Seed tuber imprinting shapes the next-generation potato microbiome.

BACKGROUND: Potato seed tubers are colonized and inhabited by soil-borne microbes, that can affect the performance of the emerging daughter plant in the next season. In this study, we investigated the intergenerational inheritance of microbiota from seed tubers to next-season daughter plants under field condition by amplicon sequencing of bacterial and fungal microbiota associated with tubers and roots, and tracked the microbial transmission from different seed tuber compartments to sprouts. RESULTS: We observed that field of production and potato genotype significantly (P < 0.01) affected the composition of the seed tuber microbiome and that these differences persisted during winter storage of the seed tubers. Remarkably, when seed tubers from different production fields were planted in a single trial field, the microbiomes of daughter tubers and roots of the emerging plants could still be distinguished (P < 0.01) according to the production field of the seed tuber. Surprisingly, we found little vertical inheritance of field-unique microbes from the seed tuber to the daughter tubers and roots, constituting less than 0.2% of their respective microbial communities. However, under controlled conditions, around 98% of the sprout microbiome was found to originate from the seed tuber and had retained their field-specific patterns. CONCLUSIONS: The field of production shapes the microbiome of seed tubers, emerging potato plants and even the microbiome of newly formed daughter tubers. Different compartments of seed tubers harbor distinct microbiomes. Both bacteria and fungi on seed tubers have the potential of being vertically transmitted to the sprouts, and the sprout subsequently promotes proliferation of a select number of microbes from the seed tuber. Recognizing the role of plant microbiomes in plant health, the initial microbiome of seed tubers specifically or planting materials in general is an overlooked trait. Elucidating the relative importance of the initial microbiome and the mechanisms by which the origin of planting materials affect microbiome assembly will pave the way for the development of microbiome-based predictive models that may predict the quality of seed tuber lots, ultimately facilitating microbiome-improved potato cultivation.

Molecular and evolutionary basis of O-antigenic polysaccharide-driven phage sensitivity in environmental pseudomonads.

IMPORTANCE: The application of plant-beneficial microorganisms to protect crop plants is a promising alternative to the usage of chemicals. However, biocontrol research often faces difficulties in implementing this approach due to the inconsistency of the bacterial inoculant to establish itself within the root microbiome. Beneficial bacterial inoculants can be decimated by the presence of their natural predators, notably bacteriophages (also called phages). Thus, it is important to gain knowledge regarding the mechanisms behind phage-bacteria interactions to overcome this challenge. Here, we evidence that the major long O-antigenic polysaccharide (O-PS, O-antigen) of the widely used model plant-beneficial bacterium Pseudomonas protegens CHA0 is the receptor of its natural predator, the phage ΦGP100. We examined the distribution of the gene cluster directing the synthesis of this O-PS and identified signatures of horizontal gene acquisitions. Altogether, our study highlights the importance of bacterial cell surface structure variation in the complex interplay between phages and their Pseudomonas hosts.

The conserved iol gene cluster in Pseudomonas is involved in rhizosphere competence.

The Pseudomonas genus has shown great potential as a sustainable solution to support agriculture through its plant-growth-promoting and biocontrol activities. However, their efficacy as bioinoculants is limited by unpredictable colonization in natural conditions. Our study identifies the iol locus, a gene cluster in Pseudomonas involved in inositol catabolism, as a feature enriched among superior root colonizers in natural soil. Further characterization revealed that the iol locus increases competitiveness, potentially caused by an observed induction of swimming motility and the production of fluorescent siderophore in response to inositol, a plant-derived compound. Public data analyses indicate that the iol locus is broadly conserved in the Pseudomonas genus and linked to diverse host-microbe interactions. Together, our findings suggest the iol locus as a potential target for developing more effective bioinoculants for sustainable agriculture.

Cell-type-specific transcriptomics reveals that root hairs and endodermal barriers play important roles in beneficial plant-rhizobacterium interactions.

