Applied microbiology of the phyllosphere.

The phyllosphere, or plant leaf surface, represents a microbial ecosystem of considerable size, holding extraordinary biodiversity and enormous potential for the discovery of new products, tools, and applications in biotechnology, agriculture, medicine, and elsewhere. This mini-review highlights the applied microbiology of the phyllosphere as an original field of study concerning itself with the genes, gene products, natural compounds, and traits that underlie phyllosphere-specific adaptations and services that have commercial and economic value for current or future innovation. Examples include plant-growth-promoting and disease-suppressive phyllobacteria, probiotics and fermented foods that support human health, as well as microbials that remedy foliar contamination with airborne pollutants, residual pesticides, or plastics. Phyllosphere microbes promote plant biomass conversion into compost, renewable energy, animal feed, or fiber. They produce foodstuffs such as thickening agents and sugar substitutes, industrial-grade biosurfactants, novel antibiotics and cancer drugs, as well as enzymes used as food additives or freezing agents. Furthermore, new developments in DNA sequence-based profiling of leaf-associated microbial communities allow for surveillance approaches in the context of food safety and security, for example, to detect enteric human pathogens on leafy greens, predict plant disease outbreaks, and intercept plant pathogens and pests on internationally traded goods. KEY POINTS: • Applied phyllosphere microbiology concerns leaf-specific adaptations for economic value • Phyllobioprospecting searches the phyllosphere microbiome for product development • Phyllobiomonitoring tracks phyllosphere microbial profiles for early risk detection.

Comparison of bacterial diversity, root exudates and soil enzymatic activities in the rhizosphere of AVP1-transgenic and nontransgenic wheat (Triticum aestivum L.).

AIMS: Soil microbial communities are among the most diverse communities that might be affected due to transgenic crops. Therefore, risk assessment studies on transgenes are essentially required as any adverse effects may depend not only on the specific gene and crop involved but also on soil conditions. METHODS AND RESULTS: The present study deals with the comparison of bacterial populations, root exudates and activities of soil enzymes in nontransgenic and AVP1-transgenic wheat rhizosphere, overexpressing vacuolar H + pyrophosphatase for salinity and drought stress tolerance. Amounts of organic acids and sugars produced as root exudates and activities of dehydrogenase, phosphatase and protease enzymes in soil solution showed no significant differences in AVP1-transgenic and nontransgenic wheat rhizosphere, except for urease and phenol oxidase activities. The higher copy number of nifH gene showed the abundance of nitrogen-fixing bacteria in the rhizosphere of AVP1-transgenic wheat compared with nontransgenic wheat. nifH gene sequence analysis indicated the common diazotrophic genera Azospirillum, Bradyrhizobium, Rhizobium and Pseudomonas in AVP1-transgenic and nontransgenic wheat except for Zoogloea detected only in nontransgenic wheat. Using 454-pyrosequencing of 16S rRNA gene from soil DNA, a total of 156, 282 sequences of 18 phyla were obtained, which represented bacterial (128,006), Archeal (7928) and unclassified (21,568) sequences. Proteobacteria, Crenarchaeota and Firmicutes were the most abundant phyla in the transgenic and nontransgenic wheat rhizosphere. Further comparison of different taxonomic units at the genus level showed similar distribution in transgenic and nontransgenic wheat rhizospheres. CONCLUSION: We conclude that the AVP1 gene in transgenic wheat has no apparent adverse effects on the soil environment and different bacterial communities. However, the bacterial community depends on several other factors, not only genetic composition of the host plants. SIGNIFICANCE OF THE STUDY: The present research supports introduction and cultivation of transgenic plants in agricultural systems without any adverse effects on indigenous bacterial communities and soil ecosystems.

Repeated Exposure of Aspergillus niger Spores to the Antifungal Bacterium Collimonas fungivorans Ter331 Selects for Delayed Spore Germination.

