Frankia umida sp. nov., isolated from root nodules of Alnus glutinosa L.

Frankia strain Ag45/Mut15T was isolated from a root nodule of Alnus glutinosa growing in a swamp at lake Grossensee, Germany. The strain forms root nodules on A. glutinosa, in which it produces hyphae and clusters of N2-fixing vesicles. N2-fixing vesicles are also produced in nitrogen-free growth medium, in addition to hyphae and sporangia. The whole-cell hydrolysates of strain Ag45/Mut15T contained meso-diaminopimelic acid in the peptidoglycan and ribose, xylose, mannose, glucose, galactose and a trace of rhamnose as cell-wall sugars. The major polar lipids were phosphatidylglycerol, phosphatidylinositol, diphosphatidylglycerol and glyco-phospholipid. The predominant (>20 %) menaquinones were MK-9(H6) and MK-9(H4). The major fatty acid profile (>10 %) consisted of iso-C16:0, C17 : 1 ω8c and C17 : 0. Pairwise 16S rRNA gene distances showed that strain Ag45/Mut15T was most closely related to Frankia torreyi CpI1T and Candidatus Frankia nodulisporulans with 16S rRNA gene similarity values of 0.001335 substitutions per site. An multilocus sequence analysis phylogeny based on atpD, dnaA, ftsZ, pgk and rpoB amino acid sequences positioned the strain within cluster 1 of Alnus- and Myrica-nodulating species, close to Candidatus F. nodulisporulans AgTrST and F. canadensis ARgP5T. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between the studied strain Ag45/Mut15T and all validly named Frankia species were below the defined threshold for prokaryotic species demarcation. Candidatus F. nodulisporulans AgTrST, which cannot be cultivated in vitro, was found to be the closest phylogenetic neighbour to strain strain Ag45/Mut15T with dDDH and ANI values of 61.8 and 97 %, respectively. Strain Ag45/Mut15T was not able to sporulate in nodule tissues like strain AgTrST.Phenotypic, physiological and phylogenomic analyses confirmed the assignment of strain Ag45/Mut15T (=DSM 114737T=LMG 326O1T) to a novel species, with Ag45/Mut15T as type strain, for which the name Frankia umida sp. nov. is proposed.

Two novel species isolated from wheat rhizospheres in Serbia: Pseudomonas serbica sp. nov. and Pseudomonas serboccidentalis sp. nov.

Pseudomonas strains IT-194P, IT-215P, IT-P366T and IT-P374T were isolated from the rhizospheres of wheat grown in soils sampled from different fields (some of them known to be disease-suppressive) located near Mionica, Serbia. Phylogenetic analysis of the 16S rRNA genes and of whole genome sequences showed that these strains belong to two potentially new species, one containing strains IT-P366T and IT-194P and clustering (whole genome analysis) next to P. umsongensis DSM16611T, and another species containing strains IT-P374T and IT-215P and clustering next to P. koreensis LMG21318T. Genome analysis confirmed the proposition of novel species, as ANI was below the threshold of 95% and dDDH below 70% for strains IT-P366T (compared with P. umsongensis DSM16611T) and IT-P374T (compared with P. koreensis LMG21318T). Unlike P. umsongensis DSM16611T, strains of P. serbica can grow on D-mannitol, but not on pectin, D-galacturonic acid, L-galactonic acid lactone and α-hydroxybutyric acid. In contrary to P. koreensis LMG21318T, strains of P. serboccidentalis can use sucrose, inosine and α-ketoglutaric acid (but not L-histidine) as carbon sources. Altogether, these results indicate the existence of two novel species for which we propose the names Pseudomonas serbica sp. nov., with the type strain IT-P366T (=CFBP 9060 T = LMG 32732 T = EML 1791 T) and Pseudomonas serboccidentalis sp. nov., with the type strain IT-P374T (=CFBP 9061 T = LMG 32734 T = EML 1792 T). Strains from this study presented a set of phytobeneficial functions modulating plant hormonal balance, plant nutrition and plant protection, suggesting a potential as Plant Growth-Promoting Rhizobacteria (PGPR).

Genomic Insights of Alnus-Infective Frankia Strains Reveal Unique Genetic Features and New Evidence on Their Host-Restricted Lifestyle.

