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Phyllosphere

The phyllosphere refers to the above-ground surfaces of plants, primarily leaves but also stems and flowers, that collectively form a vast microbial habitat colonized by epiphytic microorganisms such as bacteria, fungi, yeasts, archaea, protists, and viruses.^[1]^[2] This ecosystem, often described as the "aerial microbiome," spans an estimated terrestrial leaf surface area of 6.4 × 10^8 km² and supports a global bacterial population of approximately 10^26 cells, making it one of Earth's largest microbial environments.^[1]^[3] The term "phyllosphere" was independently coined in 1955 by F.T. Last and in 1956 by J. Ruinen to denote this distinct aerial niche, separate from the plant's internal tissues or surrounding atmosphere.^[4] Microbial communities in the phyllosphere exhibit extraordinary biodiversity, with bacterial densities typically ranging from 10^6 to 10^8 cells per square centimeter of leaf surface, dominated by phyla such as Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria.^[2]^[3] Common genera include Pseudomonas, Methylobacterium, Sphingomonas, and Bacillus, alongside fungi like Cladosporium and Alternaria, and yeasts such as Aureobasidium pullulans.^[1]^[2] These populations are shaped by host plant genotype, leaf age, geographic location, seasonal changes, and environmental stressors like UV radiation, desiccation, and nutrient scarcity from leaf exudates (e.g., sugars and amino acids).^[2]^[4] Culture-independent methods, including 16S rRNA sequencing, have revealed even greater diversity, identifying hundreds of genera and underscoring the role of unculturable microbes in this habitat.^[1] Ecologically, phyllosphere microbes are integral to plant physiology and broader environmental processes, promoting growth through nitrogen fixation, hormone production (e.g., indole-3-acetic acid), and enhanced stress tolerance via enzymes like superoxide dismutase and catalase.^[2] They contribute to biogeochemical cycles by facilitating carbon and nitrogen turnover, decomposing organic matter, and emitting volatile organic compounds that influence atmospheric chemistry.^[1]^[2] Additionally, these communities suppress pathogens through competition and antagonism, as seen with Pseudomonas fluorescens strains that inhibit frost damage or fire blight in crops.^[3] However, they also pose risks, serving as reservoirs for human pathogens like Salmonella and Escherichia coli that contaminate produce.^[1] From an applied perspective, phyllosphere microbiology has advanced agricultural and biotechnological innovations, including biocontrol agents for disease management, biofertilizers to boost crop yields, and phylloremediation for degrading pollutants like pesticides and plastics.^[2]^[3] Research since the 1990s, leveraging molecular tools like GFP tagging and metagenomics, has illuminated colonization dynamics and microbial fitness, paving the way for engineered microbiomes to enhance plant resilience amid climate change.^[1]^[4]

Definition and Habitat Characteristics

Definition

The phyllosphere refers to the total above-ground portions of plants, including leaves, stems, flowers, and fruits, conceptualized as a habitat for microorganisms.^[2] This environment supports diverse microbial life, primarily encompassing epiphytic microbes that colonize external surfaces and transient microbes that temporarily interact with the plant aerial parts.^[5] The term "phyllosphere" was independently coined by F.T. Last in 1955 and J. Ruinen in 1956 to describe the external leaf surface as an ecologically significant yet overlooked milieu for microbial growth and activity.^[4] As a microbial habitat, the phyllosphere stands out for its immense global scale, with aboveground plant biomass estimated at approximately 320 gigatons of carbon, representing about 60% of Earth's total biomass across all taxa.^[6] This vast surface area—spanning terrestrial ecosystems worldwide—positions the phyllosphere as the largest contiguous habitat for microorganisms on the planet, far exceeding other plant-associated niches in extent.^[7] The phyllosphere is distinct from other plant microbiomes, such as the rhizosphere, which encompasses the soil zone surrounding roots below ground, and the endosphere, which includes internal plant tissues across both above- and below-ground parts.^[8] While these compartments share some microbial overlap due to plant-mediated recruitment, the phyllosphere's aerial exposure subjects it to unique atmospheric influences, setting it apart in microbial ecology.^[2]