The bacterial strain Collimonas fungivorans Ter331 (CfTer331) inhibits mycelial growth and spore germination in Aspergillus niger N402 (AnN402). The mechanisms underlying this antagonistic bacterial-fungal interaction have been extensively studied, but knowledge on the long-term outcome of this interaction is currently lacking. Here, we used experimental evolution to explore the dynamics of fungal adaptation to recurrent exposure to CfTer331. Specifically, five single-spore isolates (SSIs) of AnN402 were evolved under three selection scenarios in liquid culture, i.e., (i) in the presence of CfTer331 for 80 growth cycles, (ii) in the absence of the bacterium for 80 cycles, and (iii) in the presence of CfTer331 for 40 cycles and then in its absence for 40 cycles. The evolved SSI lineages were then evaluated for phenotypic changes from the founder fungal strain, such as germinability with or without CfTer331. The analysis showed that recurrent exposure to CfTer331 selected for fungal lineages with reduced germinability and slower germination, even in the absence of CfTer331. In contrast, when AnN402 evolved in the absence of the bacteria, lineages with increased germinability and faster germination were favored. SSIs that were first evolved in the presence of CfTer331 and then in its absence showed intermediate phenotypes but overall were more similar to SSIs that evolved in the absence of CfTer331 for 80 cycles. This suggests that traits acquired from exposure to CfTer331 were reversible upon removal of the selection pressure. Overall, our study provides insights into the effects on fungi from the long-term coculture with bacteria. IMPORTANCE The use of antagonistic bacteria for managing fungal diseases is becoming increasingly popular, and thus there is a need to understand the implications of their long-term use against fungi. Most efforts have so far focused on characterizing the antifungal properties and mode of action of the bacterial antagonists, but the possible outcomes of the persisting interaction between antagonistic bacteria and fungi are not well understood. In this study, we used experimental evolution in order to explore the evolutionary aspects of an antagonistic bacterial-fungal interaction, using the antifungal bacterium Collimonas fungivorans and the fungus Aspergillus niger as a model system. We show that evolution in the presence or absence of the bacteria selects for fungal lineages with opposing and conditionally beneficial traits, such as slow and fast spore germination, respectively. Overall, our studies reveal that fungal responses to biotic factors related to antagonism could be to some extent predictable and reversible.

Optimizing viability and yield and improving stability of Gram-negative, non-spore forming plant-beneficial bacteria encapsulated by spray-drying.

This study investigates methods to commercialize safer alternatives to chemical pesticides that pose risks to human safety and the environment. Spray-drying encapsulation of the plant-protective, antifungal bacterium Collimonas arenae Cal35 in in situ cross-linked alginate microcapsules (CLAMs) was optimized to minimize losses during spray-drying and maximize yield of spray-dried powder. Only inlet temperature significantly affected survival during spray-drying, while inlet temperature, spray rate, and alginate concentration significantly affected yield of spray-dried powder. Lowering inlet temperature to 95 °C provided the greatest survival during spray-drying, while increasing inlet temperature and lowering spray rate and alginate concentration produced the highest yield. Without the CLAMs formulation, Cal35 did not survive spray-drying. When Cal35 was encapsulated in CLAMs in the presence of modified starch, shelf survival was extended to 3 weeks in a low oxygen, low humidity storage environment. Cal35 retained its antifungal activity throughout spray-drying and shelf storage, supporting its potential use as a formulated biofungicide product.

Bacterial catabolism of indole-3-acetic acid.

Indole-3-acetic acid (IAA) is a molecule with the chemical formula C10H9NO2, with a demonstrated presence in various environments and organisms, and with a biological function in several of these organisms, most notably in plants where it acts as a growth hormone. The existence of microorganisms with the ability to catabolize or assimilate IAA has long been recognized. To date, two sets of gene clusters underlying this property in bacteria have been identified and characterized: one (iac) is responsible for the aerobic degradation of IAA into catechol, and another (iaa) for the anaerobic conversion of IAA to 2-aminobenzoyl-CoA. Here, we summarize the literature on the products, reactions, and pathways that these gene clusters encode. We explore two hypotheses about the benefit that iac/iaa gene clusters confer upon their bacterial hosts: (1) exploitation of IAA as a source of carbon, nitrogen, and energy; and (2) interference with IAA-dependent processes and functions in other organisms, including plants. The evidence for both hypotheses will be reviewed for iac/iaa-carrying model strains of Pseudomonas putida, Enterobacter soli, Acinetobacter baumannii, Paraburkholderia phytofirmans, Caballeronia glathei, Aromatoleum evansii, and Aromatoleum aromaticum, more specifically in the context of access to IAA in the environments from which these bacteria were originally isolated, which include not only plants, but also soils and sediment, as well as patients in hospital environments. We end the mini-review with an outlook for iac/iaa-inspired research that addresses current gaps in knowledge, biotechnological applications of iac/iaa-encoded enzymology, and the use of IAA-destroying bacteria to treat pathologies related to IAA excess in plants and humans. KEY POINTS: • The iac/iaa gene clusters encode bacterial catabolism of the plant growth hormone IAA. • Plants are not the only environment where IAA or IAA-degrading bacteria can be found. • The iac/iaa genes allow growth at the expense of IAA; other benefits remain unknown.

