Seedling microbiota engineering using bacterial synthetic community inoculation on seeds.

Synthetic Communities (SynComs) are being developed and tested to manipulate plant microbiota and improve plant health. To date, only few studies proposed the use of SynCom on seed despite its potential for plant microbiota engineering. We developed and presented a simple and effective seedling microbiota engineering method using SynCom inoculation on seeds. The method was successful using a wide diversity of SynCom compositions and bacterial strains that are representative of the common bean seed microbiota. First, this method enables the modulation of seed microbiota composition and community size. Then, SynComs strongly outcompeted native seed and potting soil microbiota and contributed on average to 80% of the seedling microbiota. We showed that strain abundance on seed was a main driver of an effective seedling microbiota colonization. Also, selection was partly involved in seed and seedling colonization capacities since strains affiliated to Enterobacteriaceae and Erwiniaceae were good colonizers while Bacillaceae and Microbacteriaceae were poor colonizers. Additionally, the engineered seed microbiota modified the recruitment and assembly of seedling and rhizosphere microbiota through priority effects. This study shows that SynCom inoculation on seeds represents a promising approach to study plant microbiota assembly and its consequence on plant fitness.

The type VI secretion system of Stenotrophomonas rhizophila CFBP13503 limits the transmission of Xanthomonas campestris pv. campestris 8004 from radish seeds to seedlings.

Stenotrophomonas rhizophila CFBP13503 is a seedborne commensal bacterial strain, which is efficiently transmitted to seedlings and can outcompete the phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc8004). The type VI secretion system (T6SS), an interference contact-dependent mechanism, is a critical component of interbacterial competition. The involvement of the T6SS of S. rhizophila CFBP13503 in the inhibition of Xcc8004 growth and seed-to-seedling transmission was assessed. The T6SS cluster of S. rhizophila CFBP13503 and nine putative effectors were identified. Deletion of two T6SS structural genes, hcp and tssB, abolished the competitive advantage of S. rhizophila against Xcc8004 in vitro. The population sizes of these two bacterial species were monitored in seedlings after inoculation of radish seeds with mixtures of Xcc8004 and either S. rhizophila wild-type (wt) strain or isogenic hcp mutant. A significant decrease in the population size of Xcc8004 was observed during confrontation with the S. rhizophila wt in comparison with T6SS-deletion mutants in germinated seeds and seedlings. We found that the T6SS distribution among 835 genomes of the Stenotrophomonas genus is scarce. In contrast, in all available S. rhizophila genomes, T6SS clusters are widespread and mainly belong to the T6SS group i4. In conclusion, the T6SS of S. rhizophila CFBP13503 is involved in the antibiosis against Xcc8004 and reduces seedling transmission of Xcc8004 in radish. The distribution of this T6SS cluster in the S. rhizophila complex could make it possible to exploit these strains as biocontrol agents against X. campestris pv. campestris.

Defining Composition and Function of the Rhizosphere Microbiota of Barley Genotypes Exposed to Growth-Limiting Nitrogen Supplies.

