Pseudomonas syringae on Plants in Iceland Has Likely Evolved for Several Million Years Outside the Reach of Processes That Mix This Bacterial Complex across Earth's Temperate Zones.

Here we report, for the first time, the occurrence of the bacteria from the species complex Pseudomonas syringae in Iceland. We isolated this bacterium from 35 of the 38 samples of angiosperms, moss, ferns and leaf litter collected across the island from five habitat categories (boreal heath, forest, subalpine and glacial scrub, grazed pasture, lava field). The culturable populations of P. syringae on these plants varied in size across 6 orders of magnitude, were as dense as 107 cfu g-1 and were composed of strains in phylogroups 1, 2, 4, 6, 7, 10 and 13. P. syringae densities were significantly greatest on monocots compared to those on dicots and mosses and were about two orders of magnitude greater in grazed pastures compared to all other habitats. The phylogenetic diversity of 609 strains of P. syringae from Iceland was compared to that of 933 reference strains of P. syringae from crops and environmental reservoirs collected from 27 other countries based on a 343 bp sequence of the citrate synthase (cts) housekeeping gene. Whereas there were examples of identical cts sequences across multiple countries and continents among the reference strains indicating mixing among these countries and continents, the Icelandic strains grouped into monophyletic lineages that were unique compared to all of the reference strains. Based on estimates of the time of divergence of the Icelandic genetic lineages of P. syringae, the geological, botanical and land use history of Iceland, and atmospheric circulation patterns, we propose scenarios whereby it would be feasible for P. syringae to have evolved outside the reach of processes that tend to mix this bacterial complex across the planet elsewhere.

Long-term nitrogen enrichment mediates the effects of nitrogen supply and co-inoculation on a viral pathogen.

Host nutrient supply can mediate host-pathogen and pathogen-pathogen interactions. In terrestrial systems, plant nutrient supply is mediated by soil microbes, suggesting a potential role of soil microbes in plant diseases beyond soil-borne pathogens and induced plant defenses. Long-term nitrogen (N) enrichment can shift pathogenic and nonpathogenic soil microbial community composition and function, but it is unclear if these shifts affect plant-pathogen and pathogen-pathogen interactions. In a growth chamber experiment, we tested the effect of long-term N enrichment on infection by Barley Yellow Dwarf Virus (BYDV-PAV) and Cereal Yellow Dwarf Virus (CYDV-RPV), aphid-vectored RNA viruses, in a grass host. We inoculated sterilized growing medium with soil collected from a long-term N enrichment experiment (ambient, low, and high N soil treatments) to isolate effects mediated by the soil microbial community. We crossed soil treatments with a N supply treatment (low, high) and virus inoculation treatment (mock-, singly-, and co-inoculated) to evaluate the effects of long-term N enrichment on plant-pathogen and pathogen-pathogen interactions, as mediated by N availability. We measured the proportion of plants infected (i.e., incidence), plant biomass, and leaf chlorophyll content. BYDV-PAV incidence (0.96) declined with low N soil (to 0.46), high N supply (to 0.61), and co-inoculation (to 0.32). Low N soil mediated the effect of N supply on BYDV-PAV: instead of N supply reducing BYDV-PAV incidence, the incidence increased. Additionally, ambient and low N soil ameliorated the negative effect of co-inoculation on BYDV-PAV incidence. BYDV-PAV infection only reduced chlorophyll when plants were grown with low N supply and ambient N soil. There were no significant effects of long-term N soil on CYDV-RPV incidence. Soil inoculant with different levels of long-term N enrichment had different effects on host-pathogen and pathogen-pathogen interactions, suggesting that shifts in soil microbial communities with long-term N enrichment may mediate disease dynamics.

Environmental Nutrient Supply Directly Alters Plant Traits but Indirectly Determines Virus Growth Rate.

