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.

Frankia soli sp. nov., an actinobacterium isolated from soil beneath Ceanothus jepsonii.

Actinobacterial strain CjT was directly isolated from soil beneath Ceanothus jepsonii growing in the USA. The strain formed cell structures typical of the genus Frankia including extensive hyphae, vesicles and sporangia, and it effectively nodulated members of the actinorhizal Colletieae, Elaeagnaceae and Myricaceae. The whole-cell hydrolysate of strain CjT was rich in meso-diaminopimelic acid and galactose, glucose, mannose, xylose, ribose and a trace of rhamnose. Tbe polar lipid profile contained phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol and glycophospholipid. The menaquinone was predominantly MK-9(H4). The fatty acid profile predominantly consisted of C17 : 1ω8c, iso-C16 : 0, C15:0, C16 : 0 and C17 : 0. A multilocus sequence analysis phylogeny based on atp1, ftsZ, dnaK, gyrA and secA gene sequences positioned the strain within Elaeagnaceae- and Colletieae-nodulating species together with Frankia elaeagni DSM 46783T, Frankia discariae DSM 46785T and Frankia irregularis DSM 45899T. Pairwise 16S rRNA gene sequence similarities showed that strain CjT was most closely related to F. discariae DSM 46785T (99.78 %) while their digital DNA-DNA hybridization value was 41.1 %. Based on the overall analyses, strain CjT (=DSM 100623T=CECT 9041T) warrants classification as the type strain of a novel species, for which the name Frankia soli sp. nov. is proposed.

Simple colony PCR procedure for the filamentous actinobacteria Frankia.

Molecular analysis of the filamentous actinobacteria Frankia is laborious because of the slow growth rate and required biomass needed for these techniques. An efficient and simple colony PCR protocol for Frankia was developed that saved time for analysis of any Frankia strains growing on a plate. Previously, it took 5-6 weeks to get the correct size Frankia colonies on plates and then a minimum of 5 weeks of growth in liquid culture for DNA extraction. With this technique, these colonies could be screened after 5-6 weeks of growth by colony PCR. The procedure used a combination of mechanical and heat treatments and required no added buffers or chemicals. Our results demonstrate rapid and efficient PCR.

19th International Meeting on Frankia and Actinorhizal Plants.

It has been 40 years since the first meeting dedicated to Frankia and actinorhizal plants, which was held at Petersham, Massachusetts (reported in Torrey and Tjepkema, 1979). Since then biennial meetings have been organised and held in different venues around the globe (Table 1). The most recent meeting, the "19th International Meeting on Frankia and Actinorhizal Plants", organised in Hammamet, Tunisia from 17th to 19th of March, 2018, gathered scientists from Algeria, Argentina, Belgium, China, Egypt, France, India, Portugal, Senegal, Sweden, UK, USA and Tunisia. The event was a stimulating opportunity for active researchers to share many advances since the previous meeting held in Montpellier, France (Franche et al. 2016) and to discuss new perspectives in this research field.

Contrasted evolutionary constraints on carbohydrate active enzymes (CAZymes) in selected Frankia strains.

Carbohydrate active enzymes (CAZymes) are capable of breaking complex polysaccharides into simpler form. In plant-host-associated microorganisms CAZymes are known to be involved in plant cell wall degradation. However, the biology and evolution of Frankia CAZymes are largely unknown. In the present study, we took a genomic approach to evaluate the presence and putative roles of CAZymes in Frankia. The CAZymes were found to be potentially highly expressed (PHX) proteins and contained more aromatic amino acids, which increased their biosynthetic energy cost. These energy rich amino acids were present in the active sites of CAZymes aiding in their carbohydrate binding capacity. Phylogenetic and evolutionary analyses showed that, in Frankia strains with the capacity to nodulate host plants, CAZymes were evolving slower than the other PHX genes, whereas similar genes from non-nodulating (or ineffectively nodulating) Frankia strains showed little variation in their evolutionary constraints compared to other PHX genes. Thus, the present study revealed the persistence of a strong purifying selection on CAZymes of Frankia indicating their crucial role.

An update on the taxonomy of the genus Frankia Brunchorst, 1886, 174AL.