Growth- and health-promoting bacteria can boost crop productivity in a sustainable way. Pseudomonas simiae WCS417 is such a bacterium that efficiently colonizes roots, modifies the architecture of the root system to increase its size, and induces systemic resistance to make plants more resistant to pests and pathogens. Our previous work suggested that WCS417-induced phenotypes are controlled by root cell-type-specific mechanisms. However, it remains unclear how WCS417 affects these mechanisms. In this study, we transcriptionally profiled five Arabidopsis thaliana root cell types following WCS417 colonization. We found that the cortex and endodermis have the most differentially expressed genes, even though they are not in direct contact with this epiphytic bacterium. Many of these genes are associated with reduced cell wall biogenesis, and mutant analysis suggests that this downregulation facilitates WCS417-driven root architectural changes. Furthermore, we observed elevated expression of suberin biosynthesis genes and increased deposition of suberin in the endodermis of WCS417-colonized roots. Using an endodermal barrier mutant, we showed the importance of endodermal barrier integrity for optimal plant-beneficial bacterium association. Comparison of the transcriptome profiles in the two epidermal cell types that are in direct contact with WCS417-trichoblasts that form root hairs and atrichoblasts that do not-implies a difference in potential for defense gene activation. While both cell types respond to WCS417, trichoblasts displayed both higher basal and WCS417-dependent activation of defense-related genes compared with atrichoblasts. This suggests that root hairs may activate root immunity, a hypothesis that is supported by differential immune responses in root hair mutants. Taken together, these results highlight the strength of cell-type-specific transcriptional profiling to uncover "masked" biological mechanisms underlying beneficial plant-microbe associations.

Growth rate is a dominant factor predicting the rhizosphere effect.

The root microbiome is shaped by plant root activity, which selects specific microbial taxa from the surrounding soil. This influence on the microorganisms and soil chemistry in the immediate vicinity of the roots has been referred to as the rhizosphere effect. Understanding the traits that make bacteria successful in the rhizosphere is critical for developing sustainable agriculture solutions. In this study, we compared the growth rate potential, a complex trait that can be predicted from bacterial genome sequences, to functional traits encoded by proteins. We analyzed 84 paired rhizosphere- and soil-derived 16S rRNA gene amplicon datasets from 18 different plants and soil types, performed differential abundance analysis, and estimated growth rates for each bacterial genus. We found that bacteria with higher growth rate potential consistently dominated the rhizosphere, and this trend was confirmed in different bacterial phyla using genome sequences of 3270 bacterial isolates and 6707 metagenome-assembled genomes (MAGs) from 1121 plant- and soil-associated metagenomes. We then identified which functional traits were enriched in MAGs according to their niche or growth rate status. We found that predicted growth rate potential was the main feature for differentiating rhizosphere and soil bacteria in machine learning models, and we then analyzed the features that were important for achieving faster growth rates, which makes bacteria more competitive in the rhizosphere. As growth rate potential can be predicted from genomic data, this work has implications for understanding bacterial community assembly in the rhizosphere, where many uncultivated bacteria reside.

Microbes to support plant health: understanding bioinoculant success in complex conditions.

A promising, sustainable way to enhance plant health and productivity is by leveraging beneficial microbes. Beneficial microbes are natural soil residents with proven benefits for plant performance and health. When applied in agriculture to improve crop yield and performance, these microbes are commonly referred to as bioinoculants. Yet, despite their promising properties, bioinoculant efficacy can vary dramatically in the field, hampering their applicability. Invasion of the rhizosphere microbiome is a critical determinant for bioinoculant success. Invasion is a complex phenomenon that is shaped by interactions with the local, resident microbiome and the host plant. Here, we explore all of these dimensions by cross-cutting ecological theory and molecular biology of microbial invasion in the rhizosphere. We refer to the famous Chinese philosopher and strategist Sun Tzu, who believed that solutions for problems require deep understanding of the problems themselves, to review the major biotic factors determining bioinoculant effectiveness.

Sexual reproduction contributes to the evolution of resistance-breaking isolates of the spinach pathogen Peronospora effusa.

