Assembly and succession of the phyllosphere microbiome and nutrient-cycling genes during plant community development in a glacier foreland.

The phyllosphere, particularly the leaf surface of plants, harbors a diverse range of microbiomes that play a vital role in the functioning of terrestrial ecosystems. However, our understanding of microbial successions and their impact on functional genes during plant community development is limited. In this study, considering core and satellite microbial taxa, we characterized the phyllosphere microbiome and functional genes in various microhabitats (i.e., leaf litter, moss and plant leaves) across the succession of a plant community in a low-altitude glacier foreland. Our findings indicate that phyllosphere microbiomes and associated ecosystem stability increase during the succession of the plant community. The abundance of core taxa increased with plant community succession and was primarily governed by deterministic processes. In contrast, satellite taxa abundance decreased during plant community succession and was mainly governed by stochastic processes. The abundance of microbial functional genes (such as C, N, and P hydrolysis and fixation) in plant leaves generally increased during the plant community succession. However, in leaf litter and moss leaves, only a subset of functional genes (e.g., C fixation and degradation, and P mineralization) showed a tendency to increase with plant community succession. Ultimately, the community of both core and satellite taxa collaboratively influenced the characteristics of phyllosphere nutrient-cycling genes, leading to the diverse profiles and fluctuating abundance of various functional genes during plant community succession. These findings offer valuable insights into the phyllosphere microbiome and plant-microbe interactions during plant community development, advancing our understanding of the succession and functional significance of the phyllosphere microbial community.

Elucidating fungal decomposition of organic matter at sub-micrometer spatial scales using optical photothermal infrared (O-PTIR) microspectroscopy.

In microbiological studies, a common goal is to link environmental factors to microbial activities. Both environmental factors and microbial activities are typically derived from bulk samples. It is becoming increasingly clear that such bulk environmental parameters poorly represent the microscale environments microorganisms experience. Using infrared (IR) microspectroscopy, the spatial distribution of chemical compound classes can be visualized, making it a useful tool for studying the interactions between microbial cells and their microenvironments. The spatial resolution of conventional IR microspectroscopy has been limited by the diffraction limit of IR light. The recent development of optical photothermal infrared (O-PTIR) microspectroscopy has pushed the spatial resolution of IR microspectroscopy beyond this diffraction limit, allowing the distribution of chemical compound classes to be visualized at sub-micrometer spatial scales. To examine the potential and limitations of O-PTIR microspectroscopy to probe the interactions between fungal cells and their immediate environments, we imaged the decomposition of cellulose films by cells of the ectomycorrhizal fungus Paxillus involutus and compared O-PTIR results using conventional IR microspectroscopy. Whereas the data collected with conventional IR microspectroscopy indicated that P. involutus has only a very limited ability to decompose cellulose films, O-PTIR data suggested that the ability of P. involutus to decompose cellulose was substantial. Moreover, the O-PTIR method enabled the identification of a zone located outside the fungal hyphae where the cellulose was decomposed by oxidation. We conclude that O-PTIR can provide valuable new insights into the abilities and mechanisms by which microorganisms interact with their surrounding environments.IMPORTANCEInfrared (IR) microspectroscopy allows the spatial distribution of chemical compound classes to be visualized. The use of conventional IR microspectroscopy in microbiological studies has been restricted by limited spatial resolution. Recent developments in laser technology have enabled a new class of IR microspectroscopy instruments to be developed, pushing the spatial resolution beyond the diffraction limit of IR light to approximately 500 nm. This improved spatial resolution now allows microscopic observations of changes in chemical compounds to be made, making IR microspectroscopy a useful tool to investigate microscale changes in chemistry that are caused by microbial activity. We show these new possibilities using optical photothermal infrared microspectroscopy to visualize the changes in cellulose substrates caused by oxidation by the ectomycorrhizal fungus Paxillus involutus at the interface between individual fungal hyphae and cellulose substrates.

Effects of topping and non-topping on growth-regulating hormones of flue-cured tobacco (Nicotiana tabacum L.)-a proteomic analysis.