Since the recognition of the name Frankia in the Approved Lists of bacterial names (1980), few amendments have been given to the genus description. Successive editions of Bergey's Manual of Systematics of Archaea and Bacteria have broadly conflicting suprageneric treatments of the genus without any advances for subgeneric classification. This review focuses on recent results from taxongenomics and phenoarray approaches to the positioning and the structuring of the genus Frankia. Based on phylogenomic analyses, Frankia should be considered the single member of the family Frankiaceae within the monophyletic order, Frankiales. A polyphasic strategy incorporating genome to genome data and omniLog® phenoarrays, together with classical approaches, has allowed the designation and an amended description of a type strain of the type species Frankia alni, and the recognition of at least 10 novel species covering symbiotic and non symbiotic taxa within the genus. Genome to phenome data will be shortly incorporated in the scheme for proposing novel species including those recalcitrant to isolation in axenic culture.

Frankia torreyi sp. nov., the first actinobacterium of the genus Frankia Brunchorst 1886, 174AL isolated in axenic culture.

Strain CpI1T was, in 1978, the first isolate of the genus Frankia to be obtained from Comptonia peregrina root nodules. In this study, a polyphasic approach was performed to identify the taxonomic position of strain CpI1T among the members of the genus Frankia. The strain contains meso-diaminopimelic acid as the diagnostic diamino acid and galactose, glucose, mannose, rhamnose, ribose and xylose as cell wall sugars. The polar lipids were found to consist of phosphatidylinositol, diphosphatidylglycerol, glycophospholipids, phosphatidylglycerol, an aminophospholipid and unidentified phospholipids and lipids. The predominant menaquinone was identified as MK-9 (H8), while the major fatty acid are iso-C16:0 and C17:1ω 8c. The 16S rRNA gene sequence identity varies from 97.4 to 99.6% with the type strains of currently described Frankia species. Phylogenetic analyses based on 16S rRNA gene sequences and multi-locus sequence analysis (MLSA) using atp1, ftsZ, dnaK, gyrA and secA gene sequences showed that strain CpI1T is closely related to Frankia alni ACN14aT. The genome size of strain CpI1T is 7.6 Mb with a digital DNA G+C content of 72.4%. Digital DNA:DNA hybridization (values between strain CpI1T and its close phylogenetic relative F. alni ACN14aT was 44.1%, well below the threshold of 70% for distinguishing between bacterial genomic species. Based on the phenotypic, phylogenetic and genomic data, strain CpI1T (= DSM44263T = CECT9035T) warrants classification as the type strain of a novel species, for which the name Frankia torreyi sp. nov. is proposed.

Draft genome sequence of the symbiotic Frankia sp. strain BMG5.30 isolated from root nodules of Coriaria myrtifolia in Tunisia.

Frankia sp. strain BMG5.30 was isolated from root nodules of a Coriaria myrtifolia seedling on soil collected in Tunisia and represents the second cluster 2 isolate. Frankia sp. strain BMG5.30 was able to re-infect C. myrtifolia generating root nodules. Here, we report its 5.8-Mbp draft genome sequence with a G + C content of 70.03% and 4509 candidate protein-encoding genes.

Frankia communities at revegetating sites in Mt. Ontake, Japan.

In 1984 at Mt. Ontake in Japan, an earthquake caused a devastating landslide, and as a result, the vegetation on the south slope of the mountain was completely eliminated. In higher elevation (2000 m) areas, revegetation has not yet been completed even 30 years after the landslide. Revegetation progress throughout the area was heterogeneous. In the partially revegetated areas, actinorhizal plant species such as Alnus maximowiczii and Alnus matsumurae have been found. In the present study, we investigated the Frankia communities in the higher-elevation area using sequence analysis of the amplified nifH (dinitrogenase reductase) gene from nodule and soil samples collected in the disturbed region, undisturbed forest, and in the boundary between the disturbed region and the undisturbed forest. Phylogenetic analysis of partial nifH sequences revealed the presence of six clusters, each of which consisted of highly similar (> 99%) sequences. Four clusters showed significant sequence similarity to Frankia (three Alnus- and a Casuarina-infecting strains). Diversity in the Frankia community was relatively low-only one or two clusters were detected in a site. At most of the sampling sites, a dominant cluster in a nodule coincided with that in rhizosphere soil, indicating that community structure in the rhizosphere is a primary factor that determines occupancy in a nodule. No significant difference in community structure was observed between plant species. Diversity in the Frankia community varied depending on revegetation progress. Cluster A, which was the most dominant in the disturbed region, was likely to have invaded from undisturbed forest.

Frankia Diversity in Host Plant Root Nodules Is Independent of Abundance or Relative Diversity of Frankia Populations in Corresponding Rhizosphere Soils.

