The microbial-driven nitrogen cycle and its relevance for plant nutrition.

Nitrogen (N) is a vital nutrient and an essential component of biological macromolecules, such as nucleic acids and proteins. Microorganisms represent major drivers of N-cycling processes in all ecosystems, including the soil and plant environment. The availability of N is a major growth limiting factor for plants and it is significantly affected by the plant microbiome. Plants and microorganisms form complex interaction networks resulting in molecular signaling, nutrient exchange and other distinct metabolic responses. In these networks, microbial partners influence growth and N use efficiency of plants either positively or negatively. Harnessing the beneficial effects of specific players within crop microbiomes is a promising strategy to counteract the emerging threats for human and planetary health due to the overuse of industrial N fertilizers. However, in addition to N-providing activities (e.g. the well-known symbiosis of legumes and Rhizobium bacteria), other plant-microorganism interactions must be considered to obtain a complete picture of how microbial driven N-transformations might affect plant nutrition. For this, we review recent insights into the tight interplay between plants and N-cycling microorganisms focusing on microbial N-transformation processes representing N sources and sinks that ultimately shape the plant N acquisition.

From microbes to microbiomes: Applications for plant health and sustainable agriculture.

Plant-microbe interaction research has had a transformative trajectory, from individual microbial isolate studies to comprehensive analyses of plant microbiomes within the broader phytobiome framework. Acknowledging the indispensable role of plant microbiomes in shaping plant health, agriculture, and ecosystem resilience, we underscore the urgent need for sustainable crop production strategies in the face of contemporary challenges. We discuss how the synergies between advancements in 'omics technologies and artificial intelligence can help advance the profound potential of plant microbiomes. Furthermore, we propose a multifaceted approach encompassing translational considerations, transdisciplinary research initiatives, public-private partnerships, regulatory policy development, and pragmatic expectations for the practical application of plant microbiome knowledge across diverse agricultural landscapes. We advocate for strategic collaboration and intentional transdisciplinary efforts to unlock the benefits offered by plant microbiomes and address pressing global issues in food security. By emphasizing a nuanced understanding of plant microbiome complexities and fostering realistic expectations, we encourage the scientific community to navigate the transformative journey from discoveries in the laboratory to field applications. As companies specializing in agricultural microbes and microbiomes undergo shifts, we highlight the necessity of understanding how to approach sustainable agriculture with site-specific management solutions. While cautioning against over-promising, we underscore the excitement of exploring the many impacts of microbiome-plant interactions. We emphasize the importance of collaborative endeavors with societal partners to accelerate our collective capacity to harness the diverse and yet-to-be-discovered beneficial activities of plant microbiomes.

The need for an integrated multi-OMICs approach in microbiome science in the food system.

Microbiome science as an interdisciplinary research field has evolved rapidly over the past two decades, becoming a popular topic not only in the scientific community and among the general public, but also in the food industry due to the growing demand for microbiome-based technologies that provide added-value solutions. Microbiome research has expanded in the context of food systems, strongly driven by methodological advances in different -omics fields that leverage our understanding of microbial diversity and function. However, managing and integrating different complex -omics layers are still challenging. Within the Coordinated Support Action MicrobiomeSupport (https://www.microbiomesupport.eu/), a project supported by the European Commission, the workshop "Metagenomics, Metaproteomics and Metabolomics: the need for data integration in microbiome research" gathered 70 participants from different microbiome research fields relevant to food systems, to discuss challenges in microbiome research and to promote a switch from microbiome-based descriptive studies to functional studies, elucidating the biology and interactive roles of microbiomes in food systems. A combination of technologies is proposed. This will reduce the biases resulting from each individual technology and result in a more comprehensive view of the biological system as a whole. Although combinations of different datasets are still rare, advanced bioinformatics tools and artificial intelligence approaches can contribute to understanding, prediction, and management of the microbiome, thereby providing the basis for the improvement of food quality and safety.

In-depth genome analysis of Bacillus sp. BH32, a salt stress-tolerant endophyte obtained from a halophyte in a semiarid region.

