Zhilong Huoxue Tongyu Capsule regulates the macrophage polarization and inflammatory response via the let-7i/TLR9/MyD88 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Zhilong Huoxue Tongyu Capsule (ZL) is clinically prescribed for acute ischemic stroke (AIS). However, only a few studies have addressed the mechanisms of ZL in treating AIS. AIM OF THE STUDY: To explore the underlying mechanism of macrophage polarization and inflammation mediated by ZL, and to provide a reference for AIS treatment. MATERIALS AND METHODS: Sixteen SD rats were fed with different dose of ZL (0, 0.4, 0.8, and 1.6 g/kg/d) for 4 days to prepare ZL serum. After 500 ng/mL lipopolysaccharide (LPS) stimulation, RAW264.7 cells were administrated with ZL serum. Then, experiments including ELISA, flow cytometry, real-time quantitative PCR and Western blot were performed to verify the effects of ZL on macrophage polarization and inflammation. Next, let-7i inhibitor was transfected in RAW264.7 cells when treated with LPS and ZL serum to verify the regulation of ZL on the let-7i/TLR9/MyD88 signaling pathway. Moreover, the interaction between let-7i and TLR9 was confirmed by the dual-luciferase assay. RESULTS: ZL serum significantly decreased the expression of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α), and increased the expression of IL-10 and transforming growth factor ß1 (TGF-ß1) of LPS stimulated-macrophages. Furthermore, ZL serum polarized macrophages toward M2, decreased the expressions of TLR9, MyD88, and iNOS, as well as increased the expressions of let-7i, CHIL3, and Arginase-1. It is worth mentioning that the effect of ZL serum is dose-dependent. However, let-7i inhibitor restored all the above effects in LPS stimulated-macrophages. In addition, TLR9 was the target of let-7i. CONCLUSIONS: ZL targeted let-7i to inhibit TLR9 expression, thereby inhibiting the activation of the TLR9/MyD88 pathway, promoting the M2 polarization, and inhibiting the development of inflammation in AIS.

Suillus: an emerging model for the study of ectomycorrhizal ecology and evolution.

Research on mycorrhizal symbiosis has been slowed by a lack of established study systems. To address this challenge, we have been developing Suillus, a widespread ecologically and economically relevant fungal genus primarily associated with the plant family Pinaceae, into a model system for studying ectomycorrhizal (ECM) associations. Over the last decade, we have compiled extensive genomic resources, culture libraries, a phenotype database, and protocols for manipulating Suillus fungi with and without their tree partners. Our efforts have already resulted in a large number of publicly available genomes, transcriptomes, and respective annotations, as well as advances in our understanding of mycorrhizal partner specificity and host communication, fungal and plant nutrition, environmental adaptation, soil nutrient cycling, interspecific competition, and biological invasions. Here, we highlight the most significant recent findings enabled by Suillus, present a suite of protocols for working with the genus, and discuss how Suillus is emerging as an important model to elucidate the ecology and evolution of ECM interactions.

Identification of Potential Crucial Biomarkers in STEMI Through Integrated Bioinformatic Analysis. / Identificação de Potenciais Biomarcadores Cruciais em IAMCSST por Meio de Análise Bioinformática Integrada.

BACKGROUND: ST-segment elevation myocardial infarction (STEMI) is one of the leading causes of fatal cardiovascular diseases, which have been the prime cause of mortality worldwide. Diagnosis in the early phase would benefit clinical intervention and prognosis, but the exploration of the biomarkers of STEMI is still lacking. OBJECTIVES: In this study, we conducted a bioinformatics analysis to identify potential crucial biomarkers in the progress of STEMI. METHODS: We obtained GSE59867 for STEMI and stable coronary artery disease (SCAD) patients. Differentially expressed genes (DEGs) were screened with the threshold of |log2fold change| > 0.5 and p <0.05. Based on these genes, we conducted enrichment analysis to explore the potential relevance between genes and to screen hub genes. Subsequently, hub genes were analyzed to detect related miRNAs and DAVID to detect transcription factors for further analysis. Finally, GSE62646 was utilized to assess DEGs specificity, with genes demonstrating AUC results exceeding 75%, indicating their potential as candidate biomarkers. RESULTS: 133 DEGs between SCAD and STEMI were obtained. Then, the PPI network of DEGs was constructed using String and Cytoscape, and further analysis determined hub genes and 6 molecular complexes. Functional enrichment analysis of the DEGs suggests that pathways related to inflammation, metabolism, and immunity play a pivotal role in the progression from SCAD to STEMI. Besides, related-miRNAs were predicted, has-miR-124, has-miR-130a/b, and has-miR-301a/b regulated the expression of the largest number of genes. Meanwhile, Transcription factors analysis indicate that EVI1, AML1, GATA1, and PPARG are the most enriched gene. Finally, ROC curves demonstrate that MS4A3, KLRC4, KLRD1, AQP9, and CD14 exhibit both high sensitivity and specificity in predicting STEMI. CONCLUSIONS: This study revealed that immunity, metabolism, and inflammation are involved in the development of STEMI derived from SCAD, and 6 genes, including MS4A3, KLRC4, KLRD1, AQP9, CD14, and CCR1, could be employed as candidate biomarkers to STEMI.