Introduction: Until now, the mechanism underlying the impact of topping on hormone regulation in tobacco plants remains unclear, and most studies investigating the hormone signaling pathways in plants rely on genes or transcriptional pathways. Methods: This study examines the regulatory mechanisms of hormones in the roots and leaves of tobacco plants with and without topping at the protein level. Results: The results demonstrate that, compared with non-topped plants, topping leads to a decrease in the levels of IAA (auxin), ABA (abscisic acid), and GA (gibberellin) hormones in the leaves, whereas the content of the JA (jasmonic acid) hormone increases. Furthermore, in the roots, topping results in an increase in the levels of IAA, ABA, and JA hormones, along with a decrease in GA content. In the leaves, a total of 258 significantly different proteins were identified before and after topping, with 128 proteins upregulated and 130 proteins downregulated. In the roots, there were 439 proteins with significantly different quantities before and after topping, consisting of 211 upregulated proteins and 228 downregulated proteins. Notably, these proteins were closely associated with the metabolic and biosynthetic pathways of secondary metabolites, as indicated by functional categorization. Conclusions: When integrating the hormone changes and the proteomics results, it is evident that topping leads to increased metabolic activity and enhanced hormone synthesis in the root system. This research provides a theoretical foundation for further investigations into the regulation and signaling mechanisms of hormones at the protein level before and after topping in plants.

Rotation cropping and organic fertilizer jointly promote soil health and crop production.

Identifying field management practices to promote crop production, while conserving soil health is essential to maintain long-term food production in a changing world. Also, providing experimental evidence to support the use of traditional agricultural practices is necessary to secure sustainable agriculture. Here, we conducted a long-term 12-year experiment to investigate the impact of different combinations of fertilization type (control, inorganic fertilizer, organic fertilizer) and cropping regimes (continuous cropping and rotation cropping) on the crop (tobacco) production and multiple soil attributes associated with soil health, including proportions of soil-borne pathogens and decomposers, soil microbial diversity, microbial network stability and biomass, nutrient pools and microbial resource limitations. Our long-term experiment supports that the combination of organic fertilizer with rotation cropping increased crop production by at least 40% compared to the other management combinations and improved soil nutrient pools (e.g. the content of soil organic matter), improved the relative proportion of soil decomposers, and promoted bacterial and fungal network stability and biodiversity. Furthermore, this combination treatment relieved microbial resource limitation and reduced the abundance of potential fungal plant pathogens by at least 20% compared to other management combinations. In summary, we provide experimental evidence to support that the combined use of organic fertilization and rotation cropping management can help maintain long-term soil health, crop production, and economic outputs.

Secretion of Iron(III)-Reducing Metabolites during Protein Acquisition by the Ectomycorrhizal Fungus Paxillus involutus.

The ectomycorrhizal fungus Paxillus involutus decomposes proteins using a two-step mechanism, including oxidation and proteolysis. Oxidation involves the action of extracellular hydroxyl radicals (•OH) generated by the Fenton reaction. This reaction requires the presence of iron(II). Here, we monitored the speciation of extracellular iron and the secretion of iron(III)-reducing metabolites during the decomposition of proteins by P. involutus. X-ray absorption spectroscopy showed that extracellular iron was mainly present as solid iron(III) phosphates and oxides. Within 1 to 2 days, these compounds were reductively dissolved, and iron(II) complexes were formed, which remained in the medium throughout the incubation. HPLC and mass spectrometry detected five extracellular iron(III)-reducing metabolites. Four of them were also secreted when the fungus grew on a medium containing ammonium as the sole nitrogen source. NMR identified the unique iron(III)-reductant as the diarylcyclopentenone involutin. Involutin was produced from day 2, just before the elevated •OH production, preceding the oxidation of BSA. The other, not yet fully characterized iron(III)-reductants likely participate in the rapid reduction and dissolution of solid iron(III) complexes observed on day one. The production of these metabolites is induced by other environmental cues than for involutin, suggesting that they play a role beyond the Fenton chemistry associated with protein oxidation.

Regulation of fungal decomposition at single-cell level.

Filamentous fungi play a key role as decomposers in Earth's nutrient cycles. In soils, substrates are heterogeneously distributed in microenvironments. Hence, individual hyphae of a mycelium may experience very different environmental conditions simultaneously. In the current work, we investigated how fungi cope with local environmental variations at single-cell level. We developed a method based on infrared spectroscopy that allows the direct, in-situ chemical imaging of the decomposition activity of individual hyphal tips. Colonies of the ectomycorrhizal Basidiomycete Paxillus involutus were grown on liquid media, while parts of colonies were allowed to colonize lignin patches. Oxidative decomposition of lignin by individual hyphae growing under different conditions was followed for a period of seven days. We identified two sub-populations of hyphal tips: one with low decomposition activity and one with much higher activity. Active cells secreted more extracellular polymeric substances and oxidized lignin more strongly. The ratio of active to inactive hyphae strongly depended on the environmental conditions in lignin patches, but was further mediated by the decomposition activity of entire mycelia. Phenotypic heterogeneity occurring between genetically identical hyphal tips may be an important strategy for filamentous fungi to cope with heterogeneous and constantly changing soil environments.