Endophytic strains belonging to the Bacillus cereus group were isolated from the halophytes Atriplex halimus L. (Amaranthaceae) and Tamarix aphylla L. (Tamaricaceae) from costal and continental regions in Algeria. Based on their salt tolerance (up to 5%), the strains were tested for their ability to alleviate salt stress in tomato and wheat. Bacillus sp. strain BH32 showed the highest potential to reduce salinity stress (up to + 50% and + 58% of dry weight improvement, in tomato and wheat, respectively, compared to the control). To determine putative mechanisms involved in salt tolerance and plant growth promotion, the whole genome of Bacillus sp. BH32 was sequenced, annotated, and used for comparative genomics against the genomes of closely related strains. The pangenome of Bacillus sp. BH32 and its closest relative was further analyzed. The phylogenomic analyses confirmed its taxonomic position, a member of the Bacillus cereus group, with intergenomic distances (GBDP analysis) pinpointing to a new taxon (digital DNA-DNA hybridization, dDDH < 70%). Genome mining unveiled several genes involved in stress tolerance, production of anti-oxidants and genes involved in plant growth promotion as well as in the production of secondary metabolites. KEY POINTS : • Bacillus sp. BH32 and other bacterial endophytes were isolated from halophytes, to be tested on tomato and wheat and to limit salt stress adverse effects. • The strain with the highest potential was then studied at the genomic level to highlight numerous genes linked to plant growth promotion and stress tolerance. • Pangenome approaches suggest that the strain belongs to a new taxon within the Bacillus cereus group.

The plant endosphere world - bacterial life within plants.

The plant endosphere is colonized by complex microbial communities and microorganisms, which colonize the plant interior at least part of their lifetime and are termed endophytes. Their functions range from mutualism to pathogenicity. All plant organs and tissues are generally colonized by bacterial endophytes and their diversity and composition depend on the plant, the plant organ and its physiological conditions, the plant growth stage as well as on the environment. Plant-associated microorganisms, and in particular endophytes, have lately received high attention, because of the increasing awareness of the importance of host-associated microbiota for the functioning and performance of their host. Some endophyte functions are known from mostly lab assays, genome prediction and few metagenome analyses; however, we have limited understanding on in planta activities, particularly considering the diversity of micro-environments and the dynamics of conditions. In our review, we present recent findings on endosphere environments, their physiological conditions and endophyte colonization. Furthermore, we discuss microbial functions, the interaction between endophytes and plants as well as methodological limitations of endophyte research. We also provide an outlook on needs of future research to improve our understanding on the role of microbiota colonizing the endosphere on plant traits and ecosystem functioning.

Towards a unified data infrastructure to support European and global microbiome research: a call to action.

High-quality microbiome research relies on the integrity, management and quality of supporting data. Currently biobanks and culture collections have different formats and approaches to data management. This necessitates a standard data format to underpin research, particularly in line with the FAIR data standards of findability, accessibility, interoperability and reusability. We address the importance of a unified, coordinated approach that ensures compatibility of data between that needed by biobanks and culture collections, but also to ensure linkage between bioinformatic databases and the wider research community.

Major changes in grapevine wood microbiota are associated with the onset of esca, a devastating trunk disease.

Esca, a major grapevine trunk disease in old grapevines, is associated with the colonization of woody tissues by a broad range of plant pathogenic fungi. To identify which fungal and bacterial species are involved in the onset of this disease, we analysed the microbiota from woody tissues of young (10-year-old) grapevines at an early stage of esca. Using meta-barcoding, 515 fungal and 403 bacterial operational taxonomic units (OTUs) were identified in woody tissues. In situ hybridization showed that these fungi and bacteria co-inhabited in grapevine woody tissues. In non-necrotic woody tissues, fungal and bacterial microbiota varied according to organs and seasons but not diseased plant status. Phaeomoniella chlamydospora, involved in the Grapevine trunk disease, was the most abundant species in non-necrotic tissues from healthy plants, suggesting a possible non-pathogenic endophytic behaviour. Most diseased plants (70%) displayed cordons, with their central white-rot necrosis colonized essentially by two plant pathogenic fungi (Fomitiporia mediterranea: 60%-90% and P. chlamydospora: 5%-15%) and by a few bacterial taxa (Sphingomonas spp. and Mycobacterium spp.). The occurrence of a specific association of fungal and bacterial species in cordons from young grapevines expressing esca-foliar symptoms strongly suggests that that microbiota is involved in the onset of this complex disease.