Peronospora effusa causes downy mildew, the economically most important disease of cultivated spinach worldwide. To date, 19 P. effusa races have been denominated based on their capacity to break spinach resistances, but their genetic diversity and the evolutionary processes that contribute to race emergence are unknown. Here, we performed the first systematic analysis of P. effusa races showing that those emerge by both asexual and sexual reproduction. Specifically, we studied the diversity of 26 P. effusa isolates from 16 denominated races based on mitochondrial and nuclear comparative genomics. Mitochondrial genomes based on long-read sequencing coupled with diversity assessment based on short-read sequencing uncovered two mitochondrial haplogroups, each with distinct genome organization. Nuclear genome-wide comparisons of the 26 isolates revealed that 10 isolates from six races could clearly be divided into three asexually evolving groups, in concordance with their mitochondrial phylogeny. The remaining isolates showed signals of reticulated evolution and discordance between nuclear and mitochondrial phylogenies, suggesting that these evolved through sexual reproduction. Increased understanding of this pathogen's reproductive modes will provide the framework for future studies into the molecular mechanisms underlying race emergence and into the P. effusa-spinach interaction, thus assisting in sustainable production of spinach through knowledge-driven resistance breeding.

Seedborne Cercospora beticola Can Initiate Cercospora Leaf Spot from Sugar Beet (Beta vulgaris) Fruit Tissue.

Cercospora leaf spot (CLS) is a globally important disease of sugar beet (Beta vulgaris) caused by the fungus Cercospora beticola. Long-distance movement of C. beticola has been indirectly evidenced in recent population genetic studies, suggesting potential dispersal via seed. Commercial sugar beet "seed" consists of the reproductive fruit (true seed surrounded by maternal pericarp tissue) coated in artificial pellet material. In this study, we confirmed the presence of viable C. beticola in sugar beet fruit for 10 of 37 tested seed lots. All isolates harbored the G143A mutation associated with quinone outside inhibitor resistance, and 32 of 38 isolates had reduced demethylation inhibitor sensitivity (EC50 > 1 µg/ml). Planting of commercial sugar beet seed demonstrated the ability of seedborne inoculum to initiate CLS in sugar beet. C. beticola DNA was detected in DNA isolated from xylem sap, suggesting the vascular system is used to systemically colonize the host. We established nuclear ribosomal internal transcribed spacer region amplicon sequencing using the MinION platform to detect fungi in sugar beet fruit. Fungal sequences from 19 different genera were identified from 11 different sugar beet seed lots, but Fusarium, Alternaria, and Cercospora were consistently the three most dominant taxa, comprising an average of 93% relative read abundance over 11 seed lots. We also present evidence that C. beticola resides in the pericarp of sugar beet fruit rather than the true seed. The presence of seedborne inoculum should be considered when implementing integrated disease management strategies for CLS of sugar beet in the future.

Fungal social influencers: secondary metabolites as a platform for shaping the plant-associated community.

Fungal secondary metabolites (FSMs) are capable of manipulating plant community dynamics by inhibiting or facilitating the establishment of co-habitating organisms. Although production of FSMs is not crucial for survival of the producer, their absence can indirectly impair growth and/or niche competition of these fungi on the plant. The presence of FSMs with no obvious consequence on the fitness of the producer leaves questions regarding ecological impact. This review investigates how fungi employ FSMs as a platform to mediate fungal-fungal, fungal-bacterial and fungal-animal interactions associated with the plant community. We discuss how the biological function of FSMs may indirectly benefit the producer by altering the dynamics of surrounding organisms. We introduce several instances where FSMs influence antagonistic- or alliance-driven interactions. Part of our aim is to decipher the meaning of the FSM 'language' as it is widely noted to impact the surrounding community. Here, we highlight the contribution of FSMs to plant-associated interaction networks that affect the host either broadly or in ways that may have previously been unclear.

Rapid evolution of bacterial mutualism in the plant rhizosphere.

While beneficial plant-microbe interactions are common in nature, direct evidence for the evolution of bacterial mutualism is scarce. Here we use experimental evolution to causally show that initially plant-antagonistic Pseudomonas protegens bacteria evolve into mutualists in the rhizosphere of Arabidopsis thaliana within six plant growth cycles (6 months). This evolutionary transition is accompanied with increased mutualist fitness via two mechanisms: (i) improved competitiveness for root exudates and (ii) enhanced tolerance to the plant-secreted antimicrobial scopoletin whose production is regulated by transcription factor MYB72. Crucially, these mutualistic adaptations are coupled with reduced phytotoxicity, enhanced transcription of MYB72 in roots, and a positive effect on plant growth. Genetically, mutualism is associated with diverse mutations in the GacS/GacA two-component regulator system, which confers high fitness benefits only in the presence of plants. Together, our results show that rhizosphere bacteria can rapidly evolve along the parasitism-mutualism continuum at an agriculturally relevant evolutionary timescale.