Fenton reaction facilitates organic nitrogen acquisition by an ectomycorrhizal fungus.

Boreal trees rely on their ectomycorrhizal fungal symbionts to acquire growth-limiting nutrients, such as nitrogen (N), which mainly occurs as proteins complexed in soil organic matter (SOM). The mechanisms for liberating this N are unclear as ectomycorrhizal fungi have lost many genes encoding lignocellulose-degrading enzymes present in their saprotrophic ancestors. We hypothesized that hydroxyl radicals (˙ OH), produced by the ectomycorrhizal fungus Paxillus involutus during growth on SOM, are involved in liberating organic N. Paxillus involutus was grown for 7 d on N-containing or N-free substrates that represent major organic compounds of SOM. ˙ OH production, ammonium assimilation, and proteolytic activity were measured daily. ˙ OH production was strongly induced when P. involutus switched from ammonium to protein as the main N source. Extracellular proteolytic activity was initiated shortly after the oxidation. Oxidized protein substrates induced higher proteolytic activity than unmodified proteins. Dynamic modeling predicted that ˙ OH production occurs in a burst, regulated mainly by ammonium and ferric iron concentrations. We propose that the production of ˙ OH and extracellular proteolytic enzymes are regulated by similar nutritional signals. Oxidation works in concert with proteolysis, improving N liberation from proteins in SOM. Organic N mining by ectomycorrhizal fungi has, until now, only been attributed to proteolysis.

The SlZRT1 Gene Encodes a Plasma Membrane-Located ZIP (Zrt-, Irt-Like Protein) Transporter in the Ectomycorrhizal Fungus Suillus luteus.

Zinc (Zn) is an essential micronutrient but may become toxic when present in excess. In Zn-contaminated environments, trees can be protected from Zn toxicity by their root-associated micro-organisms, in particular ectomycorrhizal fungi. The mechanisms of cellular Zn homeostasis in ectomycorrhizal fungi and their contribution to the host tree's Zn status are however not yet fully understood. The aim of this study was to identify and characterize transporters involved in Zn uptake in the ectomycorrhizal fungus Suillus luteus, a cosmopolitan pine mycobiont. Zn uptake in fungi is known to be predominantly governed by members of the ZIP (Zrt/IrtT-like protein) family of Zn transporters. Four ZIP transporter encoding genes were identified in the S. luteus genome. By in silico and phylogenetic analysis, one of these proteins, SlZRT1, was predicted to be a plasma membrane located Zn importer. Heterologous expression in yeast confirmed the predicted function and localization of the protein. A gene expression analysis via RT-qPCR was performed in S. luteus to establish whether SlZRT1 expression is affected by external Zn concentrations. SlZRT1 transcripts accumulated almost immediately, though transiently upon growth in the absence of Zn. Exposure to elevated concentrations of Zn resulted in a significant reduction of SlZRT1 transcripts within the first hour after initiation of the exposure. Altogether, the data support a role as cellular Zn importer for SlZRT1 and indicate a key role in cellular Zn uptake of S. luteus. Further research is needed to understand the eventual contribution of SlZRT1 to the Zn status of the host plant.

Comparative Evaluation of Four Bacteria-Specific Primer Pairs for 16S rRNA Gene Surveys.

Bacterial taxonomic community analyses using PCR-amplification of the 16S rRNA gene and high-throughput sequencing has become a cornerstone in microbiology research. To reliably detect the members, or operational taxonomic units (OTUs), that make up bacterial communities, taxonomic surveys rely on the use of the most informative PCR primers to amplify the broad range of phylotypes present in up-to-date reference databases. However, primers specific for the domain Bacteria were often developed some time ago against database versions that are now out of date. Here we evaluated the performance of four bacterial primers for characterizing complex microbial communities in explosives contaminated and non-contaminated forest soil and by in silico evaluation against the current SILVA123 database. Primer pair 341f/785r produced the highest number of bacterial OTUs, phylogenetic richness, Shannon diversity, low non-specificity and most reproducible results, followed by 967f/1391r and 799f/1193r. Primer pair 68f/518r showed overall low coverage and a bias toward Alphaproteobacteria. In silico, primer pair 341f/785r showed the highest coverage of the domain Bacteria (96.1%) with no obvious bias toward the majority of bacterial species. This suggests the high utility of primer pair 341f/785r for soil and plant-associated bacterial microbiome studies.