Shared and host-specific microbiome diversity and functioning of grapevine and accompanying weed plants.

Weeds and crop plants select their microbiota from the same pool of soil microorganisms, however, the ecology of weed microbiomes is poorly understood. We analysed the microbiomes associated with roots and rhizospheres of grapevine and four weed species (Lamium amplexicaule L., Veronica arvensis L., Lepidium draba L. and Stellaria media L.) growing in proximity in the same vineyard using 16S rRNA gene sequencing. We also isolated and characterized 500 rhizobacteria and root endophytes from L. draba and grapevine. Microbiome data analysis revealed that all plants hosted significantly different microbiomes in the rhizosphere as well as in root compartment, however, differences were more pronounced in the root compartment. The shared microbiome of grapevine and the four weed species contained 145 OTUs (54.2%) in the rhizosphere, but only nine OTUs (13.2%) in the root compartment. Seven OTUs (12.3%) were shared in all plants and compartments. Approximately 56% of the major OTUs (>1%) showed more than 98% identity to bacteria isolated in this study. Moreover, weed-associated bacteria generally showed a higher species richness in the rhizosphere, whereas the root-associated bacteria were more diverse in the perennial plants grapevine and L. draba. Overall, weed isolates showed more plant growth-promoting characteristics compared with grapevine isolates.

Surfactin variants mediate species-specific biofilm formation and root colonization in Bacillus.

Cyclic lipopeptides (cLP) and especially surfactins produced by Bacillus spp. trigger biofilm formation and root colonization and are crucial for biocontrol activity and systemic resistance in plants. Bacillus atrophaeus 176s isolated from the moss Tortella tortuosa produces the cLP fengycins, iturins and surfactins, possesses antifungal activities and can protect tomato, lettuce and sugar beet against Rhizoctonia solani infection. In B. atrophaeus we identified for the first time the variant surfactin C, which differs from surfactin A produced by B. subtilis and B. amyloliquefaciens by an isoleucine instead of a leucine at position 7 of the lipopeptide backbone. The analysis of the complete surfactin gene clusters revealed that the dissimilarity is encoded in the adenylation domain of srfC and show that surfactin variations are distributed in a species-specific manner in bacilli. We demonstrate that the surfactin A and C with subtle structural differences have varying signal strengths on biofilm formation and root colonization and act specifically on the respective producing strain. This became evident as biofilm formation and root colonization but not swarming motility in surfactin biosynthesis mutants was restored differentially in the presence of exogenously supplemented cognate and non-cognate surfactin variants.

Agromyces aureus sp. nov., isolated from the rhizosphere of Salix caprea L. grown in a heavy-metal-contaminated soil.

A Gram-reaction-positive, motile, yellow-pigmented and rod-shaped bacterial strain, designated AR33T, was isolated from the rhizosphere of Salix caprea L. growing in a former zinc/lead mining and processing site in Austria. A polyphasic approach was applied to determine its taxonomic position. 16S rRNA gene sequence analysis, and morphological and chemotaxonomic properties showed that strain AR33T belongs to the genus Agromyces. Strain AR33T had peptidoglycan type B2γ and the major menaquinones were MK-11, MK-10 and MK-12. The main branched-chain fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. Strain AR33T showed catalase and oxidase activity and multiple heavy metal resistances to zinc, lead and cadmium. The DNA G+C content was 70.1 mol%. Levels of 16S rRNA gene sequence similarity with closely related recognized species of the genus Agromyces ranged between 98 and 99 %. However, DNA-DNA hybridization between strain AR33T and the type strains of three Agromyces species showed values lower than 42 % relatedness. Therefore, differential phenotypic characteristics together with DNA-DNA relatedness suggested that strain AR33T can be recognized as representing a distinct Agromyces species, for which the name Agromyces aureus sp. nov. is proposed. The type strain is AR33T (=DSM 101731T=LMG 29235T).

