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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., Competing Interests: The authors declare no conflict of interest.

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., (© 2024. The Author(s).)

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 10 8 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., (© 2024. The Author(s).)

The phyllosphere, a reservoir of diverse microbial life associated with plant health, harbors microbial communities that are subject to various complex ecological processes acting at multiple scales. In this study, we investigated the determinants of the spatiotemporal variation in bacterial and fungal communities within the apple tree phyllosphere, employing 16S and ITS amplicon sequencing. Our research assessed the impact of key factors-plant compartment, site, time, and cultivar-on the composition and diversity of leaf and flower microbial communities. Our analyses, based on samples collected from three cultivars in three orchards in 2022, revealed that site and time are the strongest drivers of apple tree phyllosphere microbial communities. Conversely, plant compartment and cultivar exhibited minor roles in explaining community composition and diversity. Predominantly, bacterial communities comprised Hymenobacter (25%) and Sphingomonas (10%), while the most relatively abundant fungal genera included Aureobasidium (27%) and Sporobolomyces (10%). Additionally, our results show a gradual decrease in alpha-diversity throughout the growth season. These findings emphasize the necessity to consider local microbial ecology dynamics in orchards, especially as many groups worldwide aim for the development of biocontrol strategies (e.g., by manipulating plant-microbe interactions). More research is needed to improve our understanding of the determinants of time and site-specific disparities within apple tree phyllosphere microbial communities across multiple years, locations, and cultivars., Competing Interests: The authors declare there are no competing interests.

The epiphytic and endophytic bacteria play an important role in the healthy growth of plants. Both plant species and growth environmental influence the bacterial population diversity, yet it is inconclusive whether it is the former or the latter that has a greater impact. To explore the communities of the epiphytic and endophytic microbes in Camellia oleifera, this study assessed three representative C. oleifera cultivars from three areas in Hunan, China by Illumina high-throughput sequencing. The results showed that the diversity and species richness of endophytic microbial community in leaves were significantly higher than those of microbial community in the epiphytic. The diversity and species richness of epiphytic and endophytic microbes are complex when the same cultivar was grown in different areas. The C. oleifera cultivars grown in Youxian had the highest diversity of epiphytic microbial community, but the lowest abundance, while the cultivars grown in Changsha had the highest diversity and species richness of endophytic microbes in leaves. It was concluded that the dominant phylum mainly included Proteobacteria, Actinobacteriota and Firmicutes through the analysis of the epiphytic and endophytic microbial communities of C. oleifera. The species and relative abundances of epiphytic and endophytic microbial community were extremely different at the genus level. The analysis of NMDS map and PERMANOVA shows that the species richness and diversity of microbial communities in epiphytes are greatly influenced by region. However, the community structure of endophytic microorganisms in leaves is influenced by region and cultivated varieties, but the influence of cultivars is more significant. Molecular ecological network analysis showed that the symbiotic interaction of epiphytic microbial community was more complex., (© 2024. The Author(s).)

Phyllosphere microbial communities are increasingly experiencing intense pulse disturbance events such as drought. It is currently unknown how phyllosphere communities respond to such disturbances and if they are able to recover. We explored the stability of phyllosphere communities over time, in response to drought stress, and under recovery from drought on temperate forage grasses. Compositional or functional changes were observed during the disturbance period and whether communities returned to non-stressed levels following recovery. Here, we found that phyllosphere community composition shifts as a result of simulated drought but does not fully recover after irrigation is resumed and that the degree of community response to drought is host species dependent. However, while community composition had changed, we found a high level of functional stability (resistance) over time and in the water deficit treatment. Ecological modeling enabled us to understand community assembly processes over a growing season and to determine if they were disrupted during a disturbance event. Phyllosphere community succession was characterized by a strong level of ecological drift, but drought disturbance resulted in variable selection, or, in other words, communities were diverging due to differences in selective pressures. This successional divergence of communities with drought was unique for each host species. Understanding phyllosphere responses to environmental stresses is important as climate change-induced stresses are expected to reduce crop productivity and phyllosphere functioning., Importance: Leaf surface microbiomes have the potential to influence agricultural and ecosystem productivity. We assessed their stability by determining composition, functional resistance, and resilience. Resistance is the degree to which communities remain unchanged as a result of disturbance, and resilience is the ability of a community to recover to pre-disturbance conditions. By understanding the mechanisms of community assembly and how they relate to the resistance and resilience of microbial communities under common environmental stresses such as drought, we can better understand how communities will adapt to a changing environment and how we can promote healthy agricultural microbiomes. In this study, phyllosphere compositional stability was highly related to plant host species phylogeny and, to a lesser extent, known stress tolerances. Phyllosphere community assembly and stability are a result of complex interactions of ecological processes that are differentially imposed by host species., Competing Interests: The authors declare no conflict of interest.

Leaves harbor distinct microbial communities that can have an important impact on plant health and microbial ecosystems worldwide. Nevertheless, the ecological processes that shape the composition of leaf microbial communities remain unclear, with previous studies reporting contradictory results regarding the importance of bacterial dispersal versus host selection. This discrepancy could be driven in part because leaf microbiome studies typically consider the upper and lower leaf surfaces as a single entity despite these habitats possessing considerable anatomical differences. We characterized the composition of bacterial phyllosphere communities from the upper and lower leaf surfaces across 24 plant species. Leaf surface pH and stomatal density were found to shape phyllosphere community composition, and the underside of leaves had lower richness and higher abundances of core community members than upper leaf surfaces. We found fewer endemic bacteria on the upper leaf surfaces, suggesting that dispersal is more important in shaping these communities, with host selection being a more important force in microbiome assembly on lower leaf surfaces. Our study illustrates how changing the scale in which we observe microbial communities can impact our ability to resolve and predict microbial community assembly patterns on leaf surfaces. IMPORTANCE Leaves can harbor hundreds of different bacterial species that form unique communities for every plant species. Bacterial communities on leaves are really important because they can, for example, protect their host against plant diseases. Usually, bacteria from the whole leaf are considered when trying to understand these communities; however, this study shows that the upper and lower sides of a leaf have a very different impact on how these communities are shaped. It seems that the bacteria on the lower leaf side are more closely associated with the plant host, and communities on the upper leaf side are more impacted by immigrating bacteria. This can be really important when we want to treat, for example, crops in the field with beneficial bacteria or when trying to understand host-microbe interactions on the leaves., Competing Interests: The authors declare no conflict of interest.

Analysis of microbial communities in the epiphytic phyllosphere can be challenging, especially when applying sequencing-based techniques, owing to the interference of plant-derived biomolecules such as nucleic acids. A review of recent studies on the epiphytic microbiome revealed that both mechanical and enzymatic lysis methods are widely used. Here, we evaluated the effects of the two lysis methods on DNA extraction yield, purity, integrity, and microbial 16S rRNA gene copy number per ng of template genomic DNA under different extraction conditions. Furthermore, the effect on bacterial community composition, diversity, and reproducibility was examined using 16S rRNA gene amplicon sequencing. The enzymatic lysis method yielded one to two orders of magnitude more DNA, but the DNA quality was suboptimal. Conversely, the samples prepared using the mechanical method showed high DNA purity albeit lower yield. Unexpectedly, mechanical lysis showed a higher DNA integrity number (DIN) than enzymatic lysis. The 16S rRNA amplicon sequencing results demonstrated that the samples prepared via mechanical disruption exhibited reproducibly similar microbial community compositions regardless of the extraction conditions. In contrast, the enzymatic lysis method resulted in inconsistent taxonomic compositions under different extraction conditions. This study demonstrates that mechanical DNA disruption is more suitable for epiphytic phyllosphere samples than enzymatic disruption., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Determining whether and how global change will lead to novel interactions between hosts and microbes is an important issue in ecology and evolution. Understanding the contribution of host and microbial ecologies and evolutionary histories in driving their contemporary associations is an important step towards addressing this challenge and predicting the fitness consequences of novel associations. Using shotgun metagenomic and amplicon sequencing of bacterial communities from the leaf surfaces (phyllosphere) of trees, we investigated how phylogenetic relatedness among hosts and among their associated bacteria influences the distribution of bacteria among hosts. We also evaluated whether the functional traits of trees and bacteria explained these associations across multiple host species. We show that phylogenetically similar hosts tended to associate with the same bacteria and that phylogenetically similar bacteria tended to associate with the same host species. Phylogenetic interactions between tree and bacterial taxa also explained variation in their associations. The effect of host and symbiont evolutionary histories on bacterial distribution across hosts were observed across phylogenetic scales, but prominently explained variation among higher taxonomic categories of hosts and symbionts. These results suggest that ecological variation arising early in the plant and bacterial phylogenies have been particularly important for driving their contemporary associations. Variation in bacterial functional genes associated with the biosynthesis of aromatic amino acids and compounds and with cell motility were notably important in explaining bacterial community turnover among gymnosperm and angiosperm hosts. Overall, our results suggest an influence of host and bacterial traits and evolutionary histories in driving their contemporary associations., (© 2021 John Wiley & Sons Ltd.)

