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Diazotrophic bacteria can reduce atmospheric nitrogen into ammonia enabling bioavailability of the essential element. Many diazotrophs closely associate with plant roots increasing nitrogen availability, acting as plant growth promoters. These associations have the potential to reduce the need for costly synthetic fertilizers if they could be engineered for agricultural applications. However, despite the importance of diazotrophic bacteria, genetic tools are poorly developed in a limited number of species, in turn narrowing the crops and root microbiomes that can be targeted. Here we report optimized protocols and plasmids to manipulate phylogenetically diverse diazotrophs with the goal of enabling synthetic biology and genetic engineering. Three broad-host-range plasmids can be used across multiple diazotrophs, with the identification of one specific plasmid (containing origin of replication RK2 and a kanamycin resistance marker) showing the highest degree of compatibility across bacteria tested. We then demonstrated modular expression by testing seven promoters and eleven ribosomal binding sites using proxy fluorescent proteins. Finally, we tested four small molecule inducible systems to report expression in three diazotrophs and demonstrated genome editing in Klebsiella michiganensis M5al.

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Females and males of dioecious plants have evolved sex-specific characteristics in terms of their morphological and physiological properties. However, little is known about the difference in rhizosphere microbes in dioecious plants. In this study, we used amplicon sequencing to analyze the differences in rhizosphere microbial diversity and community composition of males and females of dioecious Hippophae tibetana at different habitats, and their key factors in driving the differences were investigated. The results showed that there were differences in the diversity, community composition, and connectivity and complexity of the co-occurrence network of rhizosphere microbes between females and males of the dioecious H. tibetana at different habitats. Zoopagomycota is a unique phylum of rhizosphere fungi in the males of the dioecious H. tibetana, while Dependentiae is a unique phylum of rhizosphere bacteria in the females of the dioecious H. tibetana. Linear discriminant analysis effect size (LEfSe) analysis indicated significant enrichment of species at different levels, suggesting that these species could be potential biomarkers for females and males of H. tibetana. Spearman's analysis showed that the dominant genera of rhizosphere fungi were significantly positively correlated with soil physicochemical properties (total nitrogen and phosphorus; organic matter; available phosphorus, potassium and nitrogen; salt content; water content). PICRUSt and FUNGuild predictive analysis indicated that the function of rhizosphere fungi was different between females and males of the dioecious H. tibetana at different habitats, while metabolites were the dominant functions of rhizosphere bacteria in all samples. These results highlighted the sexual discrimination of rhizosphere microbes on the dioecious plants and provided important knowledge for females and males of the dioecious plant-microbe interaction.IMPORTANCEThis study explores the differences in rhizosphere microbes of dioecious Hippophae tibetana at different habitats and their key factors in driving the differences. Through employing amplicon sequencing techniques, we found that rhizosphere microbial communities and diversity were different between females and males of the dioecious H. tibetana at different habitats, and there notably existed unique phylum and potential biomarkers of rhizosphere microbes between females and males of the dioecious H. tibetana. Rhizosphere fungi were significantly positively correlated with soil physicochemical properties. This study reveals the differences in rhizosphere microbes of dioecious H. tibetana at different habitats and driving factors; it also contributes to our understanding of the dioecious plant-microbe interaction.