Physical and Chemical Properties

The phyllosphere, encompassing the aboveground surfaces of plants, features a complex topography that profoundly influences microbial attachment and nutrient access. Leaf surfaces are coated with a waxy cuticle, including epicuticular wax crystals, which regulates permeability and wettability, while structural elements such as stomata, trichomes, veins, and epidermal grooves create microhabitats that facilitate microbial colonization.^[2] Trichomes and vein patterns, in particular, serve as nutrient-rich sites where water droplets and exudates accumulate, promoting bacterial aggregation and biofilm formation.^[9] These surface characteristics vary by plant species and leaf age, with the tortuous arrangement of epidermal cells further affecting the spread of moisture and solutes.^[1] Chemically, the phyllosphere exhibits dynamic gradients characterized by fluctuating pH levels, typically ranging from 5 to 6, though broader variations from 4.9 to 7.2 occur depending on plant type and environmental conditions.^[10] Nutrient availability is low, with carbon primarily supplied through leachates such as sugars (e.g., glucose, fructose, sucrose at 0.2–10 μg per leaf) and organic acids, while nitrogen derives from amino acids in plant exudates.^[1]^[2] Additionally, oxidative stress arises from reactive oxygen species generated by UV exposure and plant metabolic activity, imposing selective pressure on surface colonizers.^[5] At the microscale, the phyllosphere displays significant heterogeneity due to rapid wetting and drying cycles driven by dew formation and evaporation, alongside intense UV radiation and temperature fluctuations that can elevate leaf surfaces 6–10°C above ambient air temperature through solar heating.^[11] These conditions create ephemeral oases of moisture and nutrients near stomata and trichome bases, contrasting with drier, exposed areas, and result in bacterial densities of 10⁶–10⁸ cells per cm² in favorable hotspots.^[9] Such variability underscores the habitat's oligotrophic nature, where resources are patchily distributed and subject to rapid depletion.^[1] In comparison to aquatic or soil habitats, the phyllosphere presents harsher, more ephemeral conditions marked by high desiccation risk, nutrient scarcity, and direct exposure to UV and oxidative stressors, rendering it a challenging environment that favors stress-tolerant microbial strategies.^[2]^[9]

Composition of the Phyllosphere Microbiome

Bacterial Communities

Bacterial communities dominate the phyllosphere microbiome, comprising the majority of microbial colonizers on leaf surfaces. The most prevalent phyla include Proteobacteria, particularly Alpha- and Gammaproteobacteria such as genera Sphingomonas and Pseudomonas, alongside Actinobacteria, Bacteroidetes, and Firmicutes.^[9]^[12]^[2] These groups often account for over 70% of the bacterial sequences in phyllosphere samples across diverse plant hosts, reflecting their adaptability to the aerial environment.^[13] Bacterial density in the phyllosphere typically ranges from 10^6 to 10^8 cells per cm², with higher abundances observed on younger leaves due to increased surface wetness and nutrient availability.^[3]^[1] This density varies by plant species; for instance, in rice (Oryza sativa), Pseudomonas species are often prominent in the community.^[14] In contrast, neotropical forest trees host elevated levels of Methylobacterium, a methylotrophic genus that thrives on leaf methanol emissions, representing a core component in such ecosystems.^[15] Plant-specific traits, like leaf chemistry and morphology, further shape these patterns, leading to host-dependent community structures.^[16] Functionally, phyllosphere bacteria contribute to key processes such as nitrogen fixation, where genera like Pseudomonas and Methylobacterium convert atmospheric N₂ into bioavailable forms, supporting plant nutrition in nutrient-limited settings.^[9]^[17] Certain strains, notably Pseudomonas syringae, promote ice nucleation on leaves, facilitating frost damage at subzero temperatures by lowering the freezing point threshold.^[18] Additionally, quorum sensing enables biofilm formation, allowing bacteria like Pseudomonas to coordinate colonization and enhance persistence against desiccation and UV exposure.^[19]^[20] The assembly of these communities involves a balance of stochastic and deterministic processes. Stochastic dispersal from air and soil introduces low-abundance taxa, which then establish via random colonization events.^[21] Deterministic selection by host traits, such as leaf surface hydrophobicity and volatile emissions, filters and stabilizes core members, promoting ecological succession over leaf development.^[22]^[23] This interplay results in dynamic yet predictable bacterial consortia tailored to phyllosphere conditions.

Fungal and Other Eukaryotic Communities

The fungal communities in the phyllosphere are predominantly composed of Ascomycota and Basidiomycota, which together account for the majority of detected sequences in high-throughput sequencing studies across various plant hosts.^[24] Within Ascomycota, genera such as Aureobasidium (e.g., Aureobasidium pullulans), Cladosporium, and Alternaria are frequently dominant, with A. pullulans often comprising a significant portion of the yeast-like fungal population due to its adaptation to oligotrophic leaf surfaces.^[9]^[25] Basidiomycota are represented primarily by yeasts like those in Sporobolomyces, which exhibit ballistoconidium formation for aerial dispersal and thrive in moist microhabitats on leaves.^[26] Molds such as Alternaria spp. become more prevalent on senescing leaves, where they exploit decaying plant material for colonization.^[27] Other microbial diversity in the phyllosphere includes protozoa, algae, and archaea (prokaryotes), though these groups occur at lower abundances compared to fungal and bacterial components. Protozoa, particularly amoebae, function as grazers on bacterial populations, contributing to microbial community regulation in water films on leaf surfaces.^[1] Epiphytic algae, such as those in Chlorophyta, colonize moist phyllosphere niches in humid environments like rainforests, where they form thin biofilms that influence surface wettability and nutrient availability.^[28] Archaea are sporadically detected in low relative abundance, with methanogenic taxa appearing in wetter phyllospheres of aquatic or floating macrophytes, potentially utilizing anaerobic microsites for methane production. These groups co-occur with bacteria, forming interconnected networks that shape overall phyllosphere dynamics.^[1] Specific examples illustrate the variability in fungal communities across host plants. In apple trees (Malus domestica), members of the Teratosphaeriaceae family often dominate the phyllosphere, particularly as components of the sooty blotch complex, where they form superficial mycelial mats on fruit and leaves during humid conditions.^[29] In Populus species, such as black cottonwood (Populus trichocarpa), fungal communities exhibit seasonal shifts, with yeast abundances peaking in cooler, wetter periods due to favorable conditions for spore germination and growth.^[30] Fungal structural adaptations, including hyphal networks, facilitate nutrient scavenging from leaf exudates and debris, while wind-dispersed spores enable rapid colonization and resilience in the exposed aerial environment.^[9]

