Environmental Microbiome

Chlamydiota are an ancient and hyperdiverse Phylum of obligate intracellular bacteria. The best characterized representatives are pathogens or parasites of mammals, but it is thought that their most common hosts are microeukaryotes like Amoebozoa. The diversity in taxonomy, evolution, and function of non-pathogenic Chlamydiota are slowly being described. Here we use data mining techniques and genomic analysis to extend our current knowledge of Chlamydiota diversity and its hosts, in particular the Order Parachlamydiales. We extract one Rhabdochlamydiaceae and three Simkaniaceae genomes from NCBI Short Read Archive deposits of ciliate and algal genome sequencing projects. We then use these to identify a further 14 and 8 genomes respectively amongst existing, unidentified environmental assemblies. From these data we identify two novel clades with host associated data, for which we propose the names Candidatus Sacchlamydia (Family Rhabdochlamydiaceae) and Candidatus Amphrikania (Family Simkaniaceae), as well as a third new clade of environmental MAGs Candidatus Acheromydia (Family Rhabdochlamydiaceae). The extent of uncharacterized diversity within the Rhabdochlamydiaceae and Simkaniaceae is indicated by 16 of the 22 MAGs being evolutionarily distant from currently characterised genera. Within our limited data, we observe great predicted diversity in Parachlamydiales metabolism and evolution, including the potential for metabolic and defensive symbioses as well as pathogenicity. These data provide an imperative to link genomic diversity in metagenomics data to their associated eukaryotic host, and to develop onward understanding of the functional significance of symbiosis with this hyperdiverse clade. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=161 HEIGHT=200 SRC="FIGDIR/small/533158v2_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@fd4282org.highwire.dtl.DTLVardef@1199065org.highwire.dtl.DTLVardef@156f740org.highwire.dtl.DTLVardef@829eed_HPS_FORMAT_FIGEXP M_FIG C_FIG

Following the development of high-throughput DNA sequencers, environmental prokaryotic communities were usually described by metabarcoding on short markers of the 16S domain. Among third generation sequencers, that offered the possibility to sequence the full 16s domain, the portable MinION from Oxford Nanopore was undervalued for metabarcoding because of its relatively higher error rate per read. Here we illustrate the limits and benefits of Nanopore sequencing devices by comparing the prokaryotic community structure in a mock community and 52 sediment samples from mangrove sites, inferred from full-length 16S long-reads (16S-FL, ca. 1.5 kpb) on a MinION device, with those inferred from partial 16S short-reads (16S- V4V5, ca. 0.4kpb, 16S-V4V5) on Illumina MiSeq. 16S-V4V5 and 16S-FL retrieved all the bacterial species from the mock, but Nanopore long-reads overestimated their diversity more than twice. Whether these supplementary OTUs were artefactual or not, they only accounted for ca. 10% of the reads. From the sediment samples, with a coverage-based rarefaction of reads and after singletons filtering, Mantel and Procrustean tests of co-inertia showed that bacterial community structures inferred from 16S-V4V5 and 16S- FL were significantly similar, showing both a comparable contrast between sites and a coherent sea-land orientation within sites. In our dataset, 84.7 and 98.8% of the 16S-V4V5 assigned reads were assigned strictly to the same species and genus, respectively, than those detected by 16S-FL. 16S-FL allowed to detect 92.2% of the 309 families and 87.7% of the 448 genera that were detected by the short 16S-V4V5. 16S-FL recorded 973 additional species and 392 genus not detected by 16S-V4V5 (31.5 and 10.4% of the 16S-FL reads, respectively, among which 67.8 and 79.3% were assigned), producted by both primer specificities and diffrent error rates. Thus, our results concluded to an overall similarity between 16S-V4V5 and 16S-FL sequencing strategies for this type of environmental samples.

