For more than 450 million years, arbuscular mycorrhizal fungi (AMF) have formed intimate, mutualistic symbioses with the vast majority of land plants and are major drivers in almost all terrestrial ecosystems. The obligate plant-symbiotic AMF host additional symbionts, so-called Mollicutes-related endobacteria (MRE). To uncover putative functional roles of these widespread but yet enigmatic MRE, we sequenced the genome of DhMRE living in the AMF Dentiscutata heterogama. Multilocus phylogenetic analyses showed that MRE form a previously unidentified lineage sister to the hominis group of Mycoplasma species. DhMRE possesses a strongly reduced metabolic capacity with 55% of the proteins having unknown function, which reflects unique adaptations to an intracellular lifestyle. We found evidence for transkingdom gene transfer between MRE and their AMF host. At least 27 annotated DhMRE proteins show similarities to nuclear-encoded proteins of the AMF Rhizophagus irregularis, which itself lacks MRE. Nuclear-encoded homologs could moreover be identified for another AMF, Gigaspora margarita, and surprisingly, also the non-AMF Mortierella verticillata. Our data indicate a possible origin of the MRE-fungus association in ancestors of the Glomeromycota and Mucoromycotina. The DhMRE genome encodes an arsenal of putative regulatory proteins with eukaryotic-like domains, some of them encoded in putative genomic islands. MRE are highly interesting candidates to study the evolution and interactions between an ancient, obligate endosymbiotic prokaryote with its obligate plant-symbiotic fungal host. Our data moreover may be used for further targeted searches for ancient effector-like proteins that may be key components in the regulation of the arbuscular mycorrhiza symbiosis.

The soil microbial community is highly complex and contains a high density of antibiotic-producing bacteria, making it a likely source of diverse antibiotic resistance determinants. We used functional metagenomics to search for antibiotic resistance genes in libraries generated from three different soil samples, containing 3.6 Gb of DNA in total. We identified 11 new antibiotic resistance genes: 3 conferring resistance to ampicillin, 2 to gentamicin, 2 to chloramphenicol and 4 to trimethoprim. One of the clones identified was a new trimethoprim resistance gene encoding a 26.8 kDa protein closely resembling unassigned reductases of the dihydrofolate reductase group. This protein, Tm8-3, conferred trimethoprim resistance in Escherichia coli and Sinorhizobium meliloti (γ- and α-proteobacteria respectively). We demonstrated that this gene encoded an enzyme with dihydrofolate reductase activity, with kinetic constants similar to other type I and II dihydrofolate reductases (K(m) of 8.9 µM for NADPH and 3.7 µM for dihydrofolate and IC(50) of 20 µM for trimethoprim). This is the first description of a new type of reductase conferring resistance to trimethoprim. Our results indicate that soil bacteria display a high level of genetic diversity and are a reservoir of antibiotic resistance genes, supporting the use of this approach for the discovery of novel enzymes with unexpected activities unpredictable from their amino acid sequences.

Microbial interactions occurring on and around seeds are especially important for plant fitness since seed-borne microorganisms are the initial source of inoculum for the plant microbiota. In this study, we analyze structural and functional changes occurring within the plant microbiota at these early stages of the plant cycle, namely germination and emergence. To this purpose, we performed shotgun DNA sequencing of microbial assemblages associated to seeds, germinating seeds and seedlings of two plant species: bean and radish. We observed an enrichment of Enterobacteriales and Pseudomonadales during emergence and a set of functional traits linked to copiotrophy that could be responsible for this selection as a result of an increase of nutrient availability after germination. Representative bacterial isolates of taxa that are selected in seedlings showed indeed faster bacterial growth rate in comparison to seed-associated bacteria isolates. Finally, binning of metagenomics contigs results in the reconstruction of population genomes of the major bacterial taxa associated to the samples. Together, our results demonstrate that, although seed microbiota varied across plant species, nutrient availability during germination elicits changes of the composition of microbial communities by potentially selecting microbial groups with functional traits linked to copiotrophy. The data presented here represents the first attempts to empirically assess changes in the microbial community during plant emergence and moves us toward a more holistic understanding of the plant microbiome.

The world's fourth largest food crop, potato, originates in the Andes. Here, the community composition of arbuscular mycorrhizal fungi (AMF) associated with potato in Andean ecosystems is described for the first time. AMF were studied in potato roots and rhizosphere soil at four different altitudes from 2,658 to 4,075 m above mean sea level (mamsl) and in three plant growth stages (emergence, flowering, and senescence). AMF species were distinguished by sequencing an approx. 1,500 bp nuclear rDNA region. Twenty species of AMF were identified, of which 12 came from potato roots and 15 from rhizosphere soil. Seven species were found in both roots and soil. Interestingly, altitude affected species composition with the highest altitude exhibiting the greatest species diversity. The three most common colonizers of potato roots detected were Funneliformis mosseae, an unknown Claroideoglomus sp., and Rhizophagus irregularis. Notably, the potato-associated AMF diversity observed in this Andean region is much higher than that reported for potato in other ecosystems. Potato plants were colonized by diverse species from 8 of the 11 Glomeromycota families. Identification of the AMF species is important for their potential use in sustainable management practices to improve potato production in the Andean region.