Ecological Dynamics and Interactions

Plant-Microbe Interactions

The phyllosphere hosts a range of biotic interactions between plants and microbes, encompassing mutualistic associations that enhance host fitness and antagonistic dynamics that suppress pathogens. These interactions occur primarily on leaf surfaces but can extend internally through endophytic colonization, influencing plant growth, defense, and overall health. Mutualistic microbes often provide benefits such as nutrient acquisition and stress tolerance, while antagonistic ones limit disease through direct competition or inhibitory compounds.^[9]^[31] Mutualistic interactions in the phyllosphere prominently include plant growth promotion through hormone modulation, such as indole-3-acetic acid (IAA) production by bacteria like Pseudomonas species, which stimulate root and shoot development in crops including rice and wheat.^[32]^[9] Nutrient solubilization represents another key mechanism, with phyllosphere Bacillus and Pseudomonas strains solubilizing insoluble phosphates to improve availability for plants like rice, thereby enhancing biomass accumulation under nutrient-limited conditions.^[9] Additionally, certain microbes induce systemic resistance (ISR) in plants, activating defense pathways against foliar pathogens; for instance, Bacillus amyloliquefaciens triggers jasmonic acid and ethylene signaling to protect against necrotrophic fungi in tomato.^[31]^[9] Antagonistic roles of phyllosphere microbes focus on pathogen suppression through resource competition and antimicrobial production. Bacillus species, such as B. subtilis and B. amyloliquefaciens, produce antibiotics like surfactin, iturin, and bacilysin, which inhibit phytopathogens including Botrytis cinerea and Erwinia amylovora on grapevine and apple leaves by disrupting cell membranes and metabolism.^[31] Competition for limiting nutrients, such as sugars, further limits pathogen establishment; Sphingomonas strains outcompete Pseudomonas syringae on Arabidopsis leaves by rapidly utilizing leaf exudates.^[31] Hyperparasitism, though less common among bacteria, involves microbes parasitizing pathogens, as seen with the yeast Aureobasidium pullulans hyperparasitizing Botrytis cinerea conidia on fruit surfaces through enzymatic degradation.^[9] Many phyllosphere microbes exhibit transitions from epiphytic to endophytic lifestyles, entering plant tissues via stomata or wounds to establish internal symbioses that amplify benefits. Methylobacterium species, initially colonizing leaf surfaces, can penetrate stomata to become endophytes, producing cytokinins that enhance photosynthesis and overall vigor in rice, leading to increased grain yields by up to sixfold in certain landraces.^[33]^[31] This dual habitation allows sustained hormone modulation and stress alleviation without disrupting surface communities.^[31] Host specificity governs these interactions, with plant genotype recruiting distinct microbial consortia tailored to defense needs. In the New Zealand shrub Leptospermum scoparium (mānuka), genetic traits select for a unique phyllosphere bacterial community dominated by Proteobacteria and Acidobacteria, which contributes to anti-pathogen properties through bioactive compound production and enhanced resistance to foliar diseases.^[34]^[35] This genotype-driven recruitment underscores how plants shape phyllosphere dynamics to optimize biotic exchanges.^[34]

Influence of Environmental Factors

Climatic variables profoundly shape the structure and function of phyllosphere microbial communities by selecting for taxa adapted to specific abiotic stresses. Ultraviolet (UV) radiation, particularly UV-B, alters bacterial community composition by favoring UV-resistant phenotypes, such as pigmented bacteria and isolates like Clavibacter michiganensis and Curtobacterium flaccumfaciens, which exhibit enhanced survival on leaf surfaces under high exposure.^[36] Humidity influences fungal communities by promoting spore germination and shifting composition toward saprotrophs and endophytes, with elevated air humidity accounting for 6.5% of variation in foliar fungal community composition on species like Betula pendula.^[37] Temperature extremes further drive selection, as higher temperatures in hot-dry environments correlate with increased abundance of thermotolerant taxa such as Enterobacteriaceae and Pseudomonas in poplar phyllospheres, while reducing overall diversity.^[38] Seasonal and spatial dynamics introduce temporal and geographic variability in phyllosphere assemblages. Communities exhibit higher diversity and activity in spring and early summer, with taxa like Pseudomonas dominating colonization and motility functions, transitioning to enriched carbohydrate and terpene metabolism in late summer and fall as plants senesce, and reduced activity or dormant forms persisting into winter.^[39] Along urban-rural gradients, pollution and human disturbance elevate bacterial diversity, richness, and functional gene abundance in urban settings compared to rural or natural habitats, though core taxa decrease and community assembly shifts toward deterministic environmental filtering.^[40] Biotic cross-kingdom effects, such as disturbances from pollinators and herbivores, introduce transient microbes that transiently diversify phyllosphere communities. Insect vectors like pollinators facilitate microbial dispersal across plant organs and species by carrying bacteria on their bodies, leading to equal prevalence of operational taxonomic units (OTUs) across hosts and promoting passive migration.^[41] Similarly, herbivore activity can alter community composition by depositing gut-associated microbes during feeding. These effects often impact core bacterial groups like Proteobacteria, though details vary by vector specificity.^[42] Plant developmental stages, particularly leaf age, modulate phyllosphere diversity and protective functions. Juvenile leaves harbor lower bacterial and fungal diversity and richness compared to mature leaves, where Shannon diversity increases significantly (e.g., F₂,₆₄.₇ = 3.65, p = 0.03 for bacteria), supporting more stable communities with abundant genera like Actinobacteria that provide protective roles against stresses.^[43] As leaves age, community composition shifts (PERMANOVA, F₂,₇₈ = 3.08, p = 0.002), favoring microbes adapted to changing microenvironments.^[43]