Mycorrhizal fungi form ubiquitous symbiotic associations with almost all land plants and are of key interest to evolutionary biologists and ecologists because this ancient symbiosis was essential for the colonization of land by plants - a major turning point in the evolutionary history of the earth - and the subsequent development and functioning of the terrestrial ecosystems. Within the orchid family (Orchidaceae), plants establish unique interactions with specific orchid mycorrhizal fungi. These fungal symbionts are essential for the development of orchids as they provide carbon and soil nutrients to germinating orchid seeds and the nutritional supply continues for adult orchids to different degrees. Fueled by the development of DNA sequencing techniques, the diversity of mycorrhizal and other root-associated fungi in orchid roots has been extensively reported in evolutionary and ecophysiological studies. However, the full taxonomic range of orchid-associated fungi remains to be investigated in a broad phylogenetic framework, hampering a further understanding of the evolution and ecological adaptation of orchid mycorrhizal interactions. In this study, we used the most complete DNA dataset to date to map the phylogenetic distribution and ecological lifestyles of root-associated fungi in Orchidaceae by phylogenetic reconstructions at the fungal order level. We found that a broad taxonomic range of fungi (clustered into 1898 operational taxonomic units) resided in orchid roots, belonging to at least 150 families in 28 orders in Basidiomycota and Ascomycota. These fungi were assigned to diverse ecological lifestyles including typical orchid mycorrhizal fungi ( rhizoctonia), ectomycorrhizal fungi, wood- or litter-decaying saprotrophic fungi, and other endophytes/pathogens/saprotrophs. This overview reveals that among the four different mycorrhizal types, the orchid mycorrhizal symbiosis probably involves the highest diversity of fungal taxa. We hope that our newly reconstructed phylogenetic framework of orchid-associated fungi and the assessment of their potential mycorrhizal status will benefit future ecological and evolutionary studies on orchid-fungal interactions.

Dopamine plays a critical role in animal physiology and interactions with gut microbes. In plants, dopamine is known to function in plant defense and abiotic stress tolerance; however, its role in mediating plant-microbiome interactions remains unexplored. In this study, we observed that dopamine is one of the most abundant exometabolites with natural variation in root exudates across diverse Brachypodium distachyon lines, suggesting a potential role in rhizosphere microbial assembly. To further investigate this, we colonized ten natural B. distachyon lines with a 16-member bacterial synthetic community (SynCom), collected paired metabolomic and 16S rRNA sequencing data, and performed an association analysis. Our results revealed that dopamine levels in root exudates were significantly associated with the abundance of six SynCom members in a hydroponic system. In vitro growth studies demonstrated that dopamine had a significant effect on the growth of the same six bacterial isolates. Additionally, treating soil directly with dopamine enriched Actinobacteria, consistent with both the SynCom-dopamine correlations and the isolate growth results. Collectively, our study underscores the selective influence of dopamine on rhizosphere microbial communities, with implications for precision microbiome management.

Plant roots host defined microbial communities that differ from those found in the surrounding soil and these communities shift dynamically in response to plant development and environmental changes. Whilst it is widely accepted that root exudates play a key role in the assembly and dynamics of root-associated microbial communities, the underlying mechanisms are not well understood. This is partly due to a lack of controlled experimental systems that monitor both exudate- and microbiome-dynamics simultaneously. Here, we compared two microcosm systems commonly used in either root microbiome (clay particle-based) or root exudate studies (glass bead-based) for their suitability to simultaneously monitor both aspects. We evaluated these systems based on plant performance, bacterial growth, and time-resolved community and exudate profiling. In both systems, we reveal an exudate effect, characterised by higher bacterial diversity and Pseudomonas abundances in proximity to plant roots. While clay particles impeded exudate recovery, even when plants were removed from microcosms for exudate collection, the glass bead set-up allowed us to uncover dynamic exudate shifts during bacterial community establishment. This highlighted a transient increase of glucosinolates upon root colonisation by initially dominant Pseudomonas species. Overall, the comparison proved only the glass bead-based semi-hydroponic system to be suitable for the paralleled study of exudate and root microbiome dynamics.