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Interactions between plants and soil microbial communities that benefit plant growth and enhance nutrient acquisition are driven by the selective release of metabolites from plant roots, or root exudation. To investigate these plant-microbe interactions, we developed a photoaffinity probe based on sorgoleone (sorgoleone diazirine alkyne for photoaffinity labeling, SoDA-PAL), a hydrophobic secondary metabolite and allelochemical produced in Sorghum bicolor root exudates. We applied SoDA-PAL to the identification of sorgoleone-binding proteins in Acinetobacter pittii SO1, a potential plant growth-promoting microbe isolated from sorghum rhizosphere soil. Competitive photoaffinity labeling of A. pittii whole cell lysates with SoDA-PAL identified 137 statistically enriched proteins, including putative transporters, transcriptional regulators, and a subset of proteins with predicted enzymatic functions. We performed computational protein modeling and docking with sorgoleone to prioritize candidates for experimental validation and then confirmed binding of sorgoleone to four of these proteins in vitro: the α/ß fold hydrolase SrgB (OH685_09420), a fumarylacetoacetase (OH685_02300), a lysophospholipase (OH685_14215), and an unannotated hypothetical protein (OH685_18625). Our application of this specialized sorgoleone-based probe coupled with structural bioinformatics streamlines the identification of microbial proteins involved in metabolite recognition, metabolism, and toxicity, widening our understanding of the range of cellular pathways that can be affected by a plant secondary metabolite.IMPORTANCEHere, we demonstrate that a photoaffinity-based chemical probe modeled after sorgoleone, an important secondary metabolite released by sorghum roots, can be used to identify microbial proteins that directly interact with sorgoleone. We applied this probe to the sorghum-associated bacterium Acinetobacter pittii and showed that probe labeling is dose-dependent and sensitive to competition with purified sorgoleone. Coupling the probe with proteomics and computational analysis facilitated the identification of putative sorgoleone binders, including a protein implicated in a conserved pathway essential for sorgoleone catabolism. We anticipate that discoveries seeded by this workflow will expand our understanding of the molecular mechanisms by which specific metabolites in root exudates shape the sorghum rhizosphere microbiome.

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Introduction: Microorganisms exhibit intricate interconnections with tea plants; however, despite the well-established role of microorganisms in crop growth and development, research on microbes within the tea plant remains insufficient, particularly regarding endophytic microorganisms. Methods: In this study, we collected samples of leaves and rhizosphere soils from 'Zhuyeqi', 'Baojing Huangjincha#1', 'Baiye#1', and 'Jinxuan' varieties planted. Results: Our analyses revealed significant variations in tea polyphenol contents among tea varieties, particularly with the 'Zhuyeqi' variety exhibiting higher levels of tea polyphenols (>20% contents). Microbiome studies have revealed that endophytic microbial community in tea plants exhibited higher host specificity compared to rhizospheric microbial community. Analyses of across-ecological niches of the microbial community associated with tea plants revealed that soil bacteria serve as a significant reservoir for endophytic bacteria in tea plants, Bacillus may play a crucial role in shaping the bacterial community across-ecological niche within the tea plants with higher tea polyphenol levels. In the aforementioned analyses, the microbial community of 'Zhuyeqi' exhibited a higher degree of host specificity for leaf endophytic microorganisms, the topological structure of the co-occurrence network is also more intricate, harboring a greater number of potential core microorganisms within its nodes. A closer examination was conducted on the microbial community of 'Zhuyeqi', further analyses of its endophytic bacteria indicated that its endophytic microbial community harbored a greater abundance of biomarkers, particularly among bacteria, and the enriched Methylobacterium and Sphingomonas in 'Zhuyeqi' may play distinct roles in disease resistance and drought resilience in tea plants. Conclusion: In summary, this study has shed light on the intricate relationships of tea plant varieties with their associated microbial communities, unveiling the importance of microorganisms and tea varieties with higher tea polyphenols, and offering valuable insights to the study of microorganisms and tea plants.

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Capsaicinoids are produced uniquely in pepper fruits, and its level determines the commercial quality and health-promoting properties of pepper. So, it is particularly important to increase capsaicinoids content in pepper. Rhizosphere microbiota is critical to plant growth and performance, and affected by plant varieties. However, the impact of pepper varieties with different capsaicinoids yields on the rhizosphere microbiota is poorly understood. Using high-throughput sequencing of the 16S rRNA and internal transcribed spacer (ITS) region, we investigated the rhizosphere microbial community among five pepper varieties containing different capsaicinoids. Our results demonstrated that pepper variety significantly influenced the diversity and structure of rhizosphere microbial community. Bacterial diversity in varieties with high capsaicinoids content was significantly higher than in varieties with low capsaicinoids content, while fungal diversity was opposite to bacterial diversity. The correlation analysis revealed that 19 dominant bacterial genera (e.g., Chujaibacter, Rhodanobacter, and Gemmatimonas) were significantly correlated with capsaicinoids content, and nine of them were also significantly associated with soil nutrients, whereas only one fungal genus (Podospora) was significantly correlated with capsaicinoids content. Additionally, almost all genera which significantly correlated to capsaicinoids content were biomarkers of the five pepper varieties and the correlation was well corresponding to the capsaicinoids content. Overall, our results confirmed that the variety of pepper significantly affected the rhizosphere microbial community in the fields, and bacteria and fungi responded differently to capsaicinoids, which may affect the biosynthesis of capsaicinoids and contribute to further improvement of capsaicinoids production in pepper fruits.