Research Approaches

Sampling and Cultivation Methods

Sampling phyllosphere microbes typically involves destructive or non-destructive techniques to recover epiphytic and endophytic communities from leaf surfaces. Destructive methods, such as leaf washing or homogenization, are widely used to extract microbes by immersing leaves in an elution buffer like phosphate-buffered saline (PBS) and agitating via shaking or sonication, often recovering 10^6 to 10^8 cells per gram of fresh leaf weight while preserving cell viability.^[44] These approaches, exemplified in protocols from Barillot et al. (2013), allow for multiple washes to increase recovery by up to 200%, though the first wash typically captures about one-third of the total microbiota.^[45] Homogenization further disrupts leaf tissue to access endophytes but is less common for surface-focused studies due to its invasiveness.^[46] Non-destructive methods prioritize minimal plant damage and include swabbing leaf surfaces with sterile cotton or nylon swabs dipped in buffer, impression prints by pressing leaves onto agar plates, and adhesive tape strips to lift microbes directly.^[4] Swabbing, as described by Yashiro et al. (2011), targets surface epiphytes effectively for both culturing and microscopy, while leaf printing—pioneered by Corpe (1985)—provides a quick qualitative assessment of microbial distribution without extraction.^[4] Adhesive tapes, adapted from early fungal studies by Langvad (1980), offer rapid, non-invasive sampling suitable for field conditions, though they may underrepresent deeper communities.^[47] Semi-destructive techniques, like punching small leaf discs (e.g., 1 cm diameter), balance recovery with plant preservation and are often used in standardized multi-site protocols.^[48] Cultivation of phyllosphere isolates relies on selective media to target dominant taxa, such as King's B medium for fluorescent Pseudomonas species, which promotes pigment production and growth from leaf wash dilutions.^[49] However, only 1-10% of the total bacterial community is typically culturable under standard lab conditions, with rates ranging from 0.1% to 8.4% as quantified by Rastogi et al. (2010), due to viable but non-culturable (VBNC) states induced by nutrient limitation or stress.^[50] VBNC cells, first noted in phyllosphere contexts by Dinu and Bach (2011), maintain metabolic activity but fail to form colonies, underscoring the bias toward fast-growing opportunists like Pseudomonas.^[46] Semi-selective media for pathogens, such as those for enteric bacteria outlined by Barak et al. (2011), further refine isolation but exacerbate underestimation of diversity.^[46] Historically, phyllosphere microbiology sampling evolved from early 20th-century plate counts of colony-forming units via dilution plating, as in Ruinen's 1956 identification of nitrogen-fixing bacteria, to more refined culture-dependent methods like leaf pressing reported by Dickinson and Bainbridge (1974).^[51]^[52] By the mid-20th century, quantitative assessments via washing and plating became standard, with Thompson et al. (1993) establishing seasonal baselines using such techniques.^[46] Modern protocols, influenced by Donegan et al. (1991), incorporate standardized grids for spatial sampling across sites—such as five-point patterns on leaves—to enhance reproducibility in field studies, addressing variability in earlier ad hoc approaches.^[45] These cultivation-based limitations, including low recovery of VBNC forms, are increasingly supplemented by molecular methods for comprehensive analysis.^[46]