Experimental-Evolution-Driven Identification of Arabidopsis Rhizosphere Competence Genes in Pseudomonas protegens.

Beneficial plant root-associated microorganisms carry out a range of functions that are essential for plant performance. Establishment of a bacterium on plant roots, however, requires overcoming several challenges, including competition with neighboring microorganisms and host immunity. Forward and reverse genetics have led to the identification of mechanisms that are used by beneficial microorganisms to overcome these challenges, such as the production of iron-chelating compounds, the formation of strong biofilms, or the concealment of characteristic microbial molecular patterns that trigger the host immune system. However, how such mechanisms arose from an evolutionary perspective is much less understood. To study bacterial adaptation in the rhizosphere, we employed experimental evolution to track the physiological and genetic dynamics of root-dwelling Pseudomonas protegens in the Arabidopsis thaliana rhizosphere under axenic conditions. This simplified binary one plant/one bacterium system allows for the amplification of key adaptive mechanisms for bacterial rhizosphere colonization. We identified 35 mutations, including single-nucleotide polymorphisms, insertions, and deletions, distributed over 28 genes. We found that mutations in genes encoding global regulators and in genes for siderophore production, cell surface decoration, attachment, and motility accumulated in parallel, underlining the finding that bacterial adaptation to the rhizosphere follows multiple strategies. Notably, we observed that motility increased in parallel across multiple independent evolutionary lines. All together, these results underscore the strength of experimental evolution in identifying key genes, pathways, and processes for bacterial rhizosphere colonization and a methodology for the development of elite beneficial microorganisms with enhanced root-colonizing capacities that can support sustainable agriculture in the future. IMPORTANCE Beneficial root-associated microorganisms carry out many functions that are essential for plant performance. Establishment of a bacterium on plant roots, however, requires overcoming many challenges. Previously, diverse mechanisms that are used by beneficial microorganisms to overcome these challenges were identified. However, how such mechanisms have developed from an evolutionary perspective is much less understood. Here, we employed experimental evolution to track the evolutionary dynamics of a root-dwelling pseudomonad on the root of Arabidopsis. We found that mutations in global regulators, as well as in genes for siderophore production, cell surface decoration, attachment, and motility, accumulate in parallel, emphasizing these strategies for bacterial adaptation to the rhizosphere. We identified 35 mutations distributed over 28 genes. All together, our results demonstrate the power of experimental evolution in identifying key pathways for rhizosphere colonization and a methodology for the development of elite beneficial microorganisms that can support sustainable agriculture.

A 20-kb lineage-specific genomic region tames virulence in pathogenic amphidiploid Verticillium longisporum.

Amphidiploid fungal Verticillium longisporum strains Vl43 and Vl32 colonize the plant host Brassica napus but differ in their ability to cause disease symptoms. These strains represent two V. longisporum lineages derived from different hybridization events of haploid parental Verticillium strains. Vl32 and Vl43 carry same-sex mating-type genes derived from both parental lineages. Vl32 and Vl43 similarly colonize and penetrate plant roots, but asymptomatic Vl32 proliferation in planta is lower than virulent Vl43. The highly conserved Vl43 and Vl32 genomes include less than 1% unique genes, and the karyotypes of 15 or 16 chromosomes display changed genetic synteny due to substantial genomic reshuffling. A 20 kb Vl43 lineage-specific (LS) region apparently originating from the Verticillium dahliae-related ancestor is specific for symptomatic Vl43 and encodes seven genes, including two putative transcription factors. Either partial or complete deletion of this LS region in Vl43 did not reduce virulence but led to induction of even more severe disease symptoms in rapeseed. This suggests that the LS insertion in the genome of symptomatic V. longisporum Vl43 mediates virulence-reducing functions, limits damage on the host plant, and therefore tames Vl43 from being even more virulent.

Transcriptome Signatures in Pseudomonas simiae WCS417 Shed Light on Role of Root-Secreted Coumarins in Arabidopsis-Mutualist Communication.

