Genomic and genetic analyses of diversity and plant interactions of Pseudomonas fluorescens

Background: Pseudomonas fluorescens are common soil bacteria that can improve plant health through nutrient cycling, pathogen antagonism and induction of plant defenses. The genome sequences of strains SBW25 and Pf0-1 were determined and compared to each other and with P. fluorescens Pf-5. A functional genomic in vivo expression technology (IVET) screen provided insight into genes used by P. fluorescens in its natural environment and an improved understanding of the ecological significance of diversity within this species. Results: Comparisons of three P. fluorescens genomes (SBW25, Pf0-1, Pf-5) revealed considerable divergence: 61% of genes are shared, the majority located near the replication origin. Phylogenetic and average amino acid identity analyses showed a low overall relationship. A functional screen of SBW25 defined 125 plant-induced genes including a range of functions specific to the plant environment. Orthologues of 83 of these exist in Pf0-1 and Pf-5, with 73 shared by both strains. The P. fluorescens genomes carry numerous complex repetitive DNA sequences, some resembling Miniature Inverted-repeat Transposable Elements (MITEs). In SBW25, repeat density and distribution revealed 'repeat deserts' lacking repeats, covering approximately 40% of the genome. Conclusions: P. fluorescens genomes are highly diverse. Strain-specific regions around the replication terminus suggest genome compartmentalization. The genomic heterogeneity among the three strains is reminiscent of a species complex rather than a single species. That 42% of plant-inducible genes were not shared by all strains reinforces this conclusion and shows that ecological success requires specialized and core functions. The diversity also indicates the significant size of genetic information within the Pseudomonas pan genome.

A role for parasites in stabilising the fig-pollinator mutualism

Mutualisms are interspecific interactions in which both players benefit. Explaining their maintenance is problematic, because cheaters should outcompete cooperative conspecifics, leading to mutualism instability. Monoecious figs (Ficus) are pollinated by host-specific wasps (Agaonidae), whose larvae gall ovules in their "fruits'' (syconia). Female pollinating wasps oviposit directly into Ficus ovules from inside the receptive syconium. Across Ficus species, there is a widely documented segregation of pollinator galls in inner ovules and seeds in outer ovules. This pattern suggests that wasps avoid, or are prevented from ovipositing into, outer ovules, and this results in mutualism stability. However, the mechanisms preventing wasps from exploiting outer ovules remain unknown. We report that in Ficus rubiginosa, offspring in outer ovules are vulnerable to attack by parasitic wasps that oviposit from outside the syconium. Parasitism risk decreases towards the centre of the syconium, where inner ovules provide enemy-free space for pollinator offspring. We suggest that the resulting gradient in offspring viability is likely to contribute to selection on pollinators to avoid outer ovules, and by forcing wasps to focus on a subset of ovules, reduces their galling rates. This previously unidentified mechanism may therefore contribute to mutualism persistence independent of additional factors that invoke plant defences against pollinator oviposition, or physiological constraints on pollinators that prevent oviposition in all available ovules.

Suitability of Arabidopsis thaliana as a model for host plant-Plutella xylostella-Cotesia plutellae interactions

Successful pest management is often hindered by the inherent complexity of the interactions of a pest with its environment. The use of genetically characterized model plants can allow investigation of chosen aspects of these interactions by limiting the number of variables during experimentation. However, it is important to study the generic nature of these model systems if the data generated are to be assessed in a wider context, for instance, with those systems of commercial significance. This study assesses the suitability of Arabidopsis thaliana (L.) Heynh. (Brassicaceae) as a model host plant to investigate plant-herbivore-natural enemy interactions, with Plutella xylostella (L.) (Lepidoptera: Plutellidae), the diamondback moth, and Cotesia plutellae (Kurdjumov) (Hymenoptera: Braconidae), a parasitoid of P. xylostella. The growth and development of P. xylostella and C. plutellae on an A. thaliana host plant (Columbia type) were compared to that on Brassica rapa var. pekinensis (L.) (Brassicaceae), a host crop that is widely cultivated and also commonly used as a laboratory host for P. xylostella rearing. The second part of the study investigated the potential effect of the different A. thaliana background lines, Columbia and Landsberg (used in wider scientific studies), on growth and development of P. xylostella and C. plutellae. Plutella xylostella life history parameters were found generally to be similar between the host plants investigated. However, C. plutellae were more affected by the differences in host plant. Fewer adult parasitoids resulted from development on A. thaliana compared to B. rapa, and those that did emerge were significantly smaller. Adult male C. plutellae developing on Columbia were also significantly smaller than those on Landsberg A. thaliana.