Actinorhizal plants form nitrogen-fixing root nodules in symbiosis with soil-dwelling actinobacteria within the genus Frankia, and specific Frankia taxonomic clusters nodulate plants in corresponding host infection groups. In same-soil microcosms, we observed that some host species were nodulated (Alnus glutinosa, Alnus cordata, Shepherdia argentea, Casuarina equisetifolia) while others were not (Alnus viridis, Hippophaë rhamnoides). Nodule populations were represented by eight different sequences of nifH gene fragments. Two of these sequences characterized frankiae in S. argentea nodules, and three others characterized frankiae in A. glutinosa nodules. Frankiae in A. cordata nodules were represented by five sequences, one of which was also found in nodules from A. glutinosa and C. equisetifolia, while another was detected in nodules from A. glutinosa Quantitative PCR assays showed that vegetation generally increased the abundance of frankiae in soil, independently of the target gene (i.e., nifH or the 23S rRNA gene). Targeted Illumina sequencing of Frankia-specific nifH gene fragments detected 24 unique sequences from rhizosphere soils, 4 of which were also found in nodules, while the remaining 4 sequences in nodules were not found in soils. Seven of the 24 sequences from soils represented >90% of the reads obtained in most samples; the 2 most abundant sequences from soils were not found in root nodules, and only 2 of the sequences from soils were detected in nodules. These results demonstrate large differences between detectable Frankia populations in soil and those in root nodules, suggesting that root nodule formation is not a function of the abundance or relative diversity of specific Frankia populations in soils.IMPORTANCE The nitrogen-fixing actinobacterium Frankia forms root nodules on actinorhizal plants, with members of specific Frankia taxonomic clusters nodulating plants in corresponding host infection groups. We assessed Frankia diversity in root nodules of different host plant species, and we related specific populations to the abundance and relative distribution of indigenous frankiae in rhizosphere soils. Large differences were observed between detectable Frankia populations in soil and those in root nodules, suggesting that root nodule formation is not a function of the abundance or relative diversity of specific Frankia populations in soils but rather results from plants potentially selecting frankiae from the soil for root nodule formation. These data also highlight the necessity of using a combination of different assessment tools so as to adequately address methodological constraints that could produce contradictory data sets.

In Planta Sporulation of Frankia spp. as a Determinant of Alder-Symbiont Interactions.

The Alnus genus forms symbiosis with the actinobacteria Frankia spp. and ectomycorrhizal fungi. Two types of Frankia lineages can be distinguished based on their ability to sporulate in planta Spore-positive (Sp+) strains are predominant on Alnus incana and Alnus viridis in highlands, while spore-negative (Sp-) strains are mainly associated with Alnus glutinosa in lowlands. Here, we investigated whether the Sp+ predominance in nodules is due to host selection of certain Frankia genotypes from soil communities or the result of the ecological history of the alder stand soil, as well as the effect of the sporulation genotype on the ectomycorrhizal (ECM) communities. Trapping experiments were conducted using A. glutinosa, A. incana, and A. viridis plantlets on 6 soils, differing in the alder species and the frequency of Sp+ nodules in the field. Higher diversity of Frankia spp. and variation in Sp+ frequencies were observed in the trapping than in the fields. Both indigenous and trapping species shape Frankia community structure in trapped nodules. Nodulation impediments were observed under several trapping conditions in Sp+ soils, supporting a narrower host range of Sp+ Frankia species. A. incana and A. viridis were able to associate equally with compatible Sp+ and Sp- strains in the greenhouse. Additionally, no host shift was observed for Alnus-specific ECM, and the sporulation genotype of Frankia spp. defined the ECM communities on the host roots. The symbiotic association is likely determined by the host range, the soil history, and the type of in plantaFrankia species. These results provide an insight into the biogeographical drivers of alder symbionts in the Holarctic region.IMPORTANCE Most Frankia-actinorhiza plant symbioses are capable of high rates of nitrogen fixation comparable to those found on legumes. Yet, our understanding of the ecology and distribution of Frankia spp. is still very limited. Several studies have focused on the distribution patterns of Frankia spp., demonstrating a combination of host and pedoclimatic parameters in their biogeography. However, very few have considered the in planta sporulation form of the strain, although it is a unique feature among all symbiotic plant-associated microbes. Compared with Sp- Frankia strains, Sp+ strains would be obligate symbionts that are highly dependent on the presence of a compatible host species and with lower efficiency in nitrogen fixation. Understanding the biogeographical drivers of Sp+ Frankia strains might help elucidate the ecological role of in planta sporulation and the extent to which this trait mediates host-partner interactions in the alder-Frankia-ECM fungal symbiosis.