An roGFP2-Based Bacterial Bioreporter for Redox Sensing of Plant Surfaces.

The reduction-oxidation (redox) environment of the phytobiome (i.e., the plant-microbe interface) can strongly influence the outcome of the interaction between microbial pathogens, commensals, and their host. We describe a noninvasive method using a bacterial bioreporter that responds to reactive oxygen species and redox-active chemicals to compare microenvironments perceived by microbes during their initial encounter of the plant surface. A redox-sensitive variant of green fluorescent protein (roGFP2), responsive to changes in intracellular levels of reduced and oxidized glutathione, was expressed under the constitutive SP6 and fruR promoters in the epiphytic bacterium Pantoea eucalypti 299R (Pe299R/roGFP2). Analyses of Pe299R/roGFP2 cells by ratiometric fluorometry showed concentration-dependent responses to several redox active chemicals, including hydrogen peroxide (H2O2), dithiothreitol (DTT), and menadione. Changes in intracellular redox were detected within 5 min of addition of the chemical to Pe299R/roGFP2 cells, with approximate detection limits of 25 and 6 µM for oxidation by H2O2 and menadione, respectively, and 10 µM for reduction by DTT. Caffeic acid, chlorogenic acid, and ascorbic acid mitigated the H2O2-induced oxidation of the roGFP2 bioreporter. Aqueous washes of peach and rose flower petals from young blossoms created a lower redox state in the roGFP2 bioreporter than washes from fully mature blossoms. The bioreporter also detected differences in surface washes from peach fruit at different stages of maturity and between wounded and nonwounded sites. The Pe299R/roGFP2 reporter rapidly assesses differences in redox microenvironments and provides a noninvasive tool that may complement traditional redox-sensitive chromophores and chemical analyses of cell extracts.

Persistence of Fusarium oxysporum f. sp. fragariae in Soil Through Asymptomatic Colonization of Rotation Crops.

Asymptomatic plant colonization is hypothesized to enhance persistence of pathogenic forms of Fusarium oxysporum. However, a correlation between pathogen populations on living, asymptomatic plant tissues and soilborne populations after tillage has not been demonstrated. Living and dead tissues of broccoli, lettuce, spinach, wheat, cilantro, raspberry, and strawberry plants grown in soil infested with F. oxysporum f. sp. fragariae (the cause of Fusarium wilt of strawberry) were assayed to quantify the incidence of infection and extent of colonization by this pathogen. All crops could be infected by F. oxysporum f. sp. fragariae but the extent of colonization varied between plant species. Pathogen population densities on nonliving crown tissues incorporated into the soil matrix were typically greater than those observed on living tissues. Crop-dependent differences in the inoculum density of F. oxysporum f. sp. fragariae in soil were only observed after decomposition of crop residue. Forty-four weeks after plants were incorporated into the soil, F. oxysporum f. sp. fragariae soil population densities were positively correlated with population densities on plant tissue fragments recovered at the same time point. Results indicate that asymptomatic colonization can have a significant, long-term impact on soilborne populations of Fusarium wilt pathogens. Cultural practices such as crop rotation should be leveraged to favor pathogen population decline by planting hosts that do not support extensive population growth on living or decomposing tissues.

Metabolome and Microbiome Signatures in the Roots of Citrus Affected by Huanglongbing.