Interkingdom transfer of the acne-causing agent, Propionibacterium acnes, from human to grapevine.

Here, we report the surprising and, to our knowledge, unique example of horizontal interkingdom transfer of a human opportunistic pathogen (Propionibacterium acnes) to a crop plant (the domesticated grapevine Vitis vinifera L.). Humans, like most organisms, have established a long-lasting cohabitation with a variety of microbes, including pathogens and gut-associated bacteria. Studies which have investigated the dynamics of such associations revealed numerous cases of bacterial host switches from domestic animals to humans. Much less is, however, known about the exchange of microbial symbionts between humans and plants. Fluorescent in situ hybridization localized P. acnes in the bark, in xylem fibers, and, more interestingly, inside pith tissues. Phylogenetic and population genetic analyses suggest that the establishment of the grapevine-associated P. acnes as obligate endophyte is compatible with a recent transfer event, likely during the Neolithic, when grapevine was domesticated.

Improved group-specific primers based on the full SILVA 16S rRNA gene reference database.

Quantitative PCR (qPCR) and community fingerprinting methods, such as the Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis,are well-suited techniques for the examination of microbial community structures. The use of phylum and class-specific primers can provide enhanced sensitivity and phylogenetic resolution as compared with domain-specific primers. To date, several phylum- and class-specific primers targeting the 16S ribosomal RNA gene have been published. However, many of these primers exhibit low discriminatory power against non-target bacteria in PCR. In this study, we evaluated the precision of certain published primers in silico and via specific PCR. We designed new qPCR and T-RFLP primer pairs (for the classes Alphaproteobacteria and Betaproteobacteria, and the phyla Bacteroidetes, Firmicutes and Actinobacteria) by combining the sequence information from a public dataset (SILVA SSU Ref 102 NR) with manual primer design. We evaluated the primer pairs via PCR using isolates of the above-mentioned groups and via screening of clone libraries from environmental soil samples and human faecal samples. As observed through theoretical and practical evaluation, the primers developed in this study showed a higher level of precision than previously published primers, thus allowing a deeper insight into microbial community dynamics.

Remarkable recovery and colonization behaviour of methane oxidizing bacteria in soil after disturbance is controlled by methane source only.

Little is understood about the relationship between microbial assemblage history, the composition and function of specific functional guilds and the ecosystem functions they provide. To learn more about this relationship we used methane oxidizing bacteria (MOB) as model organisms and performed soil microcosm experiments comprised of identical soil substrates, hosting distinct overall microbial diversities(i.e., full, reduced and zero total microbial and MOB diversities). After inoculation with undisturbed soil, the recovery of MOB activity, MOB diversity and total bacterial diversity were followed over 3 months by methane oxidation potential measurements and analyses targeting pmoA and 16S rRNA genes. Measurement of methane oxidation potential demonstrated different recovery rates across the different treatments. Despite different starting microbial diversities, the recovery and succession of the MOB communities followed a similar pattern across the different treatment microcosms. In this study we found that edaphic parameters were the dominant factor shaping microbial communities over time and that the starting microbial community played only a minor role in shaping MOB microbial community.

Variovorax sp. strain P1R9 applied individually or as part of bacterial consortia enhances wheat germination under salt stress conditions.

