Phyllosphere Microbiota Research Articles

Harnessing the microbial interactions from Apocynum venetum phyllosphere for natural product discovery

Natural products (NPs) afforded by living-beings, especially by microscopic species, represent invaluable and indispensable reservoirs for drug leads in clinical practice. With the rapid advancement in sequencing technology and bioinformatics, the ever-increasing number of microbial biosynthetic gene clusters (BGCs) were decrypted, while a great deal of BGCs remain cryptic or inactive under standard laboratory culture conditions. Addressing this dilemma requires innovative tactics to awaken quiescence of BGCs by releasing the potential of microbial secondary metabolism for mining novel NPs. In this study, a universal strategy was proposed to induce the expression of silent BGCs by leveraging the dynamic interactions among coexisting microbial neighbors within a microbiota. This approach involves the deconstruction/reconstruction of binary interactions among the coexisting neighbors to create a pipeline for BGCs arousing. Coupled with the acquisition of 2,760 microbial individuals from the Apocynum venetum (Luobuma, LBM) phyllosphere in a successive dilution procedure, 44 culturable isolates were screened using binary interaction, in which 12.6% pairs demonstrated potent mutual interacting effects. Furthermore, after selecting the most four promising isolates, a full-scale metabolic inspection was conducted, in which 25.3% of the interacting pairs showcased significant metabolomic variations with de-cryptic activities. Notably, with the aid of visualization of IMS technology, one of the physiologically functional entities, the bactericidal agent resistomycin, was elucidated from the core interacting pair between the co-culture of the Streptomyces sp. LBM_605 and the Rhodococcus sp. LBM_791. This study highlights the intrinsic interactions among coexisting microorganisms within a phyllosphere microbiota as novel avenues for exploring and harnessing NPs.

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.

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.

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.

Differential responses of the phyllosphere abundant and rare microbes of Eucommia ulmoides to phytohormones

Phyllosphere microbiota play a crucial role in plant productivity and adaptation, and the abundant and rare microbial taxa often possess distinct characteristics and ecological functions. However, it is unclear whether the different subcommunities of phyllosphere microbiota respond variably to the factors that influence their formation, which limits the understanding of community assembly. The effects of two phytohormones, namely, indole-3-acetic acid (IAA) and N6-(delta 2-isopentenyl)-adenine (IP), on the phyllosphere microbial subcommunities of Eucommia ulmoides were investigated using potted experiments. The results demonstrated that the phytohormones induced significant variations in the composition, diversity, and function of the abundant microbial subcommunity in the phyllosphere of E. ulmoides, however, their effects on the rare subcommunity were negligible, and their effects on the moderate subcommunity were between those of the abundant and rare taxa. The phytohormones also induced significant alterations in the phenotypic and physiological properties of E. ulmoides, which indirectly affected the phyllosphere microbial community. Leaf thickness and average leaf area were the main phenotypic variables that affected the composition of the phyllosphere microbial community. The total alkaloid content and activity of superoxide dismutase (SOD) were the main physiological variables that affected the composition of the phyllosphere microbial community. The phenotypic and physiological indices of E. ulmoides explained the variations in the phyllosphere microbial subcommunities in descending order: abundant > moderate > rare taxa. These variables explained a significant proportion of the variations in the abundant taxa, and an insignificant proportion of the variations in the rare taxa. This study improves our understanding of the assembly of the phyllosphere microbiota, which provides important theoretical knowledge for future sustainable agriculture and forestry management based on the precise regulation of phyllosphere microbiota.

Biocontrol agents modulate phyllosphere microbiota interactions against pathogen Pseudomonas syringae

The pathogen Pseudomonas syringae, responsible for a variety of diseases, poses a considerable threat to global crop yields. Emerging biocontrol strategies employ antagonistic microorganisms, utilizing phyllosphere microecology and systemic resistance to combat this disease. However, the interactions between phyllosphere microbial dynamics and the activation of the plant defense system remain poorly understood. Here we show significant alterations in phyllosphere microbiota structure and plant gene expression following the application of biocontrol agents. We reveal enhanced collaboration and integration of Sphingomonas and Methylobacterium within the microbial co-occurrence network. Notably, Sphingomonas inhibits P. syringae by disrupting pathogen chemotaxis and virulence. Additionally, both Sphingomonas and Methylobacterium activate plant defenses by upregulating pathogenesis-related gene expression through abscisic acid, ethylene, jasmonate acid, and salicylic acid signaling pathways. Our results highlighted that biocontrol agents promote plant health, from reconstructing beneficial microbial consortia to enhancing plant immunity. The findings enrich our comprehension of the synergistic interplays between phyllosphere microbiota and plant immunity, offering potential enhancements in biocontrol efficacy for crop protection.