Frankia canadensis sp. nov., isolated from root nodules of Alnus incana subspecies rugosa.

Strain ARgP5T, an actinobacterium isolated from a root nodule present on an Alnus incana subspecies rugosa shrub growing in Quebec City, Canada, was the subject of polyphasic taxonomic studies to clarify its status within the genus Frankia. 16S rRNA gene sequence similarities and ANI values between ARgP5T and type strains of species of the genus Frankiawith validly published names were 98.8 and 82 % or less, respectively. The in silico DNA G+C content was 72.4 mol%. ARgP5T is characterised by the presence of meso-A2pm, galactose, glucose, mannose, rhamnose (trace), ribose and xylose as whole-organism hydrolysates; MK-9(H8) as predominant menaquinone; diphosphatidylglycerol, phosphatidylinositol and phosphatidylglycerol as polar lipids and iso-C16 : 0 and C17 : 1ω8c as major fatty acids. The proteomic results confirmed the distinct position of ARgP5T from its closest neighbours in Frankiacluster 1. ARgP5T was found to be infective on two alder (Alnus glutinosa and Alnusalnobetula subsp. crispa) and on one bayberry (Morella pensylvanica) species and to fix nitrogen in symbiosis and in pure culture. On the basis of phylogenetic (16S rRNA gene sequence), genomic, proteomic and phenotypic results, strain ARgP5T (=DSM 45898=CECT 9033) is considered to represent a novel species within the genus Frankia for which the name Frankia canadensis sp. nov., is proposed.

Frankia irregularis sp. nov., an actinobacterium unable to nodulate its original host, Casuarina equisetifolia, but effectively nodulates members of the actinorhizal Rhamnales.

A red pigmented actinobacterium designated G2T, forming extremely branched vegetative hyphae, vesicles and mutilocular sporangia, was isolated from Casuarina equisetifolia nodules. The strain failed to nodulate its original host plant but effectively nodulated members of actinorhizal Rhamnales. The taxonomic position of G2T was determined using a polyphasic approach. The peptidoglycan of the strain contained meso-diaminopimelic acid as diagnostic diamino acid, galactose, glucose, mannose, rhamnose, ribose and xylose. The polar lipid pattern consisted of phosphatidylinositol (PI), diphosphatidylglycerol (DPG), glycophospholipids (GPL1-2), phosphatidylglycerol (PG), aminophospholipid (APL) and unknown lipids (L). The predominant menaquinones were MK-9 (H4) and MK-9 (H6) while the major fatty acids were iso-C16 : 0, C17 : 1ω8c and C15 : 0. The size of the genome of G2T was 9.5 Mb and digital DNA G+C content was 70.9 %. The 16S rRNA gene showed 97.4-99.5 % sequence identity with the type strains of species of the genus Frankia. Digital DNA -DNA hybridisation (dDDH) values between G2T and its nearest phylogenetic neighbours Frankia elaeagniand Frankia discariaewere below the threshold of 70 %. On the basis of these results, strain G2T (=DSM 45899T=CECT 9038T) is proposed to represent the type strain of a novel species Frankia irregularis sp. nov.

Frankia saprophytica sp. nov., an atypical, non-infective (Nod-) and non-nitrogen fixing (Fix-) actinobacterium isolated from Coriaria nepalensis root nodules.

Strain CN3T, a Coriaria nepalensis isolate, appears to form hyphae and sporangia typical of members fo the genus Frankia. However, it failed to form vesicles, to reduce acetylene and to induce nodules on its original host plant. A polyphasic approach was used here to determine the taxonomic status of strain CN3T. The 16S rRNA gene sequence of strain CN3T showed the highest sequence identity with Frankia asymbiotica type strain M16386T (99.4 %). Digital DNA-DNA hybridization between strains CN3T and M16386T was 25.7 %, which is clearly below the accepted cut-off point of 70 %. The G+C content of DNA was 71.8 mol%. Whole-cell hydrolysates of strain CN3T were rich in meso-diaminopimelic acid. Cell-wall sugars were composed of galactose, glucose, mannose, rhamnose and traces of ribose. The polar lipid profile contained phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, phosphoglycolipids, phospholipid, six uncharacterized glycolipids and two uncharacterized lipids. The predominant menaquinone (>25 %) was MK-9(H6). Major fatty acids (>15 %) of strain CN3T consisted of iso-C16 : 0, C17 : 1ω8c and C15 : 0. Based on 16S rRNA gene phylogeny, genome sequence analysis and phenotypic results, strain CN3T (=DSM 105290T=CECT 9314T) is proposed to represent the type strain of a novel species, Frankia saprophytica sp. nov.