The microbiota populating the rhizosphere, the interface between roots and soil, can modulate plant growth, development, and health. These microbial communities are not stochastically assembled from the surrounding soil, but their composition and putative function are controlled, at least partially, by the host plant. Here, we use the staple cereal barley as a model to gain novel insights into the impact of differential applications of nitrogen, a rate-limiting step for global crop production, on the host genetic control of the rhizosphere microbiota. Using a high-throughput amplicon sequencing survey, we determined that nitrogen availability for plant uptake is a factor promoting the selective enrichment of individual taxa in the rhizosphere of wild and domesticated barley genotypes. Shotgun sequencing and metagenome-assembled genomes revealed that this taxonomic diversification is mirrored by a functional specialization, manifested by the differential enrichment of multiple Gene Ontology terms, of the microbiota of plants exposed to nitrogen conditions limiting barley growth. Finally, a plant soil feedback experiment revealed that host control of the barley microbiota underpins the assembly of a phylogenetically diverse group of bacteria putatively required to sustain plant performance under nitrogen-limiting supplies. Taken together, our observations indicate that under nitrogen conditions limiting plant growth, host-microbe and microbe-microbe interactions fine-tune the host genetic selection of the barley microbiota at both taxonomic and functional levels. The disruption of these recruitment cues negatively impacts plant growth. IMPORTANCE The microbiota inhabiting the rhizosphere, the thin layer of soil surrounding plant roots, can promote the growth, development, and health of their host plants. Previous research indicated that differences in the genetic composition of the host plant coincide with variations in the composition of the rhizosphere microbiota. This is particularly evident when looking at the microbiota associated with input-demanding modern cultivated varieties and their wild relatives, which have evolved under marginal conditions. However, the functional significance of these differences remains to be fully elucidated. We investigated the rhizosphere microbiota of wild and cultivated genotypes of the global crop barley and determined that nutrient conditions limiting plant growth amplify the host control on microbes at the root-soil interface. This is reflected in a plant- and genotype-dependent functional specialization of the rhizosphere microbiota, which appears to be required for optimal plant growth. These findings provide novel insights into the significance of the rhizosphere microbiota for plant growth and sustainable agriculture.

Single Seed Microbiota: Assembly and Transmission from Parent Plant to Seedling.

The seed acts as the primary inoculum source for the plant microbiota. Understanding the processes involved in its assembly and dynamics during germination and seedling emergence has the potential to allow for the improvement of crop establishment. Changes in the bacterial community structure were tracked in 1,000 individual seeds that were collected throughout seed developments of beans and radishes. Seeds were associated with a dominant bacterial taxon that represented more than 75% of all reads. The identity of this taxon was highly variable between the plants and within the seeds of the same plant. We identified selection as the main ecological process governing the succession of dominant taxa during seed filling and maturation. In a second step, we evaluated the seedling transmission of seed-borne taxa in 160 individual plants. While the initial bacterial abundance on seeds was not a good predictor of seedling transmission, the identities of the seed-borne taxa modified the phenotypes of seedlings. Overall, this work revealed that individual seeds are colonized by a few bacterial taxa of highly variable identity, which appears to be important for the early stages of plant development. IMPORTANCE Seeds are key components of plant fitness and are central to the sustainability of the agri-food system. Both the seed quality for food consumption and the seed vigor in agricultural settings can be influenced by the seed microbiota. Understanding the ecological processes involved in seed microbiota assembly will inform future practices for promoting the presence of important seed microorganisms for plant health and productivity. Our results highlighted that seeds were associated with one dominant bacterial taxon of variable taxonomic identity. This variety of dominant taxa was due to (i) spatial heterogeneity between and within plants and (ii) primary succession during seed development. According to neutral models, selection was the main driver of microbial community assembly for both plant species.

Seed microbiota revealed by a large-scale meta-analysis including 50 plant species.

Seed microbiota constitutes a primary inoculum for plants that is gaining attention owing to its role for plant health and productivity. Here, we performed a meta-analysis on 63 seed microbiota studies covering 50 plant species to synthesize knowledge on the diversity of this habitat. Seed microbiota are diverse and extremely variable, with taxa richness varying from one to thousands of taxa. Hence, seed microbiota presents a variable (i.e. flexible) microbial fraction but we also identified a stable (i.e. core) fraction across samples. Around 30 bacterial and fungal taxa are present in most plant species and in samples from all over the world. Core taxa, such as Pantoea agglomerans, Pseudomonas viridiflava, P. fluorescens, Cladosporium perangustum and Alternaria sp., are dominant seed taxa. The characterization of the core and flexible seed microbiota provided here will help uncover seed microbiota roles for plant health and design effective microbiome engineering.

Transmission of Seed and Soil Microbiota to Seedling.

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.

Cross-linkage between bacterial taxonomy and gene functions: a study of metagenome-assembled genomes of gut microbiota in adult non-alcoholic fatty liver disease.

