An overview of Parafrankia (Nod+/Fix+) and Pseudofrankia (Nod+/Fix-) interactions through genome mining and experimental modeling in co-culture and co-inoculation of Elaeagnus angustifolia.

In many frankia, the ability to nodulate host plants (Nod+) and fix nitrogen (Fix+) is a common strategy. However, some frankia within the Pseudofrankia genus lack one or two of these traits. This phenomenon has been consistently observed across various actinorhizal nodule isolates, displaying Nod- and/or Fix- phenotypes. Yet, the mechanisms supporting the colonization and persistence of these inefficient frankia within nodules, both with and without symbiotic strains (Nod+/Fix+), remain unclear. It is also uncertain whether these associations burden or benefit host plants. This study delves into the ecological interactions between Parafrankia EUN1f and Pseudofrankia inefficax EuI1c, isolated from Elaeagnus umbellata nodules. EUN1f (Nod+/Fix+) and EuI1c (Nod+/Fix-) display contrasting symbiotic traits. While the prediction suggests a competitive scenario, the absence of direct interaction evidence implies that the competitive advantage of EUN1f and EuI1c is likely contingent on contextual factors such as substrate availability and the specific nature of stressors in their respective habitats. In co-culture, EUN1f outperforms EuI1c, especially under specific conditions, driven by its nitrogenase activity. Iron-depleted conditions favor EUN1f, emphasizing iron's role in microbial competition. Both strains benefit from host root exudates in pure culture, but EUN1f dominates in co-culture, enhancing its competitive traits. Nodulation experiments show that host plant preferences align with inoculum strain abundance under nitrogen-depleted conditions, while consistently favoring EUN1f in nitrogen-supplied media. This study unveils competitive dynamics and niche exclusion between EUN1f and EuI1c, suggesting that host plant may penalize less effective strains and even all strains. These findings highlight the complex interplay between strain competition and host selective pressure, warranting further research into the underlying mechanisms shaping plant-microbe-microbe interactions in diverse ecosystems. IMPORTANCE: While Pseudofrankia strains typically lack the common traits of ability to nodulate the host plant (Nod-) and/or fix nitrogen (Fix-), they are still recovered from actinorhizal nodules. The enigmatic question of how and why these unconventional strains establish themselves within nodule tissue, thriving either alongside symbiotic strains (Nod+/Fix+) or independently, while considering potential metabolic costs to the host plant, remains a perplexing puzzle. This study endeavors to unravel the competitive dynamics between Pseudofrankia inefficax strain EuI1c (Nod+/Fix-) and Parafrankia strain EU1Nf (Nod+/Fix+) through a comprehensive exploration of genomic data and empirical modeling, conducted both in controlled laboratory settings and within the host plant environment.

Blastococcus brunescens sp. nov., a member of the Geodermatophilaceae isolated from sandstone collected from the Sahara Desert in Tunisia.

The taxonomic position of strain BMG 8361T, isolated from sandstone collected in the Sahara Desert of Southern Tunisia, was refined through a polyphasic taxonomic investigation. Colonies of BMG 8361T were pale-orange coloured, irregular with a dry surface and produced a diffusible pink or brown pigment depending on media. The Gram-positive cells were catalase-positive and oxidase-negative. The strain exhibited growth at 10-40 °C and pH values ranging from 5.5 to 9.0, with optima at 28-35 °C and pH 6.5-8.0. Additionally, BMG 8361T demonstrated the ability to grow in the presence of up to 1 % NaCl (w/v) concentration. The peptidoglycan of the cell wall contained meso-diaminopimelic acid, glucose, galactose, xylose, ribose, and rhamnose. The predominant menaquinones consisted of MK-9(H4) and MK-9. The main polar lipids were phosphatidylcholine, phosphatidylinositol, glycophosphatidylinositol, diphosphatidylglycerol, phosphatidylethanolamine, and two unidentified lipids. Major cellular fatty acids were iso-C16 : 0, iso-C16 : 1 h, and C17 : 1 ω8c. Phylogenetic analyses based on both the 16S rRNA gene and whole-genome sequences assigned strain BMG 8361T within the genus Blastococcus. The highest pairwise sequence similarity observed in the 16S rRNA gene was 99.5 % with Blastococcus haudaquaticus AT 7-14T. However, when considering digital DNA-DNA hybridization and average nucleotide identity, the highest values, 48.4 and 86.58 %, respectively, were obtained with Blastococcus colisei BMG 822T. These values significantly undershoot the recommended thresholds for establishing new species, corroborating the robust support for the distinctive taxonomic status of strain BMG 8361T within the genus Blastococcus. In conjunction with the phenotyping results, this compelling evidence leads to the proposal of a novel species we named Blastococcus brunescens sp. nov. with BMG 8361T (=DSM 46845T=CECT 8880T) as the type strain.

