Microbial associates of the elm leaf beetle: uncovering the absence of resident bacteria and the influence of fungi on insect performance.

Microbial symbionts play crucial roles in the biology of many insects. While bacteria have been the primary focus of research on insect-microbe symbiosis, recent studies suggest that fungal symbionts may be just as important. The elm leaf beetle (ELB, Xanthogaleruca luteola) is a serious pest species of field elm (Ulmus minor). Using culture-dependent and independent methods, we investigated the abundance and species richness of bacteria and fungi throughout various ELB life stages and generations, while concurrently analyzing microbial communities on elm leaves. No persistent bacterial community was found to be associated with the ELB or elm leaves. By contrast, fungi were persistently present in the beetle's feeding life stages and on elm leaves. Fungal community sequencing revealed a predominance of the genera Penicillium and Aspergillus in insects and on leaves. Culture-dependent surveys showed a high prevalence of two fungal colony morphotypes closely related to Penicillium lanosocoeruleum and Aspergillus flavus. Among these, the Penicillium morphotype was significantly more abundant on feeding-damaged compared with intact leaves, suggesting that the fungus thrives in the presence of the ELB. We assessed whether the detected prevalent fungal morphotypes influenced ELB's performance by rearing insects on (i) surface-sterilized leaves, (ii) leaves inoculated with Penicillium spores, and (iii) leaves inoculated with Aspergillus spores. Insects feeding on Penicillium-inoculated leaves gained more biomass and tended to lay larger egg clutches than those consuming surface-sterilized leaves or Aspergillus-inoculated leaves. Our results demonstrate that the ELB does not harbor resident bacteria and that it might benefit from associating with Penicillium fungi.IMPORTANCEOur study provides insights into the still understudied role of microbial symbionts in the biology of the elm leaf beetle (ELB), a major pest of elms. Contrary to expectations, we found no persistent bacterial symbionts associated with the ELB or elm leaves. Our research thus contributes to the growing body of knowledge that not all insects rely on bacterial symbionts. While no persistent bacterial symbionts were detectable in the ELB and elm leaf samples, our analyses revealed the persistent presence of fungi, particularly Penicillium and Aspergillus on both elm leaves and in the feeding ELB stages. Moreover, when ELB were fed with fungus-treated elm leaves, we detected a potentially beneficial effect of Penicillium on the ELB's development and fecundity. Our results highlight the significance of fungal symbionts in the biology of this insect.

Hitching a Ride in the Phyllosphere: Surfactant Production of Pseudomonas spp. Causes Co-swarming of Pantoea eucalypti 299R.

Here, we demonstrate the beneficial effect of surfactant-producing pseudomonads on Pantoea eucalypti 299R. We conducted a series of experiments in environments of increasing complexity. P. eucalypti 299R (Pe299R), and Pseudomonas sp. FF1 (Pff1) or Pe299R and surfactant-production deficient Pseudomonas sp. FF1::ΔviscB (Pff1ΔviscB) were co-inoculated in broth, on swarming agar plates, and on plants. In broth, there were no differences in the growth dynamics of Pe299R when growing in the presence of Pff1 or Pff1ΔviscB. By contrast, on swarming agar plates, Pe299R was able to co-swarm with Pff1 which led to a significant increase in Pe299R biomass compared to Pe299R growing with Pff1ΔviscB or in monoculture. Finally in planta, and using the single-cell bioreporter for reproductive success (CUSPER), we found a temporally distinct beneficial effect of Pff1 on co-inoculated Pe299R subpopulations that did not occur in the presence of Pff1ΔviscB. We tested three additional surfactant-producing pseudomonads and their respective surfactant knockout mutants on PE299R on swarming agar showing similar results. This led us to propose a model for the positive effect of surfactant production during leaf colonization. Our results indicate that co-motility might be common during leaf colonization and adds yet another facet to the already manyfold roles of surfactants.

Priority Effects in the Apple Flower Determine If the Siderophore Desferrioxamine Is a Virulence Factor for Erwinia amylovora CFBP1430.

