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Compared to agrochemicals, bioinoculants based on plant microbiomes are a sustainable option for increasing crop yields and soil fertility. From the Mexican maize landrace “Raza cónico” (red and blue varieties), we identified yeasts and evaluated in vitro their ability to promote plant growth. Auxin production was detected from yeast isolates and confirmed using Arabidopsis thaliana plants. Inoculation tests were performed on maize, and morphological parameters were measured. Eighty-seven yeast strains were obtained (50 from blue corn and 37 from red corn). These were associated with three families of Ascomycota (Dothideaceae, Debaryomycetaceae, and Metschnikowiaceae) and five families of Basidiomycota (Sporidiobolaceae, Filobasidiaceae, Piskurozymaceae, Tremellaceae, and Rhynchogastremataceae), and, in turn, distributed in 10 genera (Clavispora, Rhodotorula, Papiliotrema, Candida, Suhomyces, Soliccocozyma, Saitozyma Holtermaniella, Naganishia, and Aeurobasidium). We identified strains that solubilized phosphate and produced siderophores, proteases, pectinases, and cellulases but did not produce amylases. Solicoccozyma sp. RY31, C. lusitaniae Y11, R. glutinis Y23, and Naganishia sp. Y52 produced auxins from L-Trp (11.9–52 µg/mL) and root exudates (1.3–22.5 µg/mL). Furthermore, they stimulated the root development of A. thaliana. Inoculation of auxin-producing yeasts caused a 1.5-fold increase in maize plant height, fresh weight, and root length compared to uninoculated controls. Overall, maize landraces harbor plant growth-promoting yeasts and have the potential for use as agricultural biofertilizers.

Medicinal plants play an important role in the discovery of new bioactive compounds with antimicrobial activity, thanks to their pharmacological properties. However, members of their microbiota can also synthesize bioactive molecules. Among these, strains belonging to the genera Arthrobacter are commonly found associated with the plant’s microenvironments, showing plant growth-promoting (PGP) activity and bioremediation properties. However, their role as antimicrobial secondary metabolite producers has not been fully explored. The aim of this work was to characterize the Arthrobacter sp. OVS8 endophytic strain, isolated from the medicinal plant Origanum vulgare L., from molecular and phenotypic viewpoints to evaluate its adaptation and influence on the plant internal microenvironments and its potential as a producer of antibacterial volatile molecules (VOCs). Results obtained from the phenotypic and genomic characterization highlight its ability to produce volatile antimicrobials effective against multidrug-resistant (MDR) human pathogens and its putative PGP role as a producer of siderophores and degrader of organic and inorganic pollutants. The outcomes presented in this work identify Arthrobacter sp. OVS8 as an excellent starting point toward the exploitation of bacterial endophytes as antibiotics sources.

International audience; Plants host a wide range of viruses and microorganisms collectively referred to as the plant microbiota. This chapter outlines the state of knowledge on the diversity of these communities of microorganisms within different plant compartments, including the rhizosphere and the phyllosphere. The ecological processes (selection, diversification, dispersal and ecological drift) involved in the assembly of the plant microbiota are also briefly introduced. Finally in the last section of this chapter, the associations between plant traits and microbiota structure are highlighted with a specific emphasis on plant adaptation to biotic stresses.