The present study aimed to use comparative genomics to explore the relationships between Frankia and actinorhizal plants using a data set made of 33 Frankia genomes. The determinants of host specificity were first explored for "Alnus-infective strains" (i.e., Frankia strains belonging to Cluster Ia). Several genes were specifically found in these strains, including an agmatine deiminase which could possibly be involved in various functions as access to nitrogen sources, nodule organogenesis or plant defense. Within "Alnus-infective strains", Sp+ Frankia genomes were compared to Sp- genomes in order to elucidate the narrower host specificity of Sp+ strains (i.e., Sp+ strains being capable of in planta sporulation, unlike Sp- strains). A total of 88 protein families were lost in the Sp+ genomes. The lost genes were related to saprophytic life (transcriptional factors, transmembrane and secreted proteins), reinforcing the proposed status of Sp+ as obligatory symbiont. The Sp+ genomes were also characterized by a loss of genetic and functional paralogs, highlighting a reduction in functional redundancy (e.g., hup genes) or a possible loss of function related to a saprophytic lifestyle (e.g., genes involved in gas vesicle formation or recycling of nutrients).

Draft Genomes of Frankia strains AiPa1 and AiPs1 Retrieved from Soil with Monocultures of Picea abies or Pinus sylvestris using Alnus incana as Capture Plant.

The genomes of two nitrogen-fixing Frankia strains, AiPa1 and AiPs1, are described as representatives of two novel candidate species. Both strains were isolated from root nodules of Alnus incana, used as capture plants in bioassays on soils from a reforested site at Karttula, Finland, that was devoid of actinorhizal plants but contained 25 year-old monocultures of spruce (Picea abies (L.) Karsten) or pine (Pinus sylvestris L.), respectively. ANI analyses indicate that each strain represents a novel Frankia species, with genome sizes of 6.98 and 7.35 Mb for AiPa1 and AiPs1, respectively. Both genomes harbored genes typical for many other symbiotic frankiae, including genes essential for nitrogen-fixation, for synthesis of hopanoid lipids and iron-sulfur clusters, as well as clusters of orthologous genes, secondary metabolite determinants and transcriptional regulators. Genomes of AiPa1 and AiPs1 had lost 475 and 112 genes, respectively, compared to those of other cultivated Alnus-infective strains with large genomes. Lost genes included one hup cluster in AiPa1 and the gvp cluster in AiPs1, suggesting that some genome erosion has started to occur in a different manner in the two strains.

Wheat genome architecture influences interactions with phytobeneficial microbial functional groups in the rhizosphere.

Wheat has undergone a complex evolutionary history, which led to allopolyploidization and the hexaploid bread wheat Triticum aestivum. However, the significance of wheat genomic architecture for beneficial plant-microbe interactions is poorly understood, especially from a functional standpoint. In this study, we tested the hypothesis that wheat genomic architecture was an overriding factor determining root recruitment of microorganisms with particular plant-beneficial traits. We chose five wheat species representing genomic profiles AA (Triticum urartu), BB {SS} (Aegilops speltoides), DD (Aegilops tauschii), AABB (Triticum dicoccon) and AABBDD (Triticum aestivum) and assessed by quantitative polymerase chain reaction their ability to interact with free-nitrogen fixers, 1-aminocyclopropane-1-carboxylate deaminase producers, 2,4-diacetylphloroglucinol producers and auxin producers via the phenylpyruvate decarboxylase pathway, in combination with Illumina MiSeq metabarcoding analysis of N fixers (and of the total bacterial community). We found that the abundance of the microbial functional groups could fluctuate according to wheat genomic profile, as did the total bacterial abundance. N fixer diversity and total bacterial diversity were also influenced significantly by wheat genomic profile. Often, rather similar results were obtained for genomes DD (Ae. tauschii) and AABBDD (T. aestivum), pointing for the first time that the D genome could be particularly important for wheat-bacteria interactions.

Microbiome Analysis of New, Insidious Cave Wall Alterations in the Apse of Lascaux Cave.

