Draft genome sequences of eight bacterial isolates from Pisum sativum leaf surfaces.

Bacterial communities in the phyllosphere, the above-ground parts of plants, are diverse yet understudied. These bacteria are important for plant health and also for inter-kingdom interactions with beneficial and pest insect species. Here, we present draft genomes of eight culturable bacterial isolates from leaf surfaces in the Pisum sativum phyllosphere.

Context-Dependent Benefits of Aphids for Bacteria in the Phyllosphere.

AbstractInsect herbivores, such as aphids, are common on plants, yet how they interact with plant microbiomes remains largely unknown. For instance, for the widespread bacterial epiphyte and potential aphid pathogen Pseudomonas syringae, aphids could impact bacterial populations by serving as secondary hosts or by altering the epiphytic habitat through feeding and/or waste secretion. Here, we examined whether the pea aphid, Acyrthosiphon pisum, could influence epiphytic populations of P. syringae. First, we quantified epiphytic growth ability without aphids and virulence to aphids across 21 diverse P. syringae strains. For eight strains that varied in these traits we then assessed the influence of aphid presence on epiphytic bacterial growth. In some cases P. syringae benefited significantly from the presence of aphids, with up to 3.8 times more cell doublings. This benefit was not correlated with strain traits but rather with initial population densities; smaller bacterial populations received relatively more benefit from aphids, and larger populations received less benefit. Honeydew, the sugary waste product of aphids, in the absence of aphids was sufficient to increase P. syringae density on leaves. We conclude that aphid honeydew can sometimes increase P. syringae epiphytic growth but that the bacteria may not benefit from using aphids as hosts.

Genetic identity and genotype × genotype interactions between symbionts outweigh species level effects in an insect microbiome.

Microbial symbionts often alter the phenotype of their host. Benefits and costs to hosts depend on many factors, including host genotype, symbiont species and genotype, and environmental conditions. Here, we present a study demonstrating genotype-by-genotype (G×G) interactions between multiple species of endosymbionts harboured by an insect, and the first to quantify the relative importance of G×G interactions compared with species interactions in such systems. In the most extensive study to date, we microinjected all possible combinations of five Hamiltonella defensa and five Fukatsuia symbiotica (X-type; PAXS) isolates into the pea aphid, Acyrthosiphon pisum. We applied several ecological challenges: a parasitoid wasp, a fungal pathogen, heat shock, and performance on different host plants. Surprisingly, genetic identity and genotype × genotype interactions explained far more of the phenotypic variation (on average 22% and 31% respectively) than species identity or species interactions (on average 12% and 0.4%, respectively). We determined the costs and benefits associated with co-infection, and how these compared to corresponding single infections. All phenotypes were highly reliant on individual isolates or interactions between isolates of the co-infecting partners. Our findings highlight the importance of exploring the eco-evolutionary consequences of these highly specific interactions in communities of co-inherited species.

Epiphytic Strains of Pseudomonas syringae Kill Diverse Aphid Species.