Frankia colletiae sp. nov., a nitrogen-fixing actinobacterium isolated from Colletia cruciata.

A nitrogen-fixing actinobacterium strain (Cc1.17T) isolated from a root nodule of Colletia cruciata was subjected to polyphasic taxonomic studies. The strain was characterized by the presence of meso-diaminopimelic acid in its peptidoglycan, galactose, glucose, mannose, rhamnose, ribose and xylose as cell-wall sugars, phosphatidylinositol, diphosphatidylglycerol, glycophospholipids, phosphatidylglycerol, glycophospholipid and uncharacterized lipids as its polar lipids, and C16 : 0, iso-C16 : 0, C17 : 1 ω9 and C18 : 1 ω9 as major fatty acids (>10 %). Strain Cc1.17T showed 16S rRNA gene sequence similarities of 97.4-99.8 % to validly named Frankia species. Phylogenetic trees based on 16S rRNA gene and genome sequences placed strain Cc1.17T in a new lineage within the genus Frankia. Digital DNA-DNA hybridization and average nucleotide identity values between strain Cc1.17T and its closest phylogenomic neighbours were well below the thresholds recommended for prokaryotic species delineation. Therefore, strain Cc1.17T (=DSM 43829T=CECT 9313T) merits recognition as the type strain of a new species for which the name Frankia colletiae sp. nov. is proposed.

Blastococcus carthaginiensis sp. nov., isolated from a monument sampled in Carthage, Tunisia.

A comprehensive polyphasic investigation was conducted to elucidate the taxonomic position of an actinobacterium, designated BMG 814T, which was isolated from the historic ruins of Carthage city in Tunisia. It grew as pink-orange pigmented colonies and displayed versatile growth capabilities, thriving within a temperature range of 20-40 °C, across a pH spectrum ranging from pH 5.5 to 10 and in the presence of up to 4 % NaCl. Chemotaxonomic investigations unveiled specific cell components, including diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, glycophosphatidylinositol, an unidentified aminoglycophospholipid, six unidentified aminolipids, two unidentified phospholipids and one unidentified lipid in its polar lipid profile. Furthermore, galactose, glucose and ribose were identified as the primary cell-wall sugars. Major menaquinones identified were MK-9(H4), MK-9(H2) and MK-9, while major fatty acids comprised iso-C15 : 0, iso-C16 : 0, C17 : 1 ω8c and C18 : 1 ω9c. Through phylogenetic analysis based on the 16S rRNA gene sequence, the strain was positioned within the genus Blastococcus, with Blastococcus capsiensis BMG 804T showing the closest relationship (99.1 %). In light of this, draft genomes for both strains, BMG 814T and BMG 804T, were sequenced in this study, and comparative analysis revealed that strain BMG 814T exhibited digital DNA-DNA hybridization and average nucleotide identity values below the recommended thresholds for demarcating new species with all available genomes of type strains of validly names species. Based on the polyphasic taxonomy assessment, strain BMG 814T (=DSM 46848T=CECT 8878T) was proposed as the type strain of a novel species named Blastococcus carthaginiensis sp. nov.

Blastococcus tunisiensis sp. nov., isolated from limestone collected in Tunisia.

A new actinobacterium strain, designated BMG 823T, was isolated from a limestone sample collected in Tunisia. Its taxonomic position was scrutinized using a polyphasic approach. Colonies of strain BMG 823T were pink orange-coloured, regular and had a moist surface. Cells are Gram-stain-positive, catalase-negative and oxidase-negative. The strain grew at pH 5.5-9, 10-40 °C and in presence of up to 4 % NaCl (w/v). Chemotaxonomically, strain BMG 823T was characterized by cell-wall type III containing meso-diaminopimelic acid as diamino acid, glucose, ribose and rhamnose as whole-cell sugars, MK-9(H4) as predominant menaquinone, and phosphatidylcholine, diphosphadidylglycerol, phosphatidethanolamine, phosphatidylcholine, phosphatidylinositol, unidentified glycolipid, unidentified aminophospholipids and unidentified glycophospholipid as major polar lipids. The fatty acid profile consisted of iso-C16 : 0 and iso-C17 : 1 ω9. Phylogenetic trees based on 16S rRNA gene and genome sequences placed strain BMG 823T within the genus Blastococcus and separated it from all type strains of validly published species. Comparison of 16S rRNA gene sequence similarity, digital DNA-DNA hybridization and average nucleotide identity indicated that strain BMG 823T was most closely related to Blastococcus litoris DSM 106127T and Blastococcus colisei BMG 822T with pairwise values well below the species differentiation thresholds. The distinct phenotypic and genotypic features of strain BMG 823T (=DSM 46838T=CECT 8881T) within the genus Blastococcus warrant its recognition as the type strain for the new species for which we propose the name Blastococcus tunisiensis sp. nov.