Lascaux Cave is a UNESCO site that was closed to the public following wall surface alterations. Most black stains that had formed on wall surface are stable or receding, but a new type of alteration visually quite different (termed dark zones) developed in Lascaux's Apse room in the last 15 years. Here, we tested the hypothesis that dark zones displayed a different microbial community than black stains previously documented in the same room, using metabarcoding (MiSeq sequencing). Indeed, dark zones, black stains and neighboring unstained parts displayed distinct microbial communities. However, similarly to what was observed in black stains, pigmented fungi such as Ochroconis (now Scolecobasidium) were more abundant and the bacteria Pseudomonas less abundant in dark zones than in unstained parts. The collembola Folsomia candida, which can disseminate microorganisms involved in black stain development, was also present on dark zones. Illumina sequencing evidenced Ochroconis (Scolecobasidium) in all collembola samples from dark zones, as in collembola from black stains. This study shows that the microbial properties of dark zones are peculiar, yet dark zones display a number of microbial resemblances with black stains, which suggests a possible role of collembola in promoting these two types of microbial alterations on wall surfaces.

Correction: Pujic et al. The Proteogenome of Symbiotic Frankia alni in Alnus glutinosa Nodules. Microorganisms 2022, 10, 651.

The authors wish to make the following corrections to this paper [...].

Draft Genome of Frankia sp. Strain R82, an Isolate from Root Nodules of Myrica gale.

The Frankia sp. strain R82 genome is described as representative of a novel candidate species within Frankia cluster 1, as indicated by average nucleotide identity (ANI) analyses, with its closest relatives being Frankia nodulisporulans AgTrs and strains Ag45/Mut15 and AgPM24 (86% identity).

Draft Genomes of Symbiotic Frankia Strains AgB32 and AgKG'84/4 from Root Nodules of Alnus Glutinosa growing under Contrasted Environmental Conditions.

The genomes of two nitrogen-fixing Frankia strains, AgB32 and AgKG'84/4, were isolated from spore-containing (spore+) and spore-free (spore-) root nodules of Alnus glutinosa, but they did not sporulate upon reinfection. The two strains are described as representatives of two novel candidate species. Phylogenomic and ANI analyses indicate that each strain represents a novel species within cluster 1, with genome sizes of 6.3 and 6.7 Mb smaller than or similar to those of other cultivated Alnus-infective cluster 1 strains. Genes essential for nitrogen-fixation, clusters of orthologous genes, secondary metabolite clusters and transcriptional regulators analyzed by comparative genomic analyses were typical of those from Alnus-infective cluster 1 cultivated strains in both genomes. Compared to other cultivated Alnus-infective strains with large genomes, those of AgB32 and AgKG'84/4 had lost 380 or 409 genes, among which one hup cluster, one shc gene and the gvp cluster, which indicates genome erosion is taking place in these two strains.

Pseudomonas ST1 and Pantoea Paga Strains Cohabit in Olive Knots.

Two bacteria belonging to the Pseudomonas and Pantoea genera were isolated from olive knots. Both bacterial strains were omnipresent in this study's olive orchard with high susceptibility of the autochthonous olive genotypes indicating coevolution of bacteria with host plants. Genomes of two endemic bacteria show conserved core genomes and genome plasticity. The Pseudomonas ST1 genome has conserved virulence-related genes including genes for quorum sensing, pilus, and flagella biosynthesis, two copies of indole acetic acid biosynthesis (IAA) operons, type I-VI secretions systems, and genes for alginate and levan biosynthesis. Development of knots depends only on the presence of the Pseudomonas ST1 strain which then allows Pantoea paga strain co-infection and cohabitation in developed knots. The two bacteria are sensitive to a large number of antimicrobials, antibiotics, H2O2, and Cu (II) salts that can be efficiently used in propagation of bacterial free olive cultivars.

Draft Genomes of Nitrogen-fixing Frankia Strains Ag45/Mut15 and AgPM24 Isolated from Root Nodules of Alnus Glutinosa.