Huanglongbing (HLB) is a severe, incurable citrus disease caused by the bacterium 'Candidatus Liberibacter asiaticus' (CLas). Although citrus leaves serve as the site of initial infection, CLas is known to migrate to and colonize the root system; however, little is known about the impact of CLas infection on root metabolism and resident microbial communities. Scions of 'Lisbon' lemon and 'Washington Navel' orange grafted onto 'Carrizo' rootstock were grafted with either CLas-infected citrus budwood or uninfected budwood. Roots were obtained from trees 46 weeks after grafting and analyzed via 1H nuclear magnetic resonance spectroscopy to identify water-soluble root metabolites and high-throughput sequencing of 16S rRNA and ITS gene amplicons to determine the relative abundance of bacterial and fungal taxa in the root rhizosphere and endosphere. In both citrus varieties, 27 metabolites were identified, of which several were significantly different between CLas(+) and control plants. CLas infection also appeared to alter the microbial community structure near and inside the roots of citrus plants. Nonmetric multidimensional scaling (NMDS) and a principal coordinate analysis (PCoA) revealed distinct metabolite and microbial profiles, demonstrating that CLas impacts the root metabolome and microbiome in a manner that is variety-specific.

iac Gene Expression in the Indole-3-Acetic Acid-Degrading Soil Bacterium Enterobacter soli LF7.

We show for soil bacterium Enterobacter soli LF7 that the possession of an indole-3-acetic acid catabolic (iac) gene cluster is causatively linked to the ability to utilize the plant hormone indole-3-acetic acid (IAA) as a carbon and energy source. Genome-wide transcriptional profiling by mRNA sequencing revealed that these iac genes, chromosomally arranged as iacHABICDEFG and coding for the transformation of IAA to catechol, were the most highly induced (>29-fold) among the relatively few (<1%) differentially expressed genes in response to IAA. Also highly induced and immediately downstream of the iac cluster were genes for a major facilitator superfamily protein (mfs) and enzymes of the ß-ketoadipate pathway (pcaIJD-catBCA), which channels catechol into central metabolism. This entire iacHABICDEFG-mfs-pcaIJD-catBCA gene set was constitutively expressed in an iacR deletion mutant, confirming the role of iacR, annotated as coding for a MarR-type regulator and located upstream of iacH, as a repressor of iac gene expression. In E. soli LF7 carrying the DNA region upstream of iacH fused to a promoterless gfp gene, green fluorescence accumulated in response to IAA at concentrations as low as 1.6 µM. The iacH promoter region also responded to chlorinated IAA, but not other aromatics tested, indicating a narrow substrate specificity. In an iacR deletion mutant, gfp expression from the iacH promoter region was constitutive, consistent with the predicted role of iacR as a repressor. A deletion analysis revealed putative -35/-10 promoter sequences upstream of iacH, as well as a possible binding site for the IacR repressor.IMPORTANCE Bacterial iac genes code for the enzymatic conversion of the plant hormone indole-3-acetic acid (IAA) to catechol. Here, we demonstrate that the iac genes of soil bacterium Enterobacter soli LF7 enable growth on IAA by coarrangement and coexpression with a set of pca and cat genes that code for complete conversion of catechol to central metabolites. This work contributes in a number of novel and significant ways to our understanding of iac gene biology in bacteria from (non-)plant environments. More specifically, we show that LF7's response to IAA involves derepression of the MarR-type transcriptional regulator IacR, which is quite fast (less than 25 min upon IAA exposure), highly specific (only in response to IAA and chlorinated IAA, and with few genes other than iac, cat, and pca induced), relatively sensitive (low micromolar range), and seemingly tailored to exploit IAA as a source of carbon and energy.

Pairwise transcriptomic analysis of the interactions between the ectomycorrhizal fungus Laccaria bicolor S238N and three beneficial, neutral and antagonistic soil bacteria.