Isolation and molecular characterization of Frankia strains resistant to some heavy metals.

Frankia strains isolated from Saudi Arabia, reported for the first time, were identified based on the morphological and molecular tools compared to those isolated from Egypt. All strains displayed typical morphological characterization of Frankia strains represented by branched hyphae, production of vesicles and sporangia. The phylogenetic analysis and relationships among Frankia strains were investigated by comparing 16S rRNA gene sequences. The analysis revealed three genetic groups which formed two clusters. The first cluster was composed of eight Frankia strains subdivided into two genetic groups (one group containing five strains; CgIT3 L2 , CgIS3 N2 , CgIS1 N1, CgIT7N2, and G5; the other group included of three strains: CgIT5L3, CgIS1 N2 , and CcI13). The second cluster was composed of only one genetic group of Frankia strain CgIS3 N1 . The strains in each genetic group exhibited similar genetic distances. All Frankia strains were able to reinfect their host of Casuarina species. For ability of these strains to resist heavy metals, our results proved that all Frankia strains isolated can resist Cu, Co, and Zn at low concentration except Pb which exhibit highly toxic effect at the same concentration used. Frankia strain G5 was proved to be the most resistant strain for heavy metals tested.

Green alder (Alnus viridis) encroachment shapes microbial communities in subalpine soils and impacts its bacterial or fungal symbionts differently.

Since the mid-twentieth century, subalpine grasslands undergo a progressive encroachment by Alnus viridis shrubs. Thanks to its rapid vegetative reproduction, its nitrogen fixing symbiosis with Frankia and its ectomycorrhizal cohorts, green alders are vigorous colonizers that quickly form mosaic of alder patches that evolves into a close canopy shrub community. To better understand how alder encroachment might influence microbial communities in this successional sequence, symbiont distribution, microbial richness and community structure in both soils and nodules were analyzed at three successional stages: grassland, mosaic and forest. Soil analyses were performed in association with measures of nitrification and denitrification, as well as DNA metabarcoding of three bacterial genes (16S rDNA, nifH and amoA) and one fungal gene (ITS1). Our results show that (i) A. viridis encroachment is associated with soil microbial community changes that are in turn, linked to certain soil properties (i.e., pH, C/N ratio and organic matter content), (ii) both taxonomic and N related functional gene structures of bacteria are modified by alder encroachment and (iii) the distribution in soils of its bacterial symbionts (Frankia) is apparently weakly influenced by alder establishment while Alnus-specific ectomyccorrhizae increase with the increase in alder shrub density.

The Influence of the Host Plant Is the Major Ecological Determinant of the Presence of Nitrogen-Fixing Root Nodule Symbiont Cluster II Frankia Species in Soil.

The actinobacterial genus Frankia establishes nitrogen-fixing root nodule symbioses with specific hosts within the nitrogen-fixing plant clade. Of four genetically distinct subgroups of Frankia, cluster I, II, and III strains are capable of forming effective nitrogen-fixing symbiotic associations, while cluster IV strains generally do not. Cluster II Frankia strains have rarely been detected in soil devoid of host plants, unlike cluster I or III strains, suggesting a stronger association with their host. To investigate the degree of host influence, we characterized the cluster II Frankia strain distribution in rhizosphere soil in three locations in northern California. The presence/absence of cluster II Frankia strains at a given site correlated significantly with the presence/absence of host plants on the site, as determined by glutamine synthetase (glnA) gene sequence analysis, and by microbiome analysis (16S rRNA gene) of a subset of host/nonhost rhizosphere soils. However, the distribution of cluster II Frankia strains was not significantly affected by other potential determinants such as host-plant species, geographical location, climate, soil pH, or soil type. Rhizosphere soil microbiome analysis showed that cluster II Frankia strains occupied only a minute fraction of the microbiome even in the host-plant-present site and further revealed no statistically significant difference in the α-diversity or in the microbiome composition between the host-plant-present or -absent sites. Taken together, these data suggest that host plants provide a factor that is specific for cluster II Frankia strains, not a general growth-promoting factor. Further, the factor accumulates or is transported at the site level, i.e., beyond the host rhizosphere. IMPORTANCE: Biological nitrogen fixation is a bacterial process that accounts for a major fraction of net new nitrogen input in terrestrial ecosystems. Transfer of fixed nitrogen to plant biomass is especially efficient via root nodule symbioses, which represent evolutionarily and ecologically specialized mutualistic associations. Frankia spp. (Actinobacteria), especially cluster II Frankia spp., have an extremely broad host range, yet comparatively little is known about the soil ecology of these organisms in relation to the host plants and their rhizosphere microbiomes. This study reveals a strong influence of the host plant on soil distribution of cluster II Frankia spp.