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Introduction: Despite their adverse environmental effects, modern agriculture relies heavily on agrochemicals to manage diseases and pests and enhance plant growth and productivity. Some of these functions could instead be fulfilled by endophytes from the plant microbiota, which have diverse activities beneficial for plant growth and health. Methods: We therefore used a microbiome-guided top-down approach to select ten bacterial strains from different taxa in the core microbiome of tomato plants in the production chain for evaluation as potential bioinoculants. High-quality genomes for each strain were obtained using Oxford Nanopore long-read and Illumina short-read sequencing, enabling the dissection of their genetic makeup to identify phyto-beneficial traits. Results: Bacterial strains included both taxa commonly used as biofertilizers and biocontrol agents (i.e. Pseudomonas and Bacillus) as well as the less studied genera Leclercia, Chryseobacterium, Glutamicibacter, and Paenarthorbacter. When inoculated in the tomato rhizosphere, these strains promoted plant growth and reduced the severity of Fusarium Crown and Root Rot and Bacterial Spot infections. Genome analysis yielded a comprehensive inventory of genes from each strain related to processes including colonization, biofertilization, phytohormones, and plant signaling. Traits directly relevant to fertilization including phosphate solubilization and acquisition of nitrogen and iron were also identified. Moreover, the strains carried several functional genes putatively involved in abiotic stress alleviation and biotic stress management, traits that indirectly foster plant health and growth. Discussion: This study employs a top-down approach to identify new plant growth-promoting rhizobacteria (PGPRs), offering an alternative to the conventional bottom-up strategy. This method goes beyond the traditional screening of the strains and thus can expand the range of potential bioinoculants available for market application, paving the way to the use of new still underexplored genera.

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Microbial communities in desert riparian forest ecosystems have developed unique adaptive strategies to thrive in harsh habitats shaped by prolonged exposure to abiotic stressors. However, the influence of drought stress on the functional and metabolic characteristics of soil rhizosphere microorganisms remains unknown. Therefore, this study aimed to investigate the effects of drought stress on soil biogeochemistry and metabolism and analyze the relationship between the biogeochemical cycle processes and network of differentially-expressed metabolites. Using metagenomics and metabolomics, this study explored the microbial functional cycle and differential metabolic pathways within desert riparian forests. The predominant biogeochemical cycles in the study area were the Carbon and Nitrogen cycles, comprising 78.90 % of C, N, Phosphorus, Sulfur and Iron cycles. Drought led to increased soil C fixation, reduced C degradation and methane metabolism, weakened denitrification, and decreased N fixation. Furthermore, drought can disrupt iron homeostasis and reduce its absorption. The differential metabolic pathways of drought stress include flavonoid biosynthesis, arachidonic acid metabolism, steroid hormone biosynthesis, and starch and sucrose degradation. Network analysis of functional genes and metabolism revealed a pronounced competitive relationship between the C cycle and metabolic network, whereas the Fe cycle and metabolic network promoted each other, optimizing resource utilization. Partial least squares analysis revealed that drought hindered the expression and metabolic processes and functional genes, whereas the rhizosphere environment facilitated metabolic expression and the functional genes. The rhizosphere effect primarily promoted metabolic processes indirectly through soil enzyme activities. The integrated multi-omics analysis further revealed that the effects of drought and the rhizosphere play a predominant role in shaping soil functional potential and the accumulation of metabolites. These insights deepen our comprehension of desert riparian forest ecosystems and offer strong support for the functionality of nutrient cycling and metabolite dynamics.