Iron is crucial for bacterial growth and virulence. Under iron-deficiency bacteria produce siderophores, iron chelators that facilitate the iron uptake into the cell via specific receptors. Erwinia amylovora, the causative agent of fire blight, produces hydroxamate-type desferrioxamine siderophores (DFO). The presented study reassesses the impact of DFO as a virulence factor of E. amylovora during its epiphytic phase on the apple flower. When inoculated in semisterile Golden Delicious flowers no difference in replication and induction of calyx necrosis could be observed between E. amylovora CFBP1430 siderophore synthesis (DfoA) or uptake (FoxR receptor) mutants and the parental strain. In addition, mutant strains only weakly induced a foxR promoter-gfpmut2 reporter construct in the flowers. When analyzing the replication of the receptor mutant in apple flowers harboring an established microbiome, either naturally, in case of orchard flowers, or by pre-inoculation of semisterile greenhouse flowers, it became evident that the mutant strain had a significantly reduced replication compared to the parental strain. The results suggest that apple flowers per se are not an iron-limiting environment for E. amylovora and that DFO is an important competition factor for the pathogen in precolonized flowers. IMPORTANCE Desferrioxamine is a siderophore produced by the fire blight pathogen E. amylovora under iron-limited conditions. In the present study, no or only weak induction of an iron-regulated promoter-GFP reporter was observed on semisterile apple flowers, and siderophore synthesis or uptake (receptor) mutants exhibited colonization of the flower and necrosis induction at parental levels. Reduced replication of the receptor mutant was observed when the flowers were precolonized by microorganisms. The results indicate that apple flowers are an iron-limited environment for E. amylovora only if precolonization with microorganisms leads to iron competition. This is an important insight for the timing of biocontrol treatments.

Fluorescent Protein Expression as a Proxy for Bacterial Fitness in a High-Throughput Assay.

Bacterial growth is classically assessed by measuring the increases in optical density of pure cultures in shaken liquid media. Measuring growth using optical density has severe limitations when studying multistrain interactions, as it is not possible to measure the growth of individual strains within mixed cultures. Here, we demonstrated that constitutively expressed fluorescent proteins can be used to track the growth of individual strains in different liquid media. Fluorescence measurements were highly correlated with optical density measurements and cell counts. This allowed us to assess bacterial growth not only in pure cultures but also in mixed bacterial cultures and determine the impact of a competitor on a focal strain, thereby assessing relative fitness. Furthermore, we were able to track the growth of two different strains simultaneously by using fluorescent proteins with differential excitation and emission wavelengths. Bacterial densities measured by fluorescence yielded more consistent data between technical replicates than optical density measurements. Our setup employs fluorescence microplate readers that allow high throughput and replication. IMPORTANCE We expand on an important limitation of the concept of measuring bacterial growth, which is classically limited to one strain at a time. By adopting our approach, it is possible to measure the growth of several bacterial strains simultaneously with high temporal resolution and in a high-throughput manner. This is important to investigate bacterial interactions, such as competition and facilitation.

Functional overlap of the Arabidopsis leaf and root microbiota.

Roots and leaves of healthy plants host taxonomically structured bacterial assemblies, and members of these communities contribute to plant growth and health. We established Arabidopsis leaf- and root-derived microbiota culture collections representing the majority of bacterial species that are reproducibly detectable by culture-independent community sequencing. We found an extensive taxonomic overlap between the leaf and root microbiota. Genome drafts of 400 isolates revealed a large overlap of genome-encoded functional capabilities between leaf- and root-derived bacteria with few significant differences at the level of individual functional categories. Using defined bacterial communities and a gnotobiotic Arabidopsis plant system we show that the isolates form assemblies resembling natural microbiota on their cognate host organs, but are also capable of ectopic leaf or root colonization. While this raises the possibility of reciprocal relocation between root and leaf microbiota members, genome information and recolonization experiments also provide evidence for microbiota specialization to their respective niche.

Pseudomonas orientalis F9 Pyoverdine, Safracin, and Phenazine Mutants Remain Effective Antagonists against Erwinia amylovora in Apple Flowers.

