sink that may impact phage therapy.

It is increasingly apparent that bacteriophages, viruses that infect bacteria and more commonly referred to as simply phages, have tropisms outside their bacterial hosts. Using live tissue culture cell imaging, we demonstrate that cell type, phage size, and morphology play a major role in phage internalization. Uptake was validated under physiological conditions using a microfluidic device. Phages adhered to mammalian tissues, with adherent phages being subsequently internalized by macropinocytosis, with functional phages accumulating intracellularly. We incorporated these results into a pharmacokinetic model demonstrating the potential impact of phage accumulation by cell layers, which represents a potential sink for circulating phages in the body. During phage therapy, high doses of phages are directly administered to a patient in order to treat a bacterial infection, thereby facilitating broad interactions between phages and mammalian cells. Understanding these interactions will have important implications on innate immune responses, phage pharmacokinetics, and the efficacy of phage therapy.

Persistent effects of management history on honeybee colony virus abundances.

Infectious diseases are a major threat to both managed and wild pollinators. One key question is how the movement or transplantation of honeybee colonies under different management regimes affects honeybee disease epidemiology. We opportunistically examined any persistent effect of colony management history following relocation by characterising the virus abundances of honeybee colonies from three management histories, representing different management histories: feral, low-intensity management, and high-intensity "industrial" management. The colonies had been maintained for one year under the same approximate 'common garden' condition. Colonies in this observational study differed in their virus abundances according to management history, with the feral population history showing qualitatively different viral abundance patterns compared to colonies from the two managed population management histories; for example, higher abundance of sacbrood virus but lower abundances of various paralysis viruses. Colonies from the high-intensity management history exhibited higher viral abundances for all viruses than colonies from the low-intensity management history. Our results provide evidence that management history has persistent impacts on honeybee disease epidemiology, suggesting that apicultural intensification could be majorly impacting on pollinator health, justifying much more substantial investigation.

Bacteriophage-Mediated Reduction of Bacterial Speck on Tomato Seedlings.

Background: One crucial first step in bacteriophage therapy is choosing a phage to apply, which involves screening for effectiveness in a meaningful way. Increasingly, research suggests that in vitro tests of phage-mediated bacterial lysis poorly translate to in planta effectiveness. Materials and Methods: We tested a seedling-based method for rapidly screening phage effectiveness in vivo. In three trials, phages were prophylactically applied to tomato seedlings in sterile conical tubes before flooding with the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. We recorded seedling disease progression and quantified endpoint bacteria and phage densities. Results: Phages replicated in all trials, but reduction of disease symptoms and endpoint P. syringae density varied across trials with different application densities. Conclusions: This resource-efficient method rapidly identified an effective phage and application density to mitigate disease on seedlings. We propose that this method could be used to screen candidate phages before testing in agricultural conditions.

Phage resistance evolution in vitro is not reflective of in vivo outcome in a plant-bacteria-phage system.

The evolution of resistance to parasites is fundamentally important to disease ecology, yet we remain unable to predict when and how resistance will evolve. This is largely due to the context-dependent nature of host-parasite interactions, as the benefit of resistance will depend on the abiotic and biotic environment. Through experimental evolution of the plant pathogenic bacterium Pseudomonas syringae and two lytic bacteriophages across two different environments (high-nutrient media and the tomato leaf apoplast), we demonstrate that de novo evolution of resistance is negligible in planta despite high levels of resistance evolution in vitro. We find no evidence supporting the evolution of phage-selected resistance in planta despite multiple passaging experiments, multiple assays for resistance, and high multiplicities of infection. Additionally, we find that phage-resistant mutants (evolved in vitro) did not realize a fitness benefit over phage-sensitive cells when grown in planta in the presence of phage, despite reduced growth of sensitive cells, evidence of phage replication in planta, and a large fitness benefit in the presence of phage observed in vitro. Thus, this context-dependent benefit of phage resistance led to different evolutionary outcomes across environments. These results underscore the importance of studying the evolution of parasite resistance in ecologically relevant environments.

Investigating the association between armour coverage and parasite infection in an estuarine population of stickleback.

Background: When threespine stickleback colonized fresh water, they repeatedly evolved reduced armour plating via changes in Eda allele frequency. This evolution is typically attributed to differential predation pressure between marine and freshwater environments. However, the chromosomal region containing Eda is associated with many other phenotypes, including schooling, antipredator behaviour, and immunity. Consequently, the evolution of armour plating may be driven by multiple selective pressures acting on Eda or linked genes. Question: Is parasite infection associated with armour phenotype? Hypothesis: Parasite load differs between stickleback armour plate morphs. Organisms: An armour-polymorphic population of threespine stickleback (Gasterosteus aculeatus), and their parasites. Field site: In June 2009 and 2012, we sampled stickleback from a single human-made salt-marsh pool in the Campbell River Estuary on Vancouver Island. Methods: We counted macroparasites on approximately 100 fish per year and counted lateral armour plates. We used generalized linear models to test for correlations between armour morph and parasite load. Results: Most parasite species were not associated with armour. The gill parasite Thersitina was more abundant on more fully armoured fish in both years. The nematode Eustrongylides also exhibited a marginally significant positive trend. If parasitic infections reduce stickleback fitness, this positive covariance between armour and infection would accelerate the loss of armour plating in stickleback colonizing fresh water.

Rapid quantification of bacteriophages and their bacterial hosts in vitro and in vivo using droplet digital PCR.

Viruses that infect bacteria, bacteriophages (phages), are well-studied across a wide range of environments and among diverse scientific fields. Nevertheless, current methods in phage research are lacking, in part due to limitations in culturability and the lack of a universal gene marker. Here, we demonstrate that droplet digital PCR (ddPCR) can be used as a repeatable and sensitive method to study bacteria-phage dynamics both in vitro and in vivo. Using fluorescent probes designed for the bacterial plant pathogen, Pseudomonas syringae, and two phages that prey upon it, we illustrate the use of ddPCR to enumerate phages, track bacteria and phage densities over time both in media co-culture and during infection of a tomato plant, compare phage time-to-lysis, and explore phage-phage competition. Overall, the ddPCR approach closley mirrors results from more traditional counts of plaque forming units (PFUs) but offers a much faster, lower waste, and more high-throughput way of studying these interactions. As such, we suggest that ddPCR will be a valuable new tool in bacteriophage research.