Genome analysis and biogeographic distribution of the earliest divergent Frankia clade in the southern hemisphere.

Coriariaceae are a small plant family of 14-17 species and subspecies that currently have a global but disjunct distribution. All species can form root nodules in symbiosis with diazotrophic Frankia cluster-2 strains, which form the earliest divergent symbiotic clade within this bacterial genus. Studies on Frankia cluster-2 mostly have focused on strains occurring in the northern hemisphere. Except for one strain from Papua New Guinea, namely Candidatus Frankia meridionalis Cppng1, no complete genome of Frankia associated with Coriaria occurring in the southern hemisphere has been published thus far, yet the majority of the Coriariaceae species occur here. We present field sampling data of novel Frankia cluster-2 strains, representing two novel species, which are associated with Coriaria arborea and Coriaria sarmentosa in New Zealand, and with Coriaria ruscifolia in Patagonia (Argentina), in addition to identifying Ca. F. meridionalis present in New Zealand. The novel Frankia species were found to be closely related to both Ca. F. meridionalis, and a Frankia species occurring in the Philippines, Taiwan, and Japan. Our data suggest that the different Frankia cluster-2 species diverged early after becoming symbiotic circa 100 million years ago.

Do DEEPER ROOTING 1 Homologs Regulate the Lateral Root Slope Angle in Cucumber (Cucumis sativus)?

The architecture of the root system is fundamental to plant productivity. The rate of root growth, the density of lateral roots, and the spatial structure of lateral and adventitious roots determine the developmental plasticity of the root system in response to changes in environmental conditions. One of the genes involved in the regulation of the slope angle of lateral roots is DEEPER ROOTING 1 (DRO1). Its orthologs and paralogs have been identified in rice, Arabidopsis, and several other species. However, nothing is known about the formation of the slope angle of lateral roots in species with the initiation of lateral root primordia within the parental root meristem. To address this knowledge gap, we identified orthologs and paralogs of the DRO1 gene in cucumber (Cucumis sativus) using a phylogenetic analysis of IGT protein family members. Differences in the transcriptional response of CsDRO1, CsDRO1-LIKE1 (CsDRO1L1), and CsDRO1-LIKE2 (CsDRO1L2) to exogenous auxin were analyzed. The results showed that only CsDRO1L1 is auxin-responsive. An analysis of promoter-reporter fusions demonstrated that the CsDRO1, CsDRO1L1, and CsDRO1L2 genes were expressed in the meristem in cell files of the central cylinder, endodermis, and cortex; the three genes displayed different expression patterns in cucumber roots with only partial overlap. A knockout of individual CsDRO1, CsDRO1L1, and CsDRO1L2 genes was performed via CRISPR/Cas9 gene editing. Our study suggests that the knockout of individual genes does not affect the slope angle formation during lateral root primordia development in the cucumber parental root.

Comparative phylogenomics and phylotranscriptomics provide insights into the genetic complexity of nitrogen-fixing root-nodule symbiosis.

Plant root-nodule symbiosis (RNS) with mutualistic nitrogen-fixing bacteria is restricted to a single clade of angiosperms, the Nitrogen-Fixing Nodulation Clade (NFNC), and is best understood in the legume family. Nodulating species share many commonalities, explained either by divergence from a common ancestor over 100 million years ago or by convergence following independent origins over that same time period. Regardless, comparative analyses of diverse nodulation syndromes can provide insights into constraints on nodulation-what must be acquired or cannot be lost for a functional symbiosis-and the latitude for variation in the symbiosis. However, much remains to be learned about nodulation, especially outside of legumes. Here, we employed a large-scale phylogenomic analysis across 88 species, complemented by 151 RNA-seq libraries, to elucidate the evolution of RNS. Our phylogenomic analyses further emphasize the uniqueness of the transcription factor NIN as a master regulator of nodulation and identify key mutations that affect its function across the NFNC. Comparative transcriptomic assessment revealed nodule-specific upregulated genes across diverse nodulating plants, while also identifying nodule-specific and nitrogen-response genes. Approximately 70% of symbiosis-related genes are highly conserved in the four representative species, whereas defense-related and host-range restriction genes tend to be lineage specific. Our study also identified over 900 000 conserved non-coding elements (CNEs), over 300 000 of which are unique to sampled NFNC species. NFNC-specific CNEs are enriched with the active H3K9ac mark and are correlated with accessible chromatin regions, thus representing a pool of candidate regulatory elements for genes involved in RNS. Collectively, our results provide novel insights into the evolution of nodulation and lay a foundation for engineering of RNS traits in agriculturally important crops.