Interactions between epiphytic bacteria and herbivorous insects are ubiquitous on plants, but little is known about their ecological implications. Aphids are devastating crop pests worldwide, so understanding how epiphytic bacteria impact aphid populations is critically important. Recent evidence demonstrates that plant-associated bacteria, such as Pseudomonas syringae, can be highly virulent to one species of aphid, the pea aphid (Acyrthosiphon pisum). Currently, we have no knowledge on how broad this phenomenon is across diverse aphid species that are of high agricultural concern. In controlled experiments using oral exposure in an artificial diet, we challenged five aphid species of agricultural importance with three strains of P. syringae that vary in virulence to the pea aphid. These strains also vary in epiphytic ability and comprise two phytopathogens and one non-plant-pathogenic strain. In general, differences in virulence to aphids remained relatively constant across strains regardless of the aphid species, except for the bird cherry-oat aphid (Rhopalosiphum padi), which is significantly less susceptible to two P. syringae strains. We demonstrate that lower infection incidence likely plays a role in the reduced susceptibility. Importantly, these data support previous results showing that interactions with epiphytic bacteria are important for aphids and may play a large, but underappreciated, role in insect population dynamics. Our study illustrates a potential role of epiphytic bacteria in the biological control of aphid pests broadly but suggests the need for more research encompassing a greater diversity of pest species.IMPORTANCE Sap-sucking aphids are insects of huge agricultural concern, not only because of direct damage caused by feeding but also because of their ability to transmit various plant pathogens. Some bacteria that grow on leaf surfaces, such as Pseudomonas syringae, can infect and kill aphids, making them potentially useful in the biological control of pest aphids. However, only one aphid species, the pea aphid (Acyrthosiphon pisum), has been tested for infection by P. syringae Here, we challenged five aphid species of agricultural importance with three strains of P. syringae that vary in virulence to the pea aphid. We found that four of these aphid species were susceptible to infection and death, suggesting that these bacteria are broadly useful for biological control. However, one aphid species was much more resistant to infection, indicating that more testing on diverse aphid species is needed.

Visual Detection and Avoidance of Pathogenic Bacteria by Aphids.

Aphids are diverse sap-sucking insects [1] that can be serious agricultural pests and vectors of plant disease [2]. Some species, including pea aphids (Acyrthosiphon pisum), are susceptible to infection by epiphytic bacteria that are commonly found on plant surfaces [3-5]. Pea aphids appear unable to recover from these infections, possibly because pea aphids are missing apparent orthologs of some immune response genes [6], and these aphids exhibit relatively low immune responses after pathogen exposure [7]. We therefore tested the ability of pea aphids to use avoidance as a non-immunological defense against Pseudomonas syringae, a widespread plant epiphyte and aphid pathogen [8, 9]. Pea aphids avoided highly virulent strains of P. syringae, but not all strains, and avoidance led to a significant reduction in infection among aphids. We found that aphids can use visual cues to detect the ultraviolet (UV)-based fluorescence of the bacterial siderophore pyoverdine [10] produced by virulent strains. Avoided epiphytic bacteria caused light leaving the surface of leaves to be richer in wavelengths that were tightly linked to both aphid visual sensitivities and the fluorescent emission spectra of pyoverdine, suggesting that pyoverdine fluorescence mediates avoidance and may be a visual cue used by aphids to detect epiphytic pathogens. Although pyoverdine production in Pseudomonas species may be a broadly reliable indicator of bacterial virulence within the phyllosphere, it was not directly responsible for virulence to aphids. Aphids may be under selection to avoid fluorescence on leaves, a phenomenon with potential use for the control of agricultural pest insects.

Entomopathogenicity to Two Hemipteran Insects Is Common but Variable across Epiphytic Pseudomonas syringae Strains.

Strains of the well-studied plant pathogen Pseudomonas syringae show large differences in their ability to colonize plants epiphytically and to inflict damage to hosts. Additionally, P. syringae can infect some sap-sucking insects and at least one P. syringae strain is highly virulent to insects, causing death to most individuals within as few as 4 days and growing to high population densities within insect hosts. The likelihood of agricultural pest insects coming into contact with transient populations of P. syringae while feeding on plants is high, yet the ecological implications of these interactions are currently not well understood as virulence has not been tested across a wide range of strains. To investigate virulence differences across strains we exposed the sweet potato whitefly, Bemisia tabaci, and the pea aphid, Acyrthosiphon pisum, both of which are cosmopolitan agricultural pests, to 12 P. syringae strains. We used oral inoculations with bacteria suspended in artificial diet in order to assay virulence while controlling for other variables such as differences in epiphytic growth ability. Generally, patterns of pathogenicity remain consistent across the two species of hemipteran insects, with bacterial strains from phylogroup II, or genomospecies 1, causing the highest rate of mortality with up to 86% of individuals dead after 72 h post infection. The rate of mortality is highly variable across strains, some significantly different from negative control treatments and others showing no discernable difference. Interestingly, one of the most pathogenic strains to both aphids and whiteflies (Cit7) is thought to be non-pathogenic on plants. We also found Cit7 to establish the highest epiphytic population after 48 h on fava beans. Between the nine P. syringae strains tested for epiphytic ability there is also much variation, but epiphytic ability was positively correlated with pathogenicity to insects, suggesting that the two traits may be linked and that strains likely to be found on plants may often be entomopathogenic. Our study highlights that there may be a use for epiphytic bacteria in the biological control of insect crop pests. It also suggests that interactions with epiphytic bacteria could be evolutionary and ecological drivers for hemipteran insects.