Sarracenia provide an optimal system for deciphering the host-microbiome interactions at various levels. We analyzed the pitcher microbiomes and metatranscriptomes of the parental species, and F1 and F2 generations from the mapping population (Sarracenia purpurea X Sarracenia psittacina) utilizing high-throughput sequencing methods. This study aimed to examine the host influences on the microbiome structure and function and to identify the key microbiome traits. Our quality datasets included 8,892,553 full-length bacterial 16s rRNA gene sequences and 65,578 assembled metatranscripts with microbial protein annotations. The correlation network of the bacterial microbiome revealed the presence of 3-7 distinct community clusters, with 8 hub and 19 connector genera. The entire microbiome consisted of viruses, bacterial, archaea, and fungi. The richness and diversity of the microbiome varied among the parental species and offspring genotypes despite being under the same greenhouse environmental conditions. We have discovered certain microbial taxa that are genotype-enriched, including the community hub and connector genera. Nevertheless, there were no significant differences observed in the functional enrichment analysis of the metatranscriptomes across the different genotypes, suggesting a functional convergence of the microbiome. Our results revealed that the pitcher microcosm harbors both rhizosphere and phyllosphere microbiomes within its boundaries, resulting in a structurally diverse and functionally complex microbiome community. A total of 50,424 microbial metatranscripts were linked to plant growth-promoting microbial proteins. We show that this complex pitcher microbiome possesses various functions that contribute to plant growth promotion, such as biofertilization, bioremediation, phytohormone signaling, stress regulation, and immune response stimulation. Additionally, the pitcher microbiome exhibits traits related to microbe-microbe interactions, such as colonization of plant systems, biofilm formation, and microbial competitive exclusion. In summary, the demonstrated taxonomical divergence and functionally convergence of the pitcher microbiome are impacted by the host genetics, making it an excellent system for discovering novel beneficial microbiome traits.

Given the impact of climate change on agriculture, the development of resilient crop cultivars is imperative. A healthy plant microbiota is key to plant productivity, influencing nutrient absorption, disease resistance, and overall vigor. The plant genetic factors controlling the assembly of microbial communities are still unknown. Here we examine if Machine Learning can predict grapevine rootstock and scion genotypes based on soil microbiota, despite environmental variability. The study utilized soil microbial bacteriome datasets from 281 vineyards across 13 countries and five continents, featuring 34 different Vitis vinifera cultivars grafted onto, often ambiguous, rootstocks. Random Forests, Adaptive Boost, Gradient Boost, Support Vector Machines, Gaussian and Bernoulli Naive Bayes, k-Nearest Neighbor, and Neural Networks algorithms were employed to predict continent, country, scion, and rootstock cultivar, under two filtering criteria: retaining sparse classes, ensuring class diversity, and excluding sparse classes assessing model robustness against overfitting. Both criteria showed remarkable F1-weighted scores (>0.8) for all classes, for most algorithms. Moreover, successful rootstock and scion genotype prediction from soil microbiomes confirms that genotypes of both plant parts shape the microbiome. These insights pave the way for identifying plant genes for use with breeding programs that enhance plant productivity and sustainability by improving the plant-microbiota relationship.

Next-generation sequencing helps describe microbial communities in rhizosphere environments, but understanding rhizosphere-plant interactions synergistic effects on plant traits and health outcomes remains challenging. This study analyses rhizosphere sRNAs ability to manipulate host gene targets in plants grown in suppressive (SP) and non-suppressive (NSP) soils with an integrated multi omics dataset. The results showed that rhizosphere sRNAs exhibited specific compositional features that may be important for rhizosphere-plant interaction. Small RNAs, less than 30 nt in size, were predominant in both samples, with a 5-prime bias towards cytosine enrichment, suggesting potential association with wheat specific argonauts. Mapping of sRNA reads to microbial metagenomes assembled draft genomes from SP and NSP soils showed sRNA loci were differentially expressed (DE) between the soils with contrasting disease suppressive capacities. In total, 96 and 132 non redundant rhizosphere sRNAs were abundant in SP and NSP rhizosphere communities, respectively. While 55 known bacterial sRNA loci were predicted from both SP and NSP metagenomes, 127 sRNAs originated from these loci were differentially expressed. Global wheat target prediction and functional analysis from DE rhizosphere sRNAs showed both soil type specific and common pathways. Upregulated NSP sRNAs target metabolic pathways, secondary metabolite biosynthesis, MAPK signalling, while SP sRNAs target glycerophospholipid metabolism, pathways such as polycomb repressive complex, starch/sucrose metabolism, and plant-pathogen interactions were targeted by both sets of sRNAs. This is the first study showing evidence for rhizosphere sRNAs and their corresponding plant transcripts in the context of biological disease suppression in agricultural soils. ImportanceSmall RNAs (sRNAs) have gained attention in host-microbe interactions due to their diverse roles in controlling biological processes. Studies have identified numerous sRNAs with novel functions across various organisms. Echoing growing evidence of sRNAs in different plant-microbe interaction, we show an evidence of rhizosphere sRNAs regulating wheat genes in soil disease suppression context. This understanding could significantly enhance our comprehension of gene regulation in biological functions, potentially paving the way for the development of microbiome-based methods to influence host traits. Understanding the microbiome communitys mechanisms in different environments offers opportunities to modify them for agriculture, including modifying farming practices, host genetics/immunity, and synthetic communities for disease suppression.