Grasslands represent a critical ecosystem important for global food production, soil carbon storage, and water regulation. Current intensification and expansion practices add to the degradation of grasslands and dramatically increase greenhouse gas emissions and pollution. Thus, new ways to sustain and improve their productivity are needed. Research efforts focus on the plant-leaf microbiome, or phyllosphere, because its microbial members impact ecosystem function by influencing pathogen resistance, plant hormone production, and nutrient availability through processes including nitrogen fixation. However, little is known about grassland phyllospheres and their response to environmental stress. In this study, globally dominant temperate and tropical forage grass species were grown in a greenhouse under current climate conditions and drought conditions that mimic future climate predictions to understand if (i) plant host taxa influence microbial community assembly, (ii) microbial communities respond to drought stress, and (iii) phyllosphere community changes correlate to changes in plant host traits and stress-response strategies. Community analysis using high-resolution sequencing revealed Gammaproteobacteria as the dominant bacterial class, which increased under severe drought stress on both temperate and tropical grasses while overall bacterial community diversity declined. Bacterial community diversity, structure, and response to drought were significantly different between grass species. This community dependence on plant host species correlated with differences in grass species traits, which became more defined under drought stress conditions, suggesting symbiotic evolutionary relationships between plant hosts and their associated microbial community. Further understanding these strategies and the functions microbes provide to plants will help us utilize microbes to promote agricultural and ecosystem productivity in the future. IMPORTANCE Globally important grassland ecosystems are at risk of degradation due to poor management practices compounded by predicted increases in severity and duration of drought over the next century. Finding new ways to support grassland productivity is critical to maintaining their ecological and agricultural benefits. Discerning how grassland microbial communities change in response to climate stress will help us understand how plant-microbe relationships may be useful to sustainably support grasslands in the future. In this study, phyllosphere community diversity and composition were significantly altered under drought conditions. The significance of our research is demonstrating how severe climate stress reduces bacterial community diversity, which previously was directly associated with decreased plant productivity. These findings guide future questions about functional plant-microbe interactions under stress conditions, greatly enhancing our understanding of how bacteria can increase food security by promoting grassland growth and resilience.

Recent work shows that the plant microbiome, particularly the initial assembly of this microbiome, influences plant health, survival, and fitness. Here, we characterize the initial assembly of the Populus microbiome across ten genotypes belonging to two poplar species in a common garden using 16S rRNA gene and ITS2 region amplicon sequencing of the leaf endosphere, leaf surface, root endosphere, and rhizosphere. We sampled these microbiomes three times throughout the first growing season and found that the composition of the microbiome changed dramatically over time across all plant-associated habitats and host genotypes. For archaea and bacteria, these changes were dominated by strong homogenizing selection (accounting for 29 to 62% of pairwise comparisons). However, fungal assembly was generally characterized by multiple ecological assembly processes (i.e., a mix of weak selective and dispersal processes). Interestingly, genotype, while a significant moderator of microbiome composition, generally explained less variation than sample date across plant-associated habitats. We defined a set of core genera that accounted for, on average, 36% of the microbiome. The relative abundance of this core community was consistent over time. Additionally, using source tracking modeling, we determined that new microbial taxa colonize from both aboveground and belowground sources, and combined with our ecological assembly null models, we found that both selective and dispersal processes explained the differences between exo- (i.e., leaf surface and rhizosphere) and endospheric microbiomes. Taken together, our results suggest that the initial assembly of the Populus microbiome is time-, genotype-, and habitat-dependent and is moderated by both selective and stochastic factors. IMPORTANCE The initial assembly of the plant microbiome may establish the trajectory of forthcoming microbiome states, which could determine the overall future health of the plant. However, while much is known about the initial microbiome assembly of grasses and agricultural crops, less is known about the initial microbiome of long-lived trees, such as poplar ( Populus spp.). Thus, a greater understanding of initial plant microbiome assembly in an ecologically and economically important plant such as Populus is highly desirable. Here, we show that the initial microbiome community composition and assembly in the first growing season of Populus is temporally dynamic and is determined by a combination of both selective and stochastic factors. Our findings could be used to prescribe ecologically informed microbial inoculations and better predict the composition of the Populus microbiome into the future and to better understand its influence on plant health.

Production of leafy vegetables for the "Ready-to-eat"-market has vastly increased the last 20 years, and consumption of these minimally processed vegetables has led to outbreaks of food-borne diseases. Contamination of leafy vegetables can occur throughout the production chain, and therefore washing of the produce has become a standard in commercial processing. This study explores the bacterial communities of spinach (Spinacia oleracea) and rocket (Diplotaxis tenuifolia) in a commercial setting in order to identify potential contamination events, and to investigate effects on bacterial load by commercial processing. Samples were taken in field, after washing of the produce and at the end of shelf-life. This study found that the bacterial community composition and diversity changed significantly from the first harvest to the end of shelf-life, where the core microbiome from the first to the last sampling constituted <2% of all OTUs. While washing of the produce had no reducing effect on bacterial load compared to unwashed, washing led to a change in species composition. As the leaves entered the cold chain after harvest, a rise was seen in the relative abundance of spoilage bacteria. E. coli was detected after the washing indicating issues of cross-contamination in the wash water., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Phyllosphere microorganisms are sensitive to fluctuations in wind, temperature, solar radiation, and rain. However, recent explorations of patterns in phyllosphere communities across time often focus on seasonal shifts and leaf senescence without measuring the contribution of environmental drivers and leaf traits. Here, we focus on the effects of rain on the phyllosphere bacterial community of the wetland macrophyte broadleaf cattail (Typha latifolia) across an entire year, specifically targeting days before and 1, 3, and 5 days after rain events. To isolate the contribution of precipitation from other factors, we covered a subset of plants to shield them from rainfall. We used targeted Illumina sequencing of the V4 region of the bacterial 16S rRNA gene to characterize phyllosphere community composition. Rain events did not have a detectable effect on phyllosphere community richness or evenness regardless of whether the leaves were covered from rain or not, suggesting that foliar microbial communities are robust to such disturbances. While climatic and leaf-based variables effectively modeled seasonal trends in phyllosphere diversity and composition, they provided more limited explanatory value at shorter time scales. These findings underscore the dominance of long-term seasonal patterns related to climatic variation as the main factor influencing the phyllosphere community.

High-resolution, spatial characterization of microbial communities is critical for the accurate understanding of microbe-microbe and microbe-plant interactions in leaf surfaces (phyllosphere). However, leaves are specially challenging surfaces for imaging methods due to their high autofluorescence. In this chapter we describe the Leaf-FISH method. Leaf-FISH is a fluorescence in situ hybridization (FISH) method specially adapted to the requirements of plant tissues. Leaf-FISH uses a combination of leaf pretreatments coupled with spectral imaging confocal microscopy and image post-processing to visualize bacterial taxa on a structural-informed context recreated from the residual background autofluorescence of the tissues. Leaf-FISH is suitable for simultaneous identification of multiple bacterial taxa using multiple taxon-specific fluorescently labeled oligonucleotide probes (combinatorial labeling).