Characteristics of the phyllosphere microbial community and its relationship with major aroma precursors during the tobacco maturation process.

Numerous bacteria, fungi and other microorganisms in the tobacco phyllosphere interstellar area participate in the physiological metabolism of plants by interacting with the host. However, there is currently little research on the characteristics of tobacco phyllosphere microbial communities, and the correlation between tobacco phyllosphere microbial communities and phyllosphere factor indicators is still unknown. Therefore, high-throughput sequencing technology based on the 16S rRNA/ITS1 gene was used to explore the diversity and composition characteristics of tobacco phyllosphere bacterial and fungal communities from different maturation processes, and to identify marker genera that distinguish phyllosphere microbial communities. In this study, the correlations between tobacco phyllosphere bacterial and fungal communities and the precursors of major aroma compounds were explored. The results showed that as the tobacco plants matured, the density of glandular trichomes on the tobacco leaves gradually decreased. The surface physicochemical properties of tobacco leaves also undergo significant changes. In addition, the overall bacterial alpha diversity in the tobacco phyllosphere area increased with maturation, while the overall fungal alpha diversity decreased. The beta diversity of bacteria and fungi in the tobacco phyllosphere area also showed significant differences. Specifically, with later top pruning time, the relative abundances of Acidisoma, Ralstonia, Bradyrhizobium, Alternaria and Talaromyces gradually increased, while the relative abundances of Pseudomonas, Filobassidium, and Tausonia gradually decreased. In the bacterial community, Acidisoma, Ralstonia, Bradyrhizobium, and Alternaria were significantly positively correlated with tobacco aroma precursors, with significant negative correlations with tobacco phyllosphere trichome morphology, while Pseudomonas showed the opposite pattern; In the fungal community, Filobasidium and Tausonia were significantly negatively correlated with tobacco aroma precursors, and significantly positively correlated with tobacco phyllosphere trichome morphology, while Alternaria showed the opposite pattern. In conclusion, the microbiota (bacteria and fungi) and aroma precursors of the tobacco phyllosphere change significantly as tobacco matures. The presence of Acidisoma, Ralstonia, Bradyrhizobium and Alternaria in the phyllosphere microbiota of tobacco may be related to the aroma precursors of tobacco.

The growth promotion in endophyte symbiotic plants does not penalise the resistance to herbivores and bacterial microbiota.

A trade-off between growth and defence against biotic stresses is common in plants. Fungal endophytes of the genus Epichloë may relieve this trade-off in their host grasses since they can simultaneously induce plant growth and produce antiherbivore alkaloids that circumvent the need for host defence. The Epichloë ability to decouple the growth-defence trade-off was evaluated by subjecting ryegrass with and without Epichloë endophytes to an exogenous treatment with gibberellin (GA) followed by a challenge with Rhopalosiphum padi aphids. In agreement with the endophyte-mediated trade-off decoupling hypothesis, the GA-derived promotion of plant growth increased the susceptibility to aphids in endophyte-free plants but did not affect the insect resistance in endophyte-symbiotic plants. In line with the unaltered insect resistance, the GA treatment did not reduce the concentration of Epichloë-derived alkaloids. The Epichloë mycelial biomass was transiently increased by the GA treatment but at the expense of hyphal integrity. The response of the phyllosphere bacterial microbiota to both GA treatment and Epichloë was also evaluated. Only Epichloë, and not the GA treatment, altered the composition of the phyllosphere microbiota and the abundance of certain bacterial taxa. Our findings clearly demonstrate that Epichloë does indeed relieve the plant growth-defence trade-off.