The recently characterized strain Pseudomonas orientalis F9, an isolate from apple flowers in a Swiss orchard, exhibits antagonistic traits against phytopathogens. At high colonization densities, it exhibits phytotoxicity against apple flowers. P. orientalis F9 harbors biosynthesis genes for the siderophore pyoverdine as well as for the antibiotics safracin and phenazine. To elucidate the role of the three compounds in biocontrol, we screened a large random knockout library of P. orientalis F9 strains for lack of pyoverdine production or in vitro antagonism. Transposon mutants that lacked the ability for fluorescence carried transposons in pyoverdine production genes. Mutants unable to antagonize Erwinia amylovora in an in vitro double-layer assay carried transposon insertions in the safracin gene cluster. As no phenazine transposon mutant could be identified using the chosen selection criteria, we constructed a site-directed deletion mutant. Pyoverdine-, safracin-, and phenazine mutants were tested for their abilities to counteract the fire blight pathogen Erwinia amylovoraex vivo on apple flowers or the soilborne pathogen Pythium ultimumin vivo in a soil microcosm. In contrast to some in vitro assays, ex vivo and in vivo assays did not reveal significant differences between parental and mutant strains in their antagonistic activities. This suggests that, ex vivo and in vivo, other factors, such as competition for resources or space, are more important than the tested antibiotics or pyoverdine for successful antagonism of P. orientalis F9 against phytopathogens in the performed assays.IMPORTANCEPseudomonas orientalis F9 is an antagonist of the economically important phytopathogen Erwinia amylovora, the causal agent of fire blight in pomme fruit. On King's B medium, P. orientalis F9 produces a pyoverdine siderophore and the antibiotic safracin. P. orientalis F9 transposon mutants lacking these factors fail to antagonize E. amylovora, depending on the in vitro assay. On isolated flowers and in soil microcosms, however, pyoverdine, safracin, and phenazine mutants control phytopathogens as clearly as their parental strains.

Pushing the limits of de novo genome assembly for complex prokaryotic genomes harboring very long, near identical repeats.

Generating a complete, de novo genome assembly for prokaryotes is often considered a solved problem. However, we here show that Pseudomonas koreensis P19E3 harbors multiple, near identical repeat pairs up to 70 kilobase pairs in length, which contained several genes that may confer fitness advantages to the strain. Its complex genome, which also included a variable shufflon region, could not be de novo assembled with long reads produced by Pacific Biosciences' technology, but required very long reads from Oxford Nanopore Technologies. Importantly, a repeat analysis, whose results we release for over 9600 prokaryotes, indicated that very complex bacterial genomes represent a general phenomenon beyond Pseudomonas. Roughly 10% of 9331 complete bacterial and a handful of 293 complete archaeal genomes represented this 'dark matter' for de novo genome assembly of prokaryotes. Several of these 'dark matter' genome assemblies contained repeats far beyond the resolution of the sequencing technology employed and likely contain errors, other genomes were closed employing labor-intense steps like cosmid libraries, primer walking or optical mapping. Using very long sequencing reads in combination with assembly algorithms capable of resolving long, near identical repeats will bring most prokaryotic genomes within reach of fast and complete de novo genome assembly.

Stabilization of cooperative virulence by the expression of an avirulent phenotype.

Pathogens often infect hosts through collective actions: they secrete growth-promoting compounds or virulence factors, or evoke host reactions that fuel the colonization of the host. Such behaviours are vulnerable to the rise of mutants that benefit from the collective action without contributing to it; how these behaviours can be evolutionarily stable is not well understood. We address this question using the intestinal pathogen Salmonella enterica serovar Typhimurium (hereafter termed S. typhimurium), which manipulates its host to induce inflammation, and thereby outcompetes the commensal microbiota. Notably, the virulence factors needed for host manipulation are expressed in a bistable fashion, leading to a slow-growing subpopulation that expresses virulence genes, and a fast-growing subpopulation that is phenotypically avirulent. Here we show that the expression of the genetically identical but phenotypically avirulent subpopulation is essential for the evolutionary stability of virulence in this pathogen. Using a combination of mathematical modelling, experimental evolution and competition experiments we found that within-host evolution leads to the emergence of mutants that are genetically avirulent and fast-growing. These mutants are defectors that exploit inflammation without contributing to it. In infection experiments initiated with wild-type S. typhimurium, defectors increase only slowly in frequency. In a genetically modified S. typhimurium strain in which the phenotypically avirulent subpopulation is reduced in size, defectors rise more rapidly, inflammation ceases prematurely, and S. typhimurium is quickly cleared from the gut. Our results establish that host manipulation by S. typhimurium is a cooperative trait that is vulnerable to the rise of avirulent defectors; the expression of a phenotypically avirulent subpopulation that grows as fast as defectors slows down this process, and thereby promotes the evolutionary stability of virulence. This points to a key role of bistable virulence gene expression in stabilizing cooperative virulence and may lead the way to new approaches for controlling pathogens.