Plants and their associated microbiota share ecological and evolutionary traits that are considered to be inseparably woven. Their coexistence foresees the use of similar metabolic pathways, leading to the generation of molecules that can cross-regulate each other’s metabolism and ultimately influence plant phenotype. However, the extent to which the microbiota contributes to the overall plant metabolic landscape remains largely unexplored. Due to their early presence in the seed, seed-borne endophytic bacteria can intimately colonize the plant’s endosphere while conferring a series of phytobeneficial services to their host. Understanding the dynamics of these endophytic communities is a crucial step toward the formulation of microbial inoculants that can modulate the functionality of the plant-associated microbiota for improved plant fitness. In this work, wheat (Triticum aestivum) roots non-inoculated and inoculated with the bacterium Herbaspirillum seropedicae strain RAM10 were analyzed to explore the impact of inoculant–endophyte–wheat interrelationships on the regulation of tryptophan (Trp) metabolism in the endosphere environment. Root inoculation with H. seropedicae led to phylum-specific changes in the cultivable seed-borne endophytic community. This modulation shifted the metabolic potential of the community in light of its capacity to modulate the levels of key Trp-related metabolites involved in both indole-3-acetic acid (IAA) biosynthesis and in the kynurenine pathway. Our results support a mode of action of H. seropedicae relying on a shift in both the composition and functionality of the seed-borne endophytic community, which may govern important processes such as root growth. We finally provide a conceptual framework illustrating that interactions among roots, inoculants, and seed-borne endophytes are critical to fine-tuning the levels of IAA in the endosphere. Understanding the outcomes of these interactions is a crucial step toward the formulation of microbial inoculants based on their joint action with seed-borne endophytic communities to promote crop growth and health in a sustainable manner.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Plant growth promoting microorganisms (PGPM) are known to establish positive relationships with plants. They can inhabit the soil, the rhizosphere and the interior of plant tissues, making up a large part of the plant microbiota. They act in favor of plant nutrition, with nitrogen fixation and/or mineral solubilization, in the production of phytohormones, in the control of pathogens, and/or in the promotion of plant tolerance to abiotic stresses. These microorganisms have provided positive results in several economically important crops and can act as an alternative to the use of chemical inputs, minimizing production costs and making agricultural practices more sustainable. Therefore, this work aimed to isolate and analyze microorganisms from soil, rhizosphere and sugarcane root endosphere, to prospect bacteria, fungi and yeasts with potential to promote plant growth. The samples were plated on NA (bacteria), PDA (filamentous fungi) and YEPD (yeast) medium. Morphologically distinct colonies were isolated in pure culture and analyzed for their ability to produce indoleacetic acid (IAA), solubilize phosphate and control the development of phytopathogen Fusarium verticillioides. A total of 281 isolates were obtained, 112 from the soil, 96 from the rhizosphere and 73 from the sugarcane root endosphere. A prevalence of bacteria (223 isolates) was observed, followed by filamentous fungi (60 isolates) and, finally, in a smaller number, yeasts (9 isolates). Among the mechanisms evaluated, there was a greater number of AIA producers isolated from the endosphere, a greater number of phosphate solubilizers isolated from the soil, and a greater number of antagonists to the phytopathogen isolated from the rhizosphere. Bacteria also stood out in all evaluated mechanisms, probably due to their prevalence in the studied environments. Few isolates showed more than one plant growth promotion mechanism. The ENDO26 bacterium, isolated from the endosphere, was the only one that showed positive results for the three growth promotion mechanisms. In addition to ENDO26, two other isolates were selected: SOL88, a yeast isolated from soil, which showed positive results for phosphate solubilization and AIA production, and RIZ85, a fungus isolated from the rhizosphere, which produced AIA and presented antagonism against the phytopathogen. These three isolates were evaluated on a plate with Arabdopsis thaliana, to verify their ability to promote plant development, however, they did not promote the plant growth in vitro. The identification of isolates and more in-depth tests with other plant species are necessary to verify the potential for using these strains as plant growth promoters. Microrganismos promotores de crescimento vegetal (MPCV) são conhecidos por estabelecerem relações positivas com as plantas. Podem habitar o solo, a rizosfera e o interior dos tecidos vegetais, constituindo grande parte da microbiota da planta. Atuam no favorecimento da nutrição vegetal, com a fixação de nitrogênio e/ou solubilização de minerais, na produção de fitohormônios, no controle de agentes patogênicos, e/ou na promoção da tolerância da planta aos estresses abióticos. Estes microrganismos têm proporcionado resultados positivos em diversas culturas de importância econômica, podendo atuar como alternativa ao uso de insumos químicos, minimizando custos de produção e tornando as práticas agrícolas mais sustentáveis. Diante disso, este trabalho teve como objetivo isolar e analisar micro- organismos de solo, rizosfera e endosfera de raiz de cana-de-açúcar, para prospectar bactérias, fungos e leveduras com potencial para promoção de crescimento vegetal. As amostras foram plaqueadas em meio NA (bactérias), BDA (fungos filamentosos) e YEPD (leveduras). As colônias morfologicamente distintas foram isoladas em cultura pura e analisadas quanto a capacidade de produzir ácido indolacético (AIA), solubilizar fosfato e controlar o desenvolvimento do fitopatógeno Fusarium verticillioides. Foram obtidos 281 isolados no total, sendo 112 do solo, 96 de rizosfera e 73 de endosfera de raiz de cana-de-açúcar. Foi observado prevalência de bactérias (223 isolados), seguido de fungos filamentosos (60 isolados) e, por último, em menor número, as leveduras (9 isolados). Dentre os mecanismos avaliados, houve maior número de produtores de AIA isolados da endosfera, um maior número de solubilizadores de fosfato isolados do solo, e um maior número de antagonistas ao fitopatógeno isolados da rizosfera. As bactérias também se destacaram em todos os mecanismos avaliados, provavelmente devido a sua prevalência nos ambientes estudados. Foram poucos os isolados que apresentaram mais de um mecanismo de promoção de crescimento vegetal. A bactéria ENDO26, isolada da endosfera, foi a única que apresentou resultado positivo para os três mecanismos. Além da ENDO26, outros dois isolados foram selecionados: SOL88, uma levedura isolada de solo, que apresentou resultado positivo para solubilização de fosfato e produção de AIA e RIZ85, fungo isolado da rizosfera, que produziu AIA e apresentou antagonismo contra o fitopatógeno. Estes três isolados foram avaliados em placa com Arabdopsis thaliana, para verificar a capacidade de promover o desenvolvimento do vegetal, porém não houve promoção de crescimento da espécie in vitro. A identificação dos isolados e testes mais aprofundados com outras espécies vegetais são necessários para verificar o potencial de emprego dessas linhagens como promotoras de crescimento vegetal. 001

11 pags., 5 figs., Lipopolysaccharides, the major outer membrane components of Gram-negative bacteria, are crucial actors of the host-microbial dialogue. They can contribute to the establishment of either symbiosis or bacterial virulence, depending on the bacterial lifestyle. Plant microbiota shows great complexity, promotes plant health and growth and assures protection from pathogens. How plants perceive LPS from plant-associated bacteria and discriminate between beneficial and pathogenic microbes is an open and urgent question. Here, we report on the structure, conformation, membrane properties and immune recognition of LPS isolated from the Arabidopsis thaliana root microbiota member Herbaspirillum sp. Root189. The LPS consists of an O-methylated and variously acetylated D-rhamnose containing polysaccharide with a rather hydrophobic surface. Plant immunology studies in A. thaliana demonstrate that the native acetylated O-antigen shields the LPS from immune recognition whereas the O-deacylated one does not. These findings highlight the role of Herbaspirillum LPS within plant-microbial crosstalk, and how O-antigen modifications influence membrane properties and modulate LPS host recognition., This study was supported by PRIN 2017 "Glytunes" (2017XZ2ZBK, 2019-2022) to AS; by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme under grant agreement No 851356 to RM. Neutron Reflectivity (NR) measurements were performed at the INTER instrument at ISIS Pulsed Neutron and Muon Source, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Didcot, UK. The authors thank the ISIS facility for provision of beam time. MACR and DS gratefully acknowl- edge financial support from the Spanish Ministry of Science, Innovation, and Universities (RTI2018-099985-B-I00), and the CIBER of Respiratory Diseases (CIBERES), an initiative from the Spanish Institute of Health Carlos III (ISCIII). AZ and LM acknowledge support from the Cluster of Excellence on Plant Sciences (CEPLAS) funded by the Deutsche For- schungsgemeinschaft (DFG, German Research Foundation) under Ger- many’s Excellence Strategy-EXC 2048/1-Project ID: 390686111 and project ZU 263/11-1 (SPP DECRyPT)