FUNDAMENTO: O infarto do miocárdio com elevação do segmento ST (IAMCSST) é uma das principais causas de doenças cardiovasculares fatais, que têm sido a principal causa de mortalidade em todo o mundo. O diagnóstico na fase inicial beneficiaria a intervenção clínica e o prognóstico, mas ainda falta a exploração dos biomarcadores do IAMCSST. OBJETIVOS: Neste estudo, conduzimos uma análise bioinformática para identificar potenciais biomarcadores cruciais no progresso do IAMCSST. MÉTODOS: Obtivemos GSE59867 para pacientes com IAMCSST e doença arterial coronariana estável (DACE). Genes diferencialmente expressos (GDEs) foram selecionados com o limiar de |log2fold change| > 0,5 e p < 0,05. Com base nesses genes, conduzimos análises de enriquecimento para explorar a relevância potencial entre genes e para rastrear genes centrais. Posteriormente, os genes centrais foram analisados para detectar miRNAs relacionados e DAVID para detectar fatores de transcrição para análise posterior. Finalmente, o GSE62646 foi utilizado para avaliar a especificidade dos GDEs, com genes demonstrando resultados de AUC superiores a 75%, indicando seu potencial como candidatos a biomarcadores. Posteriormente, os genes centrais foram analisados para detectar miRNAs relacionados e DAVID para detectar fatores de transcrição para análise posterior. Finalmente, o GSE62646 foi utilizado para avaliar a especificidade dos GDEs, com genes demonstrando resultados de AUC superiores a 75%, indicando seu potencial como candidatos a biomarcadores. RESULTADOS: 133 GDEs entre DACE e IAMCSST foram obtidos. Em seguida, a rede PPI de GDEs foi construída usando String e Cytoscape, e análises posteriores determinaram genes centrais e 6 complexos moleculares. A análise de enriquecimento funcional dos GDEs sugere que as vias relacionadas à inflamação, metabolismo e imunidade desempenham um papel fundamental na progressão de DACE para IAMCSST. Além disso, foram previstos miRNAs relacionados, has-miR-124, has-miR-130a/b e has-miR-301a/b regularam a expressão do maior número de genes. Enquanto isso, a análise dos fatores de transcrição indica que EVI1, AML1, GATA1 e PPARG são os genes mais enriquecidos. Finalmente, as curvas ROC demonstram que MS4A3, KLRC4, KLRD1, AQP9 e CD14 exibem alta sensibilidade e especificidade na previsão de IAMCSST. CONCLUSÕES: Este estudo revelou que imunidade, metabolismo e inflamação estão envolvidos no desenvolvimento de IAMCSST derivado de DACE, e 6 genes, incluindo MS4A3, KLRC4, KLRD1, AQP9, CD14 e CCR1, poderiam ser empregados como candidatos a biomarcadores para IAMCSST.

Long-term tropospheric ozone pollution disrupts plant-microbe-soil interactions in the agroecosystem.