Structural variability and niche differentiation in the rhizosphere and endosphere bacterial microbiome of field-grown poplar trees.

BACKGROUND: The plant microbiome represents one of the key determinants of plant health and productivity by providing a plethora of functional capacities such as access to low-abundance nutrients, suppression of phytopathogens, and resistance to biotic and/or abiotic stressors. However, a robust understanding of the structural composition of the bacterial microbiome present in different plant microenvironments and especially the relationship between below-ground and above-ground communities has remained elusive. In this work, we addressed hypotheses regarding microbiome niche differentiation and structural stability of the bacterial communities within different ecological plant niches. METHODS: We sampled the rhizosphere soil, root, stem, and leaf endosphere of field-grown poplar trees (Populus tremula × Populus alba) and applied 16S rRNA amplicon pyrosequencing to unravel the bacterial communities associated with the different plant habitats. RESULTS: We found that the structural variability of rhizosphere microbiomes in field-grown poplar trees (P. tremula × P. alba) is much lower than that of the endosphere microbiomes. Furthermore, our data not only confirm microbiome niche differentiation reports at the rhizosphere soil-root interface but also clearly show additional fine-tuning and adaptation of the endosphere microbiome in the stem and leaf compartment. Each plant compartment represents an unique ecological niche for the bacterial communities. Finally, we identified the core bacterial microbiome associated with the different ecological niches of Populus. CONCLUSIONS: Understanding the complex host-microbe interactions of Populus could provide the basis for the exploitation of the eukaryote-prokaryote associations in phytoremediation applications, sustainable crop production (bio-energy efficiency), and/or the production of secondary metabolites.

Performance of 16s rDNA Primer Pairs in the Study of Rhizosphere and Endosphere Bacterial Microbiomes in Metabarcoding Studies.

Next-generation sequencing technologies have revolutionized the methods for studying microbial ecology by enabling high-resolution community profiling. However, the use of these technologies in unraveling the plant microbiome remains challenging. Many bacterial 16S rDNA primer pairs also exhibit high affinity for non-target DNA such as plastid (mostly chloroplast) DNA and mitochondrial DNA. Therefore, we experimentally tested a series of commonly used primers for the analysis of plant-associated bacterial communities using 454 pyrosequencing. We evaluated the performance of all selected primer pairs in the study of the bacterial microbiomes present in the rhizosphere soil, root, stem and leaf endosphere of field-grown poplar trees (Populus tremula × Populus alba) based on (a) co-amplification of non-target DNA, (b) low amplification efficiency for pure chloroplast DNA (real-time PCR), (c) high retrieval of bacterial 16S rDNA, (d) high operational taxonomic unit (OTU) richness and Inverse Simpson diversity and (e) taxonomic assignment of reads. Results indicate that experimental evaluation of primers provide valuable information that could contribute in the selection of suitable primer pairs for 16S rDNA metabarcoding studies in plant-microbiota research. Furthermore, we show that primer pair 799F-1391R outperforms all other primer pairs in our study in the elimination of non-target DNA and retrieval of bacterial OTUs.

Lignin engineering in field-grown poplar trees affects the endosphere bacterial microbiome.

Cinnamoyl-CoA reductase (CCR), an enzyme central to the lignin biosynthetic pathway, represents a promising biotechnological target to reduce lignin levels and to improve the commercial viability of lignocellulosic biomass. However, silencing of the CCR gene results in considerable flux changes of the general and monolignol-specific lignin pathways, ultimately leading to the accumulation of various extractable phenolic compounds in the xylem. Here, we evaluated host genotype-dependent effects of field-grown, CCR-down-regulated poplar trees (Populus tremula × Populus alba) on the bacterial rhizosphere microbiome and the endosphere microbiome, namely the microbiota present in roots, stems, and leaves. Plant-associated bacteria were isolated from all plant compartments by selective isolation and enrichment techniques with specific phenolic carbon sources (such as ferulic acid) that are up-regulated in CCR-deficient poplar trees. The bacterial microbiomes present in the endosphere were highly responsive to the CCR-deficient poplar genotype with remarkably different metabolic capacities and associated community structures compared with the WT trees. In contrast, the rhizosphere microbiome of CCR-deficient and WT poplar trees featured highly overlapping bacterial community structures and metabolic capacities. We demonstrate the host genotype modulation of the plant microbiome by minute genetic variations in the plant genome. Hence, these interactions need to be taken into consideration to understand the full consequences of plant metabolic pathway engineering and its relation with the environment and the intended genetic improvement.