The nature reserve of Tehuacan-Cuicatlan in central Mexico is known for its diversity and endemism mainly in cactus plants. Although the xerophytic flora is reasonably documented, the bacterial communities associated with these species have been largely neglected. We assessed the diversity and composition of bacterial communities in bulk (non-rhizospheric) soil and the rhizosphere of three cactus plant species: Mammillaria carnea, Opuntia pilifera and Stenocereus stellatus, approached using cultivation and molecular techniques, considering the possible effect of dry and rainy seasons. Cultivation-dependent methods were focused on putative N(2)-fixers and heterotrophic aerobic bacteria, in the two media tested the values obtained for dry season samples grouped together regardless of the sample type (rhizospheric or non-rhizospheric), these groups also included the non-rhizospheric sample for rainy season, on each medium. These CFU values were smaller and significantly different from those obtained on rhizospheric samples from rainy season. Genera composition among isolates of the rhizospheric samples was very similar for each season, the most abundant taxa being α-Proteobacteria, Actinobacteria and Firmicutes. Interestingly, the genus Ochrobactrum was highly represented among rhizospheric samples, when cultured in N-free medium. The structure of the bacterial communities was approached with molecular techniques targeting partial 16S rRNA sequences such as denaturing gradient gel electrophoresis and serial analysis of ribosomal sequence tags. Under these approaches, the most represented bacterial phyla were Actinobacteria, Proteobacteria and Acidobacteria. The first two were also highly represented when using isolation techniques.

Gloria torres-cortes 2,4* the assembly of the seed microbiota involves some early microbial seed colonizers that are transmitted from the maternal plant through the vascular system, while other microbes enter through the stigma. Thus, the seed microbiota consists of microbes not only recruited from the plant vascular tissues, but also from the flower. Flowers are known to be a hub for microbial transmission between plants and insects. This floral-insect exchange opens the possibility for insect-transmitted bacteria to colonize the ovule and, subsequently, the seed to pass then into the next plant generation. In this study, we evaluated the contribution of insect pollination to the seed microbiota through high-throughput sequencing. Oilseed rape (OSR) flowers were exposed to visits and pollination by honey bees (Apis mellifera), red mason bees (Osmia bicornis), hand pollinated or left for autonomous self-pollination (ASP). Sequence analyses revealed that honey bee visitation reduced bacterial richness and diversity in seeds, but increased the variability of seed microbial structure, and introduced bee-associated taxa. In contrast, mason bee pollination had minor effects on the seed microbiota. Our study provides the first evidence that insect pollination is an ecological process involved in the transmission of bacteria from flowers to seeds. In nature, plants live in close association with a diversity of micro-and macro-organisms, both within and outside their tissues. Microbes may play beneficial roles in plant growth and development, positively affecting plant biomass or disease resistance 1-3. Although numerous studies have focused on microbial assemblages associated with different plant organs 4,5 , little is known about tripartite interactions between plants, their microbiomes and other multicellular organisms, such as pollinators. Insect visitors acquire and deposit microorganisms onto flower surfaces during nectar and pollen collection 6-9 , thus shape the flower microbiota 10-12. These flower-associated microbes are mainly fungi, followed by bacteria 10. A recent study has shown that these flower inhabitants can act as intermediaries of plant-pollinator communication; bees innately avoid flowers inhabited by bacteria but are not deterred by yeasts 13. Moreover, microorganisms transported by insects may influence plant-pollinator interactions; this is the case of yeasts transported by ants that change nectar composition 8. Furthermore, since the flower microbiota serves as one of several inocula for the plant ovule and, hence, for the seed 14 , it is possible that by affecting the microbial community of the flower (including pollen), pollinators could modify the seed microbiota. The role of insect vectors in the dispersal of bacteria and fungi to roots, stems, leaves, flowers, and fruits is well documented 15,16 , while their role in the microbial assembly of the seed has yet to be described. During seed-to-seed development, some early microbial seed colonizers are transmitted from the mother plant to the o...

Seeds are involved in the vertical transmission of microorganisms in plants and act as reservoirs for the plant microbiome. They could serve as carriers of pathogens, making the study of microbial interactions on seeds important in the emergence of plant diseases. We studied the influence of biological disturbances caused by seed transmission of two phytopathogenic agents, Alternaria brassicicola Abra43 (Abra43) and Xanthomonas campestris pv. campestris 8004 (Xcc8004), on the structure and function of radish seed microbial assemblages, as well as the nutritional overlap between Xcc8004 and the seed microbiome, to find seed microbial residents capable of outcompeting this pathogen. According to taxonomic and functional inference performed on metagenomics reads, no shift in structure and function of the seed microbiome was observed following Abra43 and Xcc8004 transmission. This lack of impact derives from a limited overlap in nutritional resources between Xcc8004 and the major bacterial populations of radish seeds. However, two native seed-associated bacterial strains belonging to Stenotrophomonas rhizophila displayed a high overlap with Xcc8004 regarding the use of resources; they might therefore limit its transmission. The strategy we used may serve as a foundation for the selection of seed indigenous bacterial strains that could limit seed transmission of pathogens.

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