Phyllosphere and rhizosphere are unique and wide-ranging habitats that harbor various microbial communities, which influence plant growth and health, and the productivity of the ecosystems. In this study, we characterized the shared microbiome of the phyllosphere and rhizosphere among three plants (Ipomoea pes-caprae, Wedelia chinensis, and Cocos nucifera), to obtain an insight into the relationships between bacteria (including diazotrophic bacteria) and fungi, present on these host plants. Quantitative PCR showed that the abundances of the microbiome in the soil samples were significantly higher than those in the phyllosphere samples, though there was an extremely low abundance of fungi in bulk soil. High-throughput sequencing showed that the alpha-diversity of bacteria and fungi was higher in the rhizosphere than the phyllosphere samples associated with the same plant, while there was no obvious shift in the alpha-diversity of diazotrophic communities between all the tested phyllosphere and soil samples. Results of the microbial composition showed that sample-specific bacteria and fungi were found among the phyllosphere and rhizosphere of the different host plants. About 10%-27% of bacteria, including diazotrophs, and fungi overlapped between the phyllosphere and the rhizosphere of these host plants. No significant difference in microbial community structure was found among the tested rhizosphere samples, and soil properties had a higher influence on the soil microbial community structures than the host plant species., (© 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Microbial assemblages such as biofilms around aquatic plants play a major role in arsenic (As) cycling, which has often been overlooked in previous studies. In this study, arsenite (As(III))-oxidizing, arsenate (As(V))-reducing and As(III)-methylating bacteria were found to coexist in the phyllosphere of Hydrilla verticillata, and their relative activities were shown to determine As speciation, accumulation and efflux. When exposed to As(III), As(III) oxidation was not observed in treatment H(III)-B, whereas treatment H(III)+B showed a significant As(III) oxidation ability, thereby indicating that epiphytic bacteria displayed a substantial As(III) oxidation ability. When exposed to As(V), the medium only contained 5.89% As(III) after 48 h of treatment H(V)-B, while an As(III) content of 86.72% was observed after treatment H(V)+B, thereby indicating that the elevated As(III) in the medium probably originated from As(V) reduction by epiphytic bacteria. Our data also indicated that oxidizing bacteria decreased the As accumulation (by approximately 64.44% compared with that of treatment H(III)-B) in plants, while reducing bacteria played a critical role in increasing As accumulation (by approximately 3.31-fold compared with that of treatment H(V)-B) in plants. Regardless of whether As(III) or As(V) was supplied, As(III) was dominant in the plant tissue (over 75%). Furthermore, the presence of epiphytic bacteria enhanced As efflux by approximately 9-fold. Metagenomic analysis revealed highly diverse As metabolism genes in epiphytic bacterial community, particularly those related to energetic metabolism (aioAB), and As resistance (arsABCR, acr3, arsM). Phylogenetic analysis of As metabolism genes revealed evidence of both vertical inheritance and horizontal gene transfer, which might have contributed to the evolution of the As metabolism genes. Taken together, our research suggested that the diversity of As metabolism genes in epiphytic bacterial community is associated with aquatic submerged macrophytes which may play an important role in As biogeochemistry in aquatic environments., Competing Interests: Declaration of competing interest None declared., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: The phyllosphere is an important microbial habitat, but our understanding of how plant hosts drive the composition of their associated leaf microbial communities and whether taxonomic associations between plants and phyllosphere microbes represent adaptive matching remains limited. In this study, we quantify bacterial functional diversity in the phyllosphere of 17 tree species in a diverse neotropical forest using metagenomic shotgun sequencing. We ask how hosts drive the functional composition of phyllosphere communities and their turnover across tree species, using host functional traits and phylogeny., Results: Neotropical tree phyllosphere communities are dominated by functions related to the metabolism of carbohydrates, amino acids, and energy acquisition, along with environmental signalling pathways involved in membrane transport. While most functional variation was observed within communities, there is non-random assembly of microbial functions across host species possessing different leaf traits. Metabolic functions related to biosynthesis and degradation of secondary compounds, along with signal transduction and cell-cell adhesion, were particularly important in driving the match between microbial functions and host traits. These microbial functions were also evolutionarily conserved across the host phylogeny., Conclusions: Functional profiling based on metagenomic shotgun sequencing offers evidence for the presence of a core functional microbiota across phyllosphere communities of neotropical trees. While functional turnover across phyllosphere communities is relatively small, the association between microbial functions and leaf trait gradients among host species supports a significant role for plant hosts as selective filters on phyllosphere community assembly. This interpretation is supported by the presence of phylogenetic signal for the microbial traits driving inter-community variation across the host phylogeny. Taken together, our results suggest that there is adaptive matching between phyllosphere microbes and their plant hosts. Video abstract.

Although the use of crop-associated bacteria as biological control agents of fungal diseases has gained increasing interest, the biotechnological potential of forest tree-associated microbes and their natural products has scarcely been investigated. The objective of this study was to identify bacteria or bacterial products with antagonistic activity against Fusarium solani and Fusarium kuroshium, causal agent of Fusarium dieback, by screening the rhizosphere and phyllosphere of three Lauraceae species. From 195 bacterial isolates, we identified 32 isolates that significantly reduced the growth of F. solani in vitro, which mostly belonged to bacterial taxa Bacillus, Pseudomonas and Actinobacteria. The antifungal activity of their volatile organic compounds (VOCs) was also evaluated. Bacterial strain Bacillus sp. CCeRi1-002, recovered from the rhizosphere of Aiouea effusa, showed the highest percentage of direct inhibition (62.5 %) of F. solani and produced diffusible compounds that significantly reduced its mycelial growth. HPLC-MS analyses on this strain allowed to tentatively identify bioactive compounds from three lipopeptide groups (iturin, surfactin and fengycin). Bacillus sp. CCeRi1-002 and another strain identified as Pseudomonas sp. significantly inhibited F. solani mycelial growth through the emission of VOCs. Chemical analysis of their volatile profiles indicated the likely presence of 2-nonanone, 2-undecanone, disulfide dimethyl and 1-butanol 3-methyl-, which had been previously reported with antifungal activity. In antagonism assays against F. kuroshium, Bacillus sp. CCeRi1-002 and its diffusible compounds exhibited significant antifungal activity and induced hyphal deformations. Our findings highlight the importance of considering bacteria associated with forest species and the need to include bacterial products in the search for potential antagonists of Fusarium dieback., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Plants are associated with hundreds of thousands of microbes that are present outside on the surfaces or colonizing inside plant organs, such as leaves and roots. Plant-associated microbiota plays a vital role in regulating various biological processes and affects a wide range of traits involved in plant growth and development, as well as plant responses to adverse environmental conditions. An increasing number of studies have illustrated the important role of microbiota in crop plant growth and environmental stress resistance, which overall assists agricultural sustainability. Beneficial bacteria and fungi have been isolated and applied, which show potential applications in the improvement of agricultural technologies, as well as plant growth promotion and stress resistance, which all lead to enhanced crop yields. The symbioses of arbuscular mycorrhizal fungi, rhizobia and Frankia species with their host plants have been intensively studied to provide mechanistic insights into the mutual beneficial relationship of plant-microbe interactions. With the advances in second generation sequencing and omic technologies, a number of important mechanisms underlying plant-microbe interactions have been unraveled. However, the associations of microbes with their host plants are more complicated than expected, and many questions remain without proper answers. These include the influence of microbiota on the allelochemical effect caused by one plant upon another via the production of chemical compounds, or how the monoculture of crops influences their rhizosphere microbial community and diversity, which in turn affects the crop growth and responses to environmental stresses. In this review, first, we systematically illustrate the impacts of beneficial microbiota, particularly beneficial bacteria and fungi on crop plant growth and development and, then, discuss the correlations between the beneficial microbiota and their host plants. Finally, we provide some perspectives for future studies on plant-microbe interactions.