Tropospheric ozone (O3 ) threatens agroecosystems, yet its long-term effects on intricate plant-microbe-soil interactions remain overlooked. This study employed two soybean genotypes of contrasting O3 -sensitivity grown in field plots exposed elevated O3 (eO3 ) and evaluated cause-effect relationships with their associated soil microbiomes and soil quality. Results revealed long-term eO3 effects on belowground soil microbiomes and soil health surpass damage visible on plants. Elevated O3 significantly disrupted belowground bacteria-fungi interactions, reduced fungal diversity, and altered fungal community assembly by impacting soybean physiological properties. Particularly, eO3 impacts on plant performance were significantly associated with arbuscular mycorrhizal fungi, undermining their contribution to plants, whereas eO3 increased fungal saprotroph proliferation, accelerating soil organic matter decomposition and soil carbon pool depletion. Free-living diazotrophs exhibited remarkable acclimation under eO3 , improving plant performance by enhancing nitrogen fixation. However, overarching detrimental consequences of eO3 negated this benefit. Overall, this study demonstrated long-term eO3 profoundly governed negative impacts on plant-soil-microbiota interactions, pointing to a potential crisis for agroecosystems. These findings highlight urgent needs to develop adaptive strategies to navigate future eO3 scenarios.

Ectomycorrhizal fungi enhance pine growth by stimulating iron-dependent mechanisms with trade-offs in symbiotic performance.

Iron (Fe) is crucial for metabolic functions of living organisms. Plants access occluded Fe through interactions with rhizosphere microorganisms and symbionts. Yet, the interplay between Fe addition and plant-mycorrhizal interactions, especially the molecular mechanisms underlying mycorrhiza-assisted Fe processing in plants, remains largely unexplored. We conducted mesocosms in Pinus plants inoculated with different ectomycorrhizal fungi (EMF) Suillus species under conditions with and without Fe coatings. Meta-transcriptomic, biogeochemical, and X-ray fluorescence imaging analyses were applied to investigate early-stage mycorrhizal roots. While Fe addition promoted Pinus growth, it concurrently reduced mycorrhiza formation rate, symbiosis-related metabolites in plant roots, and aboveground plant carbon and macronutrient content. This suggested potential trade-offs between Fe-enhanced plant growth and symbiotic performance. However, the extent of this trade-off may depend on interactions between host plants and EMF species. Interestingly, dual EMF species were more effective at facilitating plant Fe uptake by inducing diverse Fe-related functions than single-EMF species. This subsequently triggered various Fe-dependent physiological and biochemical processes in Pinus roots, significantly contributing to Pinus growth. However, this resulted in a greater carbon allocation to roots, relatively reducing the aboveground plant carbon content. Our study offers critical insights into how EMF communities rebalance benefits of Fe-induced effects on symbiotic partners.

Soil Bacterial Diversity Responds to Long-Term Establishment of Perennial Legumes in Warm-Season Grassland at Two Soil Depths.

The introduction of rhizoma peanut (RP Arachis glabrata Benth) into bahiagrass (Paspalum notatum Flüggé) may require time to develop stable plant-soil microbe interactions as the microbial legacy of the previous plant community may be long-lasting. A previous study showed that <2 years of introducing rhizoma peanut into bahiagrass pastures minimally affected soil bacterial diversity and community composition. In this study, we compared the effects of the long-term inclusion of rhizoma peanut (>8 years) into bahiagrass on soil bacterial diversity and community composition against their monocultures at 0 to 15 and 15 to 30 cm soil depths using next-generation sequencing to target bacterial 16S V3-V4 regions. We observed that a well-established RP-bahiagrass mixed stand led to a 36% increase in bacterial alpha diversity compared to the bahiagrass monoculture. There was a shift from a soil bacterial community dominated by Proteobacteria (~26%) reported in other bahiagrass and rhizoma peanut studies to a soil bacterial community dominated by Firmicutes (39%) in our study. The relative abundance of the bacterial genus Crossiella, known for its antimicrobial traits, was enhanced in the presence of RP. Differences in soil bacterial diversity and community composition were substantial between 0 to 15 and 15 to 30 cm soil layers, with N2-fixing bacteria belonging to the phylum Proteobacteria concentrated in 0 to 15 cm. Introducing RP into bahiagrass pastures is a highly sustainable alternative to mineral N fertilizer inputs. Our results provide evidence that this system also promotes greater soil microbial diversity and is associated with unique taxa that require further study to better understand their contributions to healthy pastures.

Immune landscape and regulatory mechanisms in human atherosclerotic coronary plaques: Evidence from single-cell and bulk transcriptomics.