Endophytes isolated from extremophile plants are interesting microbes for improving the stress tolerance of agricultural plants. Here, we isolated and characterized endophytic bacteria showing plant growth-promoting (PGP) traits from plants in two extreme Chilean biomes (Atacama Desert and Chilean Patagonia). Forty-two isolates were characterized as both halotolerant auxin producers (2-51 mg L-1) and 1-aminocyclopropane-1-carboxylate (ACC)-degrading bacteria (15-28 µmol αKB mg protein-1 h-1). The most efficient isolates were tested as single strains, in dual and triple consortia, or in combination with previously reported PGP rhizobacteria (Klebsiella sp. 27IJA and 8LJA) for their impact on the germination of salt-exposed (0.15 M and 0.25 M NaCl) wheat seeds. Interestingly, strain P1R9, identified as Variovorax sp., enhanced wheat germination under salt stress conditions when applied individually or as part of bacterial consortia. Under salt stress, plants inoculated with dual consortia containing the strain Variovorax sp. P1R9 showed higher biomass (41%) and reduced lipid peroxidation (33-56%) than uninoculated plants. Although the underlying mechanisms remain elusive, our data suggest that the application of Variovorax sp. P1R9, alone or as a member of PGP consortia, may improve the salt stress tolerance of wheat plants.

Bacterially induced weathering of ultramafic rock and its implications for phytoextraction.

The bioavailability of metals in soil is often cited as a limiting factor of phytoextraction (or phytomining). Bacterial metabolites, such as organic acids, siderophores, or biosurfactants, have been shown to mobilize metals, and their use to improve metal extraction has been proposed. In this study, the weathering capacities of, and Ni mobilization by, bacterial strains were evaluated. Minimal medium containing ground ultramafic rock was inoculated with either of two Arthrobacter strains: LA44 (indole acetic acid [IAA] producer) or SBA82 (siderophore producer, PO4 solubilizer, and IAA producer). Trace elements and organic compounds were determined in aliquots taken at different time intervals after inoculation. Trace metal fractionation was carried out on the remaining rock at the end of the experiment. The results suggest that the strains act upon different mineral phases. LA44 is a more efficient Ni mobilizer, apparently solubilizing Ni associated with Mn oxides, and this appeared to be related to oxalate production. SBA82 also leads to release of Ni and Mn, albeit to a much lower extent. In this case, the concurrent mobilization of Fe and Si indicates preferential weathering of Fe oxides and serpentine minerals, possibly related to the siderophore production capacity of the strain. The same bacterial strains were tested in a soil-plant system: the Ni hyperaccumulator Alyssum serpyllifolium subsp. malacitanum was grown in ultramafic soil in a rhizobox system and inoculated with each bacterial strain. At harvest, biomass production and shoot Ni concentrations were higher in plants from inoculated pots than from noninoculated pots. Ni yield was significantly enhanced in plants inoculated with LA44. These results suggest that Ni-mobilizing inoculants could be useful for improving Ni uptake by hyperaccumulator plants.

Regulon studies and in planta role of the BraI/R quorum-sensing system in the plant-beneficial Burkholderia cluster.

The genus Burkholderia is composed of functionally diverse species, and it can be divided into several clusters. One of these, designated the plant-beneficial-environmental (PBE) Burkholderia cluster, is formed by nonpathogenic species, which in most cases have been found to be associated with plants. It was previously established that members of the PBE group share an N-acyl-homoserine lactone (AHL) quorum-sensing (QS) system, designated BraI/R, that produces and responds to 3-oxo-C14-HSL (OC14-HSL). Moreover, some of them also possess a second AHL QS system, designated XenI2/R2, producing and responding to 3-hydroxy-C8-HSL (OHC8-HSL). In the present study, we performed liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis to determine which AHL molecules are produced by each QS system of this group of bacteria. The results showed that XenI2/R2 is mainly responsible for the production of OHC8-HSL and that the BraI/R system is involved in the production of several different AHLs. This analysis also revealed that Burkholderia phymatum STM815 produces greater amounts of AHLs than the other species tested. Further studies showed that the BraR protein of B. phymatum is more promiscuous than other BraR proteins, responding equally well to several different AHL molecules, even at low concentrations. Transcriptome studies with Burkholderia xenovorans LB400 and B. phymatum STM815 revealed that the BraI/R regulon is species specific, with exopolysaccharide production being the only common phenotype regulated by this system in the PBE cluster. In addition, BraI/R was shown not to be important for plant nodulation by B. phymatum strains or for endophytic colonization and growth promotion of maize by B. phytofirmans PsJN.