Plants and phyllosphere microorganisms may effectively contribute to reducing air pollution in cities through the adsorption and biodegradation of pollutants onto leaves. In this work, during all seasons, we sampled atmospheric particulate matter (PM 10 ) and leaves of southern magnolia Magnolia grandiflora and deodar cedar Cedrus deodara, two evergreen plant species widespread in the urban area of Milan where the study was carried out. We then quantified Polycyclic Aromatic Hydrocarbons (PAHs) both in PM 10 and on leaves and used sequencing of 16S rRNA gene, shotgun metagenomics and qPCR analyses to investigate the microbial communities hosted by the sampled leaves. Taxonomic and functional profiles of epiphytic bacterial communities differed between host plant species and seasons and the microbial communities on leaves harboured genes involved in the degradation of hydrocarbons. Evidence collected in this work also suggested that the abundance of hydrocarbon-degrading microorganisms on evergreen leaves increased with the concentration of hydrocarbons when atmospheric pollutants were deposited at high concentration on leaves, and that the biodegradation on the phyllosphere can contribute to the removal of PAHs from the urban air., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Growing evidence suggests that livestock manure used as organic fertilizer in agriculture may lead to the potential propagation of antibiotic resistance genes (ARGs) from "farm to fork." However, little is known about the impacts of manure fertilization on the incidence of ARGs in the plant-associated microbiomes (including rhizosphere, endosphere, and phyllosphere), which hampers our ability to assess the dissemination of antibiotic resistance in the soil-plant system. Here, we constructed a pot experiment to explore the effects of poultry and cattle manure applications on the shifts in the resistome in the plant microbiome of harvested cherry radish. A total of 144 ARGs conferring resistance to eight major classes of antibiotics were detected among all the samples. Rhizosphere and phyllosphere microbiomes harbored significantly higher diversity and abundance of ARGs than did root endophytic microbiomes of cherry radish. Manure application significantly increased the abundance of ARGs in the rhizosphere and phyllosphere but not in the endophytes of the root, which is the edible part of cherry radish. Soil and plant microbiomes changed dramatically after manure applications and clustered separately according to different sample types and treatments. Structural equation modeling revealed that bacterial abundance was the most important factor modulating the distribution patterns of soil and plant resistomes after accounting for multiple drivers. Taken together, we provide evidence that enrichment of the resistome in the rhizosphere and phyllosphere of cherry radish is more obvious than with the endosphere after manure application, suggesting that manure amendment might not enhance the dissemination of ARGs into the root of vegetables in the pot experiment. IMPORTANCE Our study provides important evidence that manure application increased the occurrence of ARGs in the rhizosphere and phyllosphere of cherry radish, compared with that in the endophytic bacterial microbiota of root, which is the edible part of cherry radish. Our findings suggest that although manure amendment is a significant route of ARGs entering agricultural soils, these manure-derived ARGs may be at low risk of migrating into the endophytes of root vegetables., (Copyright © 2020 American Society for Microbiology.)

Objective: The objectives of this research were to screen anti-quorum sensing activity of phyllosphere bacteria and quantify their antibiofilm activity against biofilm forming bacteria (Bacillus cereus, Staphylococcus aureus, Enterococcus faecalis, Salmonella typhimurium, Vibrio cholerae, Pseudomonas aeruginosa)., Results: We found 11 phyllosphere bacteria isolates with potential anti-quorum sensing activity. Most of the crude extracts from phyllosphere bacteria isolates had anti-quorum sensing activity against Chromobacterium violaceum at certain concentration (20 and 10 mg/mL), but not crude extract from isolate JB 7F. Crude extract showed the largest turbid zone (1,27 cm) using isolate JB 14B with concentration of 10 mg/mL and the narrowest turbid zone isolate (1 cm) using JB 18B with concentration of 10 mg/mL. Crude extracts showed various antibiofilm activities against all tested pathogenic bacteria, it showed the highest biofilm inhibition (90%) and destruction activities (76%) against S. aureus.

In China, the common use of antibiotics in agriculture is recognized as a potential public health risk through the increasing use of livestock derived manure as a means of fertilization. By doing so this may increase the transfer of antibiotic resistance genes (ARGs) from animals, to soils and plants. In this study two staple crops (rice and wheat) were investigated for ARG enrichment under differing fertilization regimes. Here, we applied 4 treatments, no fertilizer, mineral fertilizer, clean (reduced antibiotic practice) and dirty (current antibiotic practice) pig manure, to soil microcosms planted with either rice or wheat, to investigate fertilization effects on the abundance of ARGs in the respective phyllospheres. For both rice and wheat, samples were collected after two separate fertilization periods. In total, 162 unique ARGs and 5 mobile genetic elements (MGEs) were detected from all rice and wheat samples. The addition of both clean and dirty manure, enhanced ARG abundance significantly when compared to no fertilizer treatments (P < 0.001), though clean manure enriched ARGs to a lesser extent than dirty manure, in all rice and wheat samples (P < 0.001). The classes of ARGs recorded were different between crops, with wheat samples having a higher ARG diversity than rice. These results revealed that staple crops in China such as rice and wheat may be a reservoir for ARGs when clean and dirty pig manure is used for fertilization., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The increasing antimicrobial resistance in manure-amended soil can potentially enter food chain, representing an important vehicle for antibiotic resistance genes (ARGs) transmission into human microbiome. However, the pathways for transmission of ARGs from soil to plant remain unclear. Here, we explored the impacts of poultry and cattle manure application on the patterns of resistome in soil and lettuce microbiome including rhizosphere, root endosphere, leaf endosphere and phyllosphere, to identify the potential transmission routes of ARGs in the soil-plant system. After 90 days of cultivation, a total of 144 ARGs were detected in all samples using high-throughput quantitative PCR. Rhizosphere soil samples harbored the most diverse ARGs compared with other components of lettuce. Cattle manure application increased the abundance of ARGs in root endophyte, while poultry manure application increased ARGs in rhizosphere, root endophyte and phyllosphere, suggesting that poultry manure may have a stronger impact on lettuce resistomes. The ARG profiles were significantly correlated with the bacterial community, and the enrichment of soil and plant resistomes was strongly affected by the bacterial taxa including Solibacteres, Chloroflexi, Acidobacteria, Gemm-1 and Gemmatimonadetes, as revealed by the network analyses. Moreover, the overlaps of ARGs between lettuce tissues and soil were identified, which indicated that plant and environmental resistomes are interconnected. Our findings provide insights into the transmission routes of ARGs from manured soil to vegetables, and highlight the potential risks of plant resistome migration to the human food chain., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Describing baseline microbiota associated with agricultural commodities in the field is an important step towards improving our understanding of a wide range of important objectives from plant pathology and horticultural sustainability, to food safety. Environmental pressures on plants (wind, dust, drought, water, temperature) vary by geography and characterizing the impact of these variable pressures on phyllosphere microbiota will contribute to improved stewardship of fresh produce for both plant and human health. A higher resolution understanding of the incidence of human pathogens on food plants and co-occurring phytobiota using metagenomic approaches (metagenome tracking) may contribute to improved source attribution and risk assessment in cases where human pathogens become introduced to agro-ecologies. Between 1990 and 2007, as many as 1990 culture-confirmed Salmonella illnesses were linked to tomatoes from as many as 12 multistate outbreaks (Bell et al., 2012; Bell et al., 2015; Bennett et al., 2014; CDC, 2004; CDC, 2007; Greene et al., 2005a; Gruszynski et al., 2014). When possible, source attribution for these incidents revealed a biogeographic trend, most events were associated with eastern growing regions. To improve our understanding of potential biogeographically linked trends in contamination of tomatoes by Salmonella, we profiled microbiota from the surfaces of tomatoes from Virginia, Maryland, North Carolina and California. Bacterial profiles from California tomatoes were completely different than those of Maryland, Virginia and North Carolina (which were highly similar to each other). A statistically significant enrichment of Firmicutes taxa was observed in California phytobiota compared to the three eastern states. Rhizobiaceae, Sphingobacteriaceae and Xanthobacteraceae were the most abundant bacterial families associated with tomatoes grown in eastern states. These baseline metagenomic profiles of phyllosphere microbiota may contribute to improved understanding of how certain ecologies provide supportive resources for human pathogens on plants and how components of certain agro-ecologies may play a role in the introduction of human pathogens to plants., (Published by Elsevier Ltd.)