The genomes of two nitrogen-fixing Frankia strains, Ag45/Mut15 and AgPM24, isolated from root nodules of Alnus glutinosa are described as representatives of a novel candidate species. Phylogenomic and ANI analyses confirmed that both strains are related to cluster 1 frankiae, and that both strains belong to a novel species. At 6.4 - 6.7 Mb, their genomes were smaller than those of other cultivated Alnus-infective cluster 1 strains but larger than that of the non-cultivated Alnus-infective cluster 1 Sp+ strain AgTrS that was their closest neighbor as assessed by ANI. Comparative genomic analyses identified genes essential for nitrogen-fixation, gene composition as regards COGs, secondary metabolites clusters and transcriptional regulators typical of those from Alnus-infective cluster 1 cultivated strains in both genomes. There were 459 genes present in other cultivated Alnus-infective strains lost in the two genomes, spread over the whole of the genome, which indicates genome erosion is taking place in these two strains.

Implications of carbon catabolite repression for plant-microbe interactions.

Carbon catabolite repression (CCR) plays a key role in many physiological and adaptive responses in a broad range of microorganisms that are commonly associated with eukaryotic hosts. When a mixture of different carbon sources is available, CCR, a global regulatory mechanism, inhibits the expression and activity of cellular processes associated with utilization of secondary carbon sources in the presence of the preferred carbon source. CCR is known to be executed by completely different mechanisms in different bacteria, yeast, and fungi. In addition to regulating catabolic genes, CCR also appears to play a key role in the expression of genes involved in plant-microbe interactions. Here, we present a detailed overview of CCR mechanisms in various bacteria. We highlight the role of CCR in beneficial as well as deleterious plant-microbe interactions based on the available literature. In addition, we explore the global distribution of known regulatory mechanisms within bacterial genomes retrieved from public repositories and within metatranscriptomes obtained from different plant rhizospheres. By integrating the available literature and performing targeted meta-analyses, we argue that CCR-regulated substrate use preferences of microorganisms should be considered an important trait involved in prevailing plant-microbe interactions.

The Proteogenome of Symbiotic Frankia alni in Alnus glutinosa Nodules.

Omics are the most promising approaches to investigate microbes for which no genetic tools exist such as the nitrogen-fixing symbiotic Frankia. A proteogenomic analysis of symbiotic Frankia alni was done by comparing those proteins more and less abundant in Alnus glutinosa nodules relative to N2-fixing pure cultures with propionate as the carbon source. There were 250 proteins that were significantly overabundant in nodules at a fold change (FC) ≥ 2 threshold, and 1429 with the same characteristics in in vitro nitrogen-fixing pure culture. Nitrogenase, SuF (Fe-Su biogenesis) and hopanoid lipids synthesis determinants were the most overabundant proteins in symbiosis. Nitrogenase was found to constitute 3% of all Frankia proteins in nodules. Sod (superoxide dismutase) was overabundant, indicating a continued oxidative stress, while Kats (catalase) were not. Several transporters were overabundant including one for dicarboxylates and one for branched amino acids. The present results confirm the centrality of nitrogenase in the actinorhizal symbiosis.

Effect of Inoculation Level on the Impact of the PGPR Azospirillum lipoferum CRT1 on Selected Microbial Functional Groups in the Rhizosphere of Field Maize.

The impact of inoculated plant growth-promoting rhizobacteria (PGPR) on its host physiology and nutrition depends on inoculum level. Whether the impact of the inoculated PGPR on the indigenous rhizosphere microbiota also varies with the PGPR inoculum level is unclear. Here, we tested this issue using the PGPR Azospirillum lipoferum CRT1-maize model system, where the initial seed inoculation is known to enhance maize growth and germination, and impacts the maize rhizomicrobiota, including microbial functional groups modulating plant growth. A. lipoferum CRT1 was added to the seeds at standard (105-6 cells.seed-1) or reduced (104-5 cells.seed-1) inoculation levels, in three fields. The effect of the two PGPR formulations was assessed on maize growth and on the nifH (nitrogen fixation), acdS (ACC deaminase activity) and phlD (2,4-diacetylphloroglucinol production) microbial functional groups. The size of the three functional groups was monitored by qPCR at the six-leaf stage and the flowering stage, and the diversity of the nifH and acdS functional groups (as well as the bacterial community) were estimated by MiSeq metabarcoding at the six-leaf stage. The results showed that the benefits of the reduced inoculant formulation were significant in two out of three fields, but different (often lower) than those of the standard formulation. The effects of formulations on the size of the three functional groups differed, and depended on field site and functional group. The reduced formulation had an impact on the diversity of nifH and acdS groups at one site, whereas the standard formulation had an impact at the two other sites. Inoculation significantly impacted the total bacterial community in the three fields, but only with the reduced formulation. In conclusion, the reduced inoculant formulation impacted the indigenous rhizosphere microbiota differently, but not less efficiently, than the standard formulation.