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BACKGROUND: Within the plant kingdom, there is an exceptional amount of chemical diversity that has yet to be annotated. It is for this reason that non-targeted analysis is of interest for those working in novel natural products. To increase the number and diversity of compounds observable in root exudate extracts, several workflows which differ at three key stages were compared: 1) sample extraction, 2) chromatography, and 3) data preprocessing. RESULTS: Plants were grown in Hoagland's solution for two weeks, and exudates were initially extracted with water, followed by a 24-h regeneration period with subsequent extraction using methanol. Utilizing the second extraction showed improved results with less ion suppression and reduced retention time shifting compared to the first extraction. A single column method, utilizing a pentafluorophenyl column, paired with high-resolution mass spectrometry ionized and correctly identified 34 mock root exudate compounds, while the dual column method, incorporating a pentafluorophenyl column and a porous graphitic carbon column, retained and identified 43 compounds. In a pooled quality control sample of exudate extracts, the single column method detected 1,444 compounds. While the dual method detected fewer compounds overall (1,050), it revealed a larger number of small polar compounds. Three preprocessing methods (targeted, proprietary, and open source) successfully identified 43, 31, and 38 mock root exudate compounds to confidence level 1, respectively. SIGNIFICANCE: Enhancing signal strength and analytical method stability involves removing the high ionic strength nutrient solution before sampling root exudate extracts. Despite signal intensity loss, a dual column method enhances compound coverage, particularly for small polar metabolites. Open-source software proves a viable alternative for non-targeted analysis, even surpassing proprietary software in peak picking.

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This study investigates the diversity and composition of soil bacterial communities in the rhizosphere of Attapadi and Nelliyampathy, prominent hill stations in Palakkad district, Kerala, India. The persistent flooding and landslides in 2018 and 2019 significantly impacted agricultural productivity in these regions. Utilizing high-throughput 16S rRNA gene sequencing (Illumina MiSeq), we conducted a comprehensive analysis of soil samples. Correlative assessments between soil parameters and microbial relative abundance at the phylum level revealed noteworthy positive associations. Notably, nitrogen (N) exhibited a positive relation with Crenarchaeota, Chloroflexi, Actinobacteriota, and Acidobacteriota; pH correlated with Firmicutes; organic carbon (OC) with WPS-2; and phosphorous with Proteobacteria. A total of 31,402 operational taxonomic units (OTUs) were identified, with the highest feature counts observed in undisturbed soils from Attapadi (AUD) and Nelliyampathy (NUD) (13,007 and 12,915, respectively). Disturbed soils in Nelliyampathy (ND) and Attapadi (AD) displayed a substantial decline in microbial diversity and composition, harbouring 1409 and 4071 OTUs, respectively. Alpha and beta diversity indices further underscored the more severe impairment of ND soils compared to AD soils. Interestingly, a majority of ND samples were landslide-affected (four out of five), while flood-affected soils accounted for four out of six AD samples. This indicates that landslides exert a more pronounced impact on microbial diversity and composition than floods. The observed decline in microbial count, composition, and diversity, even after 2 years of the disaster, raises concerns about potential threats to agricultural output. The findings emphasize the need for corrective measures, including the incorporation of microbial inoculum, to restore soil fertility in post-disaster landscapes.