The emergence of the Guava Root-Knot Nematode (Meloidogyne enterolobii) poses a significant threat to tomato yields globally. This study aimed to evaluate the impact of collagen and chitin soil amendments on soil microbial composition and function (fungal and bacterial communities), and their effects on tomato plant health and M. enterolobii infection under standard (5,000 eggs plant-1) and high (50,000 eggs plant-1) inoculum pressure. Conducted in a greenhouse setting, the study investigated the effectiveness of these amendments in nurturing beneficial microbial communities across both native and agricultural soils. Both collagen and chitin were effective in reducing nematode egg counts up to 66% and 84% under standard and high inoculum pressure, respectively and enhance plant health parameters (biomass and chlorophyll content). Moreover, a microbiome shift led to an increase in bacterial (Kitasatospora, Bacillus, and Streptomyces) and fungal (Phialemonium) genera, known for their chitinase, collagenase, and plant-parasitic nematode control. Among the microbes, Streptomyces spp. were found among the core microbiome and associated with a lower disease incidence assessed through a phenotype-OTU network analysis (PhONA). Under standard inoculum a higher metabolite expression was observed with the amino acid class being the majority among the metabolite groups. The findings highlight the potential of collagen and chitin to mitigate Meloidogyne enterolobii infection by fostering beneficial soil microbial communities.

The ascomycete genus Tetracladium is best known for containing aquatic hyphomycetes, which are important decomposers in stream food webs. However, some species of Tetracladium are thought to be multifunctional and are also endobionts in plants. Suprisingly, Tetracladium sequences are increasingly being reported from metagenomics and metabarcoding studies of both plants and soils world-wide. It is not clear how these sequences are related to the described species and little is known about the non-aquatic biology of these fungi. Here, the genomes of 24 Tetracladium strains, including all described species, were sequenced and used to resolve relationships among taxa and to improve our understanding of ecological and genomic diversity in this group. All genome-sequenced Tetracladium fungi form a monophyletic group. Conspecific strains of T. furcatum from both aquatic saprotrophic and endobiont lifestyles and a putative cold-adapted clade are identified. Analysis of ITS sequences from water, soil, and plants from around the world reveals that multifunctionality may be widespread through the genus. Further, frequent reports of these fungi from extreme environments suggest they may have important but unknown roles in those ecosystems. Patterns of predicted carbohydrate active enzymes (CAZyme) and secondary metabolites in the Tetracladium genomes are more similar to each other than to other ascomycetes, regardless of ecology, suggesting a strong role for phylogeny shaping genome content in the genus. Tetracladium genomes are enriched for pectate lyase domains (including PL3-2), GH71 -1,3-glucanase domains and CBM24 -1,3-glucan/mutan binding modules, and both GH32 and CBM38, inulinase and inulin binding modules. These results indicate that these fungi are well-suited to digesting pectate and pectin in leaves when living as aquatic hyphomycetes, and inulin when living as root endobionts. Enrichment for -1,3-glucanase domains may be associated with interactions with biofilm forming microorganisms in root and submerged leaf environments.

Similar to the human gut microbiome, diverse microbes colonize the plant rhizosphere, and an imbalance of this microbial community, known as dysbiosis, may negatively impact plant health. This study aimed to investigate the influence of rhizosphere dysbiosis on above-ground plant health using tomato plants (Solanum lycopersicum L.) and the foliar bacterial spot pathogen Xanthomonas perforans as model organisms. Four-week-old tomato plants rhizospheres were treated with streptomycin (0.6 g x L-1), or water (negative control) and spray-inoculated with X. perforans (105 cells x mL-1) after 24 h. Half of the plants treated with streptomycin and X. perforans received soil microbiome transplants (SMT) from uninfected plant donors 48 h after streptomycin application. Streptomycin-treated plants showed a 26% increase in disease severity compared to plants that received no antibiotic, while plants that received the SMT had an intermediate level of disease severity. Antibiotic-treated plants showed a reduced abundance of rhizobacterial taxa like Cyanobacteria from the genus Cylindrospermum as well as down-regulation of genes related to plant primary and secondary metabolism and up-regulation of plant defense genes associated with induced systemic resistance (ISR). This study highlights the crucial role of beneficial rhizosphere microbes in disease resistance, even to foliar pathogens.