Lateral Root Initiation in Cucumber (Cucumis sativus): What Does the Expression Pattern of Rapid Alkalinization Factor 34 (RALF34) Tell Us?

In Arabidopsis, the small signaling peptide (peptide hormone) RALF34 is involved in the gene regulatory network of lateral root initiation. In this study, we aimed to understand the nature of the signals induced by RALF34 in the non-model plant cucumber (Cucumis sativus), where lateral root primordia are induced in the apical meristem of the parental root. The RALF family members of cucumber were identified using phylogenetic analysis. The sequence of events involved in the initiation and development of lateral root primordia in cucumber was examined in detail. To elucidate the role of the small signaling peptide CsRALF34 and its receptor CsTHESEUS1 in the initial stages of lateral root formation in the parental root meristem in cucumber, we studied the expression patterns of both genes, as well as the localization and transport of the CsRALF34 peptide. CsRALF34 is expressed in all plant organs. CsRALF34 seems to differ from AtRALF34 in that its expression is not regulated by auxin. The expression of AtRALF34, as well as CsRALF34, is regulated in part by ethylene. CsTHESEUS1 is expressed constitutively in cucumber root tissues. Our data suggest that CsRALF34 acts in a non-cell-autonomous manner and is not involved in lateral root initiation in cucumber.

Integrative Proteomics and Metabolomics Analysis Reveals the Role of Small Signaling Peptide Rapid Alkalinization Factor 34 (RALF34) in Cucumber Roots.

The main role of RALF small signaling peptides was reported to be the alkalization control of the apoplast for improvement of nutrient absorption; however, the exact function of individual RALF peptides such as RALF34 remains unknown. The Arabidopsis RALF34 (AtRALF34) peptide was proposed to be part of the gene regulatory network of lateral root initiation. Cucumber is an excellent model for studying a special form of lateral root initiation taking place in the meristem of the parental root. We attempted to elucidate the role of the regulatory pathway in which RALF34 is a participant using cucumber transgenic hairy roots overexpressing CsRALF34 for comprehensive, integrated metabolomics and proteomics studies, focusing on the analysis of stress response markers. CsRALF34 overexpression resulted in the inhibition of root growth and regulation of cell proliferation, specifically in blocking the G2/M transition in cucumber roots. Based on these results, we propose that CsRALF34 is not part of the gene regulatory networks involved in the early steps of lateral root initiation. Instead, we suggest that CsRALF34 modulates ROS homeostasis and triggers the controlled production of hydroxyl radicals in root cells, possibly associated with intracellular signal transduction. Altogether, our results support the role of RALF peptides as ROS regulators.

3-Pentanol glycosides from root nodules of the actinorhizal plant Alnus cremastogyne.

Alnus cremastogyne Burkill (Betulaceae), an actinorhizal plant, can enter a mutualistic symbiosis with Frankia species that leads to the formation of nitrogen fixing root nodules. Some primary metabolites (carbohydrates, dicarboxylic acids, amino acids, citrulline and amides) involved in carbon and nitrogen metabolism in actinorhizal nodules have been identified, while specialized metabolites in A. cremastogyne root nodules are yet to be characterized. In this study, we isolated and identified three undescribed 3-pentanol glycosides, i.e., 3-pentyl α-l-arabinofuranosyl-(1''→6')-ß-d-glucopyranoside, 3-pentyl α-l-rhamnopyranosyl-(1''→6')-ß-d-glucopyranoside, and 3-pentyl 6'-(3-hydroxy3-methylglutaryl)-ß-d-glucopyranoside, as well as seventeen known compounds from A. cremastogyne root nodules. 3-Pentanol glycosides are abundantly distributed in root nodules, while they are distributed in stems, roots, leaves and fruits at low/zero levels. A. cremastogyne plants treated by root nodule suspension emit 3-pentanol. This study enriches the knowledge about specialized metabolites in the actinorhizal host, and provides preliminarily information on the signal exchange in the actinorhizal symbiosis between A. cremastogyne and Frankia.

Tomato Sterol 22-desaturase Gene CYP710A11: Its Roles in Meloidogyne incognita Infection and Plant Stigmasterol Alteration.