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The rhizosphere, the soil region influenced by plant roots, represents a dynamic microenvironment where intricate interactions between plants and microorganisms shape soil health, nutrient cycling, and plant growth. Soil microorganisms are integral players in the transformation of materials, the dynamics of energy flows, and the intricate cycles of biogeochemistry. Considerable research has been dedicated to investigating the abundance, diversity, and intricacies of interactions among different microbes, as well as the relationships between plants and microbes present in the rhizosphere. Metagenomics, a powerful suite of techniques, has emerged as a transformative tool for dissecting the genetic repertoire of complex microbial communities inhabiting the rhizosphere. The review systematically navigates through various metagenomic approaches, ranging from shotgun metagenomics, enabling unbiased analysis of entire microbial genomes, to targeted sequencing of the 16S rRNA gene for taxonomic profiling. Each approach's strengths and limitations are critically evaluated, providing researchers with a nuanced understanding of their applicability in different research contexts. A central focus of the review lies in the practical applications of rhizosphere metagenomics in various fields including agriculture. By decoding the genomic content of rhizospheric microbes, researchers gain insights into their functional roles in nutrient acquisition, disease suppression, and overall plant health. The review also addresses the broader implications of metagenomic studies in advancing our understanding of microbial diversity and community dynamics in the rhizosphere. It serves as a comprehensive guide for researchers, agronomists, and policymakers, offering a roadmap for harnessing metagenomic approaches to unlock the full potential of the rhizosphere microbiome in promoting sustainable agriculture.

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Introduction: Tobacco root-knot nematode (TRKN) disease is a soil-borne disease that presents a major hazard to the cultivation of tobacco, causing significant reduction in crop quality and yield, and affecting soil microbial diversity and metabolites. However, differences in rhizosphere soil microbial communities and metabolites between healthy tobacco soils and tobacco soils with varying degrees of TRKN infection remain unclear. Methods: In this study, diseased rhizosphere soils of tobacco infected with different degrees of TRKN [severally diseased (DH) soils, moderately diseased (DM) soils, and mildly diseased (DL) soils] and healthy (H) rhizosphere soils were collected. Here, we combined microbiology with metabolomics to investigate changes in rhizosphere microbial communities and metabolism in healthy and TRKN-infected tobacco using high-throughput sequencing and LC-MS/MS platforms. Results: The results showed that the Chao1 and Shannon indices of bacterial communities in moderately and mildly diseased soils were significantly higher than healthy soils. The Proteobacteria, Actinobacteria, Ascomycota, Burkholderia, Bradyrhizobium and Dyella were enriched in the rhizosphere soil of healthy tobacco. Basidiomycota, Agaricales, Pseudeurotiaceae and Ralstonia were enriched in severally diseased soils. Besides, healthy soils exhibited a relatively complex and interconnected network of bacterial molecular ecologies, while in severally and moderately diseased soils the fungal molecular networks are relatively complex. Redundancy analysis showed that total nitrogen, nitrate nitrogen, available phosphorus, significantly affected the changes in microbial communities. In addition, metabolomics results indicated that rhizosphere soil metabolites were significantly altered after tobacco plants were infected with TRKNs. The relative abundance of organic acids was higher in severally diseased soils. Spearman's analyses showed that oleic acid, C16 sphinganine, 16-hydroxyhexadecanoic acid, D-erythro-3-methylmalate were positively correlated with Basidiomycota, Agaricales, Ralstonia. Discussion: In conclusion, this study revealed the relationship between different levels of TRKN invasion of tobacco root systems with bacteria, fungi, metabolites and soil environmental factors, and provides a theoretical basis for the biological control of TRKN disease.

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The gut microbiome of worms from composting facilities potentially harbors organisms that are beneficial to plant growth and development. In this experiment, we sought to examine the potential impacts of rhizosphere microbiomes derived from Eisenia fetida worm castings (i.e. vermicompost) on tomato (Solanum lycopersicum, L.) plant growth and physiology. Our experiment consisted of a greenhouse trial lasting 17 weeks total in which tomato plants were grown with one of three inoculant treatments: a microbial inoculant created from vermicompost (V), a microbial inoculant created from sterilized vermicompost (SV), and a no-compost control inoculant (C). We hypothesized that living microbiomes from the vermicompost inoculant treatment would enhance host plant growth and gene expression profiles compared to plants grown in sterile and control treatments. Our data showed that bacterial community composition was significantly altered in tomato rhizospheres, but fungal community composition was highly variable in each treatment. Plant phenotypes that were significantly enhanced in the vermicompost and sterile vermicompost treatments, compared to the control, included aboveground biomass and foliar δ15N nitrogen. RNA sequencing revealed distinct gene expression changes in the vermicompost treatment, including upregulation of nutrient transporter genes such as Solyc06g074995 (high affinity nitrate transporter), which exhibited a 250.2-fold increase in expression in the vermicompost treatment compared to both the sterile vermicompost and control treatments. The plant transcriptome data suggest that rhizosphere microbiomes derived from vermicompost can influence tomato gene expression and growth-related regulatory pathways, which highlights the value of RNA sequencing in uncovering molecular responses in plant microbiome studies.