Arbuscular mycorrhizal fungi, classified in the subphylum Glomeromycotina, are obligate symbionts that depend on photosynthetic products from plants. There is substantial evidence that AMF support plant and crop growth in natural and agricultural ecosystems. Fine root endophytes (FRE) also co-occur in plant roots with AMF in all but tropical environments. However, their presence remains poorly recognised, and their lifestyle and functionality remain largely unknown. Our analysis demonstrates that, in contrast to AMF, FRE colonise plants during the winter season, when photosynthesis is more challenging. It is also noteworthy that FRE was unable to colonise the roots of spring-sown crops. The results of our laboratory pot experiments demonstrated that low temperatures are not sufficient for FRE colonisation. Furthermore, we observed that FRE, in contrast to AMF, exhibited increased colonisation in soils containing metabolically inactive plants. Our results suggest that FRE does not contribute to the increase of ecosystem biomass through direct photosynthesis in summer, but may play an overlooked role in the formation of mycorrhiza-based soil ecosystems in winter. However, this winter ecosystem can be disrupted by conventional bare fallow management of the field, interrupting the annual cycle.

Drylands represent a significant part of the Earths surface and include essential and vulnerable ecosystems for the global ecological balance. The Caatinga, with its unique biodiversity adapted to the extreme conditions of this semi-arid region, offers a valuable opportunity to expand our knowledge about these ecosystems. Here, this work reveals the high microbial diversity in the soil and rhizosphere of the Caatinga, with the roots presenting more specialized communities. Bacteria such as Bacilli, Alphaproteobacteria and Firmicutes excelled in critical functions such as nutrient cycling. Interplant differences suggested the influence of root exudates. The metagenomic study of interactions between microorganisms in the rhizosphere of selected plants revealed microbial biodiversity and contributed to our understanding of nutrient cycling, plant growth and resistance to water stress. In addition, they demonstrate biotechnological potential to address global challenges such as desertification and food security.

Sorghum bicolor, an important global crop, adapted to thrive in hotter and drier conditions than maize or rice, has deep roots that interact with a unique and stratified soil microbiome that plays a crucial role in plant health, growth, and carbon storage. Microbiome studies on agricultural soils, particularly fields growing S. bicolor, have been mostly limited to surface soils (<30 cm). Here we investigated the abiotic factors of soil properties, field location, depth, and the biotic factors of sorghum type across 38 genotypes on the soil microbiome. Utilizing 16S rRNA gene amplicon sequencing, our analysis reveals significant changes in microbial composition and decreasing diversity at increasing soil depths within S. bicolor regardless of genotype or fields. Notably, specific microbial families, such as Thermogemmatisporaceae and an unclassified family within the ABS-6 order, were enriched in deeper soil layers beyond 30 cm. Additionally, microbial richness and diversity declined with depth, reaching a minimum at the 60 - 90 cm layer, with layers beyond 90 cm increasing in alpha diversity. These findings highlight the importance of soil depth in agricultural soil microbiome studies.