BACKGROUND: The reconstruction of metagenome-assembled genomes (MAGs) has emerged as a powerful approach for combining the taxonomic and functional content of microbial populations. AIM: To use this new approach to highlight mechanisms linking gut microbiota to NAFLD severity METHODS: Stool samples were collected from 96 NAFLD patients on the day of liver biopsy. Shotgun DNA sequencing of the gut microbiota was performed on an Illumina HiSeq3000 system. Contigs were binned into MAGs according to their co-abundances and tetranucleotide frequencies using Metabat v.0.32.4. Predicted protein-coding genes were clustered in orthologous groups (OGs) with DIAMOND against the EggNOG v4.5 database. Liver biopsies were read in accordance with the NASH CRN classification. RESULTS: Fifty-four patients had NASH and 44 had significant fibrosis (F ≥ 2). Sequencing of DNA extracted from stools resulted in 13.8 + 3.2 million paired-end reads per sample. Of the 4,000 reconstructed MAGs, 220 in NASH patients, 192 in non-NASH patients, 203 in F ≥ 2 patients and 230 in F0-1 patients had > 70% completeness and < 5% contamination. Within these MAGs, 28 OGs were associated with NASH, 33 with significant fibrosis, and seven with both NASH and significant fibrosis. The study of MAGs showed associations between NAFLD severity and some gut bacteria with microbiota functions related to hydrogen sulfide production, citrate transport, hemicellulose degradation, aldehyde production and vitamin B12 synthesis. CONCLUSION: Using new metagenomics methods, our study unveils potential mechanisms by which certain bacteria from the gut microbiota could protect or contribute to the development of NASH and liver fibrosis in NAFLD.

Maternal effects shape the seed mycobiome in Quercus petraea.

The tree seed mycobiome has received little attention despite its potential role in forest regeneration and health. The aim of the present study was to analyze the processes shaping the composition of seed fungal communities in natural forests as seeds transition from the mother plant to the ground for establishment. We used metabarcoding approaches and confocal microscopy to analyze the fungal communities of seeds collected in the canopy and on the ground in four natural populations of sessile oak (Quercus petraea). Ecological processes shaping the seed mycobiome were inferred using joint species distribution models. Fungi were present in seed internal tissues, including the embryo. The seed mycobiome differed among oak populations and trees within the same population. Its composition was largely influenced by the mother, with weak significant environmental influences. The models also revealed several probable interactions among fungal pathogens and mycoparasites. Our results demonstrate that maternal effects, environmental filtering and biotic interactions all shape the seed mycobiome of sessile oak. They provide a starting point for future research aimed at understanding how maternal genes and environments interact to control the vertical transmission of fungal species that could then influence seed dispersal and germination, and seedling recruitment.

Temporal dynamics of bacterial communities during seed development and maturation.

Seed microbiota acts as a starting point for the assembly of the plant microbiota and contributes to successful plant establishment. To date, the order and timing of microbial taxa immigration during seed development and maturation remained unknown. We investigated the temporal dynamics of seed bacterial communities in bean and radish. A high phylogenetic turnover was observed for both plant species with few taxa associated with all seed developmental stages. Greater heterogeneity in communities structure within each stage was observed for radish. While, about one-third of radish seed bacterial taxa were detected in buds, flowers and fruits, very few taxa seem to be transmitted by the floral route in bean. In the latter species, bacterial populations belonging to the P. fluorescens species complex were found either in buds, flowers and fruits or in seeds. The relative phylogenetic proximity of these bacterial populations combined with their habitat specificity led us to explore the genetic determinants involved in successful seed transmission in bean. Comparative genomic analyses of representatives bacterial strains revealed dozens of coding sequences specifically associated with seed-transmitted strains. This study provided a first glimpse on processes involved in seed microbiota assembly, which could be used for designing plant-beneficial microbial consortia.

Impact of a resistance gene against a fungal pathogen on the plant host residue microbiome: The case of the Leptosphaeria maculans-Brassica napus pathosystem.