Phyllosphere microbiology: at the interface between microbial individuals and the plant host.

Contents Summary 1327 I. Introduction 1327 II. Individuality and the relevance of scales for the investigation of bacteria 1328 III. Bacterial aggregation and community patterning at the single-cell resolution 1329 IV. What are the effects on the plant host? 1330 V. Future directions and current questions 1331 Acknowledgements 1332 ORCID 1332 References 1332 SUMMARY: Leaf surfaces are home to diverse bacterial communities. Within these communities, every individual cell perceives its unique environment and responds accordingly. In this insight article, the perspective of the bacterial individual is assumed in an attempt to describe how the spatially heterogeneous leaf surface determines the fate of bacteria. To investigate behaviour at scales relevant to bacteria, single-cell approaches are essential. Single-cell studies provide important lessons about how current 'omics' approaches fail to give an accurate picture of the behaviour of bacterial populations in heterogeneous environments. Upcoming techniques will soon allow us to combine the power of single-cell and omics approaches.

Fe2+ chelator proferrorosamine A: a gene cluster of Erwinia rhapontici P45 involved in its synthesis and its impact on growth of Erwinia amylovora CFBP1430.

Proferrorosamine A (proFRA) is an iron (Fe2+) chelator produced by the opportunistic plant pathogen Erwinia rhapontici P45. To identify genes involved in proFRA synthesis, transposon mutagenesis was performed. The identified 9.3 kb gene cluster, comprising seven genes, designated rosA-rosG, encodes proteins that are involved in proFRA synthesis. Based on gene homologies, a biosynthetic pathway model for proFRA is proposed. To obtain a better understanding of the effect of proFRA on non-proFRA producing bacteria, E. rhapontici P45 was co-cultured with Erwinia amylovora CFBP1430, a fire-blight-causing plant pathogen. E. rhapontici P45, but not corresponding proFRA-negative mutants, led to a pink coloration of E. amylovora CFBP1430 colonies on King's B agar, indicating accumulation of the proFRA-iron complex ferrorosamine, and growth inhibition in vitro. By saturating proFRA-containing extracts with Fe2+, the inhibitory effect was neutralized, suggesting that the iron-chelating capability of proFRA is responsible for the growth inhibition of E. amylovora CFBP1430.

Stable Fluorescent and Enzymatic Tagging of Bradyrhizobium diazoefficiens to Analyze Host-Plant Infection and Colonization.

Bradyrhizobium diazoefficiens USDA 110 (formerly named Bradyrhizobium japonicum) can fix dinitrogen when living as an endosymbiont in root nodules of soybean and some other legumes. Formation of a functional symbiosis relies on a defined developmental program mediated by controlled gene expression in both symbiotic partners. In contrast to other well-studied Rhizobium-legume model systems that have been thoroughly examined by means of genetically tagged strains, analysis of B. diazoefficiens host infection has been impaired due to the lack of suitable tagging systems. Here, we describe the construction of B. diazoefficiens strains constitutively expressing single-copy genes for fluorescent proteins (eBFP2, mTurquoise2, GFP+, sYFP2, mCherry, HcRed) and enzymes (GusA, LacZ). For stable inheritance, the constructs were recombined into the chromosome. Effectiveness and versatility of the tagged strains was demonstrated in plant infection assays. (i) The infection process was followed from root-hair attachment to colonization of nodule cells with epifluorescent microscopy. (ii) Monitoring mixed infections with two strains producing different fluorescent proteins allowed rapid analysis of nodule occupancy and revealed that the majority of nodules contained clonal populations. (iii) Microscopic analysis of nodules induced by fluorescent strains provided evidence for host-dependent control of B. diazoefficiens bacteroid morphology in nodules of Aeschynomene afraspera and Arachis hypogaea (peanut), as deduced from their altered morphology compared with bacteroids in soybean nodules.