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The rhizosphere is the hotspot for microbial enzyme activities and contributes to carbon cycling. Precipitation is an important component of global climate change that can profoundly alter belowground microbial communities. However, the impact of precipitation on conifer rhizospheric microbial populations has not been investigated in detail. In the present study, using high-throughput amplicon sequencing, we investigated the impact of precipitation on the rhizospheric soil microbial communities in two Norway Spruce clonal seed orchards, Lipová Lhota (L-site) and Prenet (P-site). P-site has received nearly double the precipitation than L-site for the last three decades. P-site documented higher soil water content with a significantly higher abundance of Aluminium (Al), Iron (Fe), Phosphorous (P), and Sulphur (S) than L-site. Rhizospheric soil metabolite profiling revealed an increased abundance of acids, carbohydrates, fatty acids, and alcohols in P-site. There was variance in the relative abundance of distinct microbiomes between the sites. A higher abundance of Proteobacteria, Acidobacteriota, Ascomycota, and Mortiellomycota was observed in P-site receiving high precipitation, while Bacteroidota, Actinobacteria, Chloroflexi, Firmicutes, Gemmatimonadota, and Basidiomycota were prevalent in L-site. The higher clustering coefficient of the microbial network in P-site suggested that the microbial community structure is highly interconnected and tends to cluster closely. The current study unveils the impact of precipitation variations on the spruce rhizospheric microbial association and opens new avenues for understanding the impact of global change on conifer rizospheric microbial associations.

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The Hyphomicrobiales (Rhizobiales) order contains soil bacteria with an irregular distribution of the Calvin-Benson-Bassham cycle (CBB). Key enzymes in the CBB cycle are ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), whose large and small subunits are encoded in cbbL and cbbS, and phosphoribulokinase (PRK), encoded by cbbP. These genes are often found in cbb operons, regulated by the LysR-type regulator CbbR. In Bradyrhizobium, pertaining to this order and bearing photosynthetic and non-photosynthetic species, the number of cbbL and cbbS copies varies, for example: zero in B. manausense, one in B. diazoefficiens, two in B. japonicum, and three in Bradyrhizobium sp. BTAi. Few studies addressed the role of CBB in Bradyrhizobium spp. symbiosis with leguminous plants. To investigate the horizontal transfer of the cbb operon among Hyphomicrobiales, we compared phylogenetic trees for concatenated cbbL-cbbP-cbbR and housekeeping genes (atpD-gyrB-recA-rpoB-rpoD). The distribution was consistent, indicating no horizontal transfer of the cbb operon in Hyphomicrobiales. We constructed a ΔcbbLS mutant in B. diazoefficiens, which lost most of the coding sequence of cbbL and has a frameshift creating a stop codon at the N-terminus of cbbS. This mutant nodulated normally but had reduced competitiveness for nodulation and long-term adhesion to soybean (Glycine max (L.) Merr.) roots, indicating a CBB requirement for colonizing soybean rhizosphere.