Plant microbiota have co-evolved with their associated plants in the entire holobiont, and their assemblages support diversity, health and productivity on our planet. Of importance is the in vitro cultivation and identification of their hub taxa for possible core microbiome modification. Therefore, we recently introduced the in situ-similis culturing strategy, based on the use of plant leaves as platform for in vitro growth of plant microbiota. Here, we augment the strategy by the incorporation of the Most Probable Number (MPN) method, based on the use of leaf strips and root segments immersed in plain semi-solid water agar, as growth milieu. Cultivability of bacterial endophytes of sunflower plants (Helianthus annuus L.) was monitored in terms of MPN population estimates, community composition through PCR-DGGE analysis and 16S rRNA gene sequencing of representative isolates. The method efficiently enriched the plant endophytes as MPN estimates exceeded log 7.0 g-1 leaf/root. Results of PCR-DGGE analyses indicated divergence in community composition of cultivable bacterial endophytes primarily attributed to the type of culture media, signaling a certain degree of plant-organ specificity and compatibility. Based on 16S rRNA gene sequencing of representative bacterial isolates, 20 genera comprising 32 species were enriched in leaf strips- and root segments-based media. They belonged to the three phyla Bacteroidetes, Firmicutes and Proteobacteria, especially Alpha- and Gammaproteobacteria. The MPN strategy furnished a diversified nutritive platform in terms of homologous/heterologous and ambient/limited oxygenic cultivation. As a result, cultivability was extended to more 8 genera - Bosea, Brevundimonas, Caulobacter, Chitinophaga, Pseudoxanthomonas, Scandinavium, Sphingobacterium, Starkeya - among them three genera, namely Caulobacter, Scandinavium and Starkeya, which were not yet reported for Sunflower, even with the use of metagenomics sequencing methods. Possible unknown species or even one new putative genus were enriched. Thus, not only potential members of the major microbiome but also the rare isolates of satellite microbiomes can be isolated using the presented in situ similis MPN-cultivation strategy. It is a feasible addition to traditional cultivation methods to probe deeper for both the major and rare taxa and fill the many cultivation gaps within plant microbiota.

The seed microbial community constitutes an initial inoculum for plant microbiota assembly. Still, the persistence of seed microbiota when seeds encounter soil during plant emergence and early growth is barely documented. We characterized the encounter event of seed and soil microbiota and how it structured seedling bacterial and fungal communities by using amplicon sequencing. We performed eight contrasting encounter events to identify drivers influencing seedling microbiota assembly. To do so, four contrasting seed lots of two Brassica napus genotypes were sown in two soils whose microbial diversity levels were manipulated by serial dilution and recolonization. Seedling root and stem microbiota were influenced by soil but not by initial seed microbiota composition or by plant genotype. A strong selection on the seed and soil communities occurred during microbiota assembly, with only 8% to 32% of soil taxa and 0.8% to 1.4% of seed-borne taxa colonizing seedlings. The recruitment of seedling microbiota came mainly from soil (35% to 72% of diversity) and not from seeds (0.3% to 15%). Soil microbiota transmission success was higher for the bacterial community than for the fungal community. Interestingly, seedling microbiota was primarily composed of initially rare taxa (from seed, soil, or unknown origin) and intermediate-abundance soil taxa. IMPORTANCE Seed microbiota can have a crucial role for crop installation by modulating dormancy, germination, seedling development, and recruitment of plant symbionts. Little knowledge is available on the fraction of the plant microbiota that is acquired through seeds. We characterize the encounter between seed and soil communities and how they colonize the seedling together. Transmission success and seedling community assemblage can be influenced by the variation of initial microbial pools, i.e., plant genotype and cropping year for seeds and diversity level for soils. Despite a supposed resident advantage of the seed microbiota, we show that transmission success is in favor of the soil microbiota. Our results also suggest that successful plant-microbiome engineering based on native seed or soil microbiota must include rare taxa.

International audience; Microorganisms are associated with all plants, recently leading to the hologenome concept. We reviewed the assembly processes of plant microbiota and analyzed its structure during the emergence of dysbioses. In particular, we discussed the Anna Karenina Principle (AKP) based on Leo Tolstoy’s assertion applied to plant microbiota: ’All healthy microbiota are alike; each disease-associated microbiota is sick in its own way.’ We propose the AKP to explain how stochastic processes in plant microbiota assembly due to several external stressors could lead to plant diseases. Finally, we propose the AKP to conceptualize plant dysbioses as a transitory loss of host capacity to regulate its microbiota, implying a loss of function that leads to a reduction of the host’s fitness.

Medicinal plants (MPs) have been used since antiquity in traditional and popular medicine, and they represent a very important source of bioactive molecules, including antibiotic, antiviral, and antifungal molecules. Such compounds are often of plant origin, but in some cases, an origin or a modification from plant microbiota has been shown. Actually, the research continues to report the production of bioactive molecules by plants, but the role of plant–endophytic interaction is emerging. Classic examples are mainly concerned with fungal endophytes; however, it has been recently shown that bacterial endophytes can also play an important role in influencing the plant metabolism related to the synthesis of bioactive compounds. In spite of this, a deep investigation on the power of MP bacterial endophytes is lacking. Here, an overview of the studies on MP bacterial microbiota and its role in the production of plant antimicrobial compounds contributing to prime host defense system and representing a huge resource for biotech and therapeutic applications is provided.