Sterols are isoprenoid-derived lipids that play essential structural and functional roles in eukaryotic cells. Plants produce a complex mixture of sterols, and changes in plant sterol profiles have been linked to plant-pathogen interactions. ß-Sitosterol and stigmasterol, in particular, have been associated with plant defense. As nematodes have lost the ability to synthesize sterols de novo, they require sterols from the host. Tomato (Solanum lycopersicum) plants infected by the plant parasitic nematode Meloidogyne incognita show a reduced level of stigmasterol and a repression of the gene CYP710A11, encoding the sterol C-22 desaturase that is responsible for the conversion of ß-sitosterol to stigmasterol. In this study, we investigated the role of the tomato sterol C-22 desaturase gene CYP710A11 in the response to infection by M. incognita. We explored the plant-nematode interaction over time by analyzing the plant sterol composition and CYP710A11 gene regulation in S. lycopersicum after M. incognita infection. The temporal gene expression analysis showed that 3 days after inoculation with M. incognita, the CYP710A11 expression was significantly suppressed in the tomato roots, while a significant decrease in the stigmasterol content was observed after 14 days. A cyp710a11 knockout mutant tomato line lacking stigmasterol was analyzed to better understand the role of CYP710A11 in nematode development. M. incognita grown in the mutant line showed reduced egg mass counts, presumably due to the impaired growth of the mutant. However, the nematodes developed as well as they did in the wild-type line. Thus, while the suppression of CYP710A11 expression during nematode development may be a defense response of the plant against the nematode, the lack of stigmasterol did not seem to affect the nematode. This study contributes to the understanding of the role of stigmasterol in the interaction between M. incognita and tomato plants and shows that the sterol C-22 desaturase is not essential for the success of M. incognita.

Salt Stress Tolerance in Casuarina glauca: Insights from the Branchlets Transcriptome.

Climate change and the accelerated rate of population growth are imposing a progressive degradation of natural ecosystems worldwide. In this context, the use of pioneer trees represents a powerful approach to reverse the situation. Among others, N2-fixing actinorhizal trees constitute important elements of plant communities and have been successfully used in land reclamation at a global scale. In this study, we have analyzed the transcriptome of the photosynthetic organs of Casuarina glauca (branchlets) to unravel the molecular mechanisms underlying salt stress tolerance. For that, C. glauca plants supplied either with chemical nitrogen (KNO3+) or nodulated by Frankia (NOD+) were exposed to a gradient of salt concentrations (200, 400, and 600 mM NaCl) and RNA-Seq was performed. An average of ca. 25 million clean reads was obtained for each group of plants, corresponding to 86,202 unigenes. The patterns of differentially expressed genes (DEGs) clearly separate two groups: (i) control- and 200 mM NaCl-treated plants, and (ii) 400 and 600 mM NaCl-treated plants. Additionally, although the number of total transcripts was relatively high in both plant groups, the percentage of significant DEGs was very low, ranging from 6 (200 mM NaCl/NOD+) to 314 (600 mM NaCl/KNO3+), mostly involving down-regulation. The vast majority of up-regulated genes was related to regulatory processes, reinforcing the hypothesis that some ecotypes of C. glauca have a strong stress-responsive system with an extensive set of constitutive defense mechanisms, complemented by a tight mechanism of transcriptional and post-transcriptional regulation. The results suggest that the robustness of the stress response system in C. glauca is regulated by a limited number of genes that tightly regulate detoxification and protein/enzyme stability, highlighting the complexity of the molecular interactions leading to salinity tolerance in this species.

Single, but not dual, attack by a biotrophic pathogen and a sap-sucking insect affects the oak leaf metabolome.

Plants interact with a multitude of microorganisms and insects, both below- and above ground, which might influence plant metabolism. Despite this, we lack knowledge of the impact of natural soil communities and multiple aboveground attackers on the metabolic responses of plants, and whether plant metabolic responses to single attack can predict responses to dual attack. We used untargeted metabolic fingerprinting (gas chromatography-mass spectrometry, GC-MS) on leaves of the pedunculate oak, Quercus robur, to assess the metabolic response to different soil microbiomes and aboveground single and dual attack by oak powdery mildew (Erysiphe alphitoides) and the common oak aphid (Tuberculatus annulatus). Distinct soil microbiomes were not associated with differences in the metabolic profile of oak seedling leaves. Single attacks by aphids or mildew had pronounced but different effects on the oak leaf metabolome, but we detected no difference between the metabolomes of healthy seedlings and seedlings attacked by both aphids and powdery mildew. Our findings show that aboveground attackers can have species-specific and non-additive effects on the leaf metabolome of oak. The lack of a metabolic signature detected by GC-MS upon dual attack might suggest the existence of a potential negative feedback, and highlights the importance of considering the impacts of multiple attackers to gain mechanistic insights into the ecology and evolution of species interactions and the structure of plant-associated communities, as well as for the development of sustainable strategies to control agricultural pests and diseases and plant breeding.