Ecological stoichiometry and resource competition theory both predict that nutrient rates and ratios can alter infectious disease dynamics. Pathogens such as viruses hijack nutrient rich host metabolites to complete multiple steps of their epidemiological cycle. As the synthesis of these molecules requires nitrogen (N) and phosphorus (P), environmental supply rates, and ratios of N and P to hosts can directly limit disease dynamics. Environmental nutrient supplies also may alter virus epidemiology indirectly by changing host phenotype or the dynamics of coinfecting pathogens. We tested whether host nutrient supplies and coinfection control pathogen growth within hosts and transmission to new hosts, either directly or through modifications of plant tissue chemistry (i.e., content and stoichiometric ratios of nutrients), host phenotypic traits, or among-pathogen interactions. We examined two widespread plant viruses (BYDV-PAV and CYDV-RPV) in cultivated oats (Avena sativa) grown along a range of N and of P supply rates. N and P supply rates altered plant tissue chemistry and phenotypic traits; however, environmental nutrient supplies and plant tissue content and ratios of nutrients did not directly alter virus titer. Infection with CYDV-RPV altered plant traits and resulted in thicker plant leaves (i.e., higher leaf mass per area) and there was a positive correlation between CYDV-RPV titer and leaf mass per area. CYDV-RPV titer was reduced by the presence of a competitor, BYDV-PAV, and higher CYDV-RPV titer led to more severe chlorotic symptoms. In our experimental conditions, virus transmission was unaffected by nutrient supply rates, co-infection, plant stoichiometry, or plant traits, although nutrient supply rates have been shown to increase infection and coinfection rates. This work provides a robust test of the role of plant nutrient content and ratios in the dynamics of globally important pathogens and reveals a more complex relationship between within-host virus growth and alterations of plant traits. A deeper understanding of the differential effects of environmental nutrient supplies on virus epidemiology and ecology is particularly relevant given the rapid increase of nutrients flowing into Earth's ecosystems as a result of human activities.

Agroecological and environmental factors influence Barley yellow dwarf viruses in grasslands in the US Pacific Northwest.

Plant pathogens can play a role in the competitive interactions between plant species and have been understudied in native prairies, which are declining globally, and in Conservation Reserve Program (CRP) lands in the United States. Barley/Cereal yellow dwarf virus (B/CYDV) are among the most economically important disease-causing agents of small grain cereal crops, such as wheat, and are known to infect over 150 Poaceae species, including many of the grass species present in prairies and CRP lands. Field surveys of Poaceae species were conducted in endangered Palouse Prairie and CRP habitats of southeastern Washington and adjacent northern Idaho, USA from 2010 to 2012 to examine for the presence of B/CYDV among plant hosts and aphid vectors. Viral species were identified via cloning and sequencing. Landscape, soil and climate data were retrieved from USDA-NASS and USDA-NRCS databases. Analyses were conducted to examine effects of diverse agroecological and environmental factors on virus prevalence. A total of 2271 grass samples representing 30 species were collected; 28 of these were infected with BYDV in at least one location. BYDV infection was detected at every CRP and prairie remnant sampled, with an overall infection of 46%. BYDV-SGV and BYDV-PAV were the only two B/CYDV species encountered, with BYDV-SGV being more prevalent. Sampling time (season) and host plant identity were the main variables explaining variation in virus prevalence among sites. BYDV was more prevalent in perennial compared to annual grass species. Aphids were encountered only once suggesting non-colonizing aphids, potentially from neighboring cereal fields, are responsible for disease spread in these habitats. BYDV prevalence increased in sampled habitats as cereal crop cover increased within a 1-km radius of a habitat patch. Results demonstrate moderate to high and persistent prevalence of BYDV in an endangered grassland habitat. Species composition and susceptibility to pathogens should be considered when creating seed mixes for CRP sites, especially in relation to agricultural crops and diseases in a region. Future work exploring host abundance, competence and habitat utilization by vectors is required to fully elucidate BYDV ecology and epidemiology in grassland habitats.

Frontiers for research on the ecology of plant-pathogenic bacteria: fundamentals for sustainability: Challenges in Bacterial Molecular Plant Pathology.

