Bioremediation of noxious metals from e-waste printed circuit boards by Frankia.

The environmental noxious e-waste was collected and physicochemical characterized by Scanning electron microscopy (SEM) along with energy dispersive X-ray spectroscopy (EDX), Atomic absorption spectrometry (AAS), and X-ray diffraction analysis (XRD) exploration to understand the presence of toxic metals like Hg, Cd, Pd, Si, Ru. Therefore, the finding provides vital knowledge about the impact of toxic metals from e-waste printed circuit boards as contaminants in the environment and its impact on humans. The Frankia sp. DDNSF-03 and Frankia casuarinae DDNSF-04 were isolated and identified, further utilized for removal of e-waste toxic metals by one and two steps bioremediation experiments executed with various e-waste concentrations. The two-step bioremediation experiment is efficient in the expression of toxic metals that were removed at a lesser concentration of e-waste. Consequently, the presence of organic acids in the Frankia primary metabolites was confirmed by FT-IR analysis besides decreasing the pH level in the Frankia growth medium. The positive control Frankia and negative control e-waste were maintained throughout the bioremediation experiments. The initial Hg 4.3, Cd 8.3, Pd 4.6 (ppm) in the e-waste and final treated with Frankia sp. DDNSF-03 Hg 0.09, Cd 5.09, Pb 0.49 (ppm), and Frankia casuarinae DDNSF-04 Hg 2.15, Cd 5.6, Pb 2.82 (ppm) concentration of toxic metals was quantified by AAS spectrum analysis. The toxic metals mercury and lead were significantly mineralized by Frankia sp. when compare the Frankia casuarinae. The above finding was confirmed the manifestation of morphological changes by an accumulation of e-waste in Frankia hyphae using SEM analysis and obtain the qualitative of toxic metals parallel peaks in EDX analysis.

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.

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.

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.

The role of Frankia inoculation in casuarina plantations in China.

Casuarina trees are planted along the coast from Hainan province in South China to the Zhoushan Islands of Zhejiang province in Southeastern China. Three key species, Casuarina equisetifolia, Casuarina cunninghamiana and Casuarina glauca, are used as windbreaks, in agroforestry systems, and for the production of timber and fuel wood. Frankia have been studied in China since 1984. Today, Frankia research fields are very wide, and cover morphology, physiology and genetic diversity, and the application of inocula for specific purposes on poor quality sites. In this paper, we review the role of Frankia inoculations in nurseries and casuarina plantations in China and discuss the benefits of inoculation.

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.

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.

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.

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.

Robust Frankia phylogeny, species delineation and intraspecies diversity based on Multi-Locus Sequence Analysis (MLSA) and Single-Locus Strain Typing (SLST) adapted to a large sample size.

Diazotrophic Actinobacteria of the genus Frankia represent a challenge to classical bacterial taxonomy as they include many unculturable strains. As a consequence, we still have a poor understanding of their diversity, evolution and biogeography. In this study, a Multi-Locus Sequence Analysis (MLSA) using atpD, dnaA, ftsZ, pgk, and rpoB loci was done on a large set of cultured and uncultured strains, compared to 16S rRNA and correlated to Average Nucleotide Identity (ANI) from available Frankia genomes. MLSA provided a robust resolution of Frankia genus phylogeny and clarified the status of unresolved species and complex of species. The robustness of single-gene topologies and their congruence with the MLSA tree were tested. Lateral Gene Transfers (LGT) were few and scattered, suggesting they had no impact on the concatenate topology. The pgk marker - providing the longest sequence, highest mean genetic divergence and least occurrence of LGT - was used to survey an unequalled number of Alnus-infective Frankia - mainly uncultured strains from a broad range of host-species and geographic origins. This marker allowed reliable Single-Locus Strain Typing (SLST) below the species level, revealed an undiscovered taxonomical diversity, and highlighted the effect of cultivation, sporulation phenotype and host plant species on symbiont richness, diversity and phylogeny.

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 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 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.

Sybr Green- and TaqMan-Based Quantitative PCR Approaches Allow Assessment of the Abundance and Relative Distribution of Frankia Clusters in Soils.

The nodule-forming actinobacterial genus Frankia can generally be divided into 4 taxonomic clusters, with clusters 1, 2, and 3 representing nitrogen-fixing strains of different host infection groups and cluster 4 representing atypical, generally non-nitrogen-fixing strains. Recently, quantitative PCR (qPCR)-based quantification methods have been developed for frankiae of clusters 1 and 3; however, similar approaches for clusters 2 and 4 were missing. We amended a database of partial 23S rRNA gene sequences of Frankia strains belonging to clusters 1 and 3 with sequences of frankiae representing clusters 2 and 4. The alignment allowed us to design primers and probes for the specific detection and quantification of these Frankia clusters by either Sybr Green- or TaqMan-based qPCR. Analyses of frankiae in different soils, all obtained from the same region in Illinois, USA, provided similar results, independent of the qPCR method applied, with abundance estimates of 10 × 105 to 15 × 105 cells (g soil)-1 depending on the soil. Diversity was higher in prairie soils (native, restored, and cultivated), with frankiae of all 4 clusters detected and those of cluster 4 dominating, while diversity in soils under Alnus glutinosa, a host plant for cluster 1 frankiae, or Betula nigra, a related nonhost plant, was restricted to cluster 1 and 3 frankiae and generally members of subgroup 1b were dominating. These results indicate that vegetation affects the basic composition of frankiae in soils, with higher diversity in prairie soils compared to much more restricted diversity under some host and nonhost trees.IMPORTANCE Root nodule formation by the actinobacterium Frankia is host plant specific and largely, but not exclusively, correlates with assignments of strains to specific clusters within the genus. Due to the lack of adequate detection and quantification tools, studies on Frankia have been limited to clusters 1 and 3 and generally excluded clusters 2 and 4. We have developed tools for the detection and quantification of clusters 2 and 4, which can now be used in combination with those developed for clusters 1 and 3 to retrieve information on the ecology of all clusters delineated within the genus Frankia Our initial results indicate that vegetation affects the basic composition of frankiae in soils, with higher diversity in prairie soils compared to much more restricted diversity under some host and nonhost trees.

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.