A tale of two lineages: how the strains of the earliest divergent symbiotic Frankia clade spread over the world.

It is currently assumed that around 100 million years ago, the common ancestor to the Fabales, Fagales, Rosales and Cucurbitales in Gondwana, developed a root nodule symbiosis with a nitrogen-fixing bacterium. The symbiotic trait evolved first in Frankia cluster-2; thus, strains belonging to this cluster are the best extant representatives of this original symbiont. Most cluster-2 strains could not be cultured to date, except for Frankia coriariae, and therefore many aspects of the symbiosis are still elusive. Based on phylogenetics of cluster-2 metagenome-assembled genomes (MAGs), it has been shown that the genomes of strains originating in Eurasia are highly conserved. These MAGs are more closely related to Frankia cluster-2 in North America than to the single genome available thus far from the southern hemisphere, i.e., from Papua New Guinea.To unravel more biodiversity within Frankia cluster-2 and predict routes of dispersal from Gondwana, we sequenced and analysed the MAGs of Frankia cluster-2 from Coriaria japonica and Coriaria intermedia growing in Japan, Taiwan and the Philippines. Phylogenetic analyses indicate there is a clear split within Frankia cluster-2, separating a continental from an island lineage. Presumably, these lineages already diverged in Gondwana.Based on fossil data on the host plants, we propose that these two lineages dispersed via at least two routes. While the continental lineage reached Eurasia together with their host plants via the Indian subcontinent, the island lineage spread towards Japan with an unknown host plant.

Legume NCRs and nodule-specific defensins of actinorhizal plants-Do they share a common origin?

The actinorhizal plant Datisca glomerata (Datiscaceae, Cucurbitales) establishes a root nodule symbiosis with actinobacteria from the earliest branching symbiotic Frankia clade. A subfamily of a gene family encoding nodule-specific defensin-like cysteine-rich peptides is highly expressed in D. glomerata nodules. Phylogenetic analysis of the defensin domain showed that these defensin-like peptides share a common evolutionary origin with nodule-specific defensins from actinorhizal Fagales and with nodule-specific cysteine-rich peptides (NCRs) from legumes. In this study, the family member with the highest expression levels, DgDef1, was characterized. Promoter-GUS studies on transgenic hairy roots showed expression in the early stage of differentiation of infected cells, and transient expression in the nodule apex. DgDef1 contains an N-terminal signal peptide and a C-terminal acidic domain which are likely involved in subcellular targeting and do not affect peptide activity. In vitro studies with E. coli and Sinorhizobium meliloti 1021 showed that the defensin domain of DgDef1 has a cytotoxic effect, leading to membrane disruption with 50% lethality for S. meliloti 1021 at 20.8 µM. Analysis of the S. meliloti 1021 transcriptome showed that, at sublethal concentrations, DgDef1 induced the expression of terminal quinol oxidases, which are associated with the oxidative stress response and are also expressed during symbiosis. Overall, the changes induced by DgDef1 are reminiscent of those of some legume NCRs, suggesting that nodule-specific defensin-like peptides were part of the original root nodule toolkit and were subsequently lost in most symbiotic legumes, while being maintained in the actinorhizal lineages.

Silicate Inhibits the Cytosolic Influx of Chloride in Protoplasts of Wheat and Affects the Chloride Transporters, TaCLC1 and TaNPF2.4/2.5.