Atherosclerosis is a chronic immuno-inflammatory disease, however, the immune landscape and regulatory mechanisms have not been clear. We detected seven principal immune cell clusters with distinct phenotypic and spatial characteristics using single-cell RNA-sequencing of aortic immune cells from patients with acute coronary syndrome and stable angina pectoris. Then we acquired 265 differentially expressed immune-related genes and the high scores were mainly found in T cells and monocytes, which were differentially regulated in atherosclerotic coronary plaques. The CCL signaling pathway was the most relevant pattern in the T cells and CCL5-CCR1 and CCL5-CCR5 ligand-receptor pairs played a vital role in the CCL signaling pathway. Further comparative analysis indicated MCH-I signaling was the most relevant pattern in the T cells and HLA ligand-related ligand-receptor pairs played a vital role. Functional analysis of the single-cell and bulk transcriptomics pointed to multiple pathways, such as antigen presentation and immune response. Nineteen common differentially expressed immune-related genes were found in both immune cells and the human peripheral blood mononuclear cells. Nine common differentially expressed transcription factors were differentially expressed in both T cell and monocyte clusters from the coronary plaques and human peripheral blood mononuclear cells and the network demonstrated that CEBPB might play an essential role in the transcriptional regulation of atherosclerosis as a hub transcription factor. The definition of immune cell diversity and heterogeneity by single-cell level analysis of aortic immune cell subsets not only unveils cell-type-specific pathways and new immune mechanisms but also discovers the functional correlation of immune cells in human atherosclerosis. Our findings provide great promise for the discovery of novel molecular mechanisms and precise therapeutic targets for atherosclerosis.

Soil Bacterial Communities Across Seven Rhizoma Peanut Cultivars (Arachis glabrata Benth.) Respond to Seasonal Variation.

Soil microorganisms play key roles in soil nutrient transformations and have a notable effect on plant growth and health. Different plant genotypes can shape soil microbial patterns via the secretion of root exudates and volatiles, but it is uncertain how a difference in soil microorganisms induced by crop cultivars will respond to short-term seasonal variations. A field experiment was conducted to assess the changes in soil bacterial communities of seven rhizoma peanut (Arachis glabrata Benth, RP) cultivars across two growing seasons, April (Spring season) and October (Fall season). Soils' bacterial communities were targeted using 16S rRNA gene amplicon sequencing. Bacterial community diversity and taxonomic composition among rhizoma peanut cultivars were significantly affected by seasons, cultivars, and their interactions (p < 0.05). Alpha diversity, as estimated by the OTU richness and Simpson index, was around onefold decrease in October than in April across most of the RP cultivars, while the soils from Arblick and Latitude had around one time higher alpha diversity in both seasons compared with other cultivars. Beta diversity differed significantly in April (R = 0.073, p < 0.01) and October (R = 0.084, p < 0.01) across seven cultivars. Bacterial dominant taxa (at phylum and genus level) were strongly affected by seasons and varied towards more dominant groups that have functional potentials involved in nutrient cycling from April to October. A large shift in water availability induced by season variations in addition to host cultivar's effects can explain the observed patterns in diversity, composition, and co-occurrence of bacterial taxa. Overall, our results demonstrate an overriding effect of short-term seasonal variations on soil bacterial communities associated with different crop cultivars. The findings suggest that season-induced shifts in environmental conditions could exert stronger impacts on soil microorganisms than the finer-scale rhizosphere effect from crop cultivars, and consequently influence largely microbe-mediated soil processes and crop health in agricultural ecosystems.

Genomic determination of breeding systems and trans-specific evolution of HD MAT genes in suilloid fungi.