Molecular and Omics Techniques

Molecular and omics techniques have revolutionized the study of phyllosphere microbiomes by enabling culture-independent profiling of microbial diversity and function, shifting focus from cultivable isolates to comprehensive community assessments.^[39] These approaches, including marker gene sequencing and shotgun-based omics, allow researchers to capture the vast genetic and metabolic potential of leaf-associated microbes without the biases of traditional culturing.^[53] Marker gene sequencing targets conserved genetic regions to characterize taxonomic composition, with 16S rRNA gene amplicon sequencing widely used for bacterial communities in the phyllosphere.^[13] High-throughput sequencing of the 16S rRNA gene has revealed core bacterial taxa, such as Proteobacteria and Actinobacteria, across diverse hosts; for instance, a study on leaves from 57 tree species in a Panamanian neotropical forest identified Sphingomonas and Methylobacterium as dominant genera, highlighting host-specific and shared phyllosphere microbiomes.^[13] For fungal communities, internal transcribed spacer (ITS) region sequencing provides resolution at the species level, uncovering Ascomycota and Basidiomycota as prevalent phyla on plant leaves.^[54] ITS-based amplicon studies on crops like switchgrass have shown seasonal shifts in epiphytic fungi, with genera such as Cladosporium and Alternaria varying in abundance due to environmental cues.^[55] Shotgun metagenomics extends beyond taxonomy by sequencing total community DNA, enabling assembly of functional gene profiles, including those involved in nutrient cycling.^[56] In the phyllosphere, this approach has detected nifH genes associated with nitrogen fixation, primarily from Alphaproteobacteria like Azorhizobium, indicating potential contributions to plant nitrogen budgets in natural ecosystems.^[56] Metatranscriptomics complements metagenomics by capturing actively expressed genes through RNA sequencing, revealing dynamic metabolic pathways; for example, analyses of perennial crop leaves like switchgrass identified upregulated genes for stress response and secondary metabolite production during seasonal transitions.^[39] Proteogenomics integrates proteomics with genomic data to map active proteins, providing insights into phyllosphere bacterial physiology under in situ conditions, such as carbon utilization and oxidative stress tolerance in lettuce leaves.^[53] Emerging tools enhance resolution of phyllosphere interactions at finer scales. Single-cell genomics isolates and sequences DNA from individual microbes, bypassing bulk community averaging to uncover rare taxa and strain-level variations in leaf microbiomes, though applications remain limited due to challenges in isolating epiphytes.^[57] CRISPR-based methods, including engineered phages and base editing, facilitate tracking of microbial interactions and functional manipulations in the phyllosphere, such as targeting specific bacterial strains to study competition or symbiosis on plant surfaces.^[58] Quantitative PCR (qPCR) offers targeted detection of pathogens, quantifying genes like those for human pathogens (e.g., invA in Salmonella) in urban landscape plant phyllospheres at levels up to 10^4 copies per gram of leaf tissue.^[59] Data analysis pipelines address the complexity of omics datasets from phyllosphere samples, employing metrics like alpha diversity (e.g., Shannon index) to quantify within-sample richness and beta diversity (e.g., Bray-Curtis dissimilarity) to compare communities across hosts or conditions.^[35] Tools such as PICRUSt predict functional profiles from 16S rRNA data, inferring pathways like nitrogen metabolism in mānuka tree phyllospheres, where core taxa contribute to predicted antibiotic biosynthesis.^[35] Biases, including PCR chimeras in amplicon sequencing, are mitigated through denoising algorithms like DADA2, ensuring accurate taxonomic assignments in high-throughput phyllosphere surveys.^[60] These analyses integrate with post-collection processing from sampling methods to provide robust interpretations of microbiome structure and function.^[61]

Applications and Future Directions

Agricultural and Biotechnological Applications

The phyllosphere microbiome offers significant potential for biocontrol applications in agriculture, where foliar sprays of beneficial bacteria suppress pathogenic fungi on crop surfaces. For instance, Pseudomonas species applied as foliar inoculants induce systemic resistance in grapevines against Botrytis cinerea, the causative agent of gray mold, by priming defense responses such as enhanced jasmonic acid signaling and reduced lesion development following pathogen challenge.^[62] In rice, actinomycetes isolated from the phyllosphere, particularly strains of Streptomyces, produce bioactive compounds like antibiotics that inhibit Pyricularia oryzae, reducing leaf blast disease severity by up to 88% in greenhouse assays.^[63] Biofertilization strategies leverage phyllosphere microbes to improve nutrient uptake through leaf-applied consortia, minimizing reliance on synthetic inputs. Phosphate-solubilizing bacteria such as Bacillus and Pseudomonas, naturally occurring in the rice phyllosphere, secrete organic acids to convert insoluble phosphates into plant-available forms, enhancing phosphorus acquisition and overall nutrient efficiency.^[64] Such foliar biofertilizers can replace 25-30% of chemical phosphorus fertilizers while boosting crop yields by 10-40% in integrated systems.^[65] In bioremediation, phyllosphere bacteria facilitate the degradation of atmospheric pollutants like polycyclic aromatic hydrocarbons (PAHs) directly on crop leaves, preventing their accumulation in food chains. Indigenous strains such as Pseudomonas and Sphingomonas on plant surfaces metabolize phenanthrene, reducing its levels by significant margins through bioaugmentation experiments on species like Ixora.^[66] Strains such as Sphingomonas sp. P2 introduced via foliar application accelerate PAH breakdown on leaves, achieving faster degradation rates than native communities and demonstrating efficacy on crops for environmental cleanup.^[67] Commercial products derived from phyllosphere microbes include microbial inoculants that enhance crop protection and industrial biopolymers. In mānuka (Leptospermum scoparium) cultivation for honey production, core phyllosphere taxa like Sphingomonas and Rhizobiales have been identified, a plant cultivated for honey production with high-methylglyoxal content and antimicrobial properties.^[35] Additionally, phyllosphere yeasts such as Pseudozyma antarctica produce mannosylerythritol lipids (MELs), glycolipid biosurfactants used as thickening and stabilizing agents in cosmetics and food formulations.^[3]