Chloride is an essential nutrient for plants, but high concentrations can be harmful. Silicon ameliorates both abiotic and biotic stresses in plants, but it is unknown if it can prevent cellular increase of chloride. Therefore, we investigated the influx of Cl- ions in two wheat cultivars different in salt sensitivity, by epifluorescence microscopy and a highly Cl--sensitive dye, MQAE, N-[ethoxycarbonylmethyl]-6-methoxy-quinolinium bromide, in absence and presence of potassium silicate, K2SiO3. The Cl--influx was higher in the salt-sensitive cv. Vinjett, than in the salt-tolerant cv. S-24, and silicate pre-treatment of protoplasts inhibited the Cl--influx in both cultivars, but more in the sensitive cv. Vinjett. To investigate if the Cl--transporters TaCLC1 and TaNPF2.4/2.5 are affected by silicate, expression analyses by RT-qPCR were undertaken of TaCLC1 and TaNPF 2.4/2.5 transcripts in the absence and presence of 100 mM NaCl, with and without the presence of K2SiO3. The results show that both transporter genes were expressed in roots and shoots of wheat seedlings, but their expressions were differently affected by silicate. The TaNPF2.4/2.5 expression in leaves was markedly depressed by silicate. These findings demonstrate that less chloride accumulates in the cytosol of leaf mesophyll by Si treatment and increases salt tolerance.

Streptomyces coriariae sp. nov., a novel streptomycete isolated from actinorhizal nodules of Coriaria intermedia.

An actinobacterial strain, CMB-FB, was isolated from surface-sterilized root nodules of a Coriaria intermedia plant growing along Halsema Highway in the province of Benguet (Luzon, Philippines). The 16S rRNA gene sequence of CMB-FB showed high sequence similarity to those of the type strains of Streptomyces rishiriensis (99.4 %), Streptomyces humidus (99.1 %), Streptomyces cacaoi subsp. asoensis (99.0 %), and Streptomyces phaeofaciens (98.6 %). The major menaquinones of CMB-FB were composed of MK-9(H4), MK-9(H6) and MK-9(H8), and there was a minor contribution of MK-9(H10). The polar lipid profile consisted of phosphatidylethanolamine, unidentified aminolipids and phospholipids, a glycophospholipid and four unidentified lipids. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. The major fatty acids were iso-C16 : 0, anteiso-C15 : 0 and anteiso-C17 : 0. The results of physiological analysis indicated that CMB-FB was mesophilic. The results of phylogenetic, genome-genome distance calculation and average nucleotide identity analysis indicated that the isolated strain represents the type strain of a novel species. On the basis of these results, strain CMB-FB (=DSM 112754T=LMG 32457T) is proposed as the type strain of the novel species Streptomyces coriariae sp. nov.

Changes in the foliar fungal community between oak leaf flushes along a latitudinal gradient in Europe.

Aim: Leaves support a large diversity of fungi, which are known to cause plant diseases, induce plant defences or influence leaf senescence and decomposition. To advance our understanding of how foliar fungal communities are structured and assembled, we assessed to what extent leaf flush and latitude can explain the within- and among-tree variation in foliar fungal communities. Location: A latitudinal gradient spanning c. 20 degrees in latitude in Europe. Taxa: The foliar fungal community associated with a foundation tree species, the pedunculate oak Quercus robur. Methods: We examined the main and interactive effects of leaf flush and latitude on the foliar fungal community by sampling 20 populations of the pedunculate oak Quercus robur across the tree's range. We used the ITS region as a target for characterization of fungal communities using DNA metabarcoding. Results: Species composition, but not species richness, differed between leaf flushes. Across the latitudinal gradient, species richness was highest in the central part of the oak's distributional range, and foliar fungal community composition shifted along the latitudinal gradient. Among fungal guilds, the relative abundance of plant pathogens and mycoparasites was lower on the first leaf flush, and the relative abundance of plant pathogens and saprotrophs decreased with latitude. Conclusions: Changes in community composition between leaf flushes and along the latitudinal gradient were mostly a result of species turnover. Overall, our findings demonstrate that leaf flush and latitude explain 5%-22% of the small- and large-scale spatial variation in the foliar fungal community on a foundation tree within the temperate region. Using space-for-time substitution, we expect that foliar fungal community structure will change with climate warming, with an increase in the abundance of plant pathogens and mycoparasites at higher latitudes, with major consequences for plant health, species interactions and ecosystem dynamics.

YABBY Genes in the Development and Evolution of Land Plants.