Spatial distribution analyses of natural phyllosphere-colonizing bacteria on Arabidopsis thaliana revealed by fluorescence in situ hybridization.

Bacterial colonizers of the aerial parts of plants, or phyllosphere, have been identified on a number of different plants using cultivation-dependent and independent methods. However, the spatial distribution at the micrometer scale of different main phylogenetic lineages is not well documented and mostly based on fluorescence-tagged model strains. In this study, we developed and applied a spatial explicit approach that allowed the use of fluorescence in situ hybridization (FISH) to study bacterial phylloplane communities of environmentally grown Arabidopsis thaliana. We found on average 5.4 × 10(6) bacteria cm(-2) leaf surface and 1.5 × 10(8) bacteria g(-1) fresh weight. Furthermore, we found that the total biomass in the phylloplane was normally distributed. About 31% of the bacteria found in the phylloplane did not hybridize to FISH probes but exhibited infrared autofluorescence indicative for aerobic anoxygenic phototrophs. Four sets of FISH probes targeting Alphaproteobacteria, Betaproteobacteria, Actinobacteria and Bacteroidetes were sufficient to identify all other major contributors of the phylloplane community based on general bacterial probing. Spatial aggregation patterns were observed for all probe-targeted populations at distances up to 7 µm, with stronger tendencies to co-aggregate for members of the same phylogenetic group. Our findings contribute to a bottom-up description of leaf surface community composition.

Nonpathogenic leaf-colonizing bacteria elicit pathogen-like responses in a colonization density-dependent manner.

Leaves are colonized by a complex mix of microbes, termed the leaf microbiota. Even though the leaf microbiota is increasingly recognized as an integral part of plant life and health, our understanding of its interactions with the plant host is still limited. Here, mature, axenically grown Arabidopsis thaliana plants were spray inoculated with six diverse leaf-colonizing bacteria. The transcriptomic changes in leaves were tracked over time and significant changes in ethylene marker (ARL2) expression were observed only 2-4 days after spray inoculation. Whole-transcriptome sequencing revealed that 4 days after inoculation, leaf transcriptional changes to colonization by nonpathogenic and pathogenic bacteria differed in strength but not in the type of response. Inoculation of plants with different densities of the nonpathogenic bacterium Williamsia sp. Leaf354 showed that high bacterial titers resulted in disease phenotypes and led to severe transcriptional reprogramming with a strong focus on plant defense. An in silico epigenetic analysis of the data was congruent with the transcriptomic analysis. These findings suggest (1) that plant responses are not rapid after spray inoculation, (2) that plant responses only differ in strength, and (3) that plants respond to high titers of nonpathogenic bacteria with pathogen-like responses.

Metabolic resource overlap impacts competition among phyllosphere bacteria.

The phyllosphere is densely colonised by microbial communities, despite sparse and heterogeneously distributed resources. The limitation of resources is expected to drive bacterial competition resulting in exclusion or coexistence based on fitness differences and resource overlap between individual colonisers. We studied the impact of resource competition by determining the effects of different bacterial colonisers on the growth of the model epiphyte Pantoea eucalypti 299R (Pe299R). Resource overlap was predicted based on genome-scale metabolic modelling. By combining results of metabolic modelling and pairwise competitions in the Arabidopsis thaliana phyllosphere and in vitro, we found that ten resources sufficed to explain fitness of Pe299R. An effect of both resource overlap and phylogenetic relationships was found on competition outcomes in vitro as well as in the phyllosphere. However, effects of resource competition were much weaker in the phyllosphere when compared to in vitro experiments. When investigating growth dynamics and reproductive success at the single-cell resolution, resource overlap and phylogenetic relationships are only weakly correlated with epiphytic Pe299R reproductive success, indicating that the leaf's spatial heterogeneity mitigates resource competition. Although the correlation is weak, the presence of competitors led to the development of Pe299R subpopulations that experienced different life histories and cell divisions. In some in planta competitions, Pe299R benefitted from the presence of epiphytes despite high resource overlap to the competitor strain suggesting other factors having stronger effects than resource competition. This study provides fundamental insights into how bacterial communities are shaped in heterogeneous environments and a framework to predict competition outcomes.