Emerging Research Areas

Recent advancements in phyllosphere microbiology have focused on engineering synthetic microbial communities (SynComs) to enhance plant resilience under stress conditions. These communities are constructed either through top-down simplification of natural assemblages, where complex phyllosphere microbiomes are reduced to key functional consortia, or bottom-up design, involving the assembly of selected strains to target specific outcomes like drought tolerance. For instance, a five-strain SynCom comprising Pseudomonas stutzeri and Bacillus mojavensis improved cotton germination and biomass under drought by producing ACC deaminase and phytohormones, demonstrating potential for scalable applications. In rice, foliar application of Bacillus megaterium PB50 isolated from drought-tolerant varieties induced systemic tolerance, increasing relative water content and reducing oxidative damage via antioxidant enzyme upregulation. These approaches leverage phyllosphere bacteria's natural roles in nutrient cycling and pathogen suppression, with emerging tools like CRISPR editing and AI-driven modeling addressing challenges in community stability and culturability. As of 2025, reviews highlight the potential of phyllosphere synthetic microbial communities to improve plant protection and nutrient efficiency in sustainable agriculture.^[68]^[69]^[70]^[68] Climate change poses significant challenges to phyllosphere stability, with research increasingly modeling the effects of altered environmental factors such as increased UV radiation and fluctuating humidity on microbial community dynamics. Elevated temperatures and drought have been shown to reduce beneficial taxa like Sphingomonas while promoting opportunistic pathogens such as Enterobacteriaceae, potentially destabilizing plant-microbe interactions and increasing disease susceptibility. Modeling efforts indicate that humidity shifts can convert non-pathogenic Pseudomonas syringae strains into ice-nucleating pathogens, disrupting community homeostasis and stomatal regulation for water uptake. Urban phyllospheres are emerging as bioindicators for pollution, with studies revealing higher antibiotic resistance gene prevalence in city greenery compared to rural sites, highlighting their role as sentinels for anthropogenic stressors like air pollution. These findings underscore the need for predictive models integrating UV and humidity data to forecast phyllosphere responses in warming scenarios.^[71]^[40]^[72] Multi-omics integration represents a promising frontier for elucidating phyllosphere functions, particularly by combining metagenomics and metabolomics to link microbial composition with plant health predictors. In tobacco leaves, this approach revealed stage-specific microbial shifts during fermentation, with Staphylococcus dominance correlating to lipid and amino acid metabolites that enhance aroma quality and stress resistance. Metagenomic profiling identified key taxa like Aspergillus and Brevundimonas influencing terpenoid and steroid production, enabling predictions of health outcomes such as improved pathogen defense and nutrient efficiency. Such integrations have also uncovered causal links in other systems, where metabolomic signatures from phyllosphere fungi predict reduced oxidative stress in hosts under abiotic pressures. By revealing functional pathways, multi-omics facilitates targeted interventions for resilient plant microbiomes.^[73]^[74]^[75] Despite progress, significant research gaps persist in phyllosphere studies, particularly regarding understudied components like viruses and archaea, the scarcity of long-term field trials, and limited exploration of global south ecosystems. Viruses in the phyllosphere remain poorly characterized, with emerging evidence of their role in biocontrol against fungal pathogens, yet comprehensive surveys are lacking due to methodological challenges in viral metagenomics. Archaea, though detected in low abundance (e.g., higher in stressed grapevine phyllospheres), are rarely functionally annotated, representing a blind spot in community assembly models. Long-term field trials are essential but infrequent, with calls for multi-year studies to assess microbiome succession and inoculant persistence under variable conditions. In the global south, tropical forest phyllospheres beyond localized Panama surveys—such as those in subtropical China—show high bacterial and fungal diversity tied to host traits and seasonality, yet broader representation from underrepresented regions is needed to capture global variability. Addressing these gaps will require expanded international collaborations and advanced sequencing.^[76]^[77]^[78]^[79]^[80]^[15]