Host-associated genomic differentiation in congeneric butterflies: now you see it, now you do not.

Ecotypic variation among populations may become associated with widespread genomic differentiation, but theory predicts that this should happen only under particular conditions of gene flow, selection and population size. In closely related species, we might expect the strength of host-associated genomic differentiation (HAD) to be correlated with the degree of phenotypic differentiation in host-adaptive traits. Using microsatellite and Amplified Fragment Length Polymorphism (AFLP) markers, and controlling for isolation by distance between populations, we sought HAD in two congeneric species of butterflies with different degrees of host plant specialization. Prior work on Euphydryas editha had shown strong interpopulation differentiation in host-adapted traits, resulting in incipient reproductive isolation among host-associated ecotypes. We show here that Euphydryas aurinia had much weaker host-associated phenotypic differentiation. Contrary to our expectations, we detected HAD in Euphydryas aurinia, but not in E. editha. Even within an E. aurinia population that fed on both hosts, we found weak but significant sympatric HAD that persisted in samples taken 9 years apart. The finding of significantly stronger HAD in the system with less phenotypic differentiation may seem paradoxical. Our findings can be explained by multiple factors, ranging from differences in dispersal or effective population size, to spatial variation in genomic or phenotypic traits and to structure induced by past histories of host-adapted populations. Other infrequently measured factors, such as differences in recombination rates, may also play a role. Our result adds to recent work as a further caution against assumptions of simple relationships between genomic and adaptive phenotypic differentiation.

Microsatellites for the marsh fritillary butterfly: de novo transcriptome sequencing, and a comparison with amplified fragment length polymorphism (AFLP) markers.

BACKGROUND: Until recently the isolation of microsatellite markers from Lepidoptera has proved troublesome, expensive and time-consuming. Following on from a previous study of Edith's checkerspot butterfly, Euphydryas editha, we developed novel microsatellite markers for the vulnerable marsh fritillary butterfly, E. aurinia. Our goal was to optimize the process in order to reduce both time and cost relative to prevailing techniques. This was accomplished by using a combination of previously developed techniques: in silico mining of a de novo assembled transcriptome sequence, and genotyping the microsatellites found there using an economic method of fluorescently labelling primers. PRINCIPAL FINDINGS: In total, we screened nine polymorphic microsatellite markers, two of which were previously published, and seven that were isolated de novo. These markers were able to amplify across geographically isolated populations throughout Continental Europe and the UK. Significant deviations from Hardy-Weinberg equilibrium were evident in some populations, most likely due to the presence of null alleles. However, we used an F(st) outlier approach to show that these markers are likely selectively neutral. Furthermore, using a set of 128 individuals from 11 populations, we demonstrate consistency in population differentiation estimates with previously developed amplified fragment length polymorphism (AFLP) markers (r = 0.68, p<0.001). SIGNIFICANCE: Rapid development of microsatellite markers for difficult taxa such as Lepidoptera, and concordant results with other putatively neutral molecular markers, demonstrate the potential of de novo transcriptional sequencing for future studies of population structure and gene flow that are desperately needed for declining species across fragmented landscapes.