Exploring the Diversity of Fungal DyPs in Mangrove Soils to Produce and Characterize Novel Biocatalysts.

The functional diversity of the New Caledonian mangrove sediments was examined, observing the distribution of fungal dye-decolorizing peroxidases (DyPs), together with the complete biochemical characterization of the main DyP. Using a functional metabarcoding approach, the diversity of expressed genes encoding fungal DyPs was investigated in surface and deeper sediments, collected beneath either Avicennia marina or Rhizophora stylosa trees, during either the wet or the dry seasons. The highest DyP diversity was observed in surface sediments beneath the R. stylosa area during the wet season, and one particular operational functional unit (OFU1) was detected as the most abundant DyP isoform. This OFU was found in all sediment samples, representing 51-100% of the total DyP-encoding sequences in 70% of the samples. The complete cDNA sequence corresponding to this abundant DyP (OFU 1) was retrieved by gene capture, cloned, and heterologously expressed in Pichia pastoris. The recombinant enzyme, called DyP1, was purified and characterized, leading to the description of its physical-chemical properties, its ability to oxidize diverse phenolic substrates, and its potential to decolorize textile dyes; DyP1 was more active at low pH, though moderately stable over a wide pH range. The enzyme was very stable at temperatures up to 50 °C, retaining 60% activity after 180 min incubation. Its ability to decolorize industrial dyes was also tested on Reactive Blue 19, Acid Black, Disperse Blue 79, and Reactive Black 5. The effect of hydrogen peroxide and sea salt on DyP1 activity was studied and compared to what is reported for previously characterized enzymes from terrestrial and marine-derived fungi.

Small-Scale Variability in Bacterial Community Structure in Different Soil Types.

Microbial spatial distribution has mostly been studied at field to global scales (i.e., ecosystem scales). However, the spatial organization at small scales (i.e., centimeter to millimeter scales), which can help improve our understanding of the impacts of spatial communities structure on microbial functioning, has received comparatively little attention. Previous work has shown that small-scale spatial structure exists in soil microbial communities, but these studies have not compared soils from geographically distant locations, nor have they utilized community ecology approaches, such as the core and satellite hypothesis and/or abundance-occupancy relationships, often used in macro-ecology, to improve the description of the spatial organization of communities. In the present work, we focused on bacterial diversity (i.e., 16S rRNA gene sequencing) occurring in micro-samples from a variety of locations with different pedo-climatic histories (i.e., from semi-arid, alpine, and temperate climates) and physicochemical properties. The forms of ecological spatial relationships in bacterial communities (i.e., occupancy-frequency and abundance-occupancy) and taxa distributions (i.e., habitat generalists and specialists) were investigated. The results showed that bacterial composition differed in the four soils at the small scale. Moreover, one soil presented a satellite mode distribution, whereas the three others presented bimodal distributions. Interestingly, numerous core taxa were present in the four soils among which 8 OTUs were common to the four sites. These results confirm that analyses of the small-scale spatial distribution are necessary to understand consequent functional processes taking place in soils, affecting thus ecosystem functioning.

Datamining and functional environmental genomics reassess the phylogenetics and functional diversity of fungal monosaccharide transporters.