Glycine betaine metabolism is enabled in Methylorubrum extorquens PA1 by alterations to dimethylglycine dehydrogenase.

Low nutrient availability is a key characteristic of the phyllosphere (the aerial surface of plants). Phyllospheric bacteria utilize a wide array of carbon sources generated by plant hosts. Glycine betaine (GB) is a plant-derived compound that can be metabolized by certain members of the phyllosphere microbiota. Metabolism of glycine betaine generates formaldehyde, an intermediate of methylotrophic metabolism, leading us to investigate how the ubiquitous plant colonizing bacterium Methylorubrum extorquens PA1 might metabolize GB encountered in its native environment. M. extorquens PA1 cannot utilize GB as a sole carbon source. Through suppressor mutation analysis, we show that M. extorquens PA1 encodes a conserved GB utilization pathway that can be activated by single point mutations conferring GB utilization as a carbon source. We identified the gene cluster encoding the GB catabolic enzymes and found that gene expression was induced in the presence of GB. We show that utilization of GB is conserved among representative Methylobacterium species and generates the one-carbon metabolism intermediate formaldehyde, which M. extorquens utilizes as a source of energy. Our results support a model where suppressor mutations in Mext_3745 or ftsH (Mext_4840) prevent the degradation of the dimethylglycine dehydrogenase subunit DgcB by the membrane integral protease FtsH, conferring the ability to utilize GB by either (i) restoring stable membrane topology of DgcB or (ii) decreasing FtsH protease activity, respectively. Both mutations alleviate the bottleneck at the second step of GB degradation catalyzed by DgcAB.IMPORTANCEOvercoming low nutrient availability is a challenge many bacteria encounter in the environment. Facultative methylotrophs are able to utilize one-carbon and multi-carbon compounds as carbon and energy sources. The utilization of plant-derived glycine betaine (GB) represents a possible source of multi-carbon and one-carbon substrates. The metabolism of glycine betaine produces formaldehyde and glycine, which may be used simultaneously by facultative methylotrophs. However, the genes required for the utilization of GB in the ubiquitous plant-associated bacterium Methylorubrum extorquens have yet to be identified or described. Our work identifies and validates the genes required for glycine betaine metabolism in M. extorquens and shows that it directly intersects with methylotrophic metabolism through the production of formaldehyde.

Characterization of phyllosphere endophytic lactic acid bacteria reveals a potential novel route to enhance silage fermentation quality.

The naturally attached phyllosphere microbiota play a crucial role in plant-derived fermentation, but the structure and function of phyllosphere endophytes remain largely unidentified. Here, we reveal the diversity, specificity, and functionality of phyllosphere endophytes in alfalfa (Medicago sativa L.) through combining typical microbial culture, high-throughput sequencing, and genomic comparative analysis. In comparison to phyllosphere bacteria (PB), the fermentation of alfalfa solely with endophytes (EN) enhances the fermentation characteristics, primarily due to the dominance of specific lactic acid bacteria (LAB) such as Lactiplantibacillus, Weissella, and Pediococcus. The inoculant with selected endophytic LAB strains also enhances the fermentation quality compared to epiphytic LAB treatment. Especially, one key endophytic LAB named Pediococcus pentosaceus EN5 shows enrichment of genes related to the mannose phosphotransferase system (Man-PTS) and carbohydrate-metabolizing enzymes and higher utilization of carbohydrates. Representing phyllosphere, endophytic LAB shows great potential of promoting ensiling and provides a novel direction for developing microbial inoculant.

Microbiome homeostasis on rice leaves is regulated by a precursor molecule of lignin biosynthesis

In terrestrial ecosystems, plant leaves provide the largest biological habitat for highly diverse microbial communities, known as the phyllosphere microbiota. However, the underlying mechanisms of host-driven assembly of these ubiquitous communities remain largely elusive. Here, we conduct a large-scale and in-depth assessment of the rice phyllosphere microbiome aimed at identifying specific host-microbe links. A genome-wide association study reveals a strong association between the plant genotype and members of four bacterial orders, Pseudomonadales, Burkholderiales, Enterobacterales and Xanthomonadales. Some of the associations are specific to a distinct host genomic locus, pathway or even gene. The compound 4-hydroxycinnamic acid (4-HCA) is identified as the main driver for enrichment of bacteria belonging to Pseudomonadales. 4-HCA can be synthesized by the host plant’s OsPAL02 from the phenylpropanoid biosynthesis pathway. A knockout mutant of OsPAL02 results in reduced Pseudomonadales abundance, dysbiosis of the phyllosphere microbiota and consequently higher susceptibility of rice plants to disease. Our study provides a direct link between a specific plant metabolite and rice phyllosphere homeostasis opening possibilities for new breeding strategies.