Impact of metal pollution on fungal diversity and community structures.

The impact of metal pollution on plant communities has been studied extensively in the past, but little is known about the effects of metal pollution on fungal communities that occur in metal-polluted soils. Metal-tolerant ecotypes of the ectomycorrhizal fungus Suillus luteus are frequently found in pioneer pine forests in the Campine region in Belgium on metal-polluted soils. We hypothesized that metal pollution would play an important role in shaping below-ground fungal communities that occur in these soils and that Suillus luteus would be a dominant player. To test these hypotheses, the fungal communities in a young pine plantation in soil polluted with zinc, and cadmium were studied using 454 amplicon pyrosequencing. Results show that zinc, cadmium and soil organic matter content were strongly correlated with the fungal community composition, but no effects on fungal diversity were observed. As hypothesized, S. luteus was found to be a dominant member of the studied fungal communities. However, other dominant fungal species, such as Sistotrema sp., Wilcoxina mikolae and Cadophora finlandica were found as well. Their presence in metal-polluted sites is discussed.

Comparison and validation of some ITS primer pairs useful for fungal metabarcoding studies.

Current metabarcoding studies aiming to characterize microbial communities generally rely on the amplification and sequencing of relatively short DNA regions. For fungi, the internal transcribed spacer (ITS) region in the ribosomal RNA (rRNA) operon has been accepted as the formal fungal barcode. Despite an increasing number of fungal metabarcoding studies, the amplification efficiency of primers is generally not tested prior to their application in metabarcoding studies. Some of the challenges that metabarcoding primers should overcome efficiently are the amplification of target DNA strands in samples rich in non-target DNA and environmental pollutants, such as humic acids, that may have been co-extracted with DNA. In the current study, three selected primer pairs were tested for their suitability as fungal metabarcoding primers. The selected primer pairs include two primer pairs that have been frequently used in fungal metabarcoding studies (ITS1F/ITS2 and ITS3/ITS4) and a primer pair (ITS86F/ITS4) that has been shown to efficiently amplify the ITS2 region of a broad range of fungal taxa in environmental soil samples. The selected primer pairs were evaluated in a 454 amplicon pyrosequencing experiment, real-time PCR (qPCR) experiments and in silico analyses. Results indicate that experimental evaluation of primers provides valuable information that could aid in the selection of suitable primers for fungal metabarcoding studies. Furthermore, we show that the ITS86F/ITS4 primer pair outperforms other primer pairs tested in terms of in silico primer efficiency, PCR efficiency, coverage, number of reads and number of species-level operational taxonomic units (OTUs) obtained. These traits push the ITS86F/ITS4 primer pair forward as highly suitable for studying fungal diversity and community structures using DNA metabarcoding.

Zinc export results in adaptive zinc tolerance in the ectomycorrhizal basidiomycete Suillus bovinus.

On Zn-polluted soils, populations of the ectomycorrhizal basidiomycete Suillus bovinus exhibit an elevated Zn tolerance when compared to populations on non-polluted sites. To elucidate the mechanism of Zn tolerance, the time-course of Zn uptake was studied in isolates with contrasting Zn tolerance. Unidirectional fluxes and subcellular compartmentation of Zn were investigated through radiotracer flux analyses. Fluorescence imaging was used to support the subcellular Zn compartmentation. After 2 h of exposure to 200 µM Zn, significantly more Zn was accumulated in Zn-sensitive isolates compared to tolerant isolates, despite similar short-term uptake kinetics and similar extracellular Zn sequestration in cell walls. In Zn-sensitive isolates twice as much Zn accumulated in the cytoplasm and 12 times more Zn in the vacuole. (65)Zn efflux analyses revealed a considerably faster Zn export in the Zn-tolerant isolate. The adaptive Zn tolerance in S. bovinus is therefore achieved by a preferential removal of Zn out of the cytoplasm, back into the apoplast, instead of the usual transfer of Zn into the vacuole. Zn exclusion in the fungal symbiont eventually contributes to a lower Zn influx in host plants.