The effect of rain on the phyllosphere community has not been extensively explored, especially in the context of spatial variation on the impact of rain throughout the tree canopy. We characterized the response of the phyllosphere bacterial community removed from leaf surfaces of the Southern Magnolia (Magnolia grandiflora) to rain across different spatial locations of the canopy. We hypothesized that: (i) rain would lead to an initial decrease in phyllosphere bacterial diversity, followed by an increase in diversity on subsequent days, but that this effect would be minimized in the lower and interior portion of the canopy, and that (ii) community beta dispersion of phyllosphere microorganisms would be lower following rain, and similarly contingent on canopy position. We used targeted next-generation sequencing of the V4 region of the bacterial 16S rRNA gene to characterize bacterial composition. We found higher bacterial richness in interior canopy and distinct composition across canopy positions. Further, the effect of rain on beta dispersion was contingent on canopy position: rain lowered dispersion in the upper canopy but increased it in the lower and interior canopy. Our results demonstrate that canopy structure should be considered when looking at the impact of rain on the collective phyllosphere community., (© FEMS 2019.)

Invasive species could benefit from being introduced to locations with more favorable species interactions, including the loss of enemies, the gain of mutualists, or the simplification of complex interaction networks. Microbiomes are an important source of species interactions with strong fitness effects on multicellular organisms, and these interactions are known to vary across regions. The highly invasive plant yellow starthistle ( Centaurea solstitialis ) has been shown to experience more favorable microbial interactions in its invasions of the Americas, but the microbiome that must contribute to this variation in interactions is unknown. We sequenced amplicons of 16S rRNA genes to characterize bacterial community compositions in the phyllosphere, ectorhizosphere, and endorhizosphere of yellow starthistle plants from seven invading populations in California, USA, and eight native populations in Europe. We tested for the differentiation of microbiomes by geography, plant compartment, and plant genotype. Bacterial communities differed significantly between native and invading plants within plant compartments, with consistently lower diversity in the microbiome of invading plants. The diversity of bacteria in roots was positively correlated with plant genotype diversity within both ranges, but this relationship did not explain microbiome differences between ranges. Our results reveal that these invading plants are experiencing either a simplified microbial environment or simplified microbial interactions as a result of the dominance of a few taxa within their microbiome. Our findings highlight several alternative hypotheses for the sources of variation that we observe in invader microbiomes and the potential for altered bacterial interactions to facilitate invasion success. IMPORTANCE Previous studies have found that introduced plants commonly experience more favorable microbial interactions in their non-native range, suggesting that changes to the microbiome could be an important contributor to invasion success. Little is known about microbiome variation across native and invading populations, however, and the potential sources of more favorable interactions are undescribed. Here, we report one of the first microbiome comparisons of plants from multiple native and invading populations, in the noxious weed yellow starthistle. We identify clear differences in composition and diversity of microbiome bacteria. Our findings raise new questions about the sources of these differences, and we outline the next generation of research that will be required to connect microbiome variation to its potential role in plant invasions., (Copyright © 2019 Lu-Irving et al.)

Aims: To describe the effects of foliar fertilizer application on the bacterial populations of almond tree leaves., Methods and Results: We applied a commercially available foliar fertilizer or a water control onto the leaves of almond trees and collected leaves after 1, 7, 14 and 56 days and examined their bacterial populations by 16S rRNA gene sequence analysis. After 1 day, we observed significant differences in 3 of the 4 predominant bacterial phyla, and 5 of the 13 predominant bacterial families. After 7 days, we observed significant differences in all of the predominant phyla, and 8 of the 13 predominant families. After 14 days, the number of significant differences decreased, and after 56 days only 2 of the 13 predominant families differed significantly., Conclusions: Foliar fertilization significantly altered the bacterial population structure of almond leaves as compared to the water control. While most of the observed perturbation was transient, significant differences remained after 56 days., Significance and Impact of the Study: This is the first report describing the effects of foliar fertilization on the bacterial populations of almond leaves and provides new insights as to how this process alters the leaf bacterial population structure., (Published 2018. This article is a U.S. Government work and is in the public domain in the USA.)

Leaves are covered by a cuticle composed of long (C11-C20) and very-long chain hydrocarbons (>C20), e.g. alkanes, fatty acids, alcohols, aldehydes, ketones and esters. In addition to these aliphatics, cyclic hydrocarbons may be present. Leaves are colonised by a variety of so-called epiphytic bacteria, which may have adapted to be able to utilise cuticle hydrocarbons. We tested the ability of a wide range of phylogenetically different epiphytic bacteria to utilise and grow on diesel and petroleum benzine and show that out of the 21 strains tested, nine had the ability to utilise diesel for growth. Only one strain was able to utilise petroleum benzine for growth. The ability to utilise hydrocarbons for growth correlated with the ability of the strains to produce surfactants and out of the 21 tested strains, 12 produced surfactants. Showing that 75% of the strains producing surfactants were able to degrade hydrocarbons. Our findings suggest that the ability to degrade hydrocarbons and to produce surfactants is highly prevalent in epiphytic bacteria. It is unclear if epiphytic bacteria utilise hydrocarbons originating from the cuticle of living leaves. The application of surfactant producing, hydrocarbon-utilising, epiphytic bacteria might serve as a method for hydrocarbon bioremediation., (© FEMS 2019.)

In this study, the effects of di-n-butyl phthalate (DBP) on the phyllosphere bacterial community of field mustard (Brassica campestris L.) at the five-leaf stage were investigated. The indigenous alpha-diversity of the phyllosphere bacteria was altered after spraying with different concentrations of DBP. Shannon diversity indices were significantly changed on day 5 after treatment at DBP concentrations > 400 mg L -1 (P > 0.05). Nevertheless, the difference between treatment and control was not significant on day 9 after DBP treatment (P > 0.05). Exposure to DBP resulted in a decrease in Proteobacteria and Firmicutes, and an increase in Actinobacteria at all sampling intervals. These changes included significant increases in the relative abundance of Paracoccus and Rhodococcus, and significant decreases in that of Pseudomonas, Exiguobacterium, an unclassified genus of Pseudomonadaceae, and an unclassified genus of Enterobacteriaceae. This study provides new evidence for the possibility of using phyllosphere microbiota to remediate DBP contamination.

Plant leaves (phyllosphere) have a great potential for colonization and microbial growth, consisting of a dynamic environment in which several factors can interfere with the microbial population structure. The use of genetically modified (GM) plants has introduced several traits in agriculture, such as the improvement of plant drought tolerance, as observed in the AtAREB1 transcription factor overexpression in soybean (Glycine max L. Merrill). The present study aimed at investigating the taxonomic and functional profile of the leaf microbial community of bacteria found in GM (drought-tolerant event 1Ea2939) and conventional (BR 16) soybean plants. Bacterial DNA was extracted from leaf samples collected from each genotype and used for microbial diversity and richness analysis through the MiSeq Illumina platform. Functional prediction was performed using the PICRUSt tool and the STAMP v 2.1.3 software. The obtainment of the GM event 1Ea2939 showed minimum effects on the microbial community and in the potential for chemical-genetic communication, i.e. in the potential for symbiotic and/or mutualistic interaction between plants and their natural microbiota.