Conjugation Dynamics of Self-Transmissible and Mobilisable Plasmids into E. coli O157:H7 on Arabidopsis thaliana Rosettes.

Many antibiotic resistance genes present in human pathogenic bacteria are believed to originate from environmental bacteria. Conjugation of antibiotic resistance conferring plasmids is considered to be one of the major reasons for the increasing prevalence of antibiotic resistances. A hotspot for plasmid-based horizontal gene transfer is the phyllosphere, i.e., the surfaces of aboveground plant parts. Bacteria in the phyllosphere might serve as intermediate hosts with transfer capability to human pathogenic bacteria. In this study, the exchange of mobilisable and self-transmissible plasmids via conjugation was evaluated. The conjugation from the laboratory strain Escherichia coli S17-1, the model phyllosphere coloniser Pantoea eucalypti 299R, and the model pathogen E. coli O157:H7 to the recipient strain E. coli O157:H7::MRE103 (EcO157:H7red) in the phyllosphere of Arabidopsis thaliana was determined. The results suggest that short-term occurrence of a competent donor is sufficient to fix plasmids in a recipient population of E. coli O157:H7red. The spread of self-transmissible plasmids was limited after initial steep increases of transconjugants that contributed up to 10% of the total recipient population. The here-presented data of plasmid transfer will be important for future modelling approaches to estimate environmental spread of antibiotic resistance in agricultural production environments.

Litterbox-A gnotobiotic Zeolite-Clay System to Investigate Arabidopsis-Microbe Interactions.

Plants are colonised by millions of microorganisms representing thousands of species withvarying effects on plant growth and health. The microbial communities found on plants arecompositionally consistent and their overall positive effect on the plant is well known. However,the effects of individual microbiota members on plant hosts and vice versa, as well as the underlyingmechanisms, remain largely unknown. Here, we describe "Litterbox", a highly controlled system toinvestigate plant-microbe interactions. Plants were grown gnotobiotically, otherwise sterile, onzeolite-clay, a soil replacement that retains enough moisture to avoid subsequent watering.Litterbox-grown plants resemble greenhouse-grown plants more closely than agar-grown plantsand exhibit lower leaf epiphyte densities (106 cfu/g), reflecting natural conditions. Apolydimethylsiloxane (PDMS) sheet was used to cover the zeolite, significantly lowering thebacterial load in the zeolite and rhizosphere. This reduced the likelihood of potential systemicresponses in leaves induced by microbial rhizosphere colonisation. We present results of exampleexperiments studying the transcriptional responses of leaves to defined microbiota members andthe spatial distribution of bacteria on leaves. We anticipate that this versatile and affordable plantgrowth system will promote microbiota research and help in elucidating plant-microbe interactionsand their underlying mechanisms.

Delivering "Chromatic Bacteria" Fluorescent Protein Tags to Proteobacteria Using Conjugation.

Recently, we published a large and versatile set of plasmids, the chromatic bacteria toolbox, to deliver eight different fluorescent protein genes and four combinations of antibiotic resistance genes to Gram-negative bacteria. Fluorescent tags are important tools for single-cell microbiology, synthetic community studies, biofilm, and host-microbe interaction studies. Using conjugation helper strain E. coli S17-1 as a donor, we show how plasmid conjugation can be used to deliver broad host range plasmids, Tn5 transposons delivery plasmids, and Tn7 transposon delivery plasmids into species belonging to the Proteobacteria. To that end, donor and recipient bacteria are grown under standard growth conditions before they are mixed and incubated under non-selective conditions. Then, transconjugants or exconjugant recipients are selected on selective media. Mutant colonies are screened using a combination of tools to ensure that the desired plasmids or transposons are present and that the colonies are not containing any surviving donors. Through conjugation, a wide range of Gram-negative bacteria can be modified without prior, often time-consuming, establishment of competent cell and electroporation procedures that need to be adjusted for every individual strain. The here presented protocol is not exclusive for the delivery of Chromatic bacteria plasmids and transposons, but can also be used to deliver other mobilizable plasmids to bacterial recipients.