Pan-metagenome reveals the abiotic stress resistome of cigar tobacco phyllosphere microbiome.

The important role of microbial associations in mediating plant protection and responses to abiotic stresses has been widely recognized. However, there have been limited studies on the functional profile of the phyllosphere microbiota from tobacco (Nicotiana tabacum), hindering our understanding of the mechanisms underlying stress resilience in this representative and easy-to-cultivate model species from the solanaceous family. To address this knowledge gap, our study employed shotgun metagenomic sequencing for the first time to analyze the genetic catalog and identify putative plant growth promoting bacteria (PGPB) candidates that confer abiotic stress resilience throughout the growth period of cigar tobacco in the phyllosphere. We identified abundant genes from specific bacterial lineages, particularly Pseudomonas, within the cigar tobacco phyllospheric microbiome. These genes were found to confer resilience against a wide range of stressors, including osmotic and drought stress, heavy metal toxicity, temperature perturbation, organic pollutants, oxidative stress, and UV light damage. In addition, we conducted a virome mining analysis on the metagenome to explore the potential roles of viruses in driving microbial adaptation to environmental stresses. Our results identified a total of 3,320 scaffolds predicted to be viral from the cigar tobacco phyllosphere metagenome, with various phages infecting Pseudomonas, Burkholderia, Enterobacteria, Ralstonia, and related viruses. Within the virome, we also annotated genes associated with abiotic stress resilience, such as alkaline phosphatase D (phoD) for nutrient solubilization and glutamate-5-semialdehyde dehydrogenase (proA) for osmolyte synthesis. These findings shed light on the unexplored roles of viruses in facilitating and transferring abiotic stress resilience in the phyllospheric microbiome through beneficial interactions with their hosts. The findings from this study have important implications for agricultural practices, as they offer potential strategies for harnessing the capabilities of the phyllosphere microbiome to enhance stress tolerance in crop plants.

Tillandsia landbeckii phyllosphere and laimosphere as refugia for bacterial life in a hyperarid desert environment

BackgroundThe lack of water is a major constraint for microbial life in hyperarid deserts. Consequently, the abundance and diversity of microorganisms in common habitats such as soil are strongly reduced, and colonization occurs primarily by specifically adapted microorganisms that thrive in particular refugia to escape the harsh conditions that prevail in these deserts. We suggest that plants provide another refugium for microbial life in hyperarid deserts. We studied the bacterial colonization of Tillandsia landbeckii (Bromeliaceae) plants, which occur in the hyperarid regions of the Atacama Desert in Chile, one of the driest and oldest deserts on Earth.ResultsWe detected clear differences between the bacterial communities being plant associated to those of the bare soil surface (PERMANOVA, R2 = 0.187, p = 0.001), indicating that Tillandsia plants host a specific bacterial community, not only dust-deposited cells. Moreover, the bacterial communities in the phyllosphere were distinct from those in the laimosphere, i.e., on buried shoots (R2 = 0.108, p = 0.001), indicating further habitat differentiation within plant individuals. The bacterial taxa detected in the phyllosphere are partly well-known phyllosphere colonizers, but in addition, some rather unusual taxa (subgroup2 Acidobacteriae, Acidiphilum) and insect endosymbionts (Wolbachia, “Candidatus Uzinura”) were found. The laimosphere hosted phyllosphere-associated as well as soil-derived taxa. The phyllosphere bacterial communities showed biogeographic patterns across the desert (R2 = 0.331, p = 0.001). These patterns were different and even more pronounced in the laimosphere (R2 = 0.467, p = 0.001), indicating that different factors determine community assembly in the two plant compartments. Furthermore, the phyllosphere microbiota underwent temporal changes (R2 = 0.064, p = 0.001).ConclusionsOur data demonstrate that T. landbeckii plants host specific bacterial communities in the phyllosphere as well as in the laimosphere. Therewith, these plants provide compartment-specific refugia for microbial life in hyperarid desert environments. The bacterial communities show biogeographic patterns and temporal variation, as known from other plant microbiomes, demonstrating environmental responsiveness and suggesting that bacteria inhabit these plants as viable microorganisms.1mJ_tDG7agf2H2TZP3DsmgVideo