Methods to ensure the health of crops owe their efficacy to the extent to which we understand the ecology and biology of environmental microorganisms and the conditions under which their interactions with plants lead to losses in crop quality or yield. However, in the pursuit of this knowledge, notions of the ecology of plant-pathogenic microorganisms have been reduced to a plant-centric and agro-centric focus. With increasing global change, i.e. changes that encompass not only climate, but also biodiversity, the geographical distribution of biomes, human demographic and socio-economic adaptations and land use, new plant health problems will emerge via a range of processes influenced by these changes. Hence, knowledge of the ecology of plant pathogens will play an increasingly important role in the anticipation and response to disease emergence. Here, we present our opinion on the major challenges facing the study of the ecology of plant-pathogenic bacteria. We argue that the discovery of markedly novel insights into the ecology of plant-pathogenic bacteria is most likely to happen within a framework of more extensive scales of space, time and biotic interactions than those that currently guide much of the research on these bacteria. This will set a context that is more propitious for the discovery of unsuspected drivers of the survival and diversification of plant-pathogenic bacteria and of the factors most critical for disease emergence, and will set the foundation for new approaches to the sustainable management of plant health. We describe the contextual background of, justification for and specific research questions with regard to the following challenges: Development of terminology to describe plant-bacterial relationships in terms of bacterial fitness. Definition of the full scope of the environments in which plant-pathogenic bacteria reside or survive. Delineation of pertinent phylogenetic contours of plant-pathogenic bacteria and naming of strains independent of their presumed life style. Assessment of how traits of plant-pathogenic bacteria evolve within the overall framework of their life history. Exploration of possible beneficial ecosystem services contributed to by plant-pathogenic bacteria.

Methodological Guidelines for Accurate Detection of Viruses in Wild Plant Species.

Ecological understanding of disease risk, emergence, and dynamics and of the efficacy of control strategies relies heavily on efficient tools for microorganism identification and characterization. Misdetection, such as the misclassification of infected hosts as healthy, can strongly bias estimates of disease prevalence and lead to inaccurate conclusions. In natural plant ecosystems, interest in assessing microbial dynamics is increasing exponentially, but guidelines for detection of microorganisms in wild plants remain limited, particularly so for plant viruses. To address this gap, we explored issues and solutions associated with virus detection by serological and molecular methods in noncrop plant species as applied to the globally important Barley yellow dwarf virus PAV (Luteoviridae), which infects wild native plants as well as crops. With enzyme-linked immunosorbent assays (ELISA), we demonstrate how virus detection in a perennial wild plant species may be much greater in stems than in leaves, although leaves are most commonly sampled, and may also vary among tillers within an individual, thereby highlighting the importance of designing effective sampling strategies. With reverse transcription-PCR (RT-PCR), we demonstrate how inhibitors in tissues of perennial wild hosts can suppress virus detection but can be overcome with methods and products that improve isolation and amplification of nucleic acids. These examples demonstrate the paramount importance of testing and validating survey designs and virus detection methods for noncrop plant communities to ensure accurate ecological surveys and reliable assumptions about virus dynamics in wild hosts.

The community ecology of pathogens: coinfection, coexistence and community composition.

Disease and community ecology share conceptual and theoretical lineages, and there has been a resurgence of interest in strengthening links between these fields. Building on recent syntheses focused on the effects of host community composition on single pathogen systems, we examine pathogen (microparasite) communities using a stochastic metacommunity model as a starting point to bridge community and disease ecology perspectives. Such models incorporate the effects of core community processes, such as ecological drift, selection and dispersal, but have not been extended to incorporate host-pathogen interactions, such as immunosuppression or synergistic mortality, that are central to disease ecology. We use a two-pathogen susceptible-infected (SI) model to fill these gaps in the metacommunity approach; however, SI models can be intractable for examining species-diverse, spatially structured systems. By placing disease into a framework developed for community ecology, our synthesis highlights areas ripe for progress, including a theoretical framework that incorporates host dynamics, spatial structuring and evolutionary processes, as well as the data needed to test the predictions of such a model. Our synthesis points the way for this framework and demonstrates that a deeper understanding of pathogen community dynamics will emerge from approaches working at the interface of disease and community ecology.

Environmental nutrient supply alters prevalence and weakens competitive interactions among coinfecting viruses.