Ectomycorrhizal fungi are surrounded by bacterial communities with which they interact physically and metabolically during their life cycle. These bacteria can have positive or negative effects on the formation and the functioning of ectomycorrhizae. However, relatively little is known about the mechanisms by which ectomycorrhizal fungi and associated bacteria interact. To understand how ectomycorrhizal fungi perceive their biotic environment and the mechanisms supporting interactions between ectomycorrhizal fungi and soil bacteria, we analysed the pairwise transcriptomic responses of the ectomycorrhizal fungus Laccaria bicolor (Basidiomycota: Agaricales) when confronted with beneficial, neutral or detrimental soil bacteria. Comparative analyses of the three transcriptomes indicated that the fungus reacted differently to each bacterial strain. Similarly, each bacterial strain produced a specific and distinct response to the presence of the fungus. Despite these differences in responses observed at the gene level, we found common classes of genes linked to cell-cell interaction, stress response and metabolic processes to be involved in the interaction of the four microorganisms.

Modeling microbial growth and dynamics.

Modeling has become an important tool for widening our understanding of microbial growth in the context of applied microbiology and related to such processes as safe food production, wastewater treatment, bioremediation, or microbe-mediated mining. Various modeling techniques, such as primary, secondary and tertiary mathematical models, phenomenological models, mechanistic or kinetic models, reactive transport models, Bayesian network models, artificial neural networks, as well as agent-, individual-, and particle-based models have been applied to model microbial growth and activity in many applied fields. In this mini-review, we summarize the basic concepts of these models using examples and applications from food safety and wastewater treatment systems. We further review recent developments in other applied fields focusing on models that explicitly include spatial relationships. Using these examples, we point out the conceptual similarities across fields of application and encourage the combined use of different modeling techniques in hybrid models as well as their cross-disciplinary exchange. For instance, pattern-oriented modeling has its origin in ecology but may be employed to parameterize microbial growth models when experimental data are scarce. Models could also be used as virtual laboratories to optimize experimental design analogous to the virtual ecologist approach. Future microbial growth models will likely become more complex to benefit from the rich toolbox that is now available to microbial growth modelers.

Artificial Surfaces in Phyllosphere Microbiology.

The study of microorganisms that reside on plant leaf surfaces, or phyllosphere microbiology, greatly benefits from the availability of artificial surfaces that mimic in one or more ways the complexity of foliage as a microbial habitat. These leaf surface proxies range from very simple, such as nutrient agars that can reveal the metabolic versatility or antagonistic properties of leaf-associated microorganisms, to the very complex, such as silicon-based casts that replicate leaf surface topography down to nanometer resolution. In this review, we summarize the various uses of artificial surfaces in experimental phyllosphere microbiology and discuss how these have advanced our understanding of the biology of leaf-associated microorganisms and the habitat they live in. We also provide an outlook into future uses of artificial leaf surfaces, foretelling a greater role for microfluidics to introduce biological and chemical gradients into artificial leaf environments, stressing the importance of artificial surfaces to generate quantitative data that support computational models of microbial life on real leaves, and rethinking the leaf surface ('phyllosphere') as a habitat that features two intimately connected but very different compartments, i.e., the leaf surface landscape ('phylloplane') and the leaf surface waterscape ('phyllotelma').

Biosynthetic genes and activity spectrum of antifungal polyynes from Collimonas fungivorans†Ter331.

The antifungal activity of bacteria from the genus Collimonas has been well documented, but the chemistry and gene functions that underlie this phenotype are still poorly understood. Screening of a random plasposon insertion library of Collimonas fungivorans Ter331 for loss-of-function mutants revealed the importance of gene cluster K, which is annotated to code for the biosynthesis of a secondary metabolite and which features genes for fatty acid desaturases and polyketide synthases. Mutants in gene cluster K had lost the ability to inhibit hyphal growth of the fungus Aspergillus niger and were no longer able to produce and secrete several metabolites that after extraction and partial purification from wildtype strain Ter331 were shown to share a putative ene-triyne moiety. Some but not all of these metabolites were able to inhibit growth of A. niger, indicating functional variation within this group of Collimonas-produced polyyne-like 'collimomycins'. Polymerase chain reaction analysis of isolates representing different Collimonas species indicated that the possession of cluster K genes correlated positively with antifungal ability, further strengthening the notion that this cluster is involved in collimomycin production. We discuss our findings in the context of other bacterially produced polyynes and the potential use of collimomycins for the control of harmful fungi.

Transcriptional profiling of Gram-positive Arthrobacter in the phyllosphere: induction of pollutant degradation genes by natural plant phenolic compounds.