Foodborne illness represents a major threat to public health and is frequently attributed to pathogenic microorganisms on fresh produce. Recurrent outbreaks often come from vegetables that are grown close to or within the ground. Therefore, the first step to understanding the public health risk of microorganisms on fresh vegetables is to identify and describe microbial communities. We investigated the phyllospheres on Chinese cabbage ( Brassica rapa subsp. pekinensis , N = 54). 16S rRNA gene amplicon sequencing targeting the V5-V6 region of 16S rRNA genes was conducted by employing the Illumina MiSeq system. Sequence quality was assessed, and phylogenetic assessments were performed using the RDP classifier implemented in QIIME with a bootstrap cutoff of 80%. Principal coordinate analysis was performed using a weighted Fast UniFrac matrix. The average number of sequence reads generated per sample was 34,584. At the phylum level, bacterial communities were composed primarily of Proteobacteria and Bacteroidetes. The most abundant genera on Chinese cabbages were Chryseobacterium, Aurantimonadaceae&#95;g, Sphingomonas , and Pseudomonas . Diverse potential pathogens, such as Pantoea, Erwinia, Klebsiella, Yersinia, Bacillus, Staphylococcus, Salmonella , and Clostridium were also detected from the samples. Although further epidemiological studies will be required to determine whether the detected potential pathogens are associated with foodborne illness, our results imply that a metagenomic approach can be used to detect pathogenic bacteria on fresh vegetables.

Rice paddies are man-made, cross-over ecologies of aquatic and terrestrial systems, which favor the proliferation of characteristic microbial communities. Moisture regimes under flooded and different levels of irrigation such as in direct seeded rice (DSR) and system of rice intensification (SRI) lead to modulation in crop physiology, soil nutrient availability, and the soil microbiome. However, the diversity of the rice phyllosphere microbiome is less investigated in terms of the influence of fertilizer application and the method of rice cultivation (conventional-flooded, DSR and SRI). Scanning electron micrographs revealed the presence of bacteria as aggregates at microsites of the leaves. Phylogenetic analysis of the dominant culturable bacterial isolates using 16S rDNA sequences revealed that they belonged to the genera - Bacillus, Brevibacillus, Pantoea, Enterobacter, Pseudomonas, Erwinia, and Streptomyces. Fertilizer application brought about a distinct modulation in the communities belonging to phyla such as Bacteriodetes, Firmicutes, and Planctomyces, besides Proteobacteria. The cyanobacterial population was much influenced by the cultivation methods, particularly the SRI. Principal component analysis (PCA), involving the culturable phyllospheric microbial groups and leaf attributes (nutrients and pigments), illustrated the importance of leaf nitrogen and zinc. Also, the communities of the phylum Firmicutes exhibited marked changes in terms of the diversity, not only due to the cultivation method, but also the application of fertilizers. Thus, the cultivation methods and fertilizer application played important roles in modulating both the structural (taxonomical) and functional attributes of the phyllosphere microbiome., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The complex role of phylloplane microorganisms is less understood than that of rhizospheric microorganisms in lieu of their pivotal role in plant's sustainability. This experiment aims to study the diversity of the culturable phylloplane bacteria of Jatropha curcas and evaluate their growth-promoting activities towards maize seedling vigor. Heterotrophic bacteria were isolated from the phylloplane of J. curcas and their 16S rRNA genes were sequenced. Sequences of the 16S rRNA gene were very similar to those of species belonging to the classes Bacillales (50%), Gammaproteobacteria (21.8%), Betaproteobacteria (15.6%), and Alphaproteobacteria (12.5%). The phylloplane bacteria preferred to utilize alcohol rather than monosaccharides and polysaccharides as a carbon source. Isolates exhibited ACC (1-aminocyclopropane-1-carboxylic acid) deaminase, phosphatase, potassium solubilization, and indole acetic acid (IAA) production activities. The phosphate-solubilizing capacity (mg of PO 4 solubilized by 10 8 cells) varied from 0.04 to 0.21. The IAA production potential (μg IAA produced by 10 8 cells in 48 h) of the isolates varied from 0.41 to 9.29. Inoculation of the isolates to maize seed significantly increased shoot and root lengths of maize seedlings. A linear regression model of the plant-growth-promoting activities significantly correlated (p < 0.01) with the growth parameters. Similarly, a correspondence analysis categorized ACC deaminase and IAA production as the major factors contributing 41% and 13.8% variation, respectively, to the growth of maize seedlings.

There is an increasing interest to use halophytes for revegetation of salt affected ecosystems, as well as in understanding their mechanisms of salt tolerance. We hypothesized that bacteria from the phyllosphere of these plants might play a key role in its high tolerance to excessive salinity. Eight endophytic bacteria belonging to Bacillus and closely related genera were isolated from phyllosphere of the halophyte Arthrocnemum macrostachyum growing in salty agricultural soils. The presence of plant-growth promoting (PGP) properties, enzymatic activities and tolerance towards NaCl was determined. Effects of inoculation on seeds germination and adult plant growth under experimental NaCl treatments (0, 510 and 1030 mM NaCl) were studied. Inoculation with a consortium including the best performing bacteria improved considerably the kinetics of germination and the final germination percentage of A. macrostachyum seeds. At high NaCl concentrations (1030 mM), inoculation of plants mitigated the effects of high salinity on plant growth and physiological performance and, in addition, this consortium appears to have increased the potential of A. macrostachyum to accumulate Na + in its shoots, thus improving sodium phytoextraction capacity. Bacteria isolated from A. macrostachyum phyllosphere seem to play an important role in plant salt tolerance under stressing salt concentrations. The combined use of A. macrostachyum and its microbiome can be an adequate tool to enhance plant adaptation and sodium phytoextraction during restoration of salt degraded soils., (© 2016 German Botanical Society and The Royal Botanical Society of the Netherlands.)

The phyllosphere of the Brazilian Atlantic Forest has been estimated to contain several million bacterial species that are associated with approximately 20000 plant species. Despite the high bacterial diversity in the phyllosphere, the function of these microorganisms and the mechanisms driving their community assembly are largely unknown. In this study, we characterized the bacterial communities in the phyllospheres of four tree species of the Atlantic Forest (Mollinedia schottiana, Ocotea dispersa, Ocotea teleiandra, and Tabebuia serratifolia) and their metaproteomes to examine the basic protein functional groups expressed in the phyllosphere. Bacterial community analyses using 16S rRNA gene sequencing confirmed prior observations that plant species harbor distinct bacterial communities and that plants of the same taxon have more similar communities than more distantly related taxa. Using LC-ESI-Q-TOF, we identified 216 nonredundant proteins, based on 3503 peptide mass spectra. Most protein families were shared among the phyllosphere communities, suggesting functional redundancy despite differences in the species compositions of the bacterial communities. Proteins involved in glycolysis and anaerobic carbohydrate metabolism, solute transport, protein metabolism, cell motility, stress and antioxidant responses, nitrogen metabolism, and iron homeostasis were among the most frequently detected. In contrast to prior studies on crop plants and Arabidopsis, a low abundance of OTUs related to Methylobacterium and no proteins associated with the metabolism of one-carbon molecules were detected in the phyllospheres of the tree species studied here. Our data suggest that even though the phyllosphere bacterial communities of different tree species are phylogenetically diverse, their metaproteomes are functionally convergent with respect to traits required for survival on leaf surfaces.