Studying the signatures of evolution can help to understand genetic processes. Here, we demonstrate how the existence of balancing selection can be used to identify the breeding systems of fungi from genomic data. The breeding systems of fungi are controlled by self-incompatibility loci that determine mating types between potential mating partners, resulting in strong balancing selection at the loci. Within the fungal phylum Basidiomycota, two such self-incompatibility loci, namely HD MAT locus and P/R MAT locus, control mating types of gametes. Loss of function at one or both MAT loci results in different breeding systems and relaxes the MAT locus from balancing selection. By investigating the signatures of balancing selection at MAT loci, one can infer a species' breeding system without culture-based studies. Nevertheless, the extreme sequence divergence among MAT alleles imposes challenges for retrieving full variants from both alleles when using the conventional read-mapping method. Therefore, we employed a combination of read-mapping and local de novo assembly to construct haplotypes of HD MAT alleles from genomes in suilloid fungi (genera Suillus and Rhizopogon). Genealogy and pairwise divergence of HD MAT alleles showed that the origins of mating types predate the split between these two closely related genera. High sequence divergence, trans-specific polymorphism, and the deeply diverging genealogy confirm the long-term functionality and multiallelic status of HD MAT locus in suilloid fungi. This work highlights a genomics approach to studying breeding systems regardless of the culturability of organisms based on the interplay between evolution and genetics.

Zhilong Huoxue Tongyu Capsule attenuates hemorrhagic transformation through the let-7f/TLR4 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Hemorrhagic transformation after acute ischemic stroke is a life-threatening disease that currently has no effective chemotherapy. Zhilong Huoxue Tongyu Capsule (ZL) is an empirical prescription of traditional Chinese medicine that is used to prevent and treat cardiovascular and cerebrovascular diseases in China. However, only a few studies have addressed the mechanisms of ZL in treating hemorrhagic transformation. AIM OF THE STUDY: To evaluate the anti-inflammatory effects of ZL on hemorrhagic transformation model rats and lipopolysaccharide (LPS)-induced RAW264.7 macrophages and to explore the underlying molecular mechanisms. MATERIALS AND METHODS: Murine RAW264.7 cells were treated with ZL and LPS (1 µg/mL), and cell viability was detected by cell counting kit-8 assay. RT-qPCR was used to detect the expression of inflammatory chemokines, microRNA let-7a/e/i/f, toll like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor kappa-B (NF-κB) p65. The protein expression levels of TLR4, MyD88, NF-κB p65, and apoptosis related molecules were determined by Western blotting. The apoptosis rate of RAW264.7 macrophages was detected by Annexin V-FITC/PI double staining. A hemorrhagic transformation model in rats was established by intraperitoneal injection of high glucose solution combined with thread embolization. Then, the model rats were observed behaviourally, pathologically, and molecularly. The gene expression of TLR4, MyD88, and NF-κB p65 was measured by RT-qPCR and used to evaluate the protective effect of ZL against hemorrhagic transformation in rats. RESULTS: ZL (5, 20, 40 µg/mL) was beneficial in cell proliferation. LPS (1 µg/mL) stimulated the production of inflammatory chemokines and inhibited the production of let-7a/e/i/f, with let-7f being influenced most strongly. Moreover, overexpression of let-7f decreased the gene and protein levels of TLR4, MyD88, and NF-κB p65, downregulated TLR4, and inhibited its transcriptional activity. ZL (5, 20, and 40 µg·mL-1) inhibited the production of TLR4, MyD88, and NF-κB p65 and promoted the production of let-7f in a concentration-dependent manner. Furthermore, the blockade of TLR4 antagonized the promoting effects of TLR4 pathway activation in cell inflammation and apoptosis by downregulating let-7f. Critically, it was confirmed in vivo and in vitro that ZL upregulated the expression of let-7f and inhibited the gene expression of TLR4, MyD88, and NF-κB p65 to reduce inflammatory cell infiltration, which determined the occurrence of hemorrhagic transformation. CONCLUSIONS: ZL can reduce inflammatory response by upregulating let-7f and subsequently inhibiting the TLR4 signaling pathway, thereby decreasing the occurrence of hemorrhagic transformation.

Microbial community dynamics responding to nutrient allocation associated with soybean cultivar 'Jake' ozone adaptation.