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The process and function that underlie the assembly of root-associated microbiomes may be strongly linked to the survival strategy of plants. However, the assembly and functional changes of root-associated microbial communities in different desert plants in natural desert ecosystems are still unclear. Thus, we studied the microbial communities and diversity of root endosphere (RE), rhizosphere soil (RS), and bulk soil (BS) among three representative desert plants (Alhagi sparsifolia, Tamarix ramosissima, and Calligonum caput-medusae) in three Xinjiang desert regions {Taklimakan (CL), Gurbantünggüt (MSW), and Kumtag (TLF)} in China. This study found that the soil properties {electrical conductivity (EC), soil organic carbon (SOC), total nitrogen (TN) and phosphorus (TP), available nitrogen (AN) and phosphorus (AP)} of C. caput-medusae were significantly lower than those of A. sparsifolia and T. ramosissima, while the root nutrients (TN and TP) of A. sparsifolia were significantly higher compared to C. caput-medusae and T. ramosissima. The beta diversity of bacteria and fungi (RE) among the three desert plants was significantly different. The common OTU numbers of bacteria and fungi in three compartments (RE, RS, and BS) of the three desert plants were ranked as RS > BS > RE. The bacterial and fungal (RE) Shannon and Simpson indexes of C. caput-medusae were significantly lower as compared to those of A. sparsifolia and T. ramosissima. Additionally, bacterial and fungal (RE and RS) node numbers and average degree of C. caput-medusae were lower than those found in A. sparsifolia and T. ramosissima. Root and soil nutrients collectively contributed to the composition of root-associated bacterial (RE, 12.4%; RS, 10.6%; BS, 16.6%) and fungal communities (RE, 34.3%; RS, 1.5%; BS, 17.7%). These findings demonstrate variations in the bacterial and fungal populations across different plant species with distinct compartments (RE, RS, and BS) in arid environments. More importantly, the study highlights how much soil and plant nutrients contribute to root-associated microbial communities.

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While biotic interactions often impose selection, species and popula- tions vary in whether they are locally adapted to biotic interactions. Evo- lutionary theory predicts that environmental conditions drive this variable local adaptation by altering the fitness impacts of species interactions. To investigate the influence of an environmental gradient on adaptation be- tween a plant and its associated rhizosphere biota, we cross-combined teosinte (Zea mays ssp. mexicana) and rhizosphere biota collected across a gradient of decreasing temperature, precipitation, and nutrients in a greenhouse common garden experiment. We measured both fitness and phenotypes expected to be influenced by biota, including concentrations of nutrients in leaves. Independent, main effects of teosinte and biota source explained most variation in teosinte fitness and traits. For example, biota from warmer sites provided population-independent fitness benefits across teosinte hosts. Effects of biota that depended on teosinte genotype were often not specific to their local hosts, and most traits had similar relation- ships to fitness across biota treatments. However, we found weak patterns of local adaptation between teosinte and biota from colder sites, suggest- ing environmental gradients may alter the importance of local adaptation in teosinte-biota interactions, as evolutionary theory predicts.

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The distribution and transmission of antibiotic resistance genes (ARGs) in agricultural soils constitute a significant threat to food safety and human health. Natural quorum sensing inhibitors (QSIs), with advantages such as low plant toxicity and low application costs, present a potential approach for mitigating ARG contamination by targeting bacterial quorum sensing systems. This study explored the impacts and mechanisms of three natural QSIs (vanillin, catechin, and tannin) on the abundance of tetracycline resistance genes (TRGs) in both rhizosphere and non-rhizosphere soils. Results illustrated a notable reduction in TRG abundance across three natural QSI treatments, with catechin displaying the most pronounced effect in the rhizosphere soil. Furthermore, the application of natural QSIs had a significant influence on the bacterial community structure and population dynamics, particularly evident in the alterations induced by catechin on bacterial interactions within the soil ecosystem. Natural QSIs inhibited the production of N-acyl homoserine lactone (AHL) signaling molecules. The primary environmental factors driving changes in bacterial community were identified as pH and NO3--N content. Through mechanisms involving the modulations of AHL concentrations and soil environmental factors, natural QSIs were found to impact bacterial population, ultimately leading to a decrease in TRG abundance. Importantly, the application of natural QSIs did not exhibit adverse effects on plant phenotypic traits. These findings serve as a useful reference for implementing natural QSIs to effectively control soil ARG contamination.