The new species Enterobacter oryziphilus sp. nov. and Enterobacter oryzendophyticus sp. nov. are key inhabitants of the endosphere of rice.

BACKGROUND: Six independent Gram-negative, facultatively anaerobic, non-spore-forming, nitrogen-fixing rod-shaped isolates were obtained from the root endosphere of rice grown at the International Rice Research Institute (IRRI) and investigated in a polyphasic taxonomic study. RESULTS: The strains produced fatty acid patterns typical for members of the family Enterobacteriaceae. Comparative sequence analyses of the 16S rRNA as well as rpoB genes allocated the strains to two well-defined groups within the genus Enterobacter, family Enterobacteriaceae. The analyses indicated Enterobacter radicincitans, Enterobacter arachidis and Enterobacter oryzae to be the closest related species. An RpoB (translated) protein comparison supported the placement in the genus Enterobacter and the relatedness of our isolates to the aforementioned species. Genomic DNA:DNA hybridization analyses and biochemical analyses provided further evidence that the novel strains belong to two new species within the genus Enterobacter. The two species can be differentiated from each other and from existing enteric species by acid production from L-rhamnose and D-melibiose, decarboxylation of ornithine and utilization of D-alanine, D-raffinose L-proline and L-aspartic acid, among other characteristics. Members of both species revealed capacities to colonise rice roots, including plant-growth-promoting capabilities such as an active supply of fixed nitrogen to the plant and solubilisation of inorganic phosphorus, next to traits allowing adaptation to the plant. CONCLUSIONS: Two novel proposed enterobacterial species, denominated Enterobacter oryziphilus sp. nov. (type strain REICA_142(T)=LMG 26429(T)=NCCB 100393(T)) and Enterobacter oryzendophyticus sp. nov. (type strain REICA_082(T)=LMG 26432(T) =NCCB 100390(T)) were isolated from rice roots. Both species are capable of promoting rice growth by supplying nitrogen and phosphorus.

Propidium monoazide-quantitative polymerase chain reaction for viable Escherichia coli and Pseudomonas aeruginosa detection from abundant background microflora.

Nucleic acid-based techniques represent a promising alternative to cultivation-based microbial water quality assessment methods. However, their application is hampered by their innate inability to differentiate between living and dead organisms. Propidium monoazide (PMA) treatment was proposed as an efficient approach for alleviating this limitation. In this study, we demonstrate the performance of PMA-quantitative polymerase chain reaction (qPCR) for the detection of indicator organisms (Escherichia coli and Pseudomonas aeruginosa) in a background of a highly abundant and complex microflora. Treatment with 10 µM PMA resulted in the complete or significant reduction of the false positive signal arising from the amplification of DNA from dead cells.

Spirosoma endophyticum sp. nov., isolated from Zn- and Cd-accumulating Salix caprea.

A Gram-reaction-negative, yellow-pigmented strain, designated EX36(T), was characterized using a polyphasic approach comprising phylogenetic, morphological and genotypic analyses. The endophytic strain was isolated from Zn/Cd-accumulating Salix caprea in Arnoldstein, Austria. Analysis of the 16S rRNA gene demonstrated that the novel strain is most closely related to members of the genus Spirosoma (95 % sequence similarity with Spirosoma linguale). The genomic DNA G+C content was 47.2 mol%. The predominant quinone was and the major cellular fatty acids were summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c), C16 : 1ω5c, iso-C17 : 0 3-OH and iso-C15 : 0. On the basis of its phenotypic and genotypic properties, strain EX36(T) should be classified as a novel species of the genus Spirosoma, for which the name Spirosoma endophyticum sp. nov. is proposed. The type strain is EX36(T) ( = DSM 26130(T) = LMG 27272(T)).