The rates and ratios of environmental nutrient supplies can determine plant community composition. However, the effect of nutrient supplies on within-host microbial interactions is poorly understood. Resource competition is a promising theory for understanding microbial interactions, because microparasites require nitrogen (N) and phosphorus (P) for synthesis of macromolecules such as nucleic acids and proteins. To better understand the effects of nutrient supplies to hosts on pathogen interactions, we singly inoculated and coinoculated Avena sativa with two virus species, barley yellow dwarf virus-PAV (BYDV-PAV) and cereal yellow dwarf virus-RPV (CYDV-RPV). Host plants were grown across a factorial combination of N and P supply rates that created a gradient of N : P supply ratios, one being replicated at low and high nutrient supply. Nutrient supply affected prevalence and the interaction strength among viruses. P addition lowered CYDV-RPV prevalence. The two viruses had a distinct competitive hierarchy: the coinoculation of BYDV-PAV lowered CYDV-RPV infection rate, but the reverse was not true. This antagonistic interaction occurred at low nutrient supply rates and disappeared at high N supply rate. Given the global scale of human alterations of N and P cycles, these results suggest that elevated nutrient supply will increase risks of virus coinfection with likely effects on virus epidemiology, virulence and evolution.

Non-random biodiversity loss underlies predictable increases in viral disease prevalence.

Disease dilution (reduced disease prevalence with increasing biodiversity) has been described for many different pathogens. Although the mechanisms causing this phenomenon remain unclear, the disassembly of communities to predictable subsets of species, which can be caused by changing climate, land use or invasive species, underlies one important hypothesis. In this case, infection prevalence could reflect the competence of the remaining hosts. To test this hypothesis, we measured local host species abundance and prevalence of four generalist aphid-vectored pathogens (barley and cereal yellow dwarf viruses) in a ubiquitous annual grass host at 10 sites spanning 2000 km along the North American West Coast. In laboratory and field trials, we measured viral infection as well as aphid fecundity and feeding preference on several host species. Virus prevalence increased as local host richness declined. Community disassembly was non-random: ubiquitous hosts dominating species-poor assemblages were among the most competent for vector production and virus transmission. This suggests that non-random biodiversity loss led to increased virus prevalence. Because diversity loss is occurring globally in response to anthropogenic changes, such work can inform medical, agricultural and veterinary disease research by providing insights into the dynamics of pathogens nested within a complex web of environmental forces.

Richness and composition of niche-assembled viral pathogen communities.

The pathogen and parasite community that inhabits every free-living organism can control host vital rates including lifespan and reproductive output. To date, however, there have been few experiments examining pathogen community assembly replicated at large-enough spatial scales to inform our understanding of pathogen dynamics in natural systems. Pathogen community assembly may be driven by neutral stochastic colonization and extinction events or by niche differentiation that constrains pathogen distributions to particular environmental conditions, hosts, or vectors. Here, we present results from a regionally-replicated experiment investigating the community of barley and cereal yellow dwarf viruses (B/CYDV's) in over 5000 experimentally planted individuals of six grass species along a 700 km latitudinal gradient along the Pacific coast of North America (USA) in response to experimentally manipulated nitrogen and phosphorus supplies. The composition of the virus community varied predictably among hosts and across nutrient-addition treatments, indicating niche differentiation among virus species. There were some concordant responses among the viral species. For example, the prevalence of most viral species increased consistently with perennial grass cover, leading to a 60% increase in the richness of the viral community within individual hosts (i.e., coinfection) in perennial-dominated plots. Furthermore, infection rates of the six host species in the field were highly correlated with vector preferences assessed in laboratory trials. Our results reveal the importance of niche differentiation in structuring virus assemblages. Virus species distributions reflected a combination of local host community composition, host species-specific vector preferences, and virus responses to host nutrition. In addition, our results suggest that heterogeneity among host species in their capacity to attract vectors or support pathogens between growing seasons can lead to positive covariation among virus species.

Do trade-offs have explanatory power for the evolution of organismal interactions?