Frankia discariae sp. nov.: an infective and effective microsymbiont isolated from the root nodule of Discaria trinervis.

Strain BCU110501T was the first isolate reported to fulfill Koch's postulates by inducing effective nodules on its host plant of origin Discaria trinervis (Rhalmnaceae). Based on 16S rRNA gene sequence similarities, the strain was found to be most closely related to the type strain of Frankia elaeagni DSM 46783T (98.6%) followed by F. alni DSM 45986T (98.2%), F. casuarinae DSM 45818T (97.8%) and F. inefficacies DSM 45817T (97.8%). Digital DNA:DNA hybridizations (dDDH) between strain BCU110501Tand the type strains of other Frankia species were clearly below the cutoff point of 70%. The G+C content of DNA is 72.36%. The cell wall of strain BCU110501T contained meso-diaminopimelic acid and the cell sugars were galactose, glucose, mannose, xylose and ribose. Polar lipids were phosphatidylinositol (PI), diphosphatidylglycerol (DPG), glycophospholipid (GPL1-3), phosphatidylglycerol (PG) and an unknown lipid (L). The major fatty acids of strain BCU110501T consisted of iso-C16:0, C17:1 w8c and C16:0. Major menaquinones were MK9 (H4), MK9 (H6) and MK9 (H2). Based on these analyses, strain BCU110501T (=DSM 46785T=CECT 9042T) should be classified as the type strain of a novel Frankia species, for which the name Frankia discariae sp. nov. is proposed.

Proposal of 'Candidatus Frankia californiensis', the uncultured symbiont in nitrogen-fixing root nodules of a phylogenetically broad group of hosts endemic to western North America.

The genus Frankia comprises a group of nitrogen-fixing actinobacteria that form root-nodule symbioses with perennial dicotyledonous plants in the nitrogen-fixing clade. These bacteria have been characterized phylogenetically and grouped into four clusters (clusters 1-4). Cluster 2 contains mostly uncultured strains that induce nodules on species of the genera Datisca (Datiscaceae), Coriaria (Coriariaceae), Ceanothus (Rhamnaceae) and several genera in the family Rosaceae (Cercocarpus, Chamaebatia, Dryas, Purshia), all of which except members of the genus Coriaria are present within the California Floristic Province (CFP) or neighbouring areas of western North America. Those strains occurring in western North America are genetically very closely related to one another, and genetically distinct from strains characterized from other locales. We hereby propose to create a 'Candidatus Frankia californiensis' species for those cluster 2 strains of the genus Frankia with both high genetic similarity and a geographical distribution in or near the CFP.

Frankia asymbiotica sp. nov., a non-infective actinobacterium isolated from Morella californica root nodule.

The taxonomic status of strain M16386T, a nitrogen-fixing but non-nodulating isolate from Morella californica, was established on the basis of a polyphasic approach. The strain grows as branched hyphae, with vesicles and non-motile productive multilocular sporangia. It metabolizes short fatty acids, TCA cycle intermediates and carbohydrates as carbon sources, and fixes nitrogen in the absence of combined nitrogen source in the growth media. Chemotaxonomic traits of strain M16386T are consistent with its affiliation to the genus Frankia. The characteristic diamino acid in the cell wall is meso-diaminopimelic acid. Strain M16386T contains phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, glycophospholipid and phospholipid as polar lipids; MK-9(H4) and MK-9(H6) as the predominant menaquinones; iso-C16 : 0 and C17 : 1ω8c as major fatty acids; and galactose, glucose, mannose, rhamnose and ribose as whole-cell sugars. Strain M16386T showed 98.2 % 16S rRNA gene sequence similarity with its closest phylogenetic neighbour, Frankia inefficaxDSM 45817T. Based on these results, strain M16386T (=DSM 100626T=CECT 9040T) is designated the type strain of a novel species of the genus Frankia,for which the name Frankia asymbiotica sp. nov. is proposed.