Elucidating the ecological networks in stone-dwelling microbiomes.

Stone surfaces are extreme environments that support microbial life. This microbial growth occurs despite unfavourable conditions associated with stone including limited sources of nutrients and water, high pH and exposure to extreme variations in temperature, humidity and irradiation. These stone-dwelling microbes are often resistant to extreme environments including exposure to desiccation, heavy metals, UV and Gamma irradiation. Here, we report on the effects of climate and stone geochemistry on microbiomes of Roman stone ruins in North Africa. Stone microbiomes were dominated by Actinobacteria, Cyanobacteria and Proteobacteria but were heavily impacted by climate variables that influenced water availability. Stone geochemistry also influenced community diversity, particularly through biologically available P, Mn and Zn. Functions associated with photosynthesis and UV protection were enriched in the metagenomes, indicating the significance of these functions for community survival on stones. Core members of the stone microbial communities were also identified and included Geodermatophilaceae, Rubrobacter, Sphingomonas and others. Our research has helped to expand the understanding of stone microbial community structure and functional capacity within the context of varying climates, geochemical properties and stone conditions.

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.

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.

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.

Actinorhizal trees and shrubs from Africa: distribution, conservation and uses.

Actinorhizal plants are a group of perennial dicotyledonous angiosperms, comprised of more than 200 species, most of which can establish root-nodule symbiosis with the nitrogen fixing actinobacteria of the genus Frankia. They are key providers of fundamental goods and services and can give a major contribution to mitigate the combined effects of climate changes, human population growth and loss of biodiversity. This aspect is particularly relevant for the developing economies of many African countries, which are highly exposed to climate and anthropogenic disturbances. In this work we have analyzed the distribution, conservation and uses of actinorhizal species native to or introduced in Africa. A total of 42 taxa distributed over six botanical families (Betulaceae, Casuarinaceae, Myricaceae, Elaeagnaceae, Rhamnaceae and Coriariaceae) were identified. The vast majority is able to thrive under a range of diverse environments and has multiple ecological and economic potential. More than half of the identified species belong to the genus Morella (Myricaceae), most of them native to Middle, Eastern and Southern Africa. Although the information about the conservation status and uses of Morella spp. is largely incomplete, the available data is indicative of their potential in e.g. forestry and agroforestry, food and medicine. Therefore, efforts should be made to upgrade actinorhizal research in Africa towards the sustainable use of biodiversity at the service of local (bio)economies.

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.

The plant-growth-promoting actinobacteria of the genus Nocardia induces root nodule formation in Casuarina glauca.

Actinorhizal plants form a symbiotic association with the nitrogen-fixing actinobacteria Frankia. These plants have important economic and ecological benefits including land reclamation, soil stabilization, and reforestation. Recently, many non-Frankia actinobacteria have been isolated from actinorhizal root nodules suggesting that they might contribute to nodulation. Two Nocardia strains, BMG51109 and BMG111209, were isolated from Casuarina glauca nodules, and they induced root nodule-like structures in original host plant promoting seedling growth. The formed root nodule-like structures lacked a nodular root at the apex, were not capable of reducing nitrogen and had their cortical cells occupied with rod-shaped Nocardiae cells. Both Nocardia strains induced root hair deformation on the host plant. BMG111209 strain induced the expression of the ProCgNin:Gus gene, a plant gene involved in the early steps of the infection process and nodulation development. Nocardia strain BMG51109 produced three types of auxins (Indole-3-acetic acid [IAA], Indole-3-Byturic Acid [IBA] and Phenyl Acetic Acid [PAA]), while Nocardia BMG111209 only produced IAA. Analysis of the Nocardia genomes identified several important predicted biosynthetic gene clusters for plant phytohormones, secondary metabolites, and novel natural products. Co-infection studies showed that Nocardia strain BMG51109 plays a role as a "helper bacteria" promoting an earlier onset of nodulation. This study raises many questions on the ecological significance and functionality of Nocardia bacteria in actinorhizal symbioses.