Oilseed rape residues are a crucial determinant of stem canker epidemiology as they support the sexual reproduction of the fungal pathogen Leptosphaeria maculans. The aim of this study was to characterize the impact of a resistance gene against L. maculans infection on residue microbial communities and to identify microorganisms interacting with this pathogen during residue degradation. We used near-isogenic lines to obtain healthy and infected host plants. The microbiome associated with the two types of plant residues was characterized by metabarcoding. A combination of linear discriminant analysis and ecological network analysis was used to compare the microbial communities and to identify microorganisms interacting with L. maculans. Fungal community structure differed between the two lines at harvest, but not subsequently, suggesting that the presence/absence of the resistance gene influences the microbiome at the base of the stem whilst the plant is alive, but that this does not necessarily lead to differential colonization of the residues by fungi. Direct interactions with other members of the community involved many fungal and bacterial amplicon sequence variants (ASVs). L. maculans appeared to play a minor role in networks, whereas one ASV affiliated to Plenodomus biglobosus (synonym Leptosphaeria biglobosa) from the Leptosphaeria species complex may be considered a keystone taxon in the networks at harvest. This approach could be used to identify and promote microorganisms with beneficial effects against residue-borne pathogens and, more broadly, to decipher the complex interactions between multispecies pathosystems and other microbial components in crop residues.

Insect pollination: an ecological process involved in the assembly of the seed microbiota.

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.

Differential dynamics of microbial community networks help identify microorganisms interacting with residue-borne pathogens: the case of Zymoseptoria tritici in wheat.

BACKGROUND: Wheat residues are a crucial determinant of the epidemiology of Septoria tritici blotch, as they support the sexual reproduction of the causal agent Zymoseptoria tritici. We aimed to characterize the effect of infection with this fungal pathogen on the microbial communities present on wheat residues and to identify microorganisms interacting with it. We used metabarcoding to characterize the microbiome associated with wheat residues placed outdoors, with and without preliminary Z. tritici inoculation, comparing the first set of residues in contact with the soil and a second set without contact with the soil, on four sampling dates in two consecutive years. RESULTS: The diversity of the tested conditions, leading to the establishment of different microbial communities according to the origins of the constitutive taxa (plant only, or plant and soil), highlighted the effect of Z. tritici on the wheat residue microbiome. Several microorganisms were affected by Z. tritici infection, even after the disappearance of the pathogen. Linear discriminant analyses and ecological network analyses were combined to describe the communities affected by the infection. The number of fungi and bacteria promoted or inhibited by inoculation with Z. tritici decreased over time and was smaller for residues in contact with the soil. The interactions between the pathogen and other microorganisms appeared to be mostly indirect, despite the strong position of the pathogen as a keystone taxon in networks. Direct interactions with other members of the communities mostly involved fungi, including other wheat pathogens. Our results provide essential information about the alterations to the microbial community in wheat residues induced by the mere presence of a fungal pathogen, and vice versa. Species already described as beneficial or biocontrol agents were found to be affected by pathogen inoculation. CONCLUSIONS: The strategy developed here can be viewed as a proof-of-concept focusing on crop residues as a particularly rich ecological compartment, with a high diversity of fungal and bacterial taxa originating from both the plant and soil compartments, and for Z. tritici-wheat as a model pathosystem. By revealing putative antagonistic interactions, this study paves the way for improving the biological control of residue-borne diseases.

rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing.