Differentiated responses of the phyllosphere bacterial community of the yellowhorn tree to precipitation and temperature regimes across Northern China.

As an ephemeral and oligotrophic environment, the phyllosphere harbors many highly diverse microorganisms. Importantly, it is known that their colonization of plant leaf surfaces is considerably influenced by a few abiotic factors related to climatic conditions. Yet how the dynamics of phyllosphere bacterial community assembly are shaped by detailed climatological elements, such as various bioclimatic variables, remains poorly understood. Using high-throughput 16S rRNA gene amplicon sequencing technology, we analyzed the bacterial communities inhabiting the leaf surfaces of an oilseed tree, yellowhorn (Xanthoceras sorbifolium), grown at four sites (Yinchuan, Otogqianqi, Tongliao, and Zhangwu) whose climatic status differs in northern China. We found that the yellowhorn phyllosphere's bacterial community was generally dominated by four phyla: Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. Nevertheless, bacterial community composition differed significantly among the four sampled site regions, indicating the possible impact of climatological factors upon the phyllosphere microbiome. Interestingly, we also noted that the α-diversities of phyllosphere microbiota showed strong positive or negative correlation with 13 bioclimatic factors (including 7 precipitation factors and 6 temperature factors). Furthermore, the relative abundances of 55 amplicon sequence variants (ASVs), including three ASVs representing two keystone taxa (the genera Curtobacterium and Streptomyces), exhibited significant yet contrary responses to the precipitation and temperature climatic variables. That pattern was consistent with all ASVs' trends of possessing opposite correlations to those two parameter classes. In addition, the total number of links and nodes, which conveys community network complexity, increased with rising values of most temperature variables. Besides that, remarkably positive relevance was found between average clustering coefficient and most precipitation variables. Altogether, these results suggest the yellowhorn phyllosphere bacterial community is capable of responding to variation in rainfall and temperature regimes in distinctive ways.

Deciphering the effect of phytohormones on the phyllosphere microbiota of Eucommia ulmoides

Phytohormones are key signals mediating plant-microbe molecular communication. However, their roles in driving phyllosphere microbiota assembly remain unclear. Here, high throughput target assays for 12 phytohormones and microbial amplicon sequencing techniques were used to reveal the effects of hormone components on phyllosphere microbiota of Eucommia ulmoides. Most of the phytohormone components in old leaves were lower than in tender leaves, such as indole-3-acetic acid (IAA), salicylic acid (SA), and jasmonic acid (JA), but the phyllosphere microbial community diversity in the older leaves was significantly higher than in the tender leaves, with more complex and aggregated microbial cooccurrence network. The E. ulmoides phyllosphere microbiota at tender and older leaf stage were dominated by the same dominant taxa at the phylum level, with Ascomycota and Basidiomycota as the main fungal taxa and Actinobacteriota, Bacteroidota, Firmicutes and Proteobacteria as the main bacterial taxa. FUNGuild and FAPROTAX functional predictions revealed that the high abundance functional groups of the E. ulmoides phyllosphere microbes were similar at tender and old leaf stages, with fungal functions mainly involving in plant pathogen, undefined saprotroph and endophyte, and bacterial functions mainly involving in chemoheterotrophy, fermentation and aerobic_chemoheterotrophy. Additionally, mantel test and variance partitioning analysis showed that IAA and N6-(delta 2-isopentenyl)-adenine (IP) were key phytohormones impacting the E. ulmoides phyllosphere microbiota, and their effects were largely interdependent. Our results improve the understanding of composition, diversity, function and influencing factors of phyllosphere microbiota, which might provide cue for sustainable agriculture and forestry management via precise regulation of the phyllosphere microbiota.