Tropospheric ozone (O3), a major air pollutant, leads to significant global yield loss in soybean [Glycine max (L.) Merr.]. Soybean cultivar 'Jake' shows O3 resilient traits in above-ground organs, but the root system remains sensitive to elevated O3 (eO3). Changing carbon (C) and nitrogen (N) resource composition during eO3 stress suggests that eO3 presumably alters belowground soil microbial communities and their driven nutrient transformation. Yet, the responses of belowground microbes to eO3 and their feedback on nutrient cycling in 'Jake' are unknown. In this study, we holistically investigated soil microbial communities associated with C and N dynamics and bacterial-fungal inter-kingdom networks in the rhizosphere and bulk soil at different developmental stages of 'Jake' grown under sub-ambient O3 [charcoal-filtered (CF) air, 12 h mean: 20 ppb] or eO3 (12 h mean: 87 ppb). The results demonstrated eO3 significantly decreased fungal diversity and complexity of microbial networks at different 'Jake' developmental stages, whereas bacterial diversity was more tolerant to eO3 in both bulk soil and rhizosphere. In the bulk soil, no O3-responsive microbial biomarkers were found to be associated with C and N content, implying eO3 may stimulate niche-based processes during 'Jake' growth. In contrast, this study identified O3-responsive microbial biomarkers that may contribute to the N acquisition (Chloroflexales) and C dynamics (Caldilineales, Thermomicrobiales, and Hypocreales) in the rhizosphere, which may support the O3 resilience of the 'Jake' cultivar. However, further investigation is required to confirm their specific contributions by determining changes in microbial gene expression. Overall, these findings conduce to an expanding knowledge base that O3 induces temporal and spatial changes in the effects of microbial and nutrient networks in the O3-tolerant agriculture ecosystems.

Direct and indirect pathways of land management effects on wetland plant litter decomposition.

Litter decomposition is a fundamental process underpinning multiple ecosystem services. Despite a long history of research on decomposition, direct and indirect effects of multiple interactive land management on wetland decomposition yet remain less well understood. Here, we used a long-term whole-ecosystem wetland experiment in south-central Florida to investigate interactive effects of land-use intensification, cattle grazing and prescribed fire on in situ wetland plant litter decomposition. We further examined the direct and indirect pathways of land management effects on litter decomposition through changes in associated litter traits, soil properties, and soil microbial attributes using structural equation models. We used the litterbag technique that quantifies decomposition rates (k-values) and recalcitrant fractions (A-values). Our results showed that land-use intensification increased k-values in ungrazed wetlands and decreased k-values in grazed wetlands, but consistently reduced A-values regardless of other treatments. Prescribed fire individually suppressed litter decomposition by reducing k and increasing A. Further, these effects occurred through altering litter, soil, and microbial properties. Our results revealed that litter traits and soil properties were the first two strongest factors in determining wetland decomposition processes. Particularly, litter P and Mg contents and soil P and K contents were the best predictors for k, while litter Ca and lignin contents and soil pH, N and water content best predicted A. Moreover, microbial traits exhibited interactive effects with litter and soil properties to affect wetland litter decomposition. Our research suggests that cattle grazing could buffer against stimulating effect of land-use intensification on decomposition rates and thus avoid nutrient releases pulses. Our study further indicates that land-use intensification and fire suppression in subtropical wetlands could promote organic matter depletion and thus nutrient loss, highlighting the need to reduce anthropogenic disturbances to natural wetlands to maintain their capacity for providing associated regulating and supporting services.

Effects of Grass-Based Crop Rotation, Nematicide, and Irrigation on the Nematode Community in Cotton.

Plant-parasitic and free-living nematodes - bacterivores, fungivores, omnivores, predators - comprise the nematode community. Nematicide application and crop rotation are important tools to manage plant-parasitic nematodes, but effects on free-living nematodes and nematode ecological indices need further study. The nematicide fluopyram was recently introduced in cotton (Gossypium hirsutum) production and its effects on the nematode community need assessment. This research was conducted in 2017 and 2018 at a long-term field site in Quincy, FL where perennial grass/sod-based (bahiagrass, Paspalum notatum) and conventional cotton rotations were established in 2000. The objective of this research was to evaluate the effects of fluopyram nematicide, crop rotation phase, and irrigation on free-living nematodes and nematode ecological indices based on three soil sampling dates each season. We did not observe consistent effects of crop rotation phase on free-living nematodes or nematode ecological indices. Only omnivores were consistently negatively impacted by fluopyram. Nematode ecological indices reflected this negative effect by exhibiting a degraded/ stressed environmental condition relative to untreated plots. Free-living nematodes were not negatively impacted by nematicide when sod-based rotation was used.

Leaf Mycobiome and Mycotoxin Profile of Warm-Season Grasses Structured by Plant Species, Geography, and Apparent Black-Stroma Fungal Structure.