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 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.

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.

Blastococcus xanthinilyticus sp. nov., isolated from monument.

A novel, non-motile, coccoid, Gram-stain-positive actinobacterium, designated BMG 862T, was isolated from a marble sample collected from the Bulla Regia monument, Northern Tunisia. Its taxonomic position was determined using a polyphasic approach. Results from chemotaxonomic analyses showed MK-9(H4), MK-8(H4) and MK-9(H2) as the predominant menaquinones. The major polar lipids comprised diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, glycophosphatidylinositol, hydroxy-phosphatidylethanolamine and three unidentified phospholipids. The fatty acids consisted of significant amounts (≥10 %) of iso-C16 : 0, C17 : 1ω8c, iso-C15 : 0 and C16 : 1ω7c. Phylogenetic analysis on the basis of 16S rRNA gene sequence comparisons showed that strain BMG 862T belongs to the genus Blastococcus, being most closely related to Blastococcus saxobsidens (=DSM 44509T) (99.5 %) and Blastococcus capsensis (=DSM 46835T=CECT 8876T) (99.3 %). The genomic DNA G+C content of the organism was 74.7 mol%. Results of DNA-DNA hybridization and physiological tests allowed differentiation of strain BMG 862T from related species. The strain was also characterized by its ability to hydrolyse xanthine. On the basis of phenotypic and molecular characteristics, strain BMG 862T (=DSM 46842T=CECT 8884T) represents the type strain of a novel species of the genus Blastococcus, for which the name Blastococcus xanthinilyticus sp. nov. is proposed.

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.

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.

Frankia coriariae sp. nov., an infective and effective microsymbiont isolated from Coriaria japonica.

The taxonomic description of a nitrogen-fixing actinobacterium, strain BMG5.1T, as a novel species within the genus Frankia was based on a polyphasic approach. The strain was isolated from the root nodules of Coriaria japonica, and it fulfilled Koch's postulates by inducing effective nodules on Coriaria spp. and Datisca spp. Based on phenotypic and chemotaxonomic features, strain BMG5.1T is distinguishable from all other species of the genus Frankia. It is characterized by the presence of phosphatidylinositol, diphosphatidylglycerol, phosphatidylglycerol and glycophospholipids in its polar lipids; galactose, glucose, mannose and a trace of ribose as cellular sugars; meso-diaminopimelic acid as cell-wall peptidoglycan; C18 : 1ω9c as major fatty acid (>30 %); and MK-9(H6) (44.7 %) as predominant isoprenolog (>30 %). The 16S rRNA gene sequence similarities and the digital DNA-DNA hybridization values between the described novel strain and strains of the other species of the genus Frankia correspond to a range of 97-98.4 % and 22.1-24 %, respectively. The DNA G+C content is 70.2  mol%. On the basis of these results, strain BMG5.1T (=CECT 9032T=DSM 100624T) is proposed as the type strain of a novel species of the genus Frankia, named Frankia coriariae sp. nov.

Frankia inefficax sp. nov., an actinobacterial endophyte inducing ineffective, non nitrogen-fixing, root nodules on its actinorhizal host plants.

Strain EuI1cT is the first actinobacterial endophyte isolated from Elaeagnus umbellata that was shown to be infective on members of Elaeagnaceae and Morella but lacking the ability to form effective root nodules on its hosts. The strain can be easily distinguished from strains of other Frankia species based on its inability to produce vesicles, the specialized thick-walled structures where nitrogen fixation occurs. Chemotaxonomically, strain EuI1cT contains phosphatidylinositol, diphosphatidylglycerol, two glycophospholipids and phosphatidylglycerol as phospholipids. The whole cell sugars were composed of glucose, galactose, mannose, ribose, rhamnose and fucose as diagnostic sugars of the species. Major fatty acids were iso-C16:0, C17:1 ω8c and C15:0 and C17:0 and the predominant menaquinones were MK-9(H6), MK-9(H8) and MK-9(H4). Analysis of the 16S rRNA gene sequence of strain EuI1cT showed 97, 97.4 and 97.9% identity with Frankia elaeagni DSM 46783T, Frankia casuarinae DSM 45818T and Frankia alni DSM 45986T, respectively. Digital DNA:DNA hybridizations with type strains of the three Frankia species with validly/effectively published names are significantly below 70%. These results warrant distinction of EuI1cT (= DSM 45817T = CECT 9037T) as the type strain of a novel species designated Frankia inefficax sp. nov.