BACKGROUND: Microbiome composition is frequently studied by the amplification and high-throughput sequencing of specific molecular markers (metabarcoding). Various hypervariable regions of the 16S rRNA gene are classically used to estimate bacterial diversity, but other universal bacterial markers with a finer taxonomic resolution could be employed. We compared specificity and sensitivity between a portion of the rpoB gene and the V3 V4 hypervariable region of the 16S rRNA gene. RESULTS: We first designed universal primers for rpoB suitable for use with Illumina sequencing-based technology and constructed a reference rpoB database of 45,000 sequences. The rpoB and V3 V4 markers were amplified and sequenced from (i) a mock community of 19 bacterial strains from both Gram-negative and Gram-positive lineages; (ii) bacterial assemblages associated with entomopathogenic nematodes. In metabarcoding analyses of mock communities with two analytical pipelines (FROGS and DADA2), the estimated diversity captured with the rpoB marker resembled the expected composition of these mock communities more closely than that captured with V3 V4. The rpoB marker had a higher level of taxonomic affiliation, a higher sensitivity (detection of all the species present in the mock communities), and a higher specificity (low rates of spurious OTU detection) than V3 V4. We compared the performance of the rpoB and V3 V4 markers in an animal ecosystem model, the infective juveniles of the entomopathogenic nematode Steinernema glaseri carrying the symbiotic bacteria Xenorhabdus poinarii. Both markers showed the bacterial community associated with this nematode to be of low diversity (< 50 OTUs), but only rpoB reliably detected the symbiotic bacterium X. poinarii. CONCLUSIONS: Our results confirm that different microbiota composition data may be obtained with different markers. We found that rpoB was a highly appropriate marker for assessing the taxonomic structure of mock communities and the nematode microbiota. Further studies on other ecosystems should be considered to evaluate the universal usefulness of the rpoB marker. Our data highlight two crucial elements that should be taken into account to ensure more reliable and accurate descriptions of microbial diversity in high-throughput amplicon sequencing analyses: i) the need to include mock communities as controls; ii) the advantages of using a multigenic approach including at least one housekeeping gene (rpoB is a good candidate) and one variable region of the 16S rRNA gene. This study will be useful to the growing scientific community describing bacterial communities by metabarcoding in diverse ecosystems.

Crop Residues in Wheat-Oilseed Rape Rotation System: a Pivotal, Shifting Platform for Microbial Meetings.

Crop residues are a crucial ecological niche with a major biological impact on agricultural ecosystems. In this study, we used a combined diachronic and synchronic field experiment based on wheat-oilseed rape rotations to test the hypothesis that plant is a structuring factor of microbial communities in crop residues, and that this effect decreases over time with their likely progressive degradation and colonisation by other microorganisms. We characterised an entire fungal and bacterial community associated with 150 wheat and oilseed rape residue samples at a plurennial scale by metabarcoding. The impact of plant species on the residue microbiota decreased over time and our data revealed turnover, with the replacement of oligotrophs, often plant-specific genera (such as pathogens) by copiotrophs, belonging to more generalist genera. Within a single cropping season, the plant-specific genera and species were gradually replaced by taxa that are likely to originate from the soil. These changes occurred more rapidly for bacteria than for fungi, known to degrade complex compounds. Overall, our findings suggest that crop residues constitute a key fully-fledged microbial ecosystem. Taking into account this ecosystem, that has been neglected for too long, is essential, not only to improve the quantitative management of residues, the presence of which can be detrimental to crop health, but also to identify groups of beneficial microorganisms. Our findings are of particular importance, because the wheat-oilseed rape rotation, in which no-till practices are frequent, is particularly widespread in the European arable cropping systems.

Functional Microbial Features Driving Community Assembly During Seed Germination and Emergence.

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.

In situ relationships between microbiota and potential pathobiota in Arabidopsis thaliana.

A current challenge in microbial pathogenesis is to identify biological control agents that may prevent and/or limit host invasion by microbial pathogens. In natura, hosts are often infected by multiple pathogens. However, most of the current studies have been performed under laboratory controlled conditions and by taking into account the interaction between a single commensal species and a single pathogenic species. The next step is therefore to explore the relationships between host-microbial communities (microbiota) and microbial members with potential pathogenic behavior (pathobiota) in a realistic ecological context. In the present study, we investigated such relationships within root-associated and leaf-associated bacterial communities of 163 ecologically contrasted Arabidopsis thaliana populations sampled across two seasons in southwest of France. In agreement with the theory of the invasion paradox, we observed a significant humped-back relationship between microbiota and pathobiota α-diversity that was robust between both seasons and plant organs. In most populations, we also observed a strong dynamics of microbiota composition between seasons. Accordingly, the potential pathobiota composition was explained by combinations of season-specific microbiota operational taxonomic units. This result suggests that the potential biomarkers controlling pathogen's invasion are highly dynamic.