Characteristics of Epicoccum latusicollum as revealed by genomic and metabolic phenomic analysis, the causal agent of tobacco Epicoccus leaf spot.

Epicoccum latusicollum is a fungus that causes a severe foliar disease on flue-cured tobacco in southwest China, resulting in significant losses in tobacco yield and quality. To better understand the organism, researchers investigated its optimal growth conditions and metabolic versatility using a combination of traditional methods and the Biolog Phenotype MicroArray technique. The study found that E. latusicollum exhibited impressive metabolic versatility, being able to metabolize a majority of carbon, nitrogen, sulfur, and phosphorus sources tested, as well as adapt to different environmental conditions, including broad pH ranges and various osmolytes. The optimal medium for mycelial growth was alkyl ester agar medium, while oatmeal agar medium was optimal for sporulation, and the optimum temperature for mycelial growth was 25°C. The lethal temperature was 40°C. The study also identified arbutin and amygdalin as optimal carbon sources and Ala-Asp and Ala-Glu as optimal nitrogen sources for E. latusicollum. Furthermore, the genome of E. latusicollum strain T41 was sequenced using Illumina HiSeq and Pacific Biosciences technologies, with 10,821 genes predicted using Nonredundant, Gene Ontology, Clusters of Orthologous Groups, Kyoto Encyclopedia of Genes and Genomes, and SWISS-PROT databases. Analysis of the metabolic functions of phyllosphere microorganisms on diseased tobacco leaves affected by E. latusicollum using the Biolog Eco microplate revealed an inability to efficiently metabolize a total of 29 carbon sources, with only tween 40 showing some metabolizing ability. The study provides new insights into the structure and function of phyllosphere microbiota and highlights important challenges for future research, as well as a theoretical basis for the integrated control and breeding for disease resistance of tobacco Epicoccus leaf spot. This information can be useful in developing new strategies for disease control and management, as well as enhancing crop productivity and quality.

Boxwood phyllosphere fungal and bacterial communities and their differential responses to film-forming anti-desiccants

BackgroundAnti-desiccant is a class of agrochemicals widely used to protect plants from water stresses, rapid temperature variations, heat and sunburn, frost and freeze damages, transplant shock, and pathogen and pest attack. Although anti-desiccants are generally considered non-toxic to organisms, it is unclear whether they may impact the phyllosphere microbial communities. In this study, three film-forming anti-desiccant products, TransFilm, Vapor Gard, and Wilt-Pruf were applied to the canopy of two boxwood cultivars ‘Vardar Valley’ and ‘Justin Brouwers’ on April 13 and August 26, 2021. Shoot samples were collected from boxwood plants treated with each of the three products, as well as nontreated control on June 16, August 26 (before the second treatment), and October 18. Microbial and plant genomic DNA was isolated together and 16S rRNA gene and the extended internal transcribed spacer regions were amplified with PCR and sequenced on a Nanopore MinION platform for bacterial and fungal identification.ResultsBacterial communities were more diverse than fungal communities. At the phylum level, the boxwood phyllosphere was dominated by Proteobacteria and Ascomycota; at the genus level, Methylobacterium and Shiraia were the most abundant bacteria and fungi, respectively. Among the three film-forming anti-desiccants, Vapor Gard and Wilt-Pruf had more impact than TransFilm on the microbial communities. Specifically, broader impacts were observed on fungal than bacterial community composition and structure, with most affected fungi being suppressed while bacteria promoted.ConclusionThis study addressed several major knowledge gaps regarding boxwood phyllosphere microbiota and the impact of anti-desiccants on plant microbiome. We identified diverse microbial communities of boxwood, a major evergreen woody crop and an iconic landscape plant. We also found differential effects of three film-forming anti-desiccants on the composition and structure of bacterial and fungal communities. These findings advanced our understanding of the associated microbiome of this landmark plant, enabling growers to fully utilize the potentials of microbiome and three anti-desiccants in improving boxwood health and productivity.

Microbial composition and diversity of the tobacco leaf phyllosphere during plant development.