Background: The increasing awareness of the role of phyllosphere microbial communities in plant health calls for a greater understanding of their structure and dynamics in natural ecosystems. Since most knowledge of tree phyllosphere bacterial communities has been gathered in tropical forests, our goal was to characterize the community structure and assembly dynamics of phyllosphere epiphytic bacterial communities in temperate forests in Quebec, Canada. We targeted five dominant tree species: Acer saccharum, Acer rubrum, Betula papyrifera, Abies balsamea, and Picea glauca. We collected 180 samples of phyllosphere communities on these species at four natural forest sites, three times during the growing season., Results: Host functional traits (i.e., wood density, leaf nitrogen content) and climate variables (summer mean temperature and precipitation) were strongly correlated with community structure. We highlight three key findings: (1) temperate tree species share a "core microbiome"; (2) significant evolutionary associations exist between groups of bacteria and host species; and (3) a greater part of the variation in phyllosphere bacterial community assembly is explained by host species identity (27 %) and species-site interaction (14 %), than by site (11 %) or time (1 %)., Conclusions: We demonstrated that host species identity is a stronger driver of temperate tree phyllosphere bacterial communities than site or time. Our results suggest avenues for future studies on the influence of host functional traits on phyllosphere community functional biogeography across terrestrial biomes.

The phyllosphere presents a unique system of discrete and easily replicable surfaces colonized primarily by bacteria. However, the biogeography of bacteria in the phyllosphere is little understood, especially at small to intermediate scales. Bacterial communities on the leaves of 91 southern magnolia (Magnolia grandiflora) trees 1-452 m apart in a small forest plot were analyzed and fragments of the 16S ribosomal RNA (rRNA) gene sequenced using the Illumina platform. Assemblages were dominated by members of the Alphaproteobacteria, Bacteroidetes, and Acidobacteria. Patterns in community composition were measured by both relative abundance (theta) and presence-absence (Jaccard) dissimilarity metrics. Distance-based Moran's eigenvector map analyses of the distance-decay relationship found a significant, positive relationship between each dissimilarity metric and significant eigenfunctions derived from geographic distance between trees, indicating trees that were closer together had more similar bacterial phyllosphere communities. Indirect gradient analyses revealed that several environmental parameters (canopy cover, tree elevation, and the slope and aspect of the ground beneath trees) were significantly related to multivariate ordination scores based on relative bacterial sequence abundances; however, these relationships were not significant when looking at the incidence of bacterial taxa. This suggests that bacterial growth and abundance in the phyllosphere is shaped by different assembly mechanisms than bacterial presence or absence. More broadly, this study demonstrates that the distance-decay relationship applies to phyllosphere communities at local scales, and that environmental parameters as well as neutral forces may both influence spatial patterns in the phyllosphere.

Bacteria on and inside leaves can influence forest tree health and resilience. The distribution and limits of a tree species' range can be influenced by various factors, with biological interactions among the most significant. We investigated the processes shaping the bacterial needle community across the species distribution of limber pine, a widespread Western conifer inhabiting a range of extreme habitats. We tested four hypotheses: (i) Needle community structure varies across sites, with site-specific factors more important to microbial assembly than host species selection; (ii) dispersal limitation structures foliar communities across the range of limber pine; (iii) the relative significance of dispersal and selection differs across sites in the tree species range; and (iv) needle age structures bacterial communities. We characterized needle communities from the needle surface and tissue of limber pine and co-occurring conifers across 16 sites in the limber pine distribution. Our findings confirmed that site characteristics shape the assembly of bacterial communities across the host species range and showed that these patterns are not driven by dispersal limitation. Furthermore, the strength of selection by the host varied by site, possibly due to differences in available microbes. Our study, by focusing on trees in their natural setting, reveals real needle bacterial dynamics in forests, which is key to understanding the balance between stochastic and deterministic processes in shaping forest tree-microbe interactions. Such understanding will be necessary to predict or manipulate these interactions to support forest ecosystem productivity or assist plant migration and adaptation in the face of global change.

Plants and microorganisms have been co-evolving and interacting for billions of years. Prior researchers have explored the plant's immune system responses and interaction with diverse microbes, but several ambiguities need further explanation. This review provides insight into mechanisms underlying plant–microbe interaction and knowledge dearth domains, along with possibilities to use beneficial microbes to improve plant growth, disease resistance, nutritional value, and productivity. Microorganisms in the phyllosphere and the rhizosphere could be beneficial or pathogenic. Host plants use their innate immune system and the antagonistic competence of plant-growth-promoting microbes against pathogens. The innate immune system of plants has two paramount protection forms involving different types of immune receptors, which assist in recognizing non-self-components. The first group of receptors is membrane-resident pattern recognition receptors (PRRs), which are responsible for sensing microbe-associated molecular patterns (MAMPs) and damage-associated molecular patterns (DAMPs). The second group consists of intracellular immune sensors, specifically resistance (R) proteins, astute in recognizing the structure or function of strain-specific pathogen effectors injected into host plant cells. Plants activate their pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) defense mechanisms to counter the infection. Plants benefit from certain microbes by promoting their growth, disease resistance, and resilience under various stress conditions in exchange for shelter and nutrients. [ABSTRACT FROM AUTHOR]

Abstract Plants and microorganisms have been co-evolving and interacting for billions of years. Prior researchers have explored the plant’s immune system responses and interaction with diverse microbes, but several ambiguities need further explanation. This review provides insight into mechanisms underlying plant–microbe interaction and knowledge dearth domains, along with possibilities to use beneficial microbes to improve plant growth, disease resistance, nutritional value, and productivity. Microorganisms in the phyllosphere and the rhizosphere could be beneficial or pathogenic. Host plants use their innate immune system and the antagonistic competence of plant-growth-promoting microbes against pathogens. The innate immune system of plants has two paramount protection forms involving different types of immune receptors, which assist in recognizing non-self-components. The first group of receptors is membrane-resident pattern recognition receptors (PRRs), which are responsible for sensing microbe-associated molecular patterns (MAMPs) and damage-associated molecular patterns (DAMPs). The second group consists of intracellular immune sensors, specifically resistance (R) proteins, astute in recognizing the structure or function of strain-specific pathogen effectors injected into host plant cells. Plants activate their pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) defense mechanisms to counter the infection. Plants benefit from certain microbes by promoting their growth, disease resistance, and resilience under various stress conditions in exchange for shelter and nutrients. Graphical Abstract

Developing an effective method for isolating bacterial genetic material from plants is a relatively challenging task and often does not yield adequately prepared material for further analyses. Previous studies often overlook connections, primarily focusing on laboratory investigations. With advancements in high-throughput sequencing techniques, we can now revisit and delve deeper into these interactions. Our study focuses on the initial phase of these investigations: genetic material isolation. Extracting bacterial DNA from aboveground plant parts, known as the phyllosphere, poses a significant challenge due to plant-derived contaminants. Existing isolation protocols frequently yield inconsistent results, necessitating continuous refinement and optimization. In our study, we developed an effective isolation protocol employing mechanical–chemical lysis, sonication, and membrane filtration. This approach yielded high-quality DNA at a concentration of 38.08 ng/µL, suitable for advanced sequencing applications. Our results underscore the effectiveness and necessity of these methods for conducting comprehensive microbiological analyses. Furthermore, our research not only lays the groundwork for further studies on lettuce microbiota, but also highlights the potential for utilizing our developed protocol in investigating other plants and their microbiomes. [ABSTRACT FROM AUTHOR]

Objetive/Background. The aim of this was to assess the diversity and antibiotic resistance of bacteria isolated from 19 crops with bacterial infection symptoms. Material and Methods: This collection was identified using 16S rRNA gene sequencing and the Biolog system. Susceptibility and minimum inhibitory concentration (MIC) for streptomycin, tetracycline, and gentamicin were determined using disk diffusion and E-test methods, respectively. Results: A total of 55 species belonging to 20 bacterial genera were identified, with Pseudomonas, Serratia, Pantoea, and Stenotrophomonas being the most abundant. Approximately 27% of the isolates were categorized as pathogenic through the hypersensitivity reaction test, including phytopathogenic species like Pseudomonas syringae, P. cichorii, Pantoea anthophila, P. stewartii, Stenotrophomonas maltophilia, Dickeya oryzae, Erwinia billingiae, Pectobacterium aroidearum, and Enterobacter cloacae subsp. dissolvens. Resistance to at least one antibiotic was detected in 60% of isolates from 17 crops, with tomatoes, heart of palm, and lettuce exhibited the highest proportion of resistant bacteria (>80%). Streptomycin resistance was most common (35%), followed by tetracycline (28%) and gentamicin (9%). Conclusions: The findings indicate the presence of antibiotic resistance in saprophytic and pathogenic bacteria associated with 17 out of 19 assessed crops, posing risks to the environment, phytosanitary conditions, and public health. [ABSTRACT FROM AUTHOR]