Phytohormones and plant-herbivore-pathogen interactions: integrating the molecular with the ecological

Current research into indirect phytopathogen–herbivore interactions (i.e., interactions mediated by the host plant) is carried out in two largely independent directions: ecological/mechanistic and molecular. We investigate the origin of these approaches and their strengths and weaknesses. Ecological studies have determined the effect of herbivores and phytopathogens on their host plants and are often correlative: the need for long-term manipulative experiments is pressing. Molecular/cellular studies have concentrated on the role of signaling pathways for systemic induced resistance, mainly involving salicylic acid and jasmonic acid, and more recently the cross-talk between these pathways. This cross-talk demonstrates how interactions between signaling mechanisms and phytohormones could mediate plant–herbivore–pathogen interactions. A bridge between these approaches may be provided by field studies using chemical induction of defense, or investigating whole-organism mechanisms of interactions among the three species. To determine the role of phytohormones in induced resistance in the field, researchers must combine ecological and molecular methods. We discuss how these methods can be integrated and present the concept of “kaleidoscopic defense.” Our recent molecular-level investigations of interactions between the herbivore Gastrophysa viridula and the rust fungus Uromyces rumicis on Rumex obtusifolius, which were well studied at the mechanistic and ecological levels, illustrate the difficulty in combining these different approaches. We suggest that the choice of the right study system (possibly wild relatives of model species) is important, and that molecular studies must consider the environmental conditions under which experiments are performed. The generalization of molecular predictions to ecologically realistic settings will be facilitated by “middle-ground studies” concentrating on the outcomes of the interactions.

Global pollinator declines: trends, impacts and drivers

Pollinators are a key component of global biodiversity, providing vital ecosystem services to crops and wild plants. There is clear evidence of recent declines in both wild and domesticated pollinators, and parallel declines in the plants that rely upon them. Here we describe the nature and extent of reported declines, and review the potential drivers of pollinator loss, including habitat loss and fragmentation, agrochemicals, pathogens, alien species, climate change and the interactions between them. Pollinator declines can result in loss of pollination services which have important negative ecological and economic impacts that could significantly affect themaintenance of wild plant diversity, wider ecosystemstability, crop production, food security and human welfare.

Lipid binding interactions of antimicrobial plant seed defence proteins: puroindoline-a and β-purothionin

The indolines and thionins are basic, amphiphilic and cysteine-rich proteins found in cereals; puroindoline-a (Pin-a) and β-purothionin (β-Pth) are members of these families in wheat (Triticum aestivum). Pin-a and β-Pth have been suggested to play a significant role in seed defence against microbial pathogens, making the interaction of these proteins with model bacterial membranes an area of potential interest. We have examined the binding of these proteins to lipid monolayers composed of 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) using a combination of neutron reflectometry, Brewster angle microscopy, and infrared spectroscopy. Results showed that both Pin-a and β-Pth interact strongly with condensed phase DPPG monolayers, but the degree of penetration was different. β-Pth was shown to penetrate the lipid acyl chain region of the monolayer and remove lipids from the air/liquid interface during the adsorption process, suggesting this protein may be able to both form membrane spanning ion channels and remove membrane phospholipids in its lytic activity. Conversely, Pin-a was shown to interact mainly with the head-group region of the condensed phase DPPG monolayer and form a 33 Å thick layer below the lipid film. The differences between the interfacial structures formed by these two proteins may be related to the differing composition of the Pin-a and β-Pth hydrophobic regions.

