Actinorhizal Research Articles

Actinorhizal plants and Frankiaceae: The overlooked future of phytoremediation.

Bioremediation of degraded soils is increasingly necessary due to rising food demand, reductions in agricultural productivity, and limitations in total available arable area. Several bioremediation strategies could be utilized to combat soil degradation, with phytoremediation emerging as a standout option due to its in situ approach and low implementation and maintenance costs compared to other methods. Phytoremediation is also a sustainable solution, which is increasingly desirable to blunt the progression of global warming. Actinorhizal plants display several desirable traits for application in phytoremediation, including the ability to revegetate saline soil and sequester heavy metals with low foliar translocation. Additionally, when grown in association with Frankiaceae endophytes, these abilities are improved and expanded to include the degradation of anthropogenic pollutants and the restoration of soil fertility. However, despite this significant potential to remediate marginalized land, the actinorhizal-Frankiaceae symbiosis remains heavily understudied and underutilized. This review aims to collate the scattered studies that demonstrate these bioremediation abilities and explain the mechanics behind such abilities to provide the necessary insight. Finally, this review will conclude with proposed future directions for utilizing this symbiosis and how it can be optimized further to facilitate improved bioremediation outcomes.

Host dependent specialized metabolism of nitrogen export in actinorhizal nodules induced by Frankia cluster-2.

Frankia cluster-2 strains are diazotrophs that engage in root nodule symbiosis with actinorhizal plants of the Cucurbitales and the Rosales. Previous studies have shown that an assimilated nitrogen source, presumably arginine, is exported to the host in nodules of Datisca glomerata (Cucurbitales), while a different metabolite is exported in the nodules of Ceanothus thyrsiflorus (Rosales). To investigate if an assimilated nitrogen form is commonly exported to the host by cluster-2 strains, and which metabolite would be exported in Ceanothus, we analysed gene expression levels, metabolite profiles, and enzyme activities in nodules. We conclude that the export of assimilated nitrogen in symbiosis seems to be a common feature for Frankia cluster-2 strains, but which source is host-dependent. The export of assimilated ammonium to the host suggests that 2-oxoglutarate is drawn from the TCA cycle at a high rate. This specialised metabolism obviates the need for the reductive branch of the TCA cycle. We found several genes encoding enzymes of the central carbon and nitrogen metabolism were lacking in Frankia cluster-2 genomes: the glyoxylate shunt and succinate semialdehyde dehydrogenase. This led to a linearization of the TCA cycle, and we hypothesize this could explain the low saprotrophic potential of Frankia cluster-2.

Tracking the trajectory of frankia research through bibliometrics: trends and future directions.

Frankia represent a unique group of filamentous, sporangia-forming bacteria, renowned for their exceptional capacity to establish symbiotic partnerships with actinorhizal plants. The objective of this paper is to offer quantitative insights into the current state of frankia research and its future potential. A comprehensive bibliometric analysis covering the years 2000-2022 was conducted using Scopus and SciVal. A steady increase in both annual publication and international collaboration has been observed, particularly since 2013. Research performance metrics for the last 5 years (2018-2022) indicate China and India as leaders with high Field-Weighted Citation Impact scores. This analysis highlighted prominent authors, research groups, and the evolving research landscape, suggesting an increasing focus on molecular and genomic aspects. The genomic era has transformed our understanding of frankia biology, highlighting their significance in diverse ecological and agricultural contexts. This study comprehensively maps the evolving landscape of frankia research, emphasizing key milestones that have catalysed international interest in frankia-actinorhizal research, expanding our perception of frankia's capabilities beyond its traditional symbiotic role. As research in this field progresses, a deeper comprehension of frankia-plant interactions, symbiotic signalling, and the intricacies of metabolic pathways holds the promise of revealing innovative techniques for optimizing nitrogen fixation and broadening the spectrum of host plants.

Root nodules of red alder (Alnus rubra) and Sitka alder (Alnus viridis ssp. sinuata) are inhabited by taxonomically diverse cultivable microbial endophytes

AbstractThe root nodules of actinorhizal plants are home to nitrogen‐fixing bacterial symbionts, known as Frankia, along with a small percentage of other microorganisms. These include fungal endophytes and non‐Frankia bacteria. The taxonomic and functional diversity of the microbial consortia within these root nodules is not well understood. In this study, we surveyed and analyzed the cultivable, non‐Frankia fungal and bacterial endophytes of root nodules from red and Sitka alder trees that grow together. We examined their taxonomic diversity, co‐occurrence, differences between hosts, and potential functional roles. For the first time, we are reporting numerous fungal endophytes of alder root nodules. These include Sporothrix guttuliformis, Fontanospora sp., Cadophora melinii, an unclassified Cadophora, Ilyonectria destructans, an unclassified Gibberella, Nectria ramulariae, an unclassified Trichoderma, Mycosphaerella tassiana, an unclassified Talaromyces, Coniochaeta sp., and Sistotrema brinkmanii. We are also reporting several bacterial genera for the first time: Collimonas, Psychrobacillus, and Phyllobacterium. Additionally, we are reporting the genus Serratia for the second time, with the first report having been recently published in 2023. Pseudomonas was the most frequently isolated bacterial genus and was found to co‐inhabit individual nodules with both fungi and bacteria. We found that the communities of fungal endophytes differed by host species, while the communities of bacterial endophytes did not.

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.

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.

Cloning of three Alnus sieboldiana type III polyketide synthases and formation of polyketides in recombinant Escherichia coli using cinnamic acid analogs as substrates

Alnus sieboldiana is an actinorhizal plant that coexists with the nitrogen-fixing actinomycete Frankia via nodules. It produces a variety of polyketides, including flavonoids, stilbenoids, and diarylheptanoids. These compounds have beneficial biological activities. Plant polyketides are produced by type III polyketide synthases (PKSIII). In this study, three A. sieboldiana PKSIIIs (AsPKSIII1, AsