Zinc oxide nanoparticles cooperate with the phyllosphere to promote grain yield and nutritional quality of rice under heatwave stress.

To address rising global food demand, the development of sustainable technologies to increase productivity is urgently needed. This study revealed that foliar application of zinc oxide nanoparticles (ZnO NPs; 30 to 80 nm, 0.67 mg/d per plant, 6 d) to rice leaves under heatwave (HW) stress increased the grain yield and nutritional quality. Compared with the HW control, the HWs+ZnO group presented increases in the grain yield, grain protein content, and amino acid content of 22.1%, 11.8%, and 77.5%, respectively. Nanoscale ZnO aggregated on the leaf surface and interacted with leaf surface molecules. Compared with that at ambient temperature, HW treatment increased the dissolution of ZnO NPs on the leaf surface by 25.9% and facilitated their translocation to mesophyll cells. The Zn in the leaves existed as both ionic Zn and particulate ZnO. Compared with the HW control, foliar application of ZnO NPs under HW conditions increased leaf nutrient levels (Zn, Mn, Cu, Fe, and Mg) by 15.8 to 416.9%, the chlorophyll content by 22.2 to 24.8%, Rubisco enzyme activity by 21.2%, and antioxidant activity by 26.7 to 31.2%. Transcriptomic analyses revealed that ZnO NPs reversed HW-induced transcriptomic dysregulation, thereby enhancing leaf photosynthesis by 74.4%. Additionally, ZnO NPs increased the diversity, stability, and enrichment of beneficial microbial taxa and protected the phyllosphere microbial community from HW damage. This work elucidates how NPs interact with the phyllosphere, highlighting the potential of NPs to promote sustainable agriculture, especially under extreme climate events (e.g., HWs).

Unravelling the microbiome of wild flowering plants: a comparative study of leaves and flowers in alpine ecosystems.

BACKGROUND: While substantial research has explored rhizosphere and phyllosphere microbiomes, knowledge on flower microbiome, particularly in wild plants remains limited. This study explores into the diversity, abundance, and composition of bacterial and fungal communities on leaves and flowers of wild flowering plants in their natural alpine habitat, considering the influence of environmental factors. METHODS: We investigated 50 wild flowering plants representing 22 families across seven locations in Austria. Sampling sites encompassed varied soil types (carbonate/silicate) and altitudes (450-2760 m). Amplicon sequencing to characterize bacterial and fungal communities and quantitative PCR to assess microbial abundance was applied, and the influence of biotic and abiotic factors assessed. RESULTS: Our study revealed distinct bacterial and fungal communities on leaves and flowers, with higher diversity and richness on leaves (228 fungal and 91 bacterial ASVs) than on flowers (163 fungal and 55 bacterial ASVs). In addition, Gammaproteobacteria on flowers and Alphaproteobacteria on leaves suggests niche specialization for plant compartments. Location significantly shaped both community composition and fungal diversity on both plant parts. Notably, soil type influenced community composition but not diversity. Altitude was associated with increased fungal species diversity on leaves and flowers. Furthermore, significant effects of plant family identity emerged within a subset of seven families, impacting bacterial and fungal abundance, fungal Shannon diversity, and bacterial species richness, particularly on flowers. CONCLUSION: This study provides novel insights into the specific microbiome of wild flowering plants, highlighting adaptations to local environments and plant-microbe coevolution. The observed specificity indicates a potential role in plant health and resilience, which is crucial for predicting how microbiomes respond to changing environments, ultimately aiding in the conservation of natural ecosystems facing climate change pressures.

The phyllosphere of Nigerian medicinal plants, Euphorbia lateriflora and Ficus thonningii is inhabited by a specific microbiota.

The microbiota of medicinal plants is known to be highly specific and can contribute to medicinal activity. However, the majority of plant species have not yet been studied. Here, we investigated the phyllosphere composition of two common Nigerian medicinal plants, Euphorbia lateriflora and Ficus thonningii, by a polyphasic approach combining analyses of metagenomic DNA and isolates. Microbial abundance estimated via qPCR using specific marker gene primers showed that all leaf samples were densely colonized, with up to 108 per gram of leaf, with higher bacterial and fungal abundance than Archaea. While no statistically significant differences between both plant species were found for abundance, amplicon sequencing of 16S rRNA and ITS genes revealed distinct microbiota compositions. Only seven of the 27 genera isolated were represented on both plants, e.g. dominant Sphingomonas spp., and numerous members of Xanthomonadaceae and Enterobacteriaceae. The most dominant fungal families on both plants were Cladosporiaceae, Mycosphaerellaceae and Trichosphaeriaceae. In addition, 225 plant-specific isolates were identified, with Pseudomonadota and Enterobacteriaceae being dominant. Interestingly, 29 isolates are likely species previously unknown, and 14 of these belong to Burkholderiales. However, a high proportion, 56% and 40% of the isolates from E. lateriflora and F. thonningii, respectively, were characterized as various Escherichia coli. The growth of most of the bacterial isolates was not influenced by extractable secondary metabolites of plants. Our results suggest that a specific and diverse microbial community inhabits the leaves of both E. lateriflora and F. thonningii, including potentially new species and producers of antimicrobials.

Coniferous Tree Species Identity and Leaf Aging Alter the Composition of Phyllosphere Communities Through Changes in Leaf Traits.

Phyllosphere microorganisms are essential for plant growth and health. Although there are an increasing number of studies showing that the composition of phyllosphere communities varies among different plant species, it remains unclear whether and how their bacterial and fungal community composition predictably varies with plant traits and leaf age. In this study, we used high-throughput sequencing to explore the diversity and composition of phyllosphere communities in needles of different ages (originating from different cohorts) for three evergreen coniferous species (Pinus koraiensis, Picea koraiensis, and Abies nephrolepis). Our results indicated that Gammaproteobacteria (bacteria) and Dothideomycetes (fungi) were dominant in newly formed needles, whereas Actinobacteria (bacteria) and Eurotiomycetes (fungi) were dominant in perennial needles. Tree species identity and needle age were the main factors explaining the variations of the α diversity (species richness of phyllosphere communities) and ß diversity (dissimilarity among phyllosphere communities). In particular, we found that leaf dry matter content, leaf mass per area, and total phosphorus content emerged as key predictors of composition and diversity of phyllosphere microbial communities, underscoring the major influence of tree species identity and needle age on phyllosphere communities through changes in plant functional traits. Finally, we found that the interaction between tree species identity and needle age also contributed significantly to explaining the diversity and composition of phyllosphere communities, probably because differences in plant functional traits or environmental conditions between new and perennial needles depend on tree growth rates and resource acquisition strategies. These findings provide new insights into the mechanisms of community assembly among different evergreen tree species and offer a better understanding of the interactions between plant traits and phyllosphere microorganisms during needle aging.

Atmospheric microplastic input into wetlands: Spatiotemporal patterns, drivers, and unique ecological impacts.

Wetlands are major microplastic sinks with a large atmospheric input. However, many details of such deposited atmospheric microplastics entering into wetlands remain unclear, including temporal patterns of input and ecological effects. We monitored the aerial microplastics during four seasons in eleven economically developed cities along the lower reaches of the Yangtze River Basin, China. The average microplastic deposition rate was 512.31 items m-2 d-1, equivalent to an annual contribution of 17.46 metric tons of plastic to the surveyed wetlands with a total area of 1652 km2. These microplastics were predominantly composed of polyamide and polyethylene terephthalate with 61.85 ± 92.29 µm sized pellets, and we obtained similar results for microplastics intercepted on moss in wetlands. Microplastic input varied between wet and dry periods, primarily influenced by wind, rainfall and ozone concentration. Civilian vehicle density and textile industry were the primary socioeconomic factors driving microplastic deposition. Further indoor microcosm experiments revealed that moss phyllosphere bacterial community structure and function were influenced by microplastic abundance and size, exemplifying the unique ecological risks of aerially deposited microplastics to wetlands. These results indicate that mosses and their phyllosphere microbiota could serve as bio-indicators of aerial microplastic characteristics and impacts.

Anoxic seagrass leaf environments as potential hotspots for toxin production and N2O emission.

Epiphytes on seagrass leaves can render parts of the leaf phyllosphere anoxic in darkness owing to leaf/epiphyte respiration and O2 diffusion constraints. In such anoxic microenvironments, anaerobic microbes can potentially produce phytotoxins and greenhouse gases, but the actual occurrence of such processes in seagrass epiphytic biofilms remain uncertain. We used microsensors to measure O2, NO, N2O and H2S concentration gradients, as well as NO and O2 dynamics within epiphytic biofilms on seagrass (Zostera marina) leaves under changing environmental conditions. The bacterial community composition of epiphytic biofilms was analyzed with 16S rRNA gene amplicon sequencing. Flavobacteriaceae and Rhodobacteraceae were dominant bacterial community members accounting for ˃50 % of the relative abundance, and sulfate-reducing bacteria (Desulfobacterota) were omnipresent in the epiphytic biofilms. We found pronounced production of NO, N2O and H2S in anoxic parts of the seagrass phyllosphere, with NO and H2S reaching maximal concentrations of 1.0 and 4.4 µmol L-1, respectively, under slow flow and hypoxic seawater conditions, while the highest N2O concentration in the epiphytic biofilms reached 5.9 µmol L-1 in hypoxic, nitrate-rich seawater. Part of the phytotoxic NO and H2S diffused into the seagrass leaves, while no NO escaped the biofilm. In contrast, N2O emission from the biofilm in hypoxic and eutrophic seawater reached 9.6 µmol N2O m-2 day-1. Such release of the potent greenhouse gas N2O from seagrass leaves with epiphytic biofilms under eutrophic conditions could potentially offset the carbon burial capacity of seagrass meadows. Ocean eutrophication can thus stimulate denitrification and sulfate reduction within anoxic leaf microenvironments, negatively impacting seagrass fitness and ecological function.

Divergence and convergence in epiphytic and endophytic phyllosphere bacterial communities of rice landraces.

Phyllosphere-associated microbes can significantly alter host plant fitness, with distinct functions provided by bacteria inhabiting the epiphytic (external surface) vs endophytic niches (internal leaf tissue). Hence, it is important to understand the assembly and stability of these phyllosphere communities, especially in field conditions. Broadly, epiphytic communities should encounter more environmental fluctuations and frequent immigration, whereas endophytic microbiota should face stronger host selection. As a result, we expect greater variability in epiphytic than endophytic communities. We analyzed the structure and stability of leaf phyllosphere microbiota of four traditionally cultivated rice landraces and one commercial variety from northeast India grown in the field for 3 consecutive years, supplemented with opportunistic sampling of eight other landraces. Epiphytic and endophytic bacterial communities shared dominant core genera such as Methylobacterium and Sphingomonas. Consistent with an overall strong environmental effect, both communities varied more across sampling years than across host landraces. Seeds sampled from a focal landrace did not support vertical transmission of phyllosphere bacteria, suggesting that both types of communities are assembled anew each generation. Despite these points of convergence, epiphytic communities had distinct composition and significantly higher microbial load and were more rich, diverse, modular, and unstable than endophytic communities. Finally, focused sampling of one landrace across developmental stages showed that the divergence between the two types of communities arose primarily at the flowering stage. Thus, our results show both convergent and divergent patterns of community assembly and composition in distinct phyllosphere niches in rice, identifying key bacterial genera and host developmental stages that may aid agricultural interventions to increase rice yield.IMPORTANCEPhyllosphere (leaf-associated) microbes significantly impact plant fitness, making it crucial to understand how these communities are assembled and maintained. While many studies have analyzed epiphytic (surface) phyllosphere communities, we have a relatively poor understanding of endophytic communities which colonize the very distinct niche formed inside leaf tissues. We found that across several rice landraces, both communities are largely colonized by the same core genera, indicating divergence at the species level across the two leaf niches and highlighting the need to understand the mechanisms underlying this divergence. Surprisingly, both epiphytic and endophytic communities were only weakly shaped by the host landrace, with a much greater role for environmental factors that likely vary over time. Thus, microbiome-based agricultural interventions for increasing productivity could perhaps be generalized across rice varieties but would need to account for the temporal instability of the microbiota. Our results thus highlight the importance of data sets such as ours-with extensive sampling across landraces and years-for understanding phyllosphere microbiota and their applications in the field.

Biocontrol agents transform the stability and functional characteristics of the grape phyllosphere microenvironment.

The spread of grape leaf diseases has a negative impact on the sustainable development of agriculture. Diseases induced by Uncinula necator significantly affect the quality of grapes. Bacillus biocontrol agents have been proven effective in disease management. However, limited research has been conducted on the impact of biocontrol agents on the assembly and potential functions of plant phyllosphere microbial communities. This study used high-throughput sequencing combined with bioinformatics analysis and culture omics technology for analysis. The results showed that biocontrol bacteria B. subtilis utilized in this study can significantly reduce the disease index of powdery mildew (p<0.05); concurrently, it exhibits a lower disease index compared to traditional fungicides. A comprehensive analysis has revealed that biocontrol bacteria have no significant impact on the diversity of phyllosphere fungi and bacteria, while fungicides can significantly reduce bacterial diversity. Additionally, biocontrol agents can increase the complexity of fungal networks and enhance the degree of modularity and stability of the bacterial network. The results also showed that the biocontrol agents, which contained a high amount of B. subtilis, were able to effectively colonize the grapevine phyllosphere, creating a microenvironment that significantly inhibits pathogenic bacteria on grape leaves while enhancing leaf photosynthetic capacity. In conclusion, biocontrol agents significantly reduce the grape powdery mildew disease index, promote a microenvironment conducive to symbiotic microorganisms and beneficial bacteria, and enhance plant photosynthetic capacity. These findings provide a basis for promoting biocontrol agents and offer valuable insights into sustainable agriculture development.

Leaf Shelters Facilitate the Colonisation of Arthropods and Enhance Microbial Diversity on Plants.

Shelter-building insects are important ecosystem engineers, playing critical roles in structuring arthropod communities. Nonetheless, the influence of leaf shelters and arthropods on plant-associated microbiota remains largely unexplored. Arthropods that visit or inhabit plants can contribute to the leaf microbial community, resulting in significant changes in plant-microbe interactions. By artificially constructing leaf shelters, we provide evidence that shelter-building insects influence not only the arthropod community structure but also impact the phyllosphere microbiota. Leaf shelters exhibited higher abundance and richness of arthropods, changing the associated arthropod community composition. These shelters also altered the composition and community structure of phyllosphere microbiota, promoting greater richness and diversity of bacteria at the phyllosphere. In leaf shelters, microbial diversity positively correlated with the richness and diversity of herbivores. These findings demonstrate the critical role of leaf shelters in structuring both arthropod and microbial communities through altered microhabitats and species interactions.

Antibiofilm activity of Morganella morganii JB8F and Pseudomonas fluorescens JB3B compound to control single and multi-species of aquaculture pathogens.

BACKGROUND: Indonesia is a country that uses half or more aquatic foods as protein intake. The increased production in aquaculture industries might cause several problems, such as bacterial disease resulting in mass mortality and economic losses. Antibiotics are no longer effective because aquaculture pathogens can form biofilm. Biofilm is a microbial community that aggregates and firmly attaches to living or non-living surfaces. Biofilm formation can be caused by environmental stress, the presence of antibiotics, and limited nutrients. Therefore, it is important to explore antibiofilm to inhibit biofilm formation and/or eradicate mature biofilm. Phyllosphere bacteria can produce bioactive compounds for antimicrobial, antibiofilm, and anti-quorum sensing. Three aquaculture pathogens were used in this study, such as Aeromonas hydrophila, Streptococcus agalactiae, and Vibrio harveyi. RESULTS: Pseudomonas fluorescens JB3B and Morganella morganii JB8F extracts could disrupt single and multi-species biofilms. Both extracts could inhibit single biofilm formation from one to seven days of incubation time. We confirmed the destruction activity on multi-species biofilm using light microscope and scanning electron microscope. Using GC-MS analysis, indole was the most active fraction of the P. fluorescens JB3B extracts and octacosane from the M. morganii JB8F extract. We also conducted a toxicity test using brine shrimp lethality assay on P. fluorescens JB3B and M. morganii JB8F extracts. P. fluorescens JB3B, M. morganii JB8F, and a mixture of both extracts were confirmed non-toxic according to the LC50 value of the brine shrimp lethality test. CONCLUSIONS: P. fluorescens JB3B and M. morganii JB8F phyllosphere extracts had antibiofilm activity to inhibit single biofilm and disrupt single and multi-species biofilm of aquaculture pathogens. Both extracts could inhibit single species biofilm until seven days of incubation. Bioactive compounds that might contribute to antibiofilm properties were found in both extracts, such as indole and phenol. P. fluorescens JB3B, M. morganii JB8F extracts, and mixture of both extracts were non-toxic against Artemia salina.

Pseudomonas fluorescens Inoculation Enhances Salix matsudana Growth by Modifying Phyllosphere Microbiomes, Surpassing Nitrogen Fertilization.

The enhancement of plant growth by soil fertilization and microbial inoculation involves different mechanisms, particularly by altering the phyllosphere microbiome. This study investigated how nitrogen (N) fertilization, Pseudomonas fluorescens strain R124 inoculation and their combined effects influence the growth of different-aged Salix matsudana cuttings by modulating N dynamics within the phyllosphere microbiome. Results showed that P. fluorescens inoculation was significantly more effective than N fertilization alone, enhancing biomass, plant nutrient uptake, soil nutrient content and root development by 90.51%, 18.18%, 72.74% and 126.20%, respectively. Crucially, the inoculation notably shifted the beta-diversity of the phyllosphere microbial community, with K-strategy fungi enhancing plant N fixation and subsequent plant growth. Cuttings from middle-aged forests displayed more robust growth than those from young-aged, associated with a varied impact on phyllosphere fungi, notably increasing the relative abundance of Myriangiales in young (76.37%) and Capnodiales in middle-aged cuttings (42.37%), which improve phyllosphere stability and plant health. These findings highlight the effectiveness of microbial inoculation over N fertilization in promoting plant growth and provide valuable insights for the sustainable management of willow plantations at different stages of development.

Trophic Transfer of Metal Oxide Nanoparticles in the Tomato-Helicoverpa armigera Food Chain: Effects on Phyllosphere Microbiota, Insect Oxidative Stress, and Gut Microbiome.

Understanding the trophic transfer and ecological cascade effects of nanofertilizers and nanopesticides in terrestrial food chains is crucial for assessing their nanotoxicity and environmental risks. Herein, the trophic transfer of La2O3 (nLa2O3) and CuO (nCuO) nanoparticles from tomato leaves to Helicoverpa armigera (Lepidoptera: Noctuidae) caterpillars and their subsequent effects on caterpillar growth and intestinal health were investigated. We found that 50 mg/L foliar nLa2O3 and nCuO were transferred from tomato leaves to H. armigera, with particulate trophic transfer factors of 1.47 and 0.99, respectively. While nCuO exposure reduced larval weight gain more (34.7%) than nLa2O3 (11.3%), owing to higher oxidative stress (e.g., MDA and H2O2) and more serious intestinal pathological damage (i.e., crumpled columnar cell and disintegrated goblet cell) by nCuO. Moreover, nCuO exposure led to a more compact antagonism between the phyllosphere and gut microbiomes compared to nLa2O3. Specifically, nCuO exposure resulted in a greater increase in pathogenic bacteria (e.g., Mycobacterium, Bacillus, and Ralstonia) and a more significant decrease in probiotics (e.g., Streptomyces and Arthrobacter) than nLa2O3, ultimately destroying larval intestinal immunity. Altogether, our findings systematically revealed the cascade effect of metal oxide nanomaterials on higher trophic consumers through alteration in the phyllosphere and insect gut microbiome interaction, thus providing insights into nanotoxicity and environmental risk assessment of nanomaterials applied in agroecosystems.

The effects of irrigation on the survival of Clostridium sporogenes in the phyllosphere and soil environments of lettuce.

This study aimed to address the gap in knowledge regarding the fate of foodborne pathogens within agro-ecosystems. It specifically focused on the surrogate microorganism Clostridium sporogenes, which was introduced into lettuce-producing environments via surface and spray irrigation methods, respectively. The concentration of C. sporogenes in the rhizosphere, phyllosphere, and non-rhizosphere soil was quantified by quantitative polymerase chain reaction (qPCR) over a 42-day trial. The surface irrigation method exhibited a more noticeable contamination effect on the soil environments, compared to the phyllosphere. The results indicated a noticeable increase in C. sporogenes concentrations during the initial 22 days, with a 10.4-fold rise (0.39-4.05 log copy numbers/g soil) in the rhizosphere and 1.9-fold increase (2.97-5.59 log copy numbers/g soil) in the non-rhizosphere. However, concentrations in both soil environments subsequently decreased, falling below the initial inoculum concentration by the end of the trial. In contrast, the spray irrigation method resulted in most of the contamination being localised on the lettuce phyllosphere, with a high C. sporogenes concentration of 9.09 log copy numbers/g leaves on day 0. This concentration exponentially decreased to a minimal 0.019 log copy numbers/g leaves by day 32. Although concentrations in both soil environments decreased over time, trace concentrations of C. sporogenes were detectable at the end of the trial, posing a potential hazard to the microbiological safety of postharvest produce. These findings shed light on the dynamics of C. sporogenes in agro-ecosystems and underscore the importance of irrigation practices that ensure the safety of those who consume fresh produce. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-04069-5.

Temporal Dynamics of Copper-Based Nanopesticide Transfer and Subsequent Modulation of the Interplay Between Host and Microbiota Across Trophic Levels.

During agricultural production, significant quantities of copper-based nanopesticides (CBNPs) may be released into terrestrial ecosystems through foliar spraying, thereby posing a potential risk of biological transmission via food chains. Consequently, we investigated the trophic transfer of two commonly available commercial CBNPs, Reap2000 (RP) and HolyCu (HC), in a plant-caterpillar terrestrial food chain and evaluated impacts on host microbiota. Upon foliar exposure (with 4 rounds of spraying, totaling 6.0 mg CBNPs per plant), leaf Cu accumulation levels were 726 ± 180 and 571 ± 121 mg kg-1 for RP and HC, respectively. HC exhibited less penetration through the cuticle compared to RP (RP: 55.5%; HC: 32.8%), possibly due to size exclusion limitations. While caterpillars accumulated higher amounts of RP, HC exhibited a slightly higher trophic transfer factor (TTF; RP: 0.69 ± 0.20; HC: 0.74 ± 0.17, p > 0.05) and was more likely to be transferred through the food chain. The application of RP promoted the dispersal of phyllosphere microbes and perturbed the original host intestinal microbiota, whereas the HC group was largely host-modulated (control: 65%; RP: 94%; HC: 34%). Integrating multiomics analyses and modeling approaches, we elucidated two pathways by which plants exert bottom-up control over caterpillar health. Beyond the direct transmission of phyllosphere microbes, the leaf microbiome recruited upon exposure to CBNPs further influenced the ingestion behavior and intestinal microbiota of caterpillars via altered leaf metabolites. Elevated Proteobacteria abundance benefited caterpillar growth with RP, while the reduction of Proteobacteria with HC increased the risk of lipid metabolism issues and gut disease. The recruited Bacteroidota in the RP phyllosphere proliferated more extensively into the caterpillar gut to enhance stress resistance. Overall, the gut microbes reshaped in RP caterpillars exerted a strong regulatory effect on host health. These findings expand our understanding of the dynamic transmission of host-microbiota interactions with foliar CBNPs exposure, and provide critical insight necessary to ensure the safety and sustainability of nanoenabled agricultural strategies.

Leaf age structures phyllosphere microbial communities in the field and greenhouse.

The structure of the leaf microbiome can alter host fitness and change in response to abiotic and biotic factors, like seasonality, climate, and leaf age. However, relatively few studies consider the influence of host age on microbial communities at a time scale of a few days, a short time scale relevant to microbes. To understand how host age modulates changes in the fungal and bacterial leaf microbiome on a short time scale, we ran independent field and greenhouse-based studies and characterized phyllosphere communities using next-generation sequencing approaches. Our field study characterized changes in the fungal and bacterial phyllosphere by examining leaves of different relative ages across individuals, whereas the greenhouse study examined changes in the fungal microbiome by absolute leaf age across individuals. Together, these results indicate that fungal communities are susceptible to change as a leaf ages as evidenced by shifts in the diversity of fungal taxa both in the field and the greenhouse. Similarly, there were increases in the diversity of fungal taxa by leaf age in the greenhouse. In bacterial communities in the field, we observed changes in the diversity, composition, and relative abundance of common taxa. These findings build upon previous literature characterizing host-associated communities at longer time scales and provide a foundation for targeted work examining how specific microbial taxa might interact with each other, such as fine-scale interactions between pathogenic and non-pathogenic species.

Life on a leaf: the epiphyte to pathogen continuum and interplay in the phyllosphere.

Epiphytic microbes are those that live for some or all of their life cycle on the surface of plant leaves. Leaf surfaces are a topologically complex, physicochemically heterogeneous habitat that is home to extensive, mixed communities of resident and transient inhabitants from all three domains of life. In this review, we discuss the origins of leaf surface microbes and how different biotic and abiotic factors shape their communities. We discuss the leaf surface as a habitat and microbial adaptations which allow some species to thrive there, with particular emphasis on microbes that occupy the continuum between epiphytic specialists and phytopathogens, groups which have considerable overlap in terms of adapting to the leaf surface and between which a single virulence determinant can move a microbial strain. Finally, we discuss the recent findings that the wheat pathogenic fungus Zymoseptoria tritici spends a considerable amount of time on the leaf surface, and ask what insights other epiphytic organisms might provide into this pathogen, as well as how Z. tritici might serve as a model system for investigating plant-microbe-microbe interactions on the leaf surface.

Impacts of eutrophication on microbial community structure in sediment, seawater, and phyllosphere of seagrass ecosystems.

Introduction: Seagrass-associated microbial communities play a crucial role in the growth and health of seagrasses. However, like seagrass meadows, seagrass-associated microbial communities are often affected by eutrophication. It remains unclear how eutrophication influences the composition and function of microbial communities associated with different parts of seagrass. Methods: We employed prokaryotic 16S rRNA gene high-throughput sequencing combining microbial community structure analysis and co-occurrence network analysis to investigate variances in microbial community compositions, potential functions and complexities across sediment, seagrass leaves, and seawater within different eutrophic areas of two adjacent seagrass meadows on Hainan Island, China. Results: Our results indicated that microbial diversity on seagrass leaves was significantly lower than in sediment but significantly higher than in seawater. Both sediment and phyllosphere microbial diversity showed no significant difference between the highly eutrophic and less eutrophic sites in each lagoon. However, sediment microbial diversity was higher in the more eutrophic lagoon, while phyllosphere microbial diversity was higher in the less eutrophic lagoon. Heavy eutrophication increased the relative abundance of phyllosphere microorganisms potentially involved in anaerobic metabolic processes, while reducing those responsible for beneficial functions like denitrification. The main factor affecting microbial diversity was organic carbon in seawater and sediment, with high organic carbon levels leading to decreased microbial diversity. The co-occurrence network analysis revealed that heavy eutrophication notably reduced the complexity and internal connections of the phyllosphere microbial community in comparison to the sediment and seawater microbial communities. Furthermore, ternary analysis demonstrated that heavy eutrophication diminished the external connections of the phyllosphere microbial community with the sediment and seawater microbial communities. Conclusion: The pronounced decrease in biodiversity and complexity of the phyllosphere microbial community under eutrophic conditions can lead to greater microbial functional loss, exacerbating seagrass decline. This study emphasizes the significance of phyllosphere microbial communities compared to sediment microbial communities in the conservation and restoration of seagrass meadows under eutrophic conditions.

Oak seedling microbiome assembly under climate warming and drought.

Despite that climate change is currently one of the most pervasive challenges, its effects on the plant-associated microbiome is still poorly studied. The aim of this study was to evaluate the impact of the independent and combinatory effect of climate warming and drought on the microbiome assembly of oak from seed to seedling. In a multifactorial experimental set up, acorns were subjected to different temperatures (15 °C, 20 °C, and 25 °C) and soil moisture levels (drought (15%) and control (60%)) from germination until the seedling stage, after which the bacterial and fungal communities associated to the rhizosphere and phyllosphere were characterized by amplicon sequencing and qPCR. The results showed a stronger effect of temperature on fungal than on bacterial diversity and the effect was more pronounced in the phyllosphere. Under drought condition, temperature had a significantly negative effect on phyllosphere fungal diversity. In the rhizosphere, temperature had a significant effect on the fungal community composition which was primarily caused by species turnover. Regardless of temperature, Actinobacteriota was significantly enriched in drought, a group of bacteria known to increase plant drought tolerance. This study provides new insights into the effect of climate change on the plant microbiome in natural ecosystems.

Diversity, functions, and antibiotic resistance genes of bacteria and fungi are examined in the bamboo plant phyllosphere that serve as food for the giant pandas.

The phyllosphere of bamboo is rich in microorganisms that can disrupt the intestinal microbiota of the giant pandas that consume them, potentially leading to their death. In the present study, the abundance, diversity, biological functions (e.g., KEGG and CAZyme), and antibiotic resistance genes (ARGs) of bacteria and fungi in two bamboo species phyllosphere (Chimonobambusa szechuanensis, CS; Bashania fangiana, BF) in Daxiangling Nature Reserve (an important part of the Giant Panda National Park) were investigated respectively by amplicon sequencing of the whole 16S rRNA and ITS1-ITS2 genes on PacBio Sequel and whole-metagenome shotgun sequencing on Illumina NovaSeq 6000 platform. The results suggested that there were respectively 18 bacterial and 34 fungi biomarkers between the phyllosphere of the two species of bamboo. Beta diversity of bacteria and fungi communities exited between the two bamboos according to the (un)weighted UniFrac distance matrix. Moreover, the functional analysis showed that the largest relative abundance was found in the genes related to metabolism and global and overview maps. Glycoside hydrolases (GHs) and glycosyl transferases (GTs) have a higher abundance in two bamboo phyllospheres. Co-occurrence network modeling suggested that bacteria and fungi communities in CS phyllosphere employed a much more complex metabolic network than that in BF, and the abundance of multidrug, tetracycline, and glycopeptide resistance genes was higher and closely correlated with other ARGs. This study references the basis for protecting bamboo resources foraged by wild giant pandas and predicts the risk of antibiotic resistance in bamboo phyllosphere bacterial and fungal microbiota in the Giant Panda National Park, China.

Multi-omics reveals the phyllosphere microbial community and material transformations in cigars.

The quality of fermented plant leaves is closely related to the interleaf microorganisms and their metabolic activities. In this experiment, a multi-omics analysis was applied to investigate the link between the structural composition of the phyllosphere microbial community and the main metabolites during the fermentation process. It was found that the whole fermentation process of cigar leaves could be divided into three stages, in which the Mid-Stage was the most active period of microbial metabolic activities and occupied an important position. Staphylococcus, Brevundimonas, Acinetobacter, Brevibacterium, Pantoea, Aspergillus, Wallemia, Meyerozyma, Sampaiozyma, Adosporium and Trichomonascus played important roles in this fermentation. Staphylococcus and Aspergillus are the microorganisms that play an important role in the fermentation process. Staphylococcus were strongly correlated with lipids and amino acids, despite its low abundance, Stenotrophomonas is importantly associated with terpene and plays a significant role throughout the process. It is worth noting that Wapper exists more characteristic fungal genera than Filler and is more rapid in fermentation progress, which implies that the details of the fermentation process should be adjusted appropriately to ensure stable quality when faced with plant leaves of different genotypes. This experiment explored the relationship between metabolites and microorganisms, and provided a theoretical basis for further optimizing the fermentation process of plant leaves and developing techniques to improve product quality. Biomarker is mostly present in the pre-fermentation phase, but the mid-fermentation phase is the most important part of the process.