Pollinator community responses to the spatial population structure of wild plants: A pan-European approach

Land-use changes can alter the spatial population structure of plant species, which may in turn affect the attractiveness of flower aggregations to different groups of pollinators at different spatial scales. To assess how pollinators respond to spatial heterogeneity of plant distributions and whether honeybees affect visitation by other pollinators we used an extensive data set comprising ten plant species and their flower visitors from five European countries. In particular we tested the hypothesis that the composition of the flower visitor community in terms of visitation frequencies by different pollinator groups were affected by the spatial plant population structure, viz. area and density measures, at a within-population (‘patch’) and among-population (‘population’) scale. We found that patch area and population density were the spatial variables that best explained the variation in visitation frequencies within the pollinator community. Honeybees had higher visitation frequencies in larger patches, while bumblebees and hoverflies had higher visitation frequencies in sparser populations. Solitary bees had higher visitation frequencies in sparser populations and smaller patches. We also tested the hypothesis that honeybees affect the composition of the pollinator community by altering the visitation frequencies of other groups of pollinators. There was a positive relationship between visitation frequencies of honeybees and bumblebees, while the relationship with hoverflies and solitary bees varied (positive, negative and no relationship) depending on the plant species under study. The overall conclusion is that the spatial structure of plant populations affects different groups of pollinators in contrasting ways at both the local (‘patch’) and the larger (‘population’) scales and, that honeybees affect the flower visitation by other pollinator groups in various ways, depending on the plant species under study. These contrasting responses emphasize the need to investigate the entire pollinator community when the effects of landscape change on plant–pollinator interactions are studied.

A method for under-sampled ecological network data analysis: plant-pollination as case study

In this paper, we develop a method, termed the Interaction Distribution (ID) method, for analysis of quantitative ecological network data. In many cases, quantitative network data sets are under-sampled, i.e. many interactions are poorly sampled or remain unobserved. Hence, the output of statistical analyses may fail to differentiate between patterns that are statistical artefacts and those which are real characteristics of ecological networks. The ID method can support assessment and inference of under-sampled ecological network data. In the current paper, we illustrate and discuss the ID method based on the properties of plant-animal pollination data sets of flower visitation frequencies. However, the ID method may be applied to other types of ecological networks. The method can supplement existing network analyses based on two definitions of the underlying probabilities for each combination of pollinator and plant species: (1), pi,j: the probability for a visit made by the i’th pollinator species to take place on the j’th plant species; (2), qi,j: the probability for a visit received by the j’th plant species to be made by the i’th pollinator. The method applies the Dirichlet distribution to estimate these two probabilities, based on a given empirical data set. The estimated mean values for pi,j and qi,j reflect the relative differences between recorded numbers of visits for different pollinator and plant species, and the estimated uncertainty of pi,j and qi,j decreases with higher numbers of recorded visits.

Rooting theories of plant community ecology in microbial interactions

Predominant frameworks for understanding plant ecology have an aboveground bias that neglects soil micro-organisms. This is inconsistent with recent work illustrating the importance of soil microbes in terrestrial ecology. Microbial effects have been incorporated into plant community dynamics using ideas of niche modification and plant–soil community feedbacks. Here, we expand and integrate qualitative conceptual models of plant niche and feedback to explore implications of microbial interactions for understanding plant community ecology. At the same time we review the empirical evidence for these processes. We also consider common mycorrhizal networks, and propose that these are best interpreted within the feedback framework. Finally, we apply our integrated model of niche and feedback to understanding plant coexistence, monodominance and invasion ecology.