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Jan 20, 2020 · The dominant taxa belonged to Pleosporaceae, Cladosporium, Alternaria, Capnodiales, Sporobolomyces and Aureobasidium pullulans. Although no ...
[28]
Phyllosphere eukaryotic microalgal communities in rainforests
The aim of this study is to identify the environmental factors that drive phyllosphere algal community composition and diversity in rainforests.
[29]
Novel fungal genera and species associated with the sooty blotch ...
The SBFS fungi colonise the epicuticular wax layer of apple fruit. Colonies that form dark mycelial mats are referred to as 'sooty blotch', whereas 'flyspeck' ...
[30]
[PDF] Weather and leaf age separately contribute to temporal shifts in ...
Jun 26, 2024 · We also observed changes in phyllosphere communities due ... Spatial from Environmental Effects on Foliar Fungal Communities of Populus. 461.
[31]
Modes of Action of Microbial Biocontrol in the Phyllosphere - Frontiers
Many Pseudomonas spp., both pathogenic as non-pathogenic, as well as other Gram-negative phyllosphere bacteria, harbor type VI secretion systems, suggesting ...
[32]
Narrow-spectrum resource-utilizing bacteria drive the stability of ...
Jul 2, 2025 · Strains exhibiting key plant beneficial functions, such as nitrogen fixation, phosphate solubilization, indoleacetic acid (IAA) synthesis, and ...
[33]
Impact of Phyllosphere Methylobacterium on Host Rice Landraces
Jul 20, 2022 · We found that inoculation with some bacteria increased rice grain production substantially while a nonnative bacterium reduced plant health.
[34]
Host selection is not a universal driver of phyllosphere community ...
Jan 31, 2025 · The specificity of the mānuka phyllosphere microbiome suggests the presence of functionally significant bacteria that are under direct, possibly ...
[35]
A core phyllosphere microbiome exists across distant populations of ...
Aug 13, 2020 · The leaf surface, or phyllosphere, harbours a microbiome comprising diverse communities of bacteria, fungi, algae, archaea, and viruses [3, 4].
[36]
Effect of Solar UV-B Radiation on a Phyllosphere Bacterial Community
Our data demonstrate that solar UV-B selection alters phyllosphere bacterial community composition and that UV tolerance is a prevalent phenotype late in the ...
[37]
The Effects of Humidity and Seasonality on Foliar and Root ...
Jul 7, 2025 · We found that increased air humidity shifts fungal community composition, but elevated soil irrigation does not.
[38]
Influences of Climate on Phyllosphere Endophytic Bacterial ...
Feb 27, 2020 · Environmental variables such as radiation, precipitation, temperature and humidity have a direct effect on stomata opening and, therefore, play ...
[39]
Seasonal activities of the phyllosphere microbiome of perennial crops
Feb 23, 2023 · It is currently unclear what functions may be associated with phyllosphere microbial dynamics and their potential interactions with plant hosts.
[40]
From nature to urbanity: exploring phyllosphere microbiome and ...
Nov 7, 2024 · Environmental stress and air pollution may directly alter specific attributes of leaves (e.g. epidermal morphology, stomatal density, and ...
[41]
Consistent host and organ occupancy of phyllosphere bacteria in a ...
Oct 17, 2019 · In the case of phyllosphere colonization via herbivores or pollinators, specific hosts or organs can be colonized as these vectors only visit ...
[42]
Impacts of global change on the phyllosphere microbiome - Zhu - 2022
Dec 18, 2021 · As such, the phyllosphere microbiome contributes to the gut microbiome of insect pollinators and therefore influences their fitness and ...
[43]
Leaf age structures phyllosphere microbial communities in the field ...
Aug 13, 2024 · Our field study characterized changes in the fungal and bacterial phyllosphere by examining leaves of different relative ages across individuals.
[44]
https://www.nature.com/articles/s41467-025-58856-6
[45]
Progress in cultivation-independent phyllosphere microbiology - PMC
Most microorganisms of the phyllosphere are nonculturable in commonly used media and culture conditions, as are those in other natural environments.
[46]
https://pmc.ncbi.nlm.nih.gov/articles/PMC3906827/
[47]
Evaluation of Different Phyllosphere Sample Types for Parallel ...
Jul 27, 2022 · Two sample types, leaf disk samples and leaf wash samples, were prepared from each leaf pool. First, a cork borer (1 cm) was used to cut one ...
[48]
Functional genomics analysis of a phyllospheric Pseudomonas spp ...
Sep 22, 2022 · The resulting wash and macerate were used for the dilution series in Petri dishes containing King's B Medium [39]. The plates were then ...
[49]
https://bmcmicrobiol.biomedcentral.com/articles/10.1186/s12866-022-02637-4
[50]
https://doi.org/10.1016/j.mimet.2010.08.006
[51]
Community proteogenomics reveals insights into the physiology of ...
To gain insight into the physiology of phyllosphere bacteria under in situ conditions, we performed a culture-independent analysis of the microbiota associated ...
[52]
The Changes of Phyllosphere Fungal Communities among Three ...
Oct 2, 2023 · High-throughput sequence analysis of ITS rDNA revealed that the dominant taxa of phyllosphere fungi are mainly Ascomycota and Basidiomycota.
[53]
Seasonal Dynamics of Core Fungi in the Switchgrass Phyllosphere ...
Oct 13, 2020 · We hypothesized that fungal communities would exhibit strong seasonal shifts, similar to that observed for bacterial communities in the ...
[54]
Metaproteogenomic analysis of microbial communities in ... - Nature
Dec 22, 2011 · In the phyllosphere, the most frequently detected nifH sequence types were those of Azorhizobium and Rhodopseudomonas. In the rhizosphere, nifH ...