Genome Sequence of the Necrotrophic Plant Pathogen Alternaria brassicicola Abra43.

Alternaria brassicicola causes dark spot (or black spot) disease, which is one of the most common and destructive fungal diseases of Brassicaceae spp. worldwide. Here, we report the draft genome sequence of strain Abra43. The assembly comprises 29 scaffolds, with an N50 value of 2.1 Mb. The assembled genome was 31,036,461 bp in length, with a G+C content of 50.85%.

Complete genomic sequence of Raphanus sativus cryptic virus 4 (RsCV4), a novel alphapartitivirus from radish.

The present work reports the discovery and complete genome sequencing of a virus from symptomless radish seedlings, classifiable as a novel member of the genus Alphapartitivirus, family Partitiviridae. Total RNA extracted from germinating seedlings was sequenced using Illumina technology. Bioinformatic analysis of the RNA-seq data revealed two contigs representing the near full-length genomic sequences of two genomic RNAs representing a new virus. Analysis of the genome sequence (excluding the polyA tail, RNA1: 1976 nt and RNA2: 1751 nt, respectively) showed a genomic organization typical of viruses classed within the Partitiviridae, with each genomic RNA encoding a single open reading frame (ORF). Phylogenetic analysis of the RNA dependent RNA polymerase (RNA1 ORF) and of the capsid protein (RNA2 ORF) clearly showed the new virus can be classified within the genus Alphapartitivirus, but sequence divergence establishes it as a new species, for which the name "Raphanus sativus cryptic virus 4" is proposed.

Ancestral acquisitions, gene flow and multiple evolutionary trajectories of the type three secretion system and effectors in Xanthomonas plant pathogens.

Deciphering the evolutionary history and transmission patterns of virulence determinants is necessary to understand the emergence of novel pathogens. The main virulence determinant of most pathogenic proteobacteria is the type three secretion system (T3SS). The Xanthomonas genus includes bacteria responsible for numerous epidemics in agroecosystems worldwide and represents a major threat to plant health. The main virulence factor of Xanthomonas is the Hrp2 family T3SS; however, this system is not conserved in all strains and it has not been previously determined whether the distribution of T3SS in this bacterial genus has resulted from losses or independent acquisitions. Based on comparative genomics of 82 genome sequences representing the diversity of the genus, we have inferred three ancestral acquisitions of the Hrp2 cluster during Xanthomonas evolution followed by subsequent losses in some commensal strains and re-acquisition in some species. While mutation was the main force driving polymorphism at the gene level, interspecies homologous recombination of large fragments expanding through several genes shaped Hrp2 cluster polymorphism. Horizontal gene transfer of the entire Hrp2 cluster also occurred. A reduced core effectome composed of xopF1, xopM, avrBs2 and xopR was identified that may allow commensal strains overcoming plant basal immunity. In contrast, stepwise accumulation of numerous type 3 effector genes was shown in successful pathogens responsible for epidemics. Our data suggest that capacity to intimately interact with plants through T3SS would be an ancestral trait of xanthomonads. Since its acquisition, T3SS has experienced a highly dynamic evolutionary history characterized by intense gene flux between species that may reflect its role in host adaptation.

Ecological patterns of seed microbiome diversity, transmission, and assembly.

Seeds are involved in the transmission of microorganisms from one plant generation to another and consequently act as the initial inoculum for the plant microbiota. The purpose of this mini-review is to provide an overview of current knowledge on the diversity, structure and role of the seed microbiota. The relative importance of the mode of transmission (vertical vs horizontal) of the microbial entities composing the seed microbiota as well as the potential connections existing between seed and other plant habitats such as the anthosphere and the spermosphere is discussed. Finally the governing processes (niche vs neutral) involved in the assembly and the dynamics of the seed microbiota are examined.