Grasses harbor diverse fungi, including some that produce mycotoxins or other secondary metabolites. Recently, Florida cattle farmers reported cattle illness, while the cattle were grazing on warm-season grass pastures, that was not attributable to common causes, such as nutritional imbalances or nitrate toxicity. To understand correlations between grass mycobiome and mycotoxin production, we investigated the mycobiomes associated with five prominent, perennial forage and weed grasses [Paspalum notatum Flügge, Cynodon dactylon (L.) Pers., Paspalum nicorae Parodi, Sporobolus indicus (L.) R. Br., and Andropogon virginicus (L.)] collected from six Florida pastures actively grazed by livestock. Black fungal stromata of Myriogenospora and Balansia were observed on P. notatum and S. indicus leaves and were investigated. High-throughput amplicon sequencing was applied to delineate leaf mycobiomes. Mycotoxins from P. notatum leaves were inspected using liquid chromatography-mass spectrometry (LC-MS/MS). Grass species, cultivars, and geographic localities interactively affected fungal community assemblies of asymptomatic leaves. Among the grass species, the greatest fungal richness was detected in the weed S. indicus. The black fungal structures of P. notatum leaves were dominated by the genus Myriogenospora, while those of S. indicus were codominated by the genus Balansia and a hypermycoparasitic fungus of the genus Clonostachys. When comparing mycotoxins detected in P. notatum leaves with and without M. atramentosa, emodin, an anthraquinone, was the only compound which was significantly different (P < 0.05). Understanding the leaf mycobiome and the mycotoxins it may produce in warm-season grasses has important implications for how these associations lead to secondary metabolite production and their subsequent impact on animal health. IMPORTANCE The leaf mycobiome of forage grasses can have a major impact on their mycotoxin contents of forage and subsequently affect livestock health. Despite the importance of the cattle industry in warm-climate regions, such as Florida, studies have been primarily limited to temperate forage systems. Our study provides a holistic view of leaf fungi considering epibiotic, endophytic, and hypermycoparasitic associations with five perennial, warm-season forage and weed grasses. We highlight that plant identity and geographic location interactively affect leaf fungal community composition. Yeasts appeared to be an overlooked fungal group in healthy forage mycobiomes. Furthermore, we detected high emodin quantities in the leaves of a widely planted forage species (P. notatum) whenever epibiotic fungi occurred. Our study demonstrated the importance of identifying fungal communities, ecological roles, and secondary metabolites in perennial, warm-season grasses and their potential for interfering with livestock health.

Mechanisms of stress tolerance and their effects on the ecology and evolution of mycorrhizal fungi.

Stress is ubiquitous and disrupts homeostasis, leading to damage, decreased fitness, and even death. Like other organisms, mycorrhizal fungi evolved mechanisms for stress tolerance that allow them to persist or even thrive under environmental stress. Such mechanisms can also protect their obligate plant partners, contributing to their health and survival under hostile conditions. Here we review the effects of stress and mechanisms of stress response in mycorrhizal fungi. We cover molecular and cellular aspects of stress and how stress impacts individual fitness, physiology, growth, reproduction, and interactions with plant partners, along with how some fungi evolved to tolerate hostile environmental conditions. We also address how stress and stress tolerance can lead to adaptation and have cascading effects on population- and community-level diversity. We argue that mycorrhizal fungal stress tolerance can strongly shape not only fungal and plant physiology, but also their ecology and evolution. We conclude by pointing out knowledge gaps and important future research directions required for both fully understanding stress tolerance in the mycorrhizal context and addressing ongoing environmental change.

Microbial Diversity and Contribution to the Formation of Volatile Compounds during Fine-Flavor Cacao Bean Fermentation.