[55]
Single-cell pigment analysis of phototrophic and phyllosphere ...
Apr 10, 2025 · Single-cell pigment analysis of phototrophic and phyllosphere bacteria using simultaneous detection of Raman and autofluorescence spectra.
[56]
CRISPR-based engineering of phages for in situ bacterial base editing
Nov 7, 2022 · Here, we describe a phage-based approach for precision editing of an individual gene within a target host organism in a community context.
[57]
Abundance of human pathogen genes in the phyllosphere of four ...
Feb 1, 2020 · Three human pathogens in phyllosphere of landscape plants were detected based on qPCR. •. The abundance of pathogenic gene of human in ...
[58]
Analysis of Phyllosphere Microorganisms and Potential Pathogens ...
In the tobacco phyllosphere, some of the microbes may have detrimental effects on plant health, while many may be neutral or even beneficial.
[59]
Phyllosphere Microbiome - Annual Reviews
May 22, 2023 · Nitrogen fixation by phyllosphere bacteria associated with ... The role of bacterial ice nucleation in frost injury to plants. Annu ...
[60]
Pseudomonas spp.-induced systemic resistance to Botrytis cinerea ...
Pseudomonas spp.-induced systemic resistance to Botrytis cinerea is associated with induction and priming of defence responses in grapevine Free. Bas W. M. ...
[61]
Leaf blast disease reduction by rice-phyllosphere actinomycetes ...
Feb 23, 2017 · Leaf blast disease reduction by rice-phyllosphere actinomycetes producing bioactive compounds. Disease Control; Published: 23 February 2017.Missing: PDF | Show results with:PDF
[62]
Nutrients and host attributes modulate the abundance and functional ...
A number of phosphate solubilizers were also obtained from the phyllosphere of rice in Kenya as reported by Mwajita et al. (2013). Phyllosphere being a nutrient ...Nutrients And Host... · 3. Results · 4. Discussion<|separator|>
[63]
Development of biofertilizers for sustainable agriculture over four ...
Studies have shown that biofertilizers, when used together with chemical fertilizers, can increase crop yields by 10%–40% and replace nearly 25%–30% of the ...Missing: foliar phyllosphere
[64]
Degradation of phenathrene on plant leaves by phyllosphere bacteria
Aug 6, 2025 · The results showed that the amount of phenanthrene was significantly reduced on leaves containing phenanthrene-degrading bacteria. These were ...
[65]
[PDF] Phylloremediation of Polyaromatic Hydrocarbonic Pollutants Using ...
Nov 23, 2018 · Phylloremediation is an effective bioremediation method which can use to remediate the air, water and soil polluted from the PAH compounds. The.<|separator|>
[66]
Phyllosphere synthetic microbial communities: a new frontier in plant ...
Jul 23, 2025 · These microbial populations colonize distinct ecological niches across various plant compartments, including the rhizosphere (soil), endosphere ...<|control11|><|separator|>
[67]
The Foliar Application of Rice Phyllosphere Bacteria induces ... - NIH
This study indicates that the foliar application of B. megaterium PB50 can induce tolerance to drought stress in rice.
[68]
Application of data integration for rice bacterial strain selection by ...
Study design. To assess PGP traits and osmotic stress responses in eight bacterial strains isolated from the phyllosphere of drought-tolerant rice varieties ...
[69]
Impacts of global change on the phyllosphere microbiome - PMC - NIH
Phyllosphere microbiomes are faced with increased stress caused by climate change, particularly by warming and drought. Climate change stresses may result in ...
[70]
Phyllosphere Community Assembly and Response to Drought ...
Phyllosphere microbes directly promote plant growth and stress tolerance through phytohormone and nutrient production and protection from UV damage (14, 41, 42) ...
[71]
Multi-omics reveals the phyllosphere microbial community and ...
Jul 31, 2024 · A multi-omics analysis was applied to investigate the link between the structural composition of the phyllosphere microbial community and the main metabolites
[72]
Integrated metabolomics and metagenomics reveal plant-microbe ...
Jun 2, 2025 · In this study, we conducted a comprehensive investigation of the relationships between tobacco metabolite profiles, microbial communities, and aroma ...
[73]
Unraveling plant–microbe interactions: can integrated omics ...
Integrating multi-omics datasets can transform our understanding of the plant microbiome from unspecified genetic influences on interacting species to specific ...
[74]
The Role of Phyllosphere Microbes and Viruses in Biocontrol ... - NIH
Oct 10, 2025 · The phyllosphere hosts diverse microbial communities that play a pivotal role in suppressing foliar pathogens through complex ecological ...Missing: hyperparasitism | Show results with:hyperparasitism
[75]
Resilience of the Natural Phyllosphere Microbiota of the Grapevine ...
... Archaea was greater in SM1 than in SM2. Moreover, the abundance of Archaea was greater in SM1 than in UD, whereas the abundance of Deltaproteobacteria was ...
[76]
Characteristics of the phyllosphere microbial community and its ...
May 9, 2024 · Archaea, viruses, and other microbes in the phyllosphere are poorly characterized due to the lack of related research. Therefore, bacteria and ...
[77]
Key Challenges in Plant Microbiome Research in the Next Decade
Summary of each segment:
[78]
Phyllosphere bacterial and fungal communities vary with host ... - NIH
Jun 9, 2022 · Our results suggest that subtropical tree phyllosphere microbial communities vary with host species identity, plant traits and seasonality.Missing: solar heating<|separator|>