Arthrobacter chlorophenolicus A6 is a Gram-positive, 4-chlorophenol-degrading soil bacterium that was recently shown to be an effective colonizer of plant leaf surfaces. The genetic basis for this phyllosphere competency is unknown. In this paper, we describe the genome-wide expression profile of A.chlorophenolicus on leaves of common bean (Phaseolus vulgaris) compared with growth on agar surfaces. In phyllosphere-grown cells, we found elevated expression of several genes known to contribute to epiphytic fitness, for example those involved in nutrient acquisition, attachment, stress response and horizontal gene transfer. A surprising result was the leaf-induced expression of a subset of the so-called cph genes for the degradation of 4-chlorophenol. This subset encodes the conversion of the phenolic compound hydroquinone to 3-oxoadipate, and was shown to be induced not only by 4-chlorophenol but also hydroquinone, its glycosylated derivative arbutin, and phenol. Small amounts of hydroquinone, but not arbutin or phenol, were detected in leaf surface washes of P.vulgaris by gas chromatography-mass spectrometry. Our findings illustrate the utility of genomics approaches for exploration and improved understanding of a microbial habitat. Also, they highlight the potential for phyllosphere-based priming of bacteria to stimulate pollutant degradation, which holds promise for the application of phylloremediation.

Re-Envisioning the Plant Disease Triangle by Integration of Host Microbiota and a Pivot in Focus to Health Outcomes.

The disease triangle is a structurally simple but conceptually rich model that is used in plant pathology and other fields of study to explain infectious disease as an outcome of the three-way relationship between a host, a pathogen, and their environment. It also serves as a guide for finding solutions to treat, predict, and prevent such diseases. With the omics-driven, evidence-based realization that the abundance and activity of a pathogen are impacted by proximity to and interaction with a diverse multitude of other microorganisms colonizing the same host, the disease triangle evolved into a tetrahedron shape, which features an added fourth dimension representing the host-associated microbiota. Another variant of the disease triangle emerged from the recently formulated pathobiome paradigm, which deviates from the classical "one pathogen" etiology of infectious disease in favor of a scenario in which disease represents a conditional outcome of complex interactions between and among a host, its microbiota (including microbes with pathogenic potential), and the environment. The result is a version of the original disease triangle where "pathogen" is substituted with "microbiota." Here, as part of a careful and concise review of the origin, history, and usage of the disease triangle, I propose a next step in its evolution, which is to replace the word "disease" in the center of the host-microbiota-environment triad with the word "health." This triangle highlights health as a desirable outcome (rather than disease as an unwanted state) and as an emergent property of host-microbiota-environment interactions. Applied to the discipline of plant pathology, the health triangle offers an expanded range of targets and approaches for the diagnosis, prediction, restoration, and maintenance of plant health outcomes. Its applications are not restricted to infectious diseases only, and its underlying framework is more inclusive of all microbial contributions to plant well-being, including those by mycorrhizal fungi and nitrogen-fixing bacteria, for which there never was a proper place in the plant disease triangle. The plant health triangle also may have an edge as an education and communication tool to convey and stress the importance of healthy plants and their associated microbiota to a broader public and stakeholdership.

Functional characterization of the bacterial iac genes for degradation of the plant hormone indole-3-acetic acid.

Pseudomonas putida 1290 is a model organism for the study of bacterial degradation of the plant hormone indole-3-acetic acid (IAA). This property is encoded by the iac gene cluster. Insertional inactivation and/or deletion of individual iac genes and heterologous expression of the gene cluster in Escherichia coli were combined with mass spectrometry to demonstrate that iac-based degradation of IAA is likely to involve 2-hydroxy-IAA, 3-hydroxy-2-oxo-IAA, and catechol as intermediates. The first gene of the cluster, iacA encodes for the first step in the pathway, and also can convert indole to indoxyl to produce the blue pigment indigo. Transcriptional profiling of iac genes in P. putida 1290 revealed that they were induced in the presence of IAA. Based on results with an iacR knockout, we propose that this gene codes for a repressor of iacA expression and that exposure to IAA relieves this repression. Transformation of P. putida KT2440 (which cannot degrade IAA) with the iac gene cluster conferred the ability to grow on IAA as a sole source of carbon and energy, but not the ability to chemotaxi towards IAA. We could show such tactic response for P. putida 1290, thus representing the first demonstration of bacterial chemotaxis towards IAA. We discuss the ecological significance of our findings, and specifically the following question: under what circumstances do bacteria with the ability to degrade, recognize, and move towards IAA have a selective advantage?