In vitro antibacterial activity of medicinal plant extracts against Escherichia coli strains from human clinical specimens and interactions with antimicrobial drugs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Trait-mediated effects on flowers: Artificial spiders deceive pollinators and decrease plant fitness

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Nicotiana tabacum as an experimental host for the study of plant-Xylella fastidiosa interactions

Xylella fastidiosa causes citrus variegated chlorosis (CVC). Information generated from the X. fastidiosa genome project is being used to study the underlying mechanisms responsible for pathogenicity. However, the lack of an experimental host other than citrus to study plant-X. fastidiosa interaction has been an obstacle to accelerated progress in this area. We present here results of three experiments that demonstrated that tobacco could be an important experimental host for X. fastidiosa. All tobacco plants inoculated with a citrus strain of X. fastidiosa expressed unequivocal symptoms, consisting of orange leaf lesions, approximately 2 months after injection of the pathogen. CVC symptoms were observed in citrus 3 to 6 months after inoculation. The pathogen was readily detected in symptomatic tobacco plants by polymerase chain reaction (PCR) and phase contrast microscopy. In addition, X. fastidiosa was reisolated on agar plates in 4 of 10 plants. Scanning electron microscopy analysis of cross sections of stems and petioles revealed the presence of rod shaped bacteria restricted to the xylem of inoculated plants. The cell size was within the limit typical of X. fastidiosa.

The importance of plant diversity in maintaining the pollinator bee, Eulaema nigrita (Hymenoptera: Apidae) in sweet passion fruit fields

The euglossine bee Eulaema nigrita plays an important role for the pollination of native and economically important plants, such as the sweet passion-fruit Passiflora alata. E. nigrita uniquely collects the nectar from the flowers of P alata, nevertheless, it needs to visit other plants to collect pollen, nectar and other resources for its survival. There are two methods to identify the species of plants used by bees in their diet: by direct observation of the bees in the flowers, and through identification of pollen grains present in brood cells, feces, or in the bees' body. In order to identify the other plants that E. nigrita visits, we analyzed samples of pollen grains removed from the bee's body in the course of the flowering period of P alata. Among our results, the flora visited by E. nigrita comprised 40 species from 32 genera and 19 families, some of them used as a pollen source or just nectar. In spite of being a polyletic species, E. nigrita exhibited preference for some plant species with poricidal anthers. P alata which has high sugar concentration nectar was the main source of nectar for this bee in the studied area. Nonetheless, the pollinic analysis indicated that others nectariferous plant species are necessary to keep the populations of E. nigrita. Studies such as this one are important since they indicate supplementary pollen-nectar sources which must be used for the conservation of the populations of E. nigrita in crops neighbouring areas. In the absence of pollinators, growers are forced to pay for hand pollination, which increases production costs; keeping pollinators in cultivated areas is still more feasible to ensure sweet passion fruit production. Rev. Biol. Trop. 60 (4): 1553-1565. Epub 2012 December 01.

Analysis of three Xanthomonas axonopodis pv. citri effector proteins in pathogenicity and their interactions with host plant proteins

Xanthomonas axonopodis pv. citri, the bacterium responsible for citrus canker, uses effector proteins secreted by a type III protein secretion system to colonize its hosts. Among the putative effector proteins identified for this bacterium, we focused on the analysis of the roles of AvrXacE1, AvrXacE2 and Xac3090 in pathogenicity and their interactions with host plant proteins. Bacterial deletion mutants in avrXacE1, avrXacE2 and xac3090 were constructed and evaluated in pathogenicity assays. The avrXacE1 and avrXacE2 mutants presented lesions with larger necrotic areas relative to the wild-type strain when infiltrated in citrus leaves. Yeast two-hybrid studies were used to identify several plant proteins likely to interact with AvrXacE1, AvrXacE2 and Xac3090. We also assessed the localization of these effector proteins fused to green fluorescent protein in the plant cell, and observed that they co-localized to the subcellular spaces in which the plant proteins with which they interacted were predicted to be confined. Our results suggest that, although AvrXacE1 localizes to the plant cell nucleus, where it interacts with transcription factors and DNA-binding proteins, AvrXacE2 appears to be involved in lesion-stimulating disease 1-mediated cell death, and Xac3090 is directed to the chloroplast where its function remains to be clarified.