Driving factors of epiphytic bacterial communities: A review.

Bacteria establish complex, compositionally consistent communities on healthy leaves. Ecological processes such as dispersal, diversification, ecological drift, and selection as well as leaf surface physicochemistry and topology impact community assembly. Since the leaf surface is an oligotrophic environment, species interactions such as competition and cooperation may be major contributors to shape community structure. Furthermore, the plant immune system impacts on microbial community composition, as plant cells respond to bacterial molecules and shape their responses according to the mixture of molecules present. Such tunability of the plant immune network likely enables the plant host to differentiate between pathogenic and non-pathogenic colonisers, avoiding costly immune responses to non-pathogenic colonisers. Plant immune responses are either systemically distributed or locally confined, which in turn affects the colonisation pattern of the associated microbiota. However, how each of these factors impacts the bacterial community is unclear. To better understand this impact, bacterial communities need to be studied at a micrometre resolution, which is the scale that is relevant to the members of the community. Here, current insights into the driving factors influencing the assembly of leaf surface-colonising bacterial communities are discussed, with a special focus on plant host immunity as an emerging factor contributing to bacterial leaf colonisation.

Replicating Arabidopsis Model Leaf Surfaces for Phyllosphere Microbiology.

Artificial surfaces are commonly used in place of leaves in phyllosphere microbiology to study microbial behaviour on plant leaf surfaces. These surfaces enable a reductionist approach to be undertaken, to enable individual environmental factors influencing microorganisms to be studied. Commonly used artificial surfaces include nutrient agar, isolated leaf cuticles, and reconstituted leaf waxes. Recently, replica surfaces mimicking the complex topography of leaf surfaces for phyllosphere microbiology studies are appearing in literature. Replica leaf surfaces have been produced in agar, epoxy, polystyrene, and polydimethylsiloxane (PDMS). However, none of these protocols are suitable for replicating fragile leaves such as of the model plant Arabidopsis thaliana. This is of importance, as A. thaliana is a model system for molecular plant genetics, molecular plant biology, and microbial ecology. To overcome this limitation, we introduce a versatile replication protocol for replicating fragile leaf surfaces into PDMS. Here we demonstrate the capacity of our replication process using optical microscopy, atomic force microscopy (AFM), and contact angle measurements to compare living and PDMS replica A. thaliana leaf surfaces. To highlight the use of our replica leaf surfaces for phyllosphere microbiology, we visualise bacteria on the replica leaf surfaces in comparison to living leaf surfaces.

Utilisation of hydrocarbons and production of surfactants by bacteria isolated from plant leaf surfaces.

Leaves are covered by a cuticle composed of long (C11-C20) and very-long chain hydrocarbons (>C20), e.g. alkanes, fatty acids, alcohols, aldehydes, ketones and esters. In addition to these aliphatics, cyclic hydrocarbons may be present. Leaves are colonised by a variety of so-called epiphytic bacteria, which may have adapted to be able to utilise cuticle hydrocarbons. We tested the ability of a wide range of phylogenetically different epiphytic bacteria to utilise and grow on diesel and petroleum benzine and show that out of the 21 strains tested, nine had the ability to utilise diesel for growth. Only one strain was able to utilise petroleum benzine for growth. The ability to utilise hydrocarbons for growth correlated with the ability of the strains to produce surfactants and out of the 21 tested strains, 12 produced surfactants. Showing that 75% of the strains producing surfactants were able to degrade hydrocarbons. Our findings suggest that the ability to degrade hydrocarbons and to produce surfactants is highly prevalent in epiphytic bacteria. It is unclear if epiphytic bacteria utilise hydrocarbons originating from the cuticle of living leaves. The application of surfactant producing, hydrocarbon-utilising, epiphytic bacteria might serve as a method for hydrocarbon bioremediation.