Mounting evidence from genomic and transcriptomic studies suggests that most genetic networks regulating the morphogenesis of land plant sporophytes were co-opted and modified from those already present in streptophyte algae and gametophytes of bryophytes sensu lato. However, thus far, no candidate genes have been identified that could be responsible for "planation", a conversion from a three-dimensional to a two-dimensional growth pattern. According to the telome theory, "planation" was required for the genesis of the leaf blade in the course of leaf evolution. The key transcription factors responsible for leaf blade development in angiosperms are YABBY proteins, which until recently were thought to be unique for seed plants. Yet, identification of a YABBY homologue in a green alga and the recent findings of YABBY homologues in lycophytes and hornworts suggest that YABBY proteins were already present in the last common ancestor of land plants. Thus, these transcriptional factors could have been involved in "planation", which fosters our understanding of the origin of leaves. Here, we summarise the current data on functions of YABBY proteins in the vegetative and reproductive development of diverse angiosperms and gymnosperms as well as in the development of lycophytes. Furthermore, we discuss a putative role of YABBY proteins in the genesis of multicellular shoot apical meristems and in the evolution of leaves in early divergent terrestrial plants.

Changes in the Plant ß-Sitosterol/Stigmasterol Ratio Caused by the Plant Parasitic Nematode Meloidogyne incognita.

Sterols play a key role in various physiological processes of plants. Commonly, stigmasterol, ß-sitosterol and campesterol represent the main plant sterols, and cholesterol is often reported as a trace sterol. Changes in plant sterols, especially in ß-sitosterol/stigmasterol levels, can be induced by different biotic and abiotic factors. Plant parasitic nematodes, such as the root-knot nematode Meloidogyne incognita, are devastating pathogens known to circumvent plant defense mechanisms. In this study, we investigated the changes in sterols of agricultural important crops, Brassica juncea (brown mustard), Cucumis sativus (cucumber), Glycine max (soybean), Solanum lycopersicum (tomato) and Zea mays (corn), 21 days post inoculation (dpi) with M. incognita. The main changes affected the ß-sitosterol/stigmasterol ratio, with an increase of ß-sitosterol and a decrease of stigmasterol in S. lycopersicum, G. max, C. sativus and Z. mays. Furthermore, cholesterol levels increased in tomato, cucumber and corn, while cholesterol levels often were below the detection limit in the respective uninfected plants. To better understand the changes in the ß-sitosterol/stigmasterol ratio, gene expression analysis was conducted in tomato cv. Moneymaker for the sterol 22C-desaturase gene CYP710A11, responsible for the conversion of ß-sitosterol to stigmasterol. Our results showed that the expression of CYP710A11 was in line with the sterol profile of tomato after M. incognita infection. Since sterols play a key role in plant-pathogen interactions, this finding opens novel insights in plant nematode interactions.

Hairy CRISPR: Genome Editing in Plants Using Hairy Root Transformation.

CRISPR/Cas-mediated genome editing is a powerful tool of plant functional genomics. Hairy root transformation is a rapid and convenient approach for obtaining transgenic roots. When combined, these techniques represent a fast and effective means of studying gene function. In this review, we outline the current state of the art reached by the combination of these approaches over seven years. Additionally, we discuss the origins of different Agrobacterium rhizogenes strains that are widely used for hairy root transformation; the components of CRISPR/Cas vectors, such as the promoters that drive Cas or gRNA expression, the types of Cas nuclease, and selectable and screenable markers; and the application of CRISPR/Cas genome editing in hairy roots. The modification of the already known vector pKSE401 with the addition of the rice translational enhancer OsMac3 and the gene encoding the fluorescent protein DsRed1 is also described.

Candidatus Frankia nodulisporulans sp. nov., an Alnus glutinosa-infective Frankia species unable to grow in pure culture and able to sporulate in-planta.

We describe a new Frankia species, for three non-isolated strains obtained from Alnus glutinosa in France and Sweden, respectively. These strains can nodulate several Alnus species (A. glutinosa, A. incana, A. alnobetula), they form hyphae, vesicles and sporangia in the root nodule cortex but have resisted all attempts at isolation in pure culture. Their genomes have been sequenced, they are significantly smaller than those of other Alnus-infective species (5Mb instead of 7.5Mb) and are very closely related to one another (ANI of 100%). The name Candidatus Frankia nodulisporulans is proposed. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and draft genome sequences reported in this study for AgTrS, AgUmASt1 and AgUmASH1 are MT023539/LR778176/LR778180 and NZ_CADCWS000000000.1/CADDZU010000001/CADDZW010000001, respectively.