Microorganisms participate in complex interactions involving different kingdoms, so rhizosphere biodiversity mapping is essential for understanding how microbes interact with each other in the soil and with roots. Although soil microbial communities are remarkably diverse and technological advances have provided a high capacity to acquire reliable sequence data, unique microbial taxa in soil, root and rhizosphere samples remain poorly described. For the first time, we organized a consortium to collect soil samples covering all Brazilian biomes, providing a comprehensive and unprecedented view of soil microbial diversity. This understanding is critical, especially within the context of climate change, which affects plant physiology, root exudation and, consequently, the composition and functionality of soil microbial communities. The interactions between soil, roots and rhizosphere are influenced by evolutionary and adaptive forces and shape the production of microbial natural products, which exhibit great therapeutic potential and Mapping and studying rhizosphere microbial biodiversity not only increases our knowledge of soil ecology but also offers valuable insights for developing sustainable practices. We employed both 16S/18S/ITS amplicon and metagenomic short-read shotgun sequencing methods to examine and catalogue the large-scale genomes of culture-independent rhizosphere microbes and their interactions with roots in six terrestrial Brazilian biomes, namely, the Amazon, Atlantic Forest, Cerrado, Caatinga, Pampa and Pantanal. Our results revealed the ubiquity of Proteobacteria, which reflects their adaptability to contrasting environments. Biomes with greater moisture availability, such as the Amazon and Pantanal, exhibited greater diversity and abundance of fast-growing bacteria, such as Proteobacteria, and nutrient cyclers, such as Thaumarchaeota. Arid and semiarid biomes, such as the Caatinga, were dominated by microorganisms tolerant to drought and nutrient-limited environments, such as Actinobacteria. Acidobacteria, which thrive in acidic, nutrient-poor soils, were very abundant in forest biomes. The Planctomycetes phylum also occurred more frequently in areas with a relatively high soil organic matter content, such as the Cerrado. Bacteroidetes was significantly more abundant in Pampa than in the other biomes. The results provide comprehensive insights into soil, root and rhizosphere biodiversity and not only enhance the knowledge of the fundamental biological processes sustaining plant life but also constitute a reliable sequencing databank to address present-day agricultural and environmental challenges.

O_LIPlants accommodate diverse microbial communities (microbiomes), which can change dynamically during plant adaptation to varying environmental conditions. However, the direction of these changes and the underlying mechanisms driving them, particularly in crops adapting to the field conditions, remain poorly understood. C_LIO_LIWe investigate the root-associated microbiome of rice (Oryza sativa L.) using 16S rRNA gene amplicon and metagenome sequencing, across four consecutive cultivation seasons in a high-yield, non-fertilized, and pesticide-free paddy field, compared to a neighboring fertilized and pesticide-treated field. C_LIO_LIOur findings reveal that root microbial community shifts and diverges based on soil fertilization status and plant developmental stages. Notably, nitrogen-fixing bacteria such as Telmatospirillum, Bradyrhizobium and Rhizomicrobium were over-represented in rice grown in the non-fertilized field, implying that the assembly of these microbes supports rice adaptation to nutrient-deficient environments. C_LIO_LIA machine learning model trained on the microbiome data successfully predicted soil fertilization status, highlighting the potential of root microbiome analysis in forecasting soil nutrition levels. Additionally, we observed significant changes in the root microbiome of ccamk mutants, which lack a master regulator of mycorrhizal symbiosis, under laboratory conditions but not in the field, suggesting a condition-dependent role for CCaMK in establishing microbiomes in paddy rice. C_LI

Trebouxiophyceae is a widespread and species-rich green algal class encompassing mostly coccoid algae with a simple ovoid or ellipsoidal outline. However, some poorly-sampled lineages have evolved more elaborate shapes or even complex thalli, adding to the classs morphological diversity. Led by new and previously established strains, this study additionally uncovered a clade of croissant-like trebouxiophytes. Phylogenetic analyses inferred from nuclear 18S rDNA and chloroplast rbcL sequences confirmed the monophyly of the microcroissant clade, which we propose to be classified as a new family, Ragelichloridaceae. This family includes two novel genera, Ragelichloris and Navichloris, and the previously described Thorsmoerkia. The position of Ragelichloridaceae within Trebouxiophyceae stayed unresolved but chloroplast phylogenomics showed that the family belongs to the broader incertae sedis group, which also includes Xylochloris and Leptosira. In addition, our study showed that the microcroissant-like genus Chlorolobion, previously classified within Chlorophyceae, is a genuine trebouxiophyte, potentially related to Ragelichloridaceae. HighlightsO_LIA new family-level clade uncovered within Trebouxiophyceae. C_LIO_LITwo new genera described. C_LIO_LIThe genus Chlorolobion shown to be a trebouxiophyte. C_LI