Characterization of Seed Mycobiota Using Culture-Dependent and Culture-Independent Approaches.

Seed fungi are potentially important for their roles in seedling microbiome assembly and seedling health, but surveys of full seed fungal communities remain limited. While culture-dependent methods have been used to characterize some members of the seed mycobiota, recent culture-independent studies have improved the ease in identifying and characterizing full seed fungal communities. In this chapter, we describe how to survey seed fungi using both traditional culture-based methods and culture-free metabarcoding. We first describe protocols for the isolation and long-term preservation of fungal strains from individual seeds and for the extraction and amplification of DNA from such fungal isolates for identification with Sanger sequencing. We also detail how to extract, amplify, and sequence fungal DNA directly from individual seeds. Finally, we provide suggestions for troubleshooting media choices, PCR inhibition by isolates and plant tissue, and PCR limitation by low fungal DNA.

The mechanics of bacterial cluster formation on plant leaf surfaces as revealed by bioreporter technology.

Bacteria that colonize the leaves of terrestrial plants often occur in clusters whose size varies from a few to thousands of cells. For the formation of such bacterial clusters, two non-mutually exclusive but very different mechanisms may be proposed: aggregation of multiple cells or clonal reproduction of a single cell. Here we assessed the contribution of both mechanisms on the leaves of bean plants that were colonized by the bacterium Pantoea agglomerans. In one approach, we used a mixture of green and red fluorescent P. agglomerans cells to populate bean leaves. We observed that this resulted in clusters made up of only one colour as well as two-colour clusters, thus providing evidence for both mechanisms. Another P. agglomerans bioreporter, designed to quantify the reproductive success of bacterial colonizers by proxy to the rate at which green fluorescent protein is diluted from dividing cells, revealed that during the first hours on the leaf surface, many bacteria were dividing, but not staying together and forming clusters, which is suggestive of bacterial relocation. Together, these findings support a dynamic model of leaf surface colonization, where both aggregative and reproductive mechanisms take place. The bioreporter-based approach we employed here should be broadly applicable towards a more quantitative and mechanistic understanding of bacterial colonization of surfaces in general.

Microbiology of the phyllosphere: a playground for testing ecological concepts.

Many concepts and theories in ecology are highly debated, because it is often difficult to design decisive tests with sufficient replicates. Examples include biodiversity theories, succession concepts, invasion theories, coexistence theories, and concepts of life history strategies. Microbiological tests of ecological concepts are rapidly accumulating, but have yet to tap into their full potential to complement traditional macroecological theories. Taking the example of microbial communities on leaf surfaces (i.e. the phyllosphere), we show that most explorations of ecological concepts in this field of microbiology focus on autecology and population ecology, while community ecology remains understudied. Notable exceptions are first tests of the island biogeography theory and of biodiversity theories. Here, the phyllosphere provides the unique opportunity to set up replicated experiments, potentially moving fields such as biogeography, macroecology, and landscape ecology beyond theoretical and observational evidence. Future approaches should take advantage of the great range of spatial scales offered by the leaf surface by iteratively linking laboratory experiments with spatial simulation models.

It's the Economy, Stupid: Applying (Micro)economic Principles to Microbiome Science.

A key challenge in microbiome science is the scale mismatch problem, which arises when the scale at which microbial communities are sampled, interrogated, and averaged is different from the scale at which individual microorganisms within those communities interact with each other and with their environment. Profiling the microbial communities in a teaspoon of soil, from a scoop of fecal matter, or along a plant leaf surface represents a scale mismatch of multiple orders of magnitude, which may limit our ability to interpret or predict species interactions and community assembly within such samples. In this Perspective, we explore how economists, who are historically and topically split along the lines of micro- and macroeconomics, deal with the scale mismatch problem, and how taking clues from (micro)economists could benefit the field of microbiomics.