Pseudomonas simiae WCS417 is a root-colonizing bacterium with well-established plant-beneficial effects. Upon colonization of Arabidopsis roots, WCS417 evades local root immune responses while triggering an induced systemic resistance (ISR) in the leaves. The early onset of ISR in roots shows similarities with the iron deficiency response, as both responses are associated with the production and secretion of coumarins. Coumarins can mobilize iron from the soil environment and have a selective antimicrobial activity that impacts microbiome assembly in the rhizosphere. Being highly coumarin-tolerant, WCS417 induces the secretion of these phenolic compounds, likely to improve its own niche establishment, while providing growth and immunity benefits for the host in return. To investigate the possible signaling function of coumarins in the mutualistic Arabidopsis-WCS417 interaction, we analyzed the transcriptome of WCS417 growing in root exudates of coumarin-producing Arabidopsis Col-0 and the coumarin-biosynthesis mutant f6'h1. We found that coumarins in F6'H1-dependent root exudates significantly affected the expression of 439 bacterial genes (8% of the bacterial genome). Of those, genes with functions related to transport and metabolism of carbohydrates, amino acids, and nucleotides were induced, whereas genes with functions related to cell motility, the bacterial mobilome, and energy production and conversion were repressed. Strikingly, most genes related to flagellar biosynthesis were down-regulated by F6'H1-dependent root exudates and we found that application of selected coumarins reduces bacterial motility. These findings suggest that coumarins' function in the rhizosphere as semiochemicals in the communication between the roots and WCS417. Collectively, our results provide important novel leads for future functional analysis of molecular processes in the establishment of plant-mutualist interactions.

Identification and characterization of Cercospora beticola necrosis-inducing effector CbNip1.

Cercospora beticola is a hemibiotrophic fungus that causes cercospora leaf spot disease of sugar beet (Beta vulgaris). After an initial symptomless biotrophic phase of colonization, necrotic lesions appear on host leaves as the fungus switches to a necrotrophic lifestyle. The phytotoxic secondary metabolite cercosporin has been shown to facilitate fungal virulence for several Cercospora spp. However, because cercosporin production and subsequent cercosporin-initiated formation of reactive oxygen species is light-dependent, cell death evocation by this toxin is only fully ensured during a period of light. Here, we report the discovery of the effector protein CbNip1 secreted by C. beticola that causes enhanced necrosis in the absence of light and, therefore, may complement light-dependent necrosis formation by cercosporin. Infiltration of CbNip1 protein into sugar beet leaves revealed that darkness is essential for full CbNip1-triggered necrosis, as light exposure delayed CbNip1-triggered host cell death. Gene expression analysis during host infection shows that CbNip1 expression is correlated with symptom development in planta. Targeted gene replacement of CbNip1 leads to a significant reduction in virulence, indicating the importance of CbNip1 during colonization. Analysis of 89 C. beticola genomes revealed that CbNip1 resides in a region that recently underwent a selective sweep, suggesting selection pressure exists to maintain a beneficial variant of the gene. Taken together, CbNip1 is a crucial effector during the C. beticola-sugar beet disease process.

The Soil-Borne Identity and Microbiome-Assisted Agriculture: Looking Back to the Future.

Looking forward includes looking back every now and then. In 2007, David Weller looked back at 30 years of biocontrol of soil-borne pathogens by Pseudomonas and signified that the progress made over decades of research has provided a firm foundation to formulate current and future research questions. It has been recognized for more than a century that soil-borne microbes play a significant role in plant growth and health. The recent application of high-throughput omics technologies has enabled detailed dissection of the microbial players and molecular mechanisms involved in the complex interactions in plant-associated microbiomes. Here, we highlight old and emerging plant microbiome concepts related to plant disease control, and address perspectives that modern and emerging microbiomics technologies can bring to functionally characterize and exploit plant-associated microbiomes for the benefit of plant health in future microbiome-assisted agriculture.

Cercospora beticola: The intoxicating lifestyle of the leaf spot pathogen of sugar beet.