The composition of the plant microbiota may be altered by ecological and evolutionary changes in the host population. Seed-associated microbiota, expected to be largely vertically transferred, have the potential to coadapt with their host over generations. Strong directional selection and changes in the genetic composition of plants during domestication and cultivation may have impacted the assembly and transmission of seed-associated microbiota. Nonetheless, the effect of plant speciation and domestication on the composition of these microbes is poorly understood. Here, we have investigated the composition of bacteria and fungi associated with the wild emmer wheat (Triticum dicoccoides) and domesticated bread wheat (Triticum aestivum). We show that vertically transmitted bacteria, but not fungi, of domesticated bread wheat species T. aestivum are less diverse and more inconsistent among individual plants compared to those of the wild emmer wheat species T. dicoccoides. We propagated wheat seeds under sterile conditions to characterize the colonization of seedlings by seed-associated microbes. Hereby, we show markedly different community compositions and diversities of leaf and root colonizers of the domesticated bread wheat compared to the wild emmer wheat. By propagating the wild emmer wheat and domesticated bread wheat in two different soils, we furthermore reveal a small effect of plant genotype on microbiota assembly. Our results suggest that domestication and prolonged breeding have impacted the vertically transferred bacteria, but only to a lesser extent have affected the soil-derived microbiota of bread wheat. IMPORTANCE Genetic and physiological changes associated with plant domestication have been studied for many crop species. Still little is known about the impact of domestication on the plant-associated microbiota. In this study, we analyze the seed-associated and soil-derived bacterial and fungal microbiota of domesticated bread wheat and wild emmer wheat. We show a significant difference in the seed-associated, but not soil-derived, bacterial communities of the wheat species. Interestingly, we find less pronounced effects on the fungal communities. Overall, this study provides novel insight into the diversity of vertically transmitted microbiota of wheat and thereby contributes to our understanding of wheat as a “metaorganism.” Insight into the wheat microbiota is of fundamental importance for the development of improved crops.

Genetic and physiological changes associated with plant domestication have been studied for many crop species. Still little is known about the impact of domestication on the plant-associated microbiota. In this study, we analyze the seed-associated and soil-derived bacterial and fungal microbiota of domesticated bread wheat and wild emmer wheat. We show a significant difference in the seed-associated, but not soil-derived, bacterial communities of the wheat species. Interestingly, we find less pronounced effects on the fungal communities. Overall, this study provides novel insight into the diversity of vertically transmitted microbiota of wheat and thereby contributes to our understanding of wheat as a “metaorganism.” Insight into the wheat microbiota is of fundamental importance for the development of improved crops., The composition of the plant microbiota may be altered by ecological and evolutionary changes in the host population. Seed-associated microbiota, expected to be largely vertically transferred, have the potential to coadapt with their host over generations. Strong directional selection and changes in the genetic composition of plants during domestication and cultivation may have impacted the assembly and transmission of seed-associated microbiota. Nonetheless, the effect of plant speciation and domestication on the composition of these microbes is poorly understood. Here, we have investigated the composition of bacteria and fungi associated with the wild emmer wheat (Triticum dicoccoides) and domesticated bread wheat (Triticum aestivum). We show that vertically transmitted bacteria, but not fungi, of domesticated bread wheat species T. aestivum are less diverse and more inconsistent among individual plants compared to those of the wild emmer wheat species T. dicoccoides. We propagated wheat seeds under sterile conditions to characterize the colonization of seedlings by seed-associated microbes. Hereby, we show markedly different community compositions and diversities of leaf and root colonizers of the domesticated bread wheat compared to the wild emmer wheat. By propagating the wild emmer wheat and domesticated bread wheat in two different soils, we furthermore reveal a small effect of plant genotype on microbiota assembly. Our results suggest that domestication and prolonged breeding have impacted the vertically transferred bacteria, but only to a lesser extent have affected the soil-derived microbiota of bread wheat.

There is growing recognition of the role that the microbiome plays in the health and physiology of many plant species. However, considerably less research has been conducted on the postharvest microbiome of produce and the impact that postharvest processing may have on its composition. Here, amplicon sequencing was used to study the effect of washing, waxing, and low-temperature storage at 2 &deg, C for six months on the bacterial and fungal communities of apple calyx-end, stem-end, and peel tissues. The results of the present work reveal that tissue-type is the main factor defining fungal and bacterial diversity and community composition on apple fruit. Both postharvest treatments and low temperature storage had a strong impact on the fungal and bacterial diversity and community composition of these tissue types. Distinct spatial and temporal changes in the composition and diversity of the microbiota were observed in response to various postharvest management practices. The greatest impact was attributed to sanitation practices with major differences among unwashed, washed and washed-waxed apples. The magnitude of the differences, however, was tissue-specific, with the greatest impact occurring on peel tissues. Temporally, the largest shift occurred during the first two months of low-temperature storage, although fungi were more affected by storage time than bacteria. In general, fungi and bacteria were impacted equally by sanitation practices, especially the epiphytic microflora of peel tissues. This research provides a foundation for understanding the impact of postharvest management practices on the microbiome of apple and its potential subsequent effects on postharvest disease management and food safety.

High-throughput cultivation methods have recently been developed to accelerate the recovery of microorganisms reluctant to cultivation. They simulate in situ environmental conditions for the isolation of environmental microbiota through the exchange of growth substrates during cultivation. Here, we introduce leaf-based culture media adopting the concept of the plant being the master architect of the composition of its microbial community. Pre-physical treatments of sunflower plant leaves, namely punching, freezing, and/or autoclavation, allowed the diffusion of electrolytes and other nutrients to configure the leaf surface as a natural pad, i.e., creating an “in situ similis” environment suitable for the growth of rarely isolated microbiota. We used surface inoculation and membrane-filtration methods to assess the culturability of endophytic bacteria from the sunflower phyllosphere and rhizosphere. Both methods supported excellent colony-forming unit (CFU) development when compared to standard R2A medium, with a special affinity to support better growth of epiphytic and endophytic populations of the phyllosphere compared with the rhizosphere. A 16S rRNA gene analysis of >122 representative isolates indicated the cultivation of a diverse set of microorganisms by application of the new methods. It indicated the predominance of 13 genera of >30 potential species, belonging to Firmicutes, Proteobacteria, and Actinobacteria, and especially genera not commonly reported for sunflower, e.g., Rhizobium, Aureimonas, Sphingomonas, Paracoccus, Stenotrophomonas, Pantoea, Kosakonia, and Erwinia. The strategy successfully extended diversity and richness in the endophyllosphere compared to the endorhizosphere, while CFUs grown on the standard R2A medium mainly pertain to Firmicutes, especially Bacillus spp. MALDI-TOF MS analysis clustered the isolates according to their niche and potential functions, where the majority of isolates of the endorhizosphere were clustered away from those of the endophyllosphere. Isolates identified as Gammaproteobacteria and Alphaproteobacteria were distinguishably sub-clustered, which was in contrast to the heterogeneous isolates of Firmicutes (Bacillus spp.). In conclusion, leaf in situ similis cultivation is an effective strategy to support the future application of culturomics of plant microbiota. This is an effort to access novel isolates that are more adapted and competitive in their natural environments, especially those subjected to abiotic stresses like those prevailing in arid/semi-arid zones, and, consequently, to support the application of agro-biotechnologies, among other technologies, to improving agriculture in such zones.