Cacao demand is continuously increasing, and variations in cacao prices have been associated with the aroma of fermented cacao beans. However, the role of microorganisms in the formation of volatile-aroma compounds during fermentation remains unclear. Microbial diversity in Nacional × Trinitario cacao was characterized during spontaneous fermentation by using culture-based methods and next-generation sequencing (NGS) of DNA amplicons. Cacao beans that were spontaneously fermented for 0, 24, 48, 72 and 96 h were UV-sterilized prior to the inoculation of the microbial isolates obtained by the culture-based methods. The volatile formation in inoculated cacao beans was evaluated by GC-MS. The species isolated during fermentation included yeast, such as Saccharomyces cerevisiae and Candida metapsilosis; lactic acid bacteria (LAB), such as Limosilactobacillus fermentum and Liquorilactobacillus nagelii; acetic acid bacteria (AAB), such as Acetobacter pasteurianus, Acetobacter ghanensis and Acetobacter syzygii, as well as other species, such as Bacillus subtilis and Bacillus amyloliquefaciens. Additionally, NGS revealed an abundance of environmental microorganisms, including Escherichia spp., Pantoea spp., Staphylococcus spp., Botrytis spp., Tetrapisispora spp. and Pichia spp., among others. During the lab-scale fermentation, the inoculation of S. cerevisiae mostly yielded alcohols, while LAB and AAB produced volatiles associated with floral, almond and fruity notes throughout the fermentation, but AAB also produced acetic acid with a sour aroma. Similarly, the inoculation of C. metapsilosis and Bacillus spp. in 96 h fermented cacao beans yielded esters with floral aromas. This is the first report describing the role of microorganisms in volatile formation during fine-flavor cacao fermentation.

Comparative transcriptomics of fungal endophytes in co-culture with their moss host Dicranum scoparium reveals fungal trophic lability and moss unchanged to slightly increased growth rates.

Mosses harbor fungi whose interactions within their hosts remain largely unexplored. Trophic ranges of fungal endophytes from the moss Dicranum scoparium were hypothesized to encompass saprotrophism. This moss is an ideal host to study fungal trophic lability because of its natural senescence gradient, and because it can be grown axenically. Dicranum scoparium was co-cultured with each of eight endophytic fungi isolated from naturally occurring D. scoparium. Moss growth rates, and gene expression levels (RNA sequencing) of fungi and D. scoparium, were compared between axenic and co-culture treatments. Functional lability of two fungal endophytes was tested by comparing their RNA expression levels when colonizing living vs dead gametophytes. Growth rates of D. scoparium were unchanged, or increased, when in co-culture. One fungal isolate (Hyaloscyphaceae sp.) that promoted moss growth was associated with differential expression of auxin-related genes. When grown with living vs dead gametophytes, Coniochaeta sp. switched from having upregulated carbohydrate transporter activity to upregulated oxidation-based degradation, suggesting an endophytism to saprotrophism transition. However, no such transition was detected for Hyaloscyphaceae sp. Individually, fungal endophytes did not negatively impact growth rates of D. scoparium. Our results support the long-standing hypothesis that some fungal endophytes can switch to saprotrophism.

Soil bacterial community response to rhizoma peanut incorporation into Florida pastures.

Incorporating legumes is one option for improving pasture fertility, sustainability, and biodiversity. Diazotrophic microorganisms, including rhizobia that form symbioses with legumes, represent a small fraction of the total soil microbial community. Yet, they can offset nitrogen (N) fertilizer inputs through their ability to convert atmospheric N2 into plant-usable N via biological N2 fixation (BNF). This study used amplicon sequencing of 16S rRNA genes to investigate soil bacterial community composition and diversity in grazed 'Argentine' bahiagrass (Paspalum notatum Flügge) pastures where N fertilizer was supplanted with legume-derived N from BNF in some treatments. Treatments consisted of bahiagrass fertilized with (a) mineral N (224 kg N ha-1  yr-1 ), (b) combination mineral N (34 kg N ha-1  yr-1 ) and legume-derived N via cool-season clover (CSC) (Trifolium spp.) mix, or (c) combination mineral N (34 kg N ha-1  yr-1 ) and legume-derived N via CSC mix and strips of Ecoturf rhizoma peanut (Arachis glabrata Benth.). Bradyrhizobium spp. relative abundance was 44% greater in the mixed pasture. Other bacterial genera with BNF or denitrification potentials were greater in pastures with legumes, whereas sequences assigned to genera associated with high litter turnover were greater in bahiagrass pastures receiving only mineral N. Soil bacteria alpha diversity was greater in pastures receiving 34 kg ha-1  yr-1 N fertilizer application and the CSC mix than in pastures with the CSC mix and rhizoma peanut strips. Our results demonstrate soil microbial community shifts that may affect soil C and N cycling in pastures common to the southeastern United States.