The concept of a trade-off has long played a prominent role in understanding the evolution of organismal interactions such as mutualism, parasitism, and competition. Given the complexity inherent to interactions between different evolutionary entities, ecological factors may especially limit the power of trade-off models to predict evolutionary change. Here, we use four case studies to examine the importance of ecological context for the study of trade-offs in organismal interactions: (1) resource-based mutualisms, (2) parasite transmission and virulence, (3) plant biological invasions, and (4) host range evolution in parasites and parasitoids. In the first two case studies, mechanistic trade-off models have long provided a strong theoretical framework but face the challenge of testing assumptions under ecologically realistic conditions. Work under the second two case studies often has a strong ecological grounding, but faces challenges in identifying or quantifying the underlying genetic mechanism of the trade-off. Attention is given to recent studies that have bridged the gap between evolutionary mechanism and ecological realism. Finally, we explore the distinction between ecological factors that mask the underlying evolutionary trade-offs, and factors that actually change the trade-off relationship between fitness-related traits important to organismal interactions.

A single-stranded conformational polymorphism (SSCP)-derived quantitative variable to monitor the virulence of a Barley yellow dwarf virus-PAV (BYDV-PAV) isolate during adaptation to the TC14 resistant wheat line.

A standardized single-stranded conformational polymorphism (SSCP) procedure is proposed as an alternative to the time-consuming biological characterization of Barley yellow dwarf virus-PAV (BYDV-PAV) isolates. Using this procedure, six of 21 overlapping regions used to scan the viral genome gave patterns specific to '4E' (avirulent) or '4T' ('4E'-derived virulent) isolates. The calibration of samples and integration of SSCP patterns corresponding to the nucleotide region 1482-2023 allowed the estimation of P(T) values that reflect the proportions of a '4T'-specific band. Analysis of the biological (area under the pathogen progress curve) and molecular (P(T)) data suggested a positive linear relation between these variables. Moreover, sequence analysis of the nucleotide region 1482-2023 highlighted the presence of a nucleotide polymorphism (C/A(1835)) which can be considered as a candidate for virus-host interactions linked to the monitored virulence. According to these parameters, P(T) values associated with '4E'- and '4T'-derived populations show that: (i) long-term infection of a BYDV-PAV isolate on the 'TC14' resistant host leads to the fixation of virulent individuals in viral populations; and (ii) the introduction of susceptible hosts in successive 'TC14' infections results in the maintenance of low virulence of the populations. Thus, the presented study demonstrates that SSCP is a useful tool for monitoring viral populations during the host adaptation process. The described impact of host alternation provides new opportunities for the use of the 'TC14' resistance source in BYDV-resistant breeding programmes. This study is part of the global effort made by the scientific community to propose sustainable alternatives to the chemical control of this viral disease.

A quantitative proteomic approach using two-dimensional gel electrophoresis and isotope-coded affinity tag labeling for studying human 20S proteasome heterogeneity.

Mammalian proteasomes are macromolecular complexes formed of a catalytic 20S core associated to two regulatory complexes. The 20S core complex consists of four stacked rings of seven alpha or beta subunits. Three beta subunits contain a catalytic site and can be replaced by three interferon gamma-inducible counterparts to form the immunoproteasome. Cells may constitutively possess a mixture of both 20S proteasome types leading to a heterogeneous proteasome population. Purified rat 20S proteasome has been separated in several chromatographic fractions indicating an even higher degree of complexity in 20S proteasome subunit composition. This complexity may arise from the presence of subunit isoforms, as previously detected in purified human erythrocyte 20S proteasome. In this study, we have used a quantitative proteomic approach based on two-dimensional gel electrophoresis and isotope-coded affinity tag (ICAT) labeling to quantify the variations in subunit composition, including subunit isoforms, of 20S proteasomes purified from different cells. The protocol has been adapted to the analysis of low quantities of 20S proteasome complexes. The strategy has then been validated using standard proteins and has been applied to the comparison of 20S proteasomes from erythrocytes and U937 cancer cells. The results obtained show that this approach represents a valuable tool for the study of 20S proteasome heterogeneity.