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Phyllosphere
Phyllosphere
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The plant aerial surface, mostly occupied by leaves, is inhabited by diverse microorganisms, forming the phyllosphere

In microbiology, the phyllosphere is the total above-ground surface of a plant when viewed as a habitat for microorganisms.[1][2][3] The phyllosphere can be further subdivided into the caulosphere (stems), phylloplane (leaves), anthosphere (flowers), and carposphere (fruits). The below-ground microbial habitats (i.e. the thin-volume of soil surrounding root or subterranean stem surfaces) are referred to as the rhizosphere and laimosphere. Most plants host diverse communities of microorganisms including bacteria, fungi, archaea, and protists. Some are beneficial to the plant, while others function as plant pathogens and may damage the host plant or even kill it.

The phyllosphere microbiome

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The leaf surface, or phyllosphere, harbours a microbiome comprising diverse communities of bacteria, archaea, fungi, algae and viruses.[4][5] Microbial colonizers are subjected to diurnal and seasonal fluctuations of heat, moisture, and radiation. In addition, these environmental elements affect plant physiology (such as photosynthesis, respiration, water uptake etc.) and indirectly influence microbiome composition.[6] Rain and wind also cause temporal variation to the phyllosphere microbiome.[7]

The phyllosphere includes the total aerial (above-ground) surface of a plant, and as such includes the surface of the stem, flowers and fruit, but most particularly the leaf surfaces. Compared with the rhizosphere and the endosphere the phyllosphere is nutrient poor and its environment more dynamic.

Interactions between plants and their associated microorganisms in many of these microbiomes can play pivotal roles in host plant health, function, and evolution.[8] Interactions between the host plant and phyllosphere bacteria have the potential to drive various aspects of host plant physiology.[9][2][10] However, as of 2020 knowledge of these bacterial associations in the phyllosphere remains relatively modest, and there is a need to advance fundamental knowledge of phyllosphere microbiome dynamics.[11][12]

The assembly of the phyllosphere microbiome, which can be strictly defined as epiphytic bacterial communities on the leaf surface, can be shaped by the microbial communities present in the surrounding environment (i.e., stochastic colonisation) and the host plant (i.e., biotic selection).[4][13][12] However, although the leaf surface is generally considered a discrete microbial habitat,[14][15] there is no consensus on the dominant driver of community assembly across phyllosphere microbiomes. For example, host-specific bacterial communities have been reported in the phyllosphere of co-occurring plant species, suggesting a dominant role of host selection.[15][16][17][12]

Conversely, microbiomes of the surrounding environment have also been reported to be the primary determinant of phyllosphere community composition.[14][18][19][20] As a result, the processes that drive phyllosphere community assembly are not well understood but unlikely to be universal across plant species. However, the existing evidence does indicate that phyllosphere microbiomes exhibiting host-specific associations are more likely to interact with the host than those primarily recruited from the surrounding environment.[9][21][22][23][12]

Spatial scales matter
Trinidad
A leaf
The area of Trinidad is about 5000 sq km (2000 sq mi). Compared to the size of a human, this is about the same relative area as a typical leaf compared to the size of a bacterium. Imagine a human somewhere on Trinidad without legs to move, and neither eyes to see nor ears to hear, retaining only the ability to smell and touch. This is a parallel to how an individual bacterium perceives a leaf. There is no ability to perceive anything beyond its most immediate surroundings. Bacteria need water for movement and they perceive only "signals, such as sugars, amino acids or volatiles, diffusing to their occupied site". This microhabitat determines the experience of the individual bacterium and how it responds.[24]

Overall, there remains high species richness in phyllosphere communities. Fungal communities are highly variable in the phyllosphere of temperate regions and are more diverse than in tropical regions.[25] There can be up to 107 microbes per square centimetre present on the leaf surfaces of plants, and the bacterial population of the phyllosphere on a global scale is estimated to be 1026 cells.[26] The population size of the fungal phyllosphere is likely to be smaller.[27]

Phyllosphere microbes from different plants appear to be somewhat similar at high levels of taxa, but at the lower levels taxa there remain significant differences. This indicates microorganisms may need finely tuned metabolic adjustment to survive in phyllosphere environment.[25] Pseudomonadota seems to be the dominant colonizers, with Bacteroidota and Actinomycetota also predominant in phyllospheres.[28] Although there are similarities between the rhizosphere and soil microbial communities, very little similarity has been found between phyllosphere communities and microorganisms floating in open air (aeroplankton).[29][6]

The search for a core microbiome in host-associated microbial communities is a useful first step in trying to understand the interactions that may be occurring between a host and its microbiome.[30][31] The prevailing core microbiome concept is built on the notion that the persistence of a taxon across the spatiotemporal boundaries of an ecological niche is directly reflective of its functional importance within the niche it occupies; it therefore provides a framework for identifying functionally critical microorganisms that consistently associate with a host species.[30][32][33][12]

A leaf from a healthy Arabidopsis plant (left) and a leaf from a dysbiosis mutant plant (right)[34]

Divergent definitions of "core microbiome" have arisen across scientific literature with researchers variably identifying "core taxa" as those persistent across distinct host microhabitats [35][36] and even different species.[17][21] Given the functional divergence of microorganisms across different host species [17] and microhabitats,[37] defining core taxa sensu stricto as those persistent across broad geographic distances within tissue- and species-specific host microbiomes, represents the most biologically and ecologically appropriate application of this conceptual framework.[38][12] Tissue- and species-specific core microbiomes across host populations separated by broad geographical distances have not been widely reported for the phyllosphere using the stringent definition established by Ruinen.[2][12]

Example: The manuka phyllosphere

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Relative abundance of core phyllosphere taxa in manuka
Manuka is a flowering scrub. The chart shows an abundance-occupancy distribution identifying core phyllosphere taxa in non-rarefied (green) and rarefied (purple) datasets. Each point represents a taxon plotted by its mean logarithmic relative abundance and occupancy. Taxa (pink) with an occupancy of 1 (i.e., detected in all 89 phyllosphere samples) were considered members of the core microbiome.[12]

The flowering tea tree commonly known as manuka is indigenous to New Zealand.[39] Manuka honey, produced from the nectar of manuka flowers, is known for its non-peroxide antibacterial properties.[40][41] These non-peroxide antibacterial properties have been principally linked to the accumulation of the three-carbon sugar dihydroxyacetone (DHA) in the nectar of the manuka flower, which undergoes a chemical conversion to methylglyoxal (MGO) in mature honey.[42][43][44] However, the concentration of DHA in the nectar of manuka flowers is notoriously variable, and the antimicrobial efficacy of manuka honey consequently varies from region to region and from year to year.[45][46][47] Despite extensive research efforts, no reliable correlation has been identified between DHA production and climatic,[48] edaphic,[49] or host genetic factors.[50][12]

{A} The heatmap on the left illustrates how the composition of operational taxonomic units (OTUs) in the manuka phyllosphere and associated soil communities differed significantly. No core soil microbiome was detected.
(B) The chart on the right shows how OTUs in phyllosphere and associated soil communities differed in relative abundances.[12]

Microorganisms have been studied in the manuka rhizosphere and endosphere.[51][52][53] Earlier studies primarily focussed on fungi, and a 2016 study provided the first investigation of endophytic bacterial communities from three geographically and environmentally distinct manuka populations using fingerprinting techniques and revealed tissue-specific core endomicrobiomes.[54][12] A 2020 study identified a habitat-specific and relatively abundant core microbiome in the manuka phyllosphere, which was persistent across all samples. In contrast, non-core phyllosphere microorganisms exhibited significant variation across individual host trees and populations that was strongly driven by environmental and spatial factors. The results demonstrated the existence of a dominant and ubiquitous core microbiome in the phyllosphere of manuka.[12]

See also

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References

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Revisions and contributorsEdit on WikipediaRead on Wikipedia

Phyllosphere

View on Grokipedia
from Grokipedia
The phyllosphere refers to the above-ground surfaces of , primarily leaves but also stems and flowers, that collectively form a vast microbial habitat colonized by epiphytic microorganisms such as , fungi, yeasts, , protists, and viruses. This ecosystem, often described as the "aerial ," spans an estimated terrestrial leaf surface area of 6.4 × 10^8 km² and supports a global bacterial of approximately 10^26 cells, making it one of Earth's largest microbial environments. 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. Microbial communities in the phyllosphere exhibit extraordinary , with bacterial densities typically ranging from 10^6 to 10^8 cells per square centimeter of surface, dominated by phyla such as Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. Common genera include , , , and , alongside fungi like and , and yeasts such as . These populations are shaped by host plant , age, geographic location, seasonal changes, and environmental stressors like UV radiation, , and nutrient scarcity from leaf exudates (e.g., sugars and ). 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. Ecologically, phyllosphere microbes are integral to and broader environmental processes, promoting growth through , hormone production (e.g., ), and enhanced stress tolerance via enzymes like and . They contribute to biogeochemical cycles by facilitating carbon and nitrogen turnover, decomposing organic matter, and emitting volatile organic compounds that influence . Additionally, these communities suppress pathogens through competition and antagonism, as seen with strains that inhibit frost damage or in crops. However, they also pose risks, serving as reservoirs for human pathogens like and that contaminate produce. From an applied perspective, phyllosphere 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. Research since the 1990s, leveraging molecular tools like GFP tagging and , has illuminated colonization dynamics and microbial fitness, paving the way for engineered microbiomes to enhance resilience amid .

Definition and Habitat Characteristics

Definition

The phyllosphere refers to the total above-ground portions of , including leaves, stems, flowers, and fruits, conceptualized as a for microorganisms. 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. The term "phyllosphere" was independently coined by F.T. Last in 1955 and J. Ruinen in 1956 to describe the external surface as an ecologically significant yet overlooked milieu for microbial growth and activity. 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. This vast surface area—spanning terrestrial ecosystems worldwide—positions the phyllosphere as the largest contiguous for microorganisms on the planet, far exceeding other plant-associated niches in extent. The phyllosphere is distinct from other plant microbiomes, such as the , which encompasses the soil zone surrounding roots below ground, and the endosphere, which includes internal plant tissues across both above- and below-ground parts. 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.

Physical and Chemical Properties

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

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 such as genera Sphingomonas and Pseudomonas, alongside Actinobacteria, Bacteroidetes, and Firmicutes. 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. 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. This density varies by plant species; for instance, in (Oryza sativa), Pseudomonas species are often prominent in the community. In contrast, neotropical forest trees host elevated levels of Methylobacterium, a methylotrophic genus that thrives on leaf emissions, representing a core component in such ecosystems. Plant-specific traits, like leaf chemistry and morphology, further shape these patterns, leading to host-dependent community structures. 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. Certain strains, notably Pseudomonas syringae, promote ice nucleation on leaves, facilitating frost damage at subzero temperatures by lowering the freezing point threshold. Additionally, quorum sensing enables biofilm formation, allowing bacteria like Pseudomonas to coordinate colonization and enhance persistence against desiccation and UV exposure. The assembly of these communities involves a balance of and deterministic processes. dispersal from air and introduces low-abundance taxa, which then establish via random events. Deterministic selection by host traits, such as leaf surface hydrophobicity and volatile emissions, filters and stabilizes core members, promoting over leaf development. 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 and , which together account for the majority of detected sequences in high-throughput sequencing studies across various hosts. Within , genera such as Aureobasidium (e.g., ), , and are frequently dominant, with A. pullulans often comprising a significant portion of the yeast-like fungal due to its to oligotrophic surfaces. are represented primarily by yeasts like those in Sporobolomyces, which exhibit ballistoconidium formation for aerial dispersal and thrive in moist microhabitats on leaves. Molds such as spp. become more prevalent on senescing leaves, where they exploit decaying material for colonization. Other microbial diversity in the phyllosphere includes , , and (prokaryotes), though these groups occur at lower abundances compared to fungal and bacterial components. , particularly amoebae, function as grazers on bacterial populations, contributing to microbial community regulation in water films on surfaces. Epiphytic , such as those in , colonize moist phyllosphere niches in humid environments like rainforests, where they form thin biofilms that influence surface wettability and nutrient availability. are sporadically detected in low relative abundance, with methanogenic taxa appearing in wetter phyllospheres of aquatic or floating macrophytes, potentially utilizing anaerobic microsites for production. These groups co-occur with , forming interconnected networks that shape overall phyllosphere dynamics. 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. In species, such as black cottonwood (), fungal communities exhibit seasonal shifts, with yeast abundances peaking in cooler, wetter periods due to favorable conditions for and growth. Fungal structural adaptations, including hyphal networks, facilitate scavenging from exudates and , while wind-dispersed spores enable rapid colonization and resilience in the exposed aerial environment.

Ecological Dynamics and Interactions

Plant-Microbe Interactions

The phyllosphere hosts a range of biotic interactions between and microbes, encompassing mutualistic associations that enhance host fitness and antagonistic dynamics that suppress pathogens. These interactions occur primarily on surfaces but can extend internally through endophytic , influencing plant growth, defense, and overall health. Mutualistic microbes often provide benefits such as acquisition and stress tolerance, while antagonistic ones limit through direct or inhibitory compounds. Mutualistic interactions in the phyllosphere prominently include plant growth promotion through hormone modulation, such as (IAA) production by like Pseudomonas species, which stimulate and shoot development in crops including and . Nutrient solubilization represents another key mechanism, with phyllosphere Bacillus and Pseudomonas strains solubilizing insoluble phosphates to improve availability for like , thereby enhancing accumulation under nutrient-limited conditions. Additionally, certain microbes induce systemic resistance (ISR) in , activating defense pathways against foliar pathogens; for instance, Bacillus amyloliquefaciens triggers and signaling to protect against necrotrophic fungi in . Antagonistic roles of phyllosphere microbes focus on pathogen suppression through resource and production. Bacillus , such as B. subtilis and B. amyloliquefaciens, produce antibiotics like surfactin, iturin, and bacilysin, which inhibit phytopathogens including and Erwinia amylovora on grapevine and apple leaves by disrupting cell membranes and metabolism. Competition for limiting nutrients, such as sugars, further limits pathogen establishment; strains outcompete on leaves by rapidly utilizing leaf exudates. Hyperparasitism, though less common among bacteria, involves microbes parasitizing , as seen with the hyperparasitizing conidia on fruit surfaces through enzymatic degradation. Many phyllosphere microbes exhibit transitions from epiphytic to endophytic lifestyles, entering plant tissues via stomata or wounds to establish internal symbioses that amplify benefits. species, initially colonizing surfaces, can penetrate stomata to become endophytes, producing cytokinins that enhance and overall vigor in , leading to increased grain yields by up to sixfold in certain landraces. This dual habitation allows sustained hormone modulation and stress alleviation without disrupting surface communities. 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. This genotype-driven recruitment underscores how plants shape phyllosphere dynamics to optimize biotic exchanges.

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. (UV) radiation, particularly UV-B, alters bacterial community composition by favoring UV-resistant phenotypes, such as pigmented and isolates like and Curtobacterium flaccumfaciens, which exhibit enhanced survival on surfaces under high exposure. influences fungal communities by promoting and shifting composition toward saprotrophs and endophytes, with elevated air accounting for 6.5% of variation in foliar fungal community composition on species like . extremes further drive selection, as higher temperatures in hot-dry environments correlate with increased abundance of thermotolerant taxa such as and in poplar phyllospheres, while reducing overall diversity. 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. 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. Biotic cross-kingdom effects, such as disturbances from pollinators and , introduce transient microbes that transiently diversify phyllosphere communities. Insect vectors like pollinators facilitate microbial dispersal across organs and by carrying on their bodies, leading to equal prevalence of operational taxonomic units (OTUs) across hosts and promoting passive migration. 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. 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 ), supporting more stable communities with abundant genera like Actinobacteria that provide protective roles against stresses. As leaves age, community composition shifts (PERMANOVA, F₂,₇₈ = 3.08, p = 0.002), favoring microbes adapted to changing microenvironments.

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 buffer like (PBS) and agitating via shaking or , often recovering 10^6 to 10^8 cells per gram of fresh leaf weight while preserving cell viability. 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 . Homogenization further disrupts leaf tissue to access endophytes but is less common for surface-focused studies due to its invasiveness. Non-destructive methods prioritize minimal plant damage and include swabbing leaf surfaces with sterile or swabs dipped in buffer, impression prints by pressing leaves onto plates, and strips to lift microbes directly. Swabbing, as described by Yashiro et al. (2011), targets surface epiphytes effectively for both culturing and , while leaf printing—pioneered by Corpe (1985)—provides a quick qualitative assessment of microbial distribution without extraction. 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. 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. Cultivation of phyllosphere isolates relies on selective media to target dominant taxa, such as King's B medium for fluorescent species, which promotes production and growth from wash dilutions. However, only 1-10% of the total community is typically culturable under standard lab conditions, with rates ranging from 0.1% to 8.4% as quantified by et al. (2010), due to viable but non-culturable (VBNC) states induced by limitation or stress. 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 . Semi-selective media for pathogens, such as those for enteric outlined by Barak et al. (2011), further refine isolation but exacerbate underestimation of diversity. Historically, phyllosphere sampling evolved from early 20th-century plate counts of colony-forming units via dilution , as in Ruinen's 1956 identification of nitrogen-fixing , to more refined culture-dependent methods like pressing reported by Dickinson and Bainbridge (). By the mid-20th century, quantitative assessments via washing and became standard, with Thompson et al. (1993) establishing seasonal baselines using such techniques. 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 in field studies, addressing variability in earlier approaches. These cultivation-based limitations, including low recovery of VBNC forms, are increasingly supplemented by molecular methods for comprehensive analysis.

Molecular and Omics Techniques

Molecular and 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. These approaches, including sequencing and shotgun-based , allow researchers to capture the vast genetic and metabolic potential of leaf-associated microbes without the biases of traditional culturing. 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. 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 and as dominant genera, highlighting host-specific and shared phyllosphere microbiomes. For fungal communities, (ITS) region sequencing provides resolution at the species level, uncovering Ascomycota and Basidiomycota as prevalent phyla on plant leaves. ITS-based amplicon studies on crops like switchgrass have shown seasonal shifts in epiphytic fungi, with genera such as and varying in abundance due to environmental cues. Shotgun metagenomics extends beyond taxonomy by sequencing total community DNA, enabling assembly of functional gene profiles, including those involved in nutrient cycling. 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. 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. 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. Emerging tools enhance resolution of phyllosphere interactions at finer scales. Single-cell 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. 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 on plant surfaces. Quantitative PCR (qPCR) offers targeted detection of pathogens, quantifying genes like those for human pathogens (e.g., invA in ) in urban landscape plant phyllospheres at levels up to 10^4 copies per gram of tissue. Data analysis pipelines address the complexity of datasets from phyllosphere samples, employing metrics like (e.g., Shannon index) to quantify within-sample richness and (e.g., Bray-Curtis dissimilarity) to compare communities across hosts or conditions. 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 . Biases, including PCR chimeras in amplicon sequencing, are mitigated through denoising algorithms like DADA2, ensuring accurate taxonomic assignments in high-throughput phyllosphere surveys. These analyses integrate with post-collection processing from sampling methods to provide robust interpretations of structure and function.

Applications and Future Directions

Agricultural and Biotechnological Applications

The phyllosphere offers significant potential for biocontrol applications in , where foliar sprays of beneficial suppress pathogenic fungi on crop surfaces. For instance, species applied as foliar inoculants induce systemic resistance in grapevines against , the causative agent of gray mold, by priming defense responses such as enhanced signaling and reduced lesion development following challenge. In , actinomycetes isolated from the phyllosphere, particularly strains of , produce bioactive compounds like antibiotics that inhibit Pyricularia oryzae, reducing leaf blast disease severity by up to 88% in greenhouse assays. Biofertilization strategies leverage phyllosphere microbes to improve nutrient uptake through leaf-applied consortia, minimizing reliance on synthetic inputs. Phosphate-solubilizing such as and , naturally occurring in the phyllosphere, secrete organic acids to convert insoluble phosphates into plant-available forms, enhancing acquisition and overall nutrient efficiency. Such foliar biofertilizers can replace 25-30% of chemical fertilizers while boosting crop yields by 10-40% in integrated systems. In , phyllosphere 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 and on plant surfaces metabolize , reducing its levels by significant margins through experiments on species like . 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. Commercial products derived from phyllosphere microbes include microbial inoculants that enhance crop protection and industrial . In mānuka (Leptospermum scoparium) cultivation for production, core phyllosphere taxa like and Rhizobiales have been identified, a plant cultivated for production with high-methylglyoxal content and properties. Additionally, phyllosphere yeasts such as Pseudozyma antarctica produce mannosylerythritol lipids (MELs), biosurfactants used as thickening and stabilizing agents in and food formulations.

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. 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 on microbial community dynamics. Elevated temperatures and drought have been shown to reduce beneficial taxa like while promoting opportunistic pathogens such as , potentially destabilizing plant-microbe interactions and increasing disease susceptibility. Modeling efforts indicate that humidity shifts can convert non-pathogenic strains into ice-nucleating pathogens, disrupting community and stomatal regulation for water uptake. Urban phyllospheres are emerging as bioindicators for , with studies revealing higher resistance prevalence in city greenery compared to rural sites, highlighting their role as sentinels for anthropogenic stressors like . These findings underscore the need for predictive models integrating UV and humidity data to forecast phyllosphere responses in warming scenarios. 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. Despite progress, significant research gaps persist in phyllosphere studies, particularly regarding understudied components like viruses and , 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. , 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 succession and inoculant persistence under variable conditions. In the global south, tropical forest phyllospheres beyond localized surveys—such as those in subtropical —show high bacterial and fungal diversity tied to host traits and , yet broader representation from underrepresented regions is needed to capture global variability. Addressing these gaps will require expanded international collaborations and advanced sequencing.

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