Global microbial kingdom analysis conducted simultaneously on multiple plants shows that cereals, legumes, and Brassicaceae establish similar prokaryotic and similar eukaryotic communities inside and on the root surface. While the bacterial microbiota is recruited from the surrounding soil, its profile is influenced by the root fraction more so than by soil or plant species. However, in contrast, the fungal microbiota is most strongly influenced by soil. This was observed in two different soils and for all plant species examined, indicating conserved adaptation of microbial communities to plants. Microbiota structure is established within 2 weeks of plant growth in soil and remains stable thereafter. We observed a remarkable similarity in the structure of a plant’s phyllosphere and root microbiotas and show by reciprocal soil swap experiments that both fractions are colonized from the soil in which the plant is grown. Thus, the phyllosphere is continuously colonized by the soil microbiota., Plant roots influence the soil microbiota via physical interaction, secretion, and plant immunity. However, it is unclear whether the root fraction or soil is more important in determining the structure of the prokaryotic or eukaryotic community and whether this varies between plant species. Furthermore, the leaf (phyllosphere) and root microbiotas have a large overlap; however, it is unclear whether this results from colonization of the phyllosphere by the root microbiota. Soil, rhizosphere, rhizoplane, and root endosphere prokaryote-, eukaryote-, and fungus-specific microbiotas of four plant species were analyzed with high-throughput sequencing. The strengths of factors controlling microbiota structure were determined using permutational multivariate analysis of variance (PERMANOVA) statistics. The origin of the phyllosphere microbiota was investigated using a soil swap experiment. Global microbial kingdom analysis conducted simultaneously on multiple plants shows that cereals, legumes, and Brassicaceae establish similar prokaryotic and similar eukaryotic communities inside and on the root surface. While the bacterial microbiota is recruited from the surrounding soil, its profile is influenced by the root itself more so than by soil or plant species. However, in contrast, the fungal microbiota is most strongly influenced by soil. This was observed in two different soils and for all plant species examined. Microbiota structure is established within 2 weeks of plant growth in soil and remains stable thereafter. A reciprocal soil swap experiment shows that the phyllosphere is colonized from the soil in which the plant is grown.

International audience; Microbes encompass tremendous biodiversity, provide support to all living forms, including humans, and play an important role in many ecosystem services. The rules that govern microorganism community assembly are increasingly revealed due to key advances in molecular and analytical methods but their understanding remain a key challenge in microbial ecology. The existence of biogeographic patterns within microbial communities has been established and explained in relation to landscape-scale processes, including selection, drift, dispersal and mutation. The effect of habitat patchiness on microorganisms' assembly rules remains though incompletely understood. Here, we review how landscape ecology principles can be adapted to explore new perspectives on the mechanisms that determine microbial community structure. To provide a general overview, we characterize microbial landscapes, the spatial and temporal scales of the mechanisms that drive microbial assembly and the feedback between microorganisms and landscape structure. We provide evidence for the effects of landscape heterogeneity, landscape fragmentation and landscape dynamics on microbial community structure, and show that predictions made for macro-organisms at least partly also apply to microorganisms. We explain why emerging metacommunity approaches in microbial ecology should include explicit characterization of landscape structure in their development and interpretation. We also explain how biotic interactions, such as competition, prey-predator or mutualist relations may influence the microbial landscape and may be involved in the above-mentioned feedback process. However, we argue that the application of landscape ecology to the microbial world cannot simply involve transposing existing theoretical frameworks. This is due to the particularity of these organisms, in terms of size, generation time, and for some of them, tight interaction with hosts. These characteristics imply dealing with unusual and dependent space and time scales of effect. Evolutionary processes have also a strong importance in microorganisms' response to their landscapes. Lastly, microorganisms' activity and distribution induce feedback effects on the landscape that have to be taken into account. The transposition of the landscape ecology framework to microorganisms provides many challenging research directions for microbial ecology.

Two Pythium‐infested soils were used to compare the wheat root and rhizosphere soil microbial communities from plants grown in the field or in greenhouse trials and their stability in the presence of biocontrol agents. Bacteria showed the highest diversity at early stages of wheat growth in both field and greenhouse trials, while fungal diversity increased later on, at 12 weeks of the crop cycle. The microbial communities were stable in roots and rhizosphere samples across both soil types used in this study. Such stability was also observed irrespective of the cultivation system (field or greenhouse) or addition of biocontrol coatings to wheat seeds to control Pythium disease (in this study soil infected with Pythium sp. clade F was tested). In greenhouse plant roots, Archaeorhizomyces, Debaryomyces, Delftia, and unclassified Pseudeurotiaceae were significantly reduced when compared to plant roots obtained from the field trials. Some operational taxonomic units (OTUs) represented genetic determinants clearly transmitted vertically by seed endophytes (specific OTUs were found in plant roots) and the plant microbiota was enriched over time by OTUs from the rhizosphere soil. This study provided key information regarding the microbial communities associated with wheat roots and rhizosphere soils at different stages of plant growth and the role that Paenibacillus and Streptomyces strains play as biocontrol agents in supporting plant growth in infested soils., The microbial communities were stable in wheat roots and rhizosphere soils exposed to Pythium and protected by biocontrol agents, whether the plants were grown in the field or greenhouse trials.