Sugar transporters are essential components of carbon metabolism and have been extensively studied to control sugar uptake by yeasts and filamentous fungi used in fermentation processes. Based on published information on characterized fungal sugar porters, we show that this protein family encompasses phylogenetically distinct clades. While several clades encompass transporters that seemingly specialized on specific "sugar-related" molecules (e.g., myo-inositol, charged sugar analogs), others include mostly either mono- or di/oligosaccharide low-specificity transporters. To address the issue of substrate specificity of sugar transporters, that protein primary sequences do not fully reveal, we screened "multi-species" soil eukaryotic cDNA libraries for mannose transporters, a sugar that had never been used to select transporters. We obtained 19 environmental transporters, mostly from Basidiomycota and Ascomycota. Among them, one belonged to the unusual "Fucose H+ Symporter" family, which is only known in Fungi for a rhamnose transporter in Aspergillus niger. Functional analysis of the 19 transporters by expression in yeast and for two of them in Xenopus laevis oocytes for electrophysiological measurements indicated that most of them showed a preference for D-mannose over other tested D-C6 (glucose, fructose, galactose) or D-C5 (xylose) sugars. For the several glucose and fructose-negative transporters, growth of the corresponding recombinant yeast strains was prevented on mannose in the presence of one of these sugars that may act by competition for the binding site. Our results highlight the potential of environmental genomics to figure out the functional diversity of key fungal protein families and that can be explored in a context of biotechnology. KEY POINTS: • Most fungal sugar transporters accept several sugars as substrates. • Transporters, belonging to 2 protein families, were isolated from soil cDNA libraries. • Environmental transporters featured novel substrate specificities.

Field Site-Specific Effects of an Azospirillum Seed Inoculant on Key Microbial Functional Groups in the Rhizosphere.

The beneficial effects of plant growth-promoting Rhizobacteria (PGPR) entail several interaction mechanisms with the plant or with other root-associated microorganisms. These microbial functions are carried out by multiple taxa within functional groups and contribute to rhizosphere functioning. It is likely that the inoculation of additional PGPR cells will modify the ecology of these functional groups. We also hypothesized that the inoculation effects on functional groups are site specific, similarly as the PGPR phytostimulation effects themselves. To test this, we assessed in the rhizosphere of field-grown maize the effect of seed inoculation with the phytostimulatory PGPR Azospirillum lipoferum CRT1 on the size and/or diversity of selected microbial functional groups important for plant growth, using quantitative polymerase chain reaction and/or Illumina MiSeq metabarcoding. The functional groups included bacteria able to fix nitrogen (a key nutrient for plant growth), producers of 1-aminocyclopropane-1-carboxylate (ACC) deaminase (which modulate ethylene metabolism in plant and stimulate root growth), and producers of 2,4-diacetylphloroglucinol (an auxinic signal enhancing root branching). To test the hypothesis that such ecological effects were site-specific, the functional groups were monitored at three different field sites, with four sampling times over two consecutive years. Despite poor inoculant survival, inoculation enhanced maize growth. It also increased the size of functional groups in the three field sites, at the maize six-leaf and flowering stages for diazotrophs and only at flowering stage for ACC deaminase and 2,4-diacetylphloroglucinol producers. Sequencing done in the second year revealed that inoculation modified the composition of diazotrophs (and of the total bacterial community) and to a lesser extent of ACC deaminase producers. This study revealed an ecological impact that was field specific (even though a few taxa were impacted in all fields) and of unexpected magnitude with the phytostimulatory Azospirillum inoculant, when considering microbial functional groups. Further methodological developments are needed to monitor additional functional groups important for soil functioning and plant growth under optimal or stress conditions.

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.

Proposal of 'Candidatus Frankia alpina', the uncultured symbiont of Alnus alnobetula and A. incana that forms spore-containing nitrogen-fixing root nodules.

The members of the genus Frankia are, with a few exceptions, a group of nitrogen-fixing symbiotic actinobacteria that nodulate mostly woody dicotyledonous plants belonging to three orders, eight families and 23 genera of pioneer dicots. These bacteria have been characterized phylogenetically and grouped into four molecular clusters. One of the clusters, cluster 1 contains strains that induce nodules on Alnus spp. (Betulaceae), Myrica spp., Morella spp. and Comptonia spp. (Myricaceae) that have global distributions. Some of these strains produce not only hyphae and vesicles, as other cluster 1 strains do, but also numerous sporangia in their host symbiotic tissues, hence their phenotype being described as spore-positive (Sp+). While Sp+ strains have resisted repeated attempts at cultivation, their genomes have recently been characterized and found to be different from those of all described species, being markedly smaller than their phylogenetic neighbours. We thus hereby propose to create a 'Candidatus Frankia alpina' species for some strains present in nodules of Alnus alnobetula and A. incana that grow in alpine environments at high altitudes or in subarctic environments at high latitudes.