Phyllosphere-associated microorganisms affect host plant's nutrients availability, its growth and ecological functions. Tobacco leaves provide a wide-area habitat for microbial life. Previous studies have mainly focused on phyllosphere microbiota at one time point of tobacco growth process, but more is unknown about dynamic changes in phyllospheric microbial composition from earlier to the late stage of plant development. In the current study, we had determined the bacterial and fungal communities succession of tobacco growth stages (i.e., seedling, squaring, and maturing) by using both 16S rRNA sequencing for bacterial and ITS sequencing for fungi. Our results demonstrated that among tobacco growth stages, the phyllospheric bacterial communities went through more distinct succession than the fungal communities did. Proteobacteria and Actinobacteria exerted the most influence in tobacco development from seedling to squaring stages. At maturing stage, Proteobacteria and Actinobacteria dominance was gradually replaced by Firmicutes and Bacteroidetes. Network analysis revealed that Proteobacteria, as the core phyllospheric microbia, played essential role in stabilizing the whole bacterial network during tobacco development, and consequently rendered it to more profound ecological functions. During tobacco development, the contents of leaf sugar, nicotine, nitrogen and potassium were significantly correlated with either bacterial or fungal communities, and these abiotic factors accounted for 39.3 and 51.5% of the total variation, respectively. We overall evinced that the development of tobacco phyllosphere is accompanied by variant dynamics of phyllospheric microbial community.

The mature phyllosphere microbiome of grapevine is associated with resistance against Plasmopara viticola.

Phyllosphere microbiota represents a substantial but hardly explored reservoir for disease resistance mechanisms. The goal of our study was to understand the link between grapevine cultivars susceptibility to Plasmopara viticola, one of the most devastating leaf pathogens in viticulture, and the phyllosphere microbiota. Therefore, we analyzed a 16S rRNA gene library for the dominant phyllosphere bacterial phyla Alphaproteobacteria of seven Vitis genotypes at different developmental stages, i.e., flowering and harvesting, via amplicon sequencing. Young leaves had significantly higher Alphaproteobacterial richness and diversity without significant host-specificity. In contrast, the microbial communities of mature leaves were structurally distinct in accordance with P. viticola resistance levels. This statistically significant link between mature bacterial phyllosphere communities and resistant phenotypes was corroborated by beta diversity metrics and network analysis. Beyond direct host-driven effects via the provision of microhabitats, we found evidence that plants recruit for specific bacterial taxa that were likely playing a fundamental role in mediating microbe-microbe interactions and structuring clusters within mature communities. Our results on grape-microbiota interaction provide insights for targeted biocontrol and breeding strategies.

Out of site, out of mind: Changes in feather moss phyllosphere microbiota in mine offsite boreal landscapes.

Plant-microbe interactions play a crucial role in maintaining biodiversity and ecological services in boreal forest biomes. Mining for minerals, and especially the emission of heavy metal-enriched dust from mine sites, is a potential threat to biodiversity in offsite landscapes. Understanding the impacts of mining on surrounding phyllosphere microbiota is especially lacking. To investigate this, we characterized bacterial and fungal communities in the phyllosphere of feather moss Pleurozium schreberi (Brid). Mitt in boreal landscapes near six gold mine sites at different stages of the mine lifecycle. We found that (1) both mining stage and ecosystem type are drivers of the phyllosphere microbial community structure in mine offsite landscapes; (2) Bacterial alpha diversity is more sensitive than fungal alpha diversity to mining stage, while beta diversity of both groups is impacted; (3) mixed and deciduous forests have a higher alpha diversity and a distinct microbial community structure when compared to coniferous and open canopy ecosystems; (4) the strongest effects are detectable within 0.2 km from operating mines. These results confirmed the presence of offsite effects of mine sites on the phyllosphere microbiota in boreal forests, as well as identified mining stage and ecosystem type as drivers of these effects. Furthermore, the footprint was quantified at 0.2 km, providing a reference distance within which mining companies and policy makers should pay more attention during ecological assessment and for the development of mitigation strategies. Further studies are needed to assess how these offsite effects of mines affect the functioning of boreal ecosystems.