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

A fast-growing field of research focuses on microbial biocontrol within the phyllosphere. Phyllosphere microorganisms possess biocontrol capacity with good range of adaptation to the phyllosphere environment and inhibit the expansion of microbial pathogens, thus sustaining plant health. These biocontrol factors are often categorized in direct, microbe-microbe, and indirect, host-microbe, interactions. This review gives an summary of the modes of action of microbial adaptation and biocontrol within the phyllosphere, the genetic basis of the mechanisms and samples of experiments which will detect these mechanisms in laboratory and field experiments. Detailed insights in such mechanisms are key for the rational design of novel microbial biocontrol strategies and increase crop protection and production. Such novel biocontrol strategies are much needed in today's world to ensure sufficient food production to feed the growing world population. [ABSTRACT FROM AUTHOR]

Peru is one of the leading countries that produce and export specialty coffees, favorably positioned in the international markets for its physical and organoleptic cup qualities. In recent years, yellow coffee rust caused by the phytopathogenic fungus Hemileia vastatrix stands out as one of the main phytosanitary diseases that affect coffee culture yields. Many studies have demonstrated bacteria antagonistic activity against a number of phytopathogen fungi. In this context, the aim of this work was to select and characterize phyllospheric bacteria isolated from Coffea arabica with antagonistic features against coffee rust to obtain biocontrollers. For that purpose, a total of 82 phyllospheric bacteria were isolated from two coffee leaf rust-susceptible varieties, typica and caturra roja, and one tolerant variety, catimor. Of all the isolates, 15% were endophytic and 85% were epiphytes. Among all the isolates, 14 were capable of inhibiting the mycelial radial growth of Mycena citricolor, and Colletotrichum sp. 16S rRNA gene sequence-based analysis showed that 9 isolates were related to Achromobacter insuavis, 2 were related to Luteibacter anthropi and 1 was related to Rodococcus ceridiohylli, Achromobacter marplatensis and Pseudomonas parafulva. A total of 7 representative bacteria of each group were selected based on their antagonistic activity and tested in germination inhibition assays of coffee rust uredinospores. The CRRFLT7 and TRFLT8 isolates showed a high inhibition percentage of urediniospores germination (81% and 82%, respectively), similar to that obtained with the chemical control (91%). An experimental field assay showed a good performance of both strains against rust damage too, making them a promising alternative for coffee leaf rust biocontrol. [ABSTRACT FROM AUTHOR]

Switchgrass is a promising feedstock for biofuel production, with potential for leveraging its native microbial community to increase productivity and resilience to environmental stress. Here, we characterized the bacterial, archaeal and fungal diversity of the leaf microbial community associated with four switchgrass (Panicum virgatum) genotypes, subjected to two harvest treatments (annual harvest and unharvested control), and two fertilization levels (fertilized and unfertilized control), based on 16S rRNA gene and internal transcribed spacer (ITS) region amplicon sequencing. Leaf surface and leaf endosphere bacterial communities were significantly different with Alphaproteobacteria enriched in the leaf surface and Gammaproteobacteria and Bacilli enriched in the leaf endosphere. Harvest treatment significantly shifted presence/absence and abundances of bacterial and fungal leaf surface community members: Gammaproteobacteria were significantly enriched in harvested and Alphaproteobacteria were significantly enriched in unharvested leaf surface communities. These shifts were most prominent in the upland genotype DAC where the leaf surface showed the highest enrichment of Gammaproteobacteria, including taxa with 100% identity to those previously shown to have phytopathogenic function. Fertilization did not have any significant impact on bacterial or fungal communities. We also identified bacterial and fungal taxa present in both the leaf surface and leaf endosphere across all genotypes and treatments. These core taxa were dominated by Methylobacterium, Enterobacteriaceae, and Curtobacterium, in addition to Aureobasidium, Cladosporium, Alternaria and Dothideales. Local core leaf bacterial and fungal taxa represent promising targets for plant microbe engineering and manipulation across various genotypes and harvest treatments. Our study showcases, for the first time, the significant impact that harvest treatment can have on bacterial and fungal taxa inhabiting switchgrass leaves and the need to include this factor in future plant microbial community studies.

Bacteria from the leaf surface and the leaf tissue have been attributed with several beneficial properties for their plant host. Though physically connected, the microbial ecology of these compartments has mostly been studied separately such that we lack an integrated understanding of the processes shaping their assembly. We sampled leaf epiphytes and endophytes from the same individuals of sugar maple across the northern portion of its range to evaluate if their community composition was driven by similar processes within and across populations differing in plant traits and overall abiotic environment. Leaf compartment explained most of the variation in community diversity and composition across samples. Leaf epiphytic communities were driven more by host and site characteristics than endophytic communities, whose community composition was more idiosyncratic across samples. Our results suggest a greater importance of priority effects and opportunistic colonization in driving community assembly of leaf endophytes. Understanding the comparative assembly of bacterial communities at the surface and inside plant leaves may be particularly useful for leveraging their respective potential for improving the health of plants in natural and anthropized ecosystems. [ABSTRACT FROM AUTHOR]

Key message The datasets describe bacterial and fungal communities of European beech (Fagus sylvatica) leaves collected along a vertical gradient in a gallery forest throughout the growing season. They also describe communities in the surrounding environment of beech trees. Dataset access is at https://doi.org/10.7910/DVN/FFHAQU , and associated metadata are available at https://metadata-afs.nancy.inra.fr/geonetwork/srv/fre/catalog.search#/metadata/f17fe848-fc3e-4297-be11-9871b35a1be4 . Both can be used to uncover the dynamics and assembly processes of phyllosphere microbial communities in forest ecosystems.

Peru is one of the leading countries that produce and export specialty coffees, favorably positioned in the international markets for its physical and organoleptic cup qualities. In recent years, yellow coffee rust caused by the phytopathogenic fungus Hemileia vastatrix stands out as one of the main phytosanitary diseases that affect coffee culture yields. Many studies have demonstrated bacteria antagonistic activity against a number of phytopathogen fungi. In this context, the aim of this work was to select and characterize phyllospheric bacteria isolated from Coffea arabica with antagonistic features against coffee rust to obtain biocontrollers. For that purpose, a total of 82 phyllospheric bacteria were isolated from two coffee leaf rust-susceptible varieties, typica and caturra roja, and one tolerant variety, catimor. Of all the isolates, 15% were endophytic and 85% were epiphytes. Among all the isolates, 14 were capable of inhibiting the mycelial radial growth of Mycena citricolor, and Colletotrichum sp. 16S rRNA gene sequence-based analysis showed that 9 isolates were related to Achromobacter insuavis, 2 were related to Luteibacter anthropi and 1 was related to Rodococcus ceridiohylli, Achromobacter marplatensis and Pseudomonas parafulva. A total of 7 representative bacteria of each group were selected based on their antagonistic activity and tested in germination inhibition assays of coffee rust uredinospores. The CRRFLT7 and TRFLT8 isolates showed a high inhibition percentage of urediniospores germination (81% and 82%, respectively), similar to that obtained with the chemical control (91%). An experimental field assay showed a good performance of both strains against rust damage too, making them a promising alternative for coffee leaf rust biocontrol.