The spermosphere is the transient, immediate zone of soil around imbibing and germinating seeds. It represents a habitat where there is contact between seed-associated microbes and soil microbes, but is studied less compared to other plant habitats. Previous studies on spermosphere microbiology were primarily culture-based or did not sample the spermosphere soil as initially defined in space and time. Thus, the objectives of this study were to develop an efficient strategy to collect spermosphere soils around imbibing soybean and cotton in non-sterile soil and investigate changes in microbial communities. The method employed sufficiently collected spermosphere soil as initially defined in space by constraining the soil sampled with a cork borer and confining the soil to a 12-well microtiter plate. Spermosphere prokaryote composition changed over time and depended on the crop within six hours after seeds were sown. By 12 to 18 hours, crops had unique microbial communities in spermosphere soils. Prokaryote evenness dropped following seed imbibition with the proliferation of copiotrophic soil bacteria. Due to their long history of plant growth promotion, prokaryote OTUs in Bacillus, Paenibacillus, Burkholderia, Massilia, Azospirillum, and Pseudomonas were notable genera enriched. Fungi and prokaryotes were hub taxa in cotton and soybean spermosphere networks. Additionally, the enriched taxa were not hubs in networks, suggesting other taxa besides those enriched may be important for spermosphere communities. Overall, this study advances knowledge in the assembly of the plant microbiome early in a plants life, which may have plant health implications in more mature plant growth stages.

Drylands comprise 45% of Earths land area and contain ecologically critical soil surface communities known as biocrusts. Biocrusts are composed extremotolerant organisms including cyanobacteria, microfungi, algae, lichen, and bryophytes. Fungi in biocrusts help aggregate these communities and may form symbiotic relationships with nearby plants. Climate change threatens biocrusts, particularly moss biocrusts, but its effects on the biocrust mycobiome remain unknown. Here, we performed a culture-dependent and metabarcoding survey of the moss biocrust mycobiome across an aridity gradient to determine whether local climate influences fungal community composition. As the local aridity index increased, fungal communities exhibited greater homogeneity in beta diversity. At arid and hyper-arid sites, communities shifted toward more extremotolerant taxa. We identified a significant proportion of fungal reads and cultures from biocrusts that could not be classified. Rhodotorula mucilaginosa and R. paludigena were significantly enriched following surface sterilization of healthy biocrust mosses. This aligns with their known roles as plant endophytes. We also observed septate endophyte colonization in the photosynthetic tissues of mosses from arid climates. Collectively, these results suggest that the biocrust mycobiome will undergo significant shifts in diversity due to climate change, favoring extremotolerant taxa as climate conditions intensify. The survey results also highlight taxa with the potential to serve as bioinoculants for enhancing biocrust resilience to climate change. These findings offer valuable insights into the potential impacts of climate change on drylands and provide crucial information for biocrust conservation.

The traditional milpa system is a polyculture originating in Mesoamerica, whose core is maize (Zea mays L.), associated with squash (Cucurbita spp.) and beans (Phaseolus vulgaris L.). In recent years, milpa-type crops have decreased owing to climate change, rapid population growth, and the excessive use of agrochemicals; therefore, the application of plant growth-promoting rhizobacteria (PGPR) to counteract these negative effects has been little explored. In this study, a maize crop in a milpa system was fertilized with the PGPR Pseudomonas fluorescens UM270, and the endophytic root microbiome (endobiome) of maize was assessed by 16S rRNA and internal transcribed spacer regions (ITS) sequencing. The results showed that UM270 the rhizosphere inoculation of P. fluorescens UM270 did not increase alpha diversity in either monoculture or the milpa, but it did alter the endophytic microbiome of maize plant roots by stimulating the presence of bacterial operational taxonomic units (OTUs) of the genera Burkholderia and Pseudomonas (in a monoculture), whereas in the milpa system, the PGPR stimulated a greater endophytic diversity and the presence of genera such as Burkholderia, Variovorax, and N-fixing rhizobia genera, including Rhizobium, Mesorhizobium and Bradyrhizobium. No clear association was found between fungal diversity and the presence of strain UM270, but beneficial fungi such as Rizophagus irregularis and Exophiala pisciphila were detected in the milpa system. In addition, network analysis revealed unique interactions with species like Stenotrophomonas sp., Burkholderia xenovorans, and Sphingobium yanoikuyae, which would potentially be playing a beneficial role with the plant. To the best of our knowledge, this is the first study in which the root microbiome of maize growing under a milpa model was assessed by bio-inoculation with PGPRs.