Cercospora leaf spot, caused by the fungal pathogen Cercospora beticola, is the most destructive foliar disease of sugar beet worldwide. This review discusses C. beticola genetics, genomics, and biology and summarizes our current understanding of the molecular interactions that occur between C. beticola and its sugar beet host. We highlight the known virulence arsenal of C. beticola as well as its ability to overcome currently used disease management strategies. Finally, we discuss future prospects for the study and management of C. beticola infections in the context of newly employed molecular tools to uncover additional information regarding the biology of this pathogen. TAXONOMY: Cercospora beticola Sacc.; Kingdom Fungi, Phylum Ascomycota, Class Dothideomycetes, Order Capnodiales, Family Mycosphaerellaceae, Genus Cercospora. HOST RANGE: Well-known pathogen of sugar beet (Beta vulgaris subsp. vulgaris) and most species of the Beta genus. Reported as pathogenic on other members of the Chenopodiaceae (e.g., lamb's quarters, spinach) as well as members of the Acanthaceae (e.g., bear's breeches), Apiaceae (e.g., Apium), Asteraceae (e.g., chrysanthemum, lettuce, safflower), Brassicaceae (e.g., wild mustard), Malvaceae (e.g., Malva), Plumbaginaceae (e.g., Limonium), and Polygonaceae (e.g., broad-leaved dock) families. DISEASE SYMPTOMS: Leaves infected with C. beticola exhibit circular lesions that are coloured tan to grey in the centre and are often delimited by tan-brown to reddish-purple rings. As disease progresses, spots can coalesce to form larger necrotic areas, causing severely infected leaves to wither and die. At the centre of these spots are black spore-bearing structures (pseudostromata). Older leaves often show symptoms first and younger leaves become infected as the disease progresses. MANAGEMENT: Application of a mixture of fungicides with different modes of action is currently performed although elevated resistance has been documented in most employed fungicide classes. Breeding for high-yielding cultivars with improved host resistance is an ongoing effort and prudent cultural practices, such as crop rotation, weed host management, and cultivation to reduce infested residue levels, are widely used to manage disease. USEFUL WEBSITE: https://www.ncbi.nlm.nih.gov/genome/11237?genome_assembly_id=352037.

Genome comparisons suggest an association between Ceratocystis host adaptations and effector clusters in unique transposable element families.

Ceratocystis fimbriata is a host specific fungal pathogen of sweet potato (Ipomoea batatas). The closely related species, C. manginecans, is an important pathogen of trees (e.g. Acacia mangium and Mangifera indica) but has never been isolated from tuber crops. The genetic factors that determine the host range and host specificity of these species have not been determined. The aim of this study was to compare the genomes of C. fimbriata and C. manginecans in order to identify species-specific genetic differences that could be associated with host specificity. This included whole-genome alignments as well as comparisons of gene content and transposable elements (TEs). The genomes of the two species were found to be very similar, sharing similar catalogues of CAZymes, peptidases and lipases. However, the genomes of the two species also varied, harbouring species-specific genes (e.g. small secreted effectors, nutrient processing proteins and stress response proteins). A portion of the TEs identified (17%) had a unique distribution in each species. Transposable elements appeared to have played a prominent role in the divergence of the two species because they were strongly associated with chromosomal translocations and inversions as well as with unique genomic regions containing species-specific genes. Two large effector clusters, with unique TEs in each species, were identified. These effectors displayed non-synonymous mutations and deletions, conserved within a species, and could serve as mutational hot-spots for the development of host specificity in the two species.

A secreted LysM effector protects fungal hyphae through chitin-dependent homodimer polymerization.

Plants trigger immune responses upon recognition of fungal cell wall chitin, followed by the release of various antimicrobials, including chitinase enzymes that hydrolyze chitin. In turn, many fungal pathogens secrete LysM effectors that prevent chitin recognition by the host through scavenging of chitin oligomers. We previously showed that intrachain LysM dimerization of the Cladosporium fulvum effector Ecp6 confers an ultrahigh-affinity binding groove that competitively sequesters chitin oligomers from host immune receptors. Additionally, particular LysM effectors are found to protect fungal hyphae against chitinase hydrolysis during host colonization. However, the molecular basis for the protection of fungal cell walls against hydrolysis remained unclear. Here, we determined a crystal structure of the single LysM domain-containing effector Mg1LysM of the wheat pathogen Zymoseptoria tritici and reveal that Mg1LysM is involved in the formation of two kinds of dimers; a chitin-dependent dimer as well as a chitin-independent homodimer. In this manner, Mg1LysM gains the capacity to form a supramolecular structure by chitin-induced oligomerization of chitin-independent Mg1LysM homodimers, a property that confers protection to fungal cell walls against host chitinases.