Many studies on plant biostimulants and organic fertilizers have been focused on the ability of these products to increase crop productivity and ameliorate crop tolerance to abiotic stresses. However, little information is available on their effect on plant microbiota, whereas it is well known that microorganisms associated with plant play crucial roles on the health and productivity of their host. The aim of this study was to evaluate the effect of a vegetal-derived protein hydrolysate (PH), a vegetal-derived PH enriched with copper (Cu-PH), and a tropical plant extract enriched with micronutrients (PE) on shoot growth and the epiphytic bacterial population of lettuce plants and the ability of these products to enhance the growth of beneficial or harmful bacteria. The three plant-derived products enhanced shoot biomass of lettuce plants indicating a biostimulant effect of the products. Data obtained using culture-independent (Terminal Restriction Fragment Length Polymorphism and Next Generation Sequencing) and culture-dependent approaches indicated that foliar application of commercial products altered the composition of the microbial population and stimulated the growth of specific bacteria belonging toPantoea,Pseudomonas,Acinetobacter, andBacillusgenus. Data presented in this work demonstrated that some of these strains exhibited potential plant growth-promoting properties and/or biocontrol activity against fungi and bacteria phytopathogens includingFusarium,Trichoderma, andErwiniaspecies. No indication of potential health risks associated to the enrichment of human or plant bacterial pathogens emerged by the analysis of the microbiota of treated and no-treated plants. Overall, the findings presented in this study indicate that the commercial organic-based products can enhance the growth of beneficial bacteria occurring in the plant microbiota and signals produced by these bacteria can act synergistically with the organic compounds to enhance plant growth and productivity.

Plant-inhabiting microorganisms interact directly with each other, forming complex microbial interaction networks. These interactions can either prevent or facilitate the establishment of new microbial species, such as a pathogen infecting the plant. Here, our aim was to identify the most likely interactions between Erysiphe alphitoides, the causal agent of oak powdery mildew, and other foliar microorganisms of pedunculate oak (Quercus robur L.). We combined metabarcoding techniques and a Bayesian method of network inference to decipher these interactions. Our results indicate that infection with E. alphitoides is accompanied by significant changes in the composition of the foliar fungal and bacterial communities. They also highlight 13 fungal operational taxonomic units (OTUs) and 13 bacterial OTUs likely to interact directly with E. alphitoides. Half of these OTUs, including the fungal endophytes Mycosphaerella punctiformis and Monochaetia kansensis, could be antagonists of E. alphitoides according to the inferred microbial network. Further studies will be required to validate these potential interactions experimentally. Overall, we showed that a combination of metabarcoding and network inference, by highlighting potential antagonists of pathogen species, could potentially improve the biological control of plant diseases.

Plant microbiota support the diversity and productivity of plants. Thus, cultivation-dependent approaches are indispensable for in vitro manipulation of hub taxa. Despite recent advances in high-throughput methods, cultivability is lagging behind other environmental microbiomes, notably the human microbiome. As a plant-based culturing strategy, we developed culture media based on a broth of cooked aqueous mixtures of host plants. This improved the in vitro growth of representative isolates of plant microbiota and extended the in situ recovery of plant microbiota. With clover, 16S rRNA gene sequencing of representative isolates confirmed the predominance of Firmicutes, Alphaproteobacteria and Gammaproteobacteria, and less frequently Bacteroidetes and Actinobacteria. Whereas bovine-based culture media (modified R2A) confined the diversity to Firmicutes, the plant broth-based culture media revealed a wider scope of endophytes beyond rhizobia, i.e., multiple genera such as Chryseobacterium, Cronobacter, Kosakonia, Tsukamurella, and a potentially/presumptive novel species. Matrix-assisted laser desorption/ionization time-of-flight (MADI-TOF) analysis clustered isolates according to their plant niches, the endo-phyllosphere/endo-rhizosphere. We recommend the plant broth for simplicity, reproducibility and perdurable storage, supporting future culturomics applications, good laboratory practice (GLP) and good manufacturing practice (GMP). The strategy creates an &ldquo, in-situ-similis&rdquo, vegan nutritional matrix to analyze microbial diversity and reveal novel microbial resources pertinent to biotechnological and environmental applications.

Plants are colonised by millions of microorganisms representing thousands of species withvarying effects on plant growth and health. The microbial communities found on plants arecompositionally consistent and their overall positive effect on the plant is well known. However,the effects of individual microbiota members on plant hosts and vice versa, as well as the underlyingmechanisms, remain largely unknown. Here, we describe &ldquo, Litterbox&rdquo, a highly controlled system toinvestigate plant&ndash, microbe interactions. Plants were grown gnotobiotically, otherwise sterile, onzeolite-clay, a soil replacement that retains enough moisture to avoid subsequent watering.Litterbox-grown plants resemble greenhouse-grown plants more closely than agar-grown plantsand exhibit lower leaf epiphyte densities (106 cfu/g), reflecting natural conditions. Apolydimethylsiloxane (PDMS) sheet was used to cover the zeolite, significantly lowering thebacterial load in the zeolite and rhizosphere. This reduced the likelihood of potential systemicresponses in leaves induced by microbial rhizosphere colonisation. We present results of exampleexperiments studying the transcriptional responses of leaves to defined microbiota members andthe spatial distribution of bacteria on leaves. We anticipate that this versatile and affordable plantgrowth system will promote microbiota research and help in elucidating plant-microbe interactionsand their underlying mechanisms.