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Plant growth-promoting bacteria (PGPB) are of increased interest as they offer sustainable alternatives to the more common chemical fertilisers. Research, however, has increased into the use of PGPB as bioinoculants to improve yields. Legumes are known to interact with diazotroph PGPB which increase nutrient uptake, prevent pathogenic infections, and actively fix nitrogen. This study aimed to comprehensively describe PGPB associated with legumes grown in Namibia through analysis of the site-specific bacterial microbiomes. In the present study, we used the 16S rRNA sequencing approach to determine the structure of rhizosphere, root, and seed endosphere microbiomes of five drought-tolerant legume species: Macrotyloma uniflorum, Vigna radiata, Vigna aconitifolia, Vigna unguiculata and Lablab purpureus. Several important phyla were identified including Actinobacteriota, Bacteroidota, Firmicutes, Proteobacteria and Verrucomicrobiota. Overall, Proteobacteria was the most abundant phylum followed by Actinobacteria. The most important genera identified were Bacillus, Mesorhizobium, Pseudomonas, Bradyrhizobium and the Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium group. The relative abundance of these genera varied across sample types and legume species. This study identified important diazotrophs across all the legume species. Bacillus, an important PGPB, was found to be the most abundant genus among all the niches analysed and legume species, while Rhizobium spp. was particularly enriched in roots. This study ultimately provides previously undescribed information on legume-associated bacterial communities in Namibia.

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Rising atmospheric carbon dioxide (CO2) and soil heavy metal pollution affect crop safety and production. Exposure to elevated CO2 (ECO2) increases cadmium (Cd) uptake in some crops like wheat and rice, however, it remains unclear how ECO2 affects Cd uptake by Brassica napus. Here, we investigated the responses of B. napus seedlings exposed to ECO2 and Cd through analyses of physiology, transcriptome, metabolome, and rhizosphere microbes. Compared with Cd-stress alone (Cd50_ACO2), ECO2 boosted the uptake of Cd by B. napus roots by 38.78% under coupled stresses (Cd50_ECO2). The biomass and leaf chlorophyll a content increased by 38.49% and 79.66% respectively in Cd50_ECO2 relative to Cd50_ACO2. Activities of superoxide dismutase (SOD) and peroxidase (POD) enhanced by 8.42% and 185.01%, respectively, while glutathione (GSH) and ascorbic acid (AsA) contents increased by 16.44% and 52.48%, and abundances of rhizosphere microbes changed significantly under coupled stresses (Cd50_ECO2) relative to Cd-stress alone (Cd50_ACO2). Also, the upregulation of glutathione, glutathione transferase genes, and heavy metal ATPase expression promoted the detoxification effect of rapeseed on Cd. Changes in the expression of transcription factors like MAPK, WRKY, BAK1 and PR1, as well as changes in metabolic pathways like ß-alanine, may be involved in the regulatory mechanism of stress response. These findings provide new insights for studying the regulatory mechanism of rapeseed under ECO2 on soil Cd stress, and also provide a basis for further research on Cd tolerant rapeseed varieties in the future climate context.

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Aspergillus and Fusarium are two economically important genera of fungi. They cause significant yield losses and contamination of crops with mycotoxins. In this study we aimed to evaluate the impact of 2,4-diacetylphloroglucinol (2,4-DAPG) on Aspergillus and Fusarium fungi. It is hypothesized that two fungal genera, which have different ecological strategies, react differently to stress caused by a secondary metabolite produced by rhizosphere Pseudomonas species. We found that 2,4-DAPG was able to reduce biofilm formation of Aspergillus and Fusarium, as reflected in biomass and its chemical composition. Furthermore, subinhibitory concentrations of 2,4-DAPG increased the levels of ergosterol and polysaccharides (α- and ß-glucans, chitin) in the cell membrane and cell wall of Aspergillus, while decreasing them in Fusarium. 2,4-DAPG altered the production of secondary metabolites, especially mycotoxins and extracellular proteases. The production of ochratoxin A was decreased in A. ochraceus, and T-2 toxin and zearalenone, on the contrary, were increased in F. culmorum and F. sporotrichioides, respectively. Thus, using 2,4-DAPG we demonstrated that the ecological role of fungi determines their reaction to antibiotic substances produced by the plant microbiome. Our data contributes to understanding the molecular mechanisms behind symbiotic relationships in natural communities, which are mediated by the biosynthesis of antibiotics.