Plant genotype drives the development of plant phenotypes and the assembly of plant microbiota. The potential influence of the plant phenotypic characters on its microbiota is not well characterized and the co-occurrence interrelations for specific microbial taxa and plant phenotypic characters are poorly understood. We established a common garden experiment, which quantifies prokaryotic and fungal communities in the phyllosphere and rhizosphere of six spruce (Picea spp.) tree species, through Illumina amplicon sequencing. We tested for relationships between bacterial/archaeal and fungal communities and for the phenotypic characters of their plant hosts. Host phenotypic characters including leaf length, leaf water content, leaf water storage capacity, leaf dry mass per area, leaf nitrogen content, leaf phosphorous content, leaf potassium content, leaf δ13C values, stomatal conductance, net photosynthetic rate, intercellular carbon dioxide concentration, and transpiration rate were significantly correlated with the diversity and composition of the bacterial/archaeal and fungal communities. These correlations between plant microbiota and suites of host plant phenotypic characters suggest that plant genotype shape its microbiota by driving the development of plant phenotypes. This will advance our understanding of plant-microbe associations and the drivers of variation in plant and ecosystem function.

Natural control of phytopathogenic microorganisms is assumed as a priority function of the commensal plant microbiota. In this study, the suitability of fluorescent pseudomonads in the phyllosphere of crop plants as natural control agents was evaluated. Under field conditions, ears of winter wheat were found to be colonized with high consistency and at a high density by pseudomonads at the late milk dough stage. Isolates of these bacteria were evaluated for their potential to protect the plants from phytopathogenic Alternaria and Fusarium fungi. More Pseudomonas isolates were antagonistically active against alternaria than against fusaria in the dual culture test. The alternaria responded species-specifically and more sensitively to bacterial antagonism than the strain-specific reacting fusaria. A total of 110 randomly selected Pseudomonas isolates were screened for genes involved in the biosynthesis of the antibiotics 2,4-diacetylphloroglucinol, phenazine-1-carboxylic acid, pyoluteorin, and pyrrolnitrin. The key gene for production of the phloroglucinol was found in none of these isolates. At least one of the genes, encoding the biosynthesis of the other antibiotics was detected in 81% of the isolates tested. However, the antagonistic effect found in the dual culture assay was not necessarily associated with the presence of these antibiotic genes. Wheat grains as natural substrate were inoculated with selected antagonistic Pseudomonas isolates and Alternaria and Fusarium strains, respectively. The fungal growth was only slightly delayed, but the mycotoxin production was significantly reduced in most of these approaches. In conclusion, the distribution of phytopathogenic fungi of the genera Alternaria and Fusarium in the field is unlikely to be inhibited by naturally occurring pseudomonads, also because the bacterial antagonists were not evenly distributed in the field. However, pseudomonads can reduce the production of Alternaria and Fusarium mycotoxins in wheat grains and thus have the potential to improve the crop quality.

Since the colonization of land by ancestral plant lineages 450 million years ago, plants and their associated microbes have been interacting with each other, forming an assemblage of species that is often referred to as a “holobiont.” Selective pressure acting on holobiont components has likely shaped plant-associated microbial communities and selected for host-adapted microorganisms that impact plant fitness. However, the high microbial densities detected on plant tissues, together with the fast generation time of microbes and their more ancient origin compared to their host, suggest that microbe-microbe interactions are also important selective forces sculpting complex microbial assemblages in the phyllosphere, rhizosphere, and plant endosphere compartments. Reductionist approaches conducted under laboratory conditions have been critical to decipher the strategies used by specific microbes to cooperate and compete within or outside plant tissues. Nonetheless, our understanding of these microbial interactions in shaping more complex plant-associated microbial communities, along with their relevance for host health in a more natural context, remains sparse. Using examples obtained from reductionist and community-level approaches, we discuss the fundamental role of microbe-microbe interactions (prokaryotes and micro-eukaryotes) for microbial community structure and plant health. We provide a conceptual framework illustrating that interactions among microbiota members are critical for the establishment and the maintenance of host-microbial homeostasis.

Background Within the root endosphere, fungi are known to be important for plant nutrition and resistance to stresses. However, description and understanding of the rules governing community assembly in the fungal fraction of the plant microbiome remains scarce. Methods We used an innovative DNA- and RNA-based analysis of co-extracted nucleic acids to reveal the complexity of the fungal community colonizing the roots of an Agrostis stolonifera population. The normalized RNA/DNA ratio, designated the ‘mean expression ratio’, was used as a functional trait proxy. The link between this trait and phylogenetic relatedness was measured using the Blomberg’s K statistic. Results Fungal communities were highly diverse. Only ∼1.5% of the 635 OTUs detected were shared by all individuals, however these accounted for 33% of the sequence number. The endophytic fungal communities in plant roots exhibit phylogenetic clustering that can be explained by a plant host effect acting as environmental filter. The ‘mean expression ratio’ displayed significant but divergent phylogenetic signals between fungal phyla. Discussion These results suggest that environmental filtering by the host plant favours the co-existence of related and similar OTUs within the Basidiomycota community assembly, whereas the Ascomycota and Glomeromycota communities seem to be impacted by competitive interactions which promote the co-existence of phylogenetically related but ecologically dissimilar OTUs.

We examined whether the microbiota of two related aromatic thyme species, Thymus vulgaris and Thymus citriodorus, differs in relation to the composition of the respective essential oil (EO). A total of 576 bacterial isolates were obtained from three districts (leaves, roots and rhizospheric soil). They were taxonomically characterized and inspected for tolerance to the EO from the two thyme species. A district-related taxonomic pattern was found. In particular, high taxonomic diversity among the isolates from leaves was detected. Moreover, data obtained revealed a differential pattern of resistance of the isolates to EOs extracted from T. vulgaris and T. citriodorus , which was interpreted in terms of differing chemical composition of the EO of their respective host plants. In conclusion, we suggest that bacterial colonization of leaves in Thymus spp. is influenced by the EO present in leaf glandular tissue as one of the selective forces shaping endophytic community composition.

Plants are surrounded with microorganisms whose abundance is promoted by the release of plant organic compounds and by the presence of niches favourable to microbial development and activities. These microorganisms thrive in three main plant compartments, i.e., spermosphere, rhizosphere and phyllosphere, which are interconnected. They are recruited from the environment (soil, atmosphere) and from the mother plant via the seed. Plants indeed modulate the composition and activities of the hosted microbial populations through complex communication trackways relying on trophic interactions and/or molecular signalization. The tuning of these interactions by the plant favours beneficial microbial populations and activities while depressing deleterious ones, which have a major impact on plant growth and health. This review presents the current knowledge of the plant communication with associated microorganisms in the spermosphere, rhizosphere and phyllosphere and of plant and microbial traits involved. Possible prospects of application of this knowledge for monitoring plant–microbe interactions in agroecological systems with reduced chemical inputs are discussed.

With a continuous increase in the production and use in everyday life applications of engineered nanomaterials, concerns have appeared in the past decades related to their possible environmental toxicity and impact on edible plants (and therefore, upon human health). Soybean is one of the most commercially-important crop plants, and a perfect model for nanomaterials accumulation studies, due to its high biomass production and ease of cultivation. In this review, we aim to summarize the most recent research data concerning the impact of engineered nanomaterials on the soya bean, covering both inorganic (metal and metal-oxide nanoparticles) and organic (carbon-based) nanomaterials. The interactions between soybean plants and engineered nanomaterials are discussed in terms of positive and negative impacts on growth and production, metabolism and influences on the root-associated microbiota. Current data clearly suggests that under specific conditions, nanomaterials can negatively influence the development and metabolism of soybean plants. Moreover, in some cases, a possible risk of trophic transfer and transgenerational impact of engineered nanomaterials are suggested. Therefore, comprehensive risk-assessment studies should be carried out prior to any mass productions of potentially hazardous materials.

International audience; The classic understanding of organisms focuses on genes as the main source of species evolution and diversification. The recent concept of genetic accommodation questions this gene centric view by emphasizing the importance of phenotypic plasticity on evolutionary trajectories. Recent discoveries on epigenetics and symbiotic microbiota demonstrated their deep impact on plant survival, adaptation and evolution thus suggesting a novel comprehension of the plant phenotype. In addition, interplays between these two phenomena controlling plant plasticity can be suggested. Because epigenetic and plant-associated (micro-) organisms are both key sources of phenotypic variation allowing environmental adjustments, we argue that they must be considered in terms of evolution. This ‘non-conventional’ set of mediators of phenotypic variation can be seen as a toolbox for plant adaptation to environment over short, medium and long time-scales

Summary Roots of healthy plants are inhabited by soil-derived bacteria, fungi, and oomycetes that have evolved independently in distinct kingdoms of life. How these microorganisms interact and to what extent those interactions affect plant health are poorly understood. We examined root-associated microbial communities from three Arabidopsis thaliana populations and detected mostly negative correlations between bacteria and filamentous microbial eukaryotes. We established microbial culture collections for reconstitution experiments using germ-free A. thaliana. In plants inoculated with mono- or multi-kingdom synthetic microbial consortia, we observed a profound impact of the bacterial root microbiota on fungal and oomycetal community structure and diversity. We demonstrate that the bacterial microbiota is essential for plant survival and protection against root-derived filamentous eukaryotes. Deconvolution of 2,862 binary bacterial-fungal interactions ex situ, combined with community perturbation experiments in planta, indicate that biocontrol activity of bacterial root commensals is a redundant trait that maintains microbial interkingdom balance for plant health., Graphical Abstract, Highlights • Roots of healthy plants are colonized by multi-kingdom microbial consortia • Bacterial Root Commensals (BRCs) shape fungal and oomycetal community structure • BRCs protect plants against fungi and oomycetes • Biocontrol activity of BRCs is a redundant trait and essential for plant survival, An interkingdom analysis of the microbes associated with Arabidopsis roots explains their functional contributions to plant survival.

Next generation sequencing (NGS) technologies have impressively accelerated research in biological science during the last years by enabling the production of large volumes of sequence data to a drastically lower price per base, compared to traditional sequencing methods. The recent and ongoing developments in the field allow addressing research questions in plant-microbe biology that were not conceivable just a few years ago. The present review provides an overview of NGS technologies and their usefulness for the analysis of microorganisms that live in association with plants. Possible limitations of the different sequencing systems, in particular sources of errors and bias, are critically discussed and methods are disclosed that help to overcome these shortcomings. A focus will be on the application of NGS methods in metagenomic studies, including the analysis of microbial communities by amplicon sequencing, which can be considered as a targeted metagenomic approach. Different applications of NGS technologies are exemplified by selected research articles that address the biology of the plant associated microbiota to demonstrate the worth of the new methods.

Complex microbial communities are associated with plants and can improve their resilience under harsh environmental conditions. In particular, plants and their associated communities have developed complex adaptation strategies against cold stress. Although changes in plant-associated microbial community structure have been analysed in different cold regions, scarce information is available on possible common taxonomic and functional features of microbial communities across cold environments. In this review, we discuss recent advances in taxonomic and functional characterization of plant-associated microbial communities in three main cold regions, such as alpine, Arctic and Antarctica environments. Culture-independent and culture-dependent approaches are analysed, in order to highlight the main factors affecting the taxonomic structure of plant-associated communities in cold environments. Moreover, biotechnological applications of plant-associated microorganisms from cold environments are proposed for agriculture, industry and medicine, according to biological functions and cold adaptation strategies of bacteria and fungi. Although further functional studies may improve our knowledge, the existing literature suggest that plants growing in cold environments harbor complex, host-specific and cold-adapted microbial communities, which may play key functional roles in plant growth and survival under cold conditions., Review on plant-associated microbial communities in alpine, Arctic and Antarctic regions: factors affecting the taxonomic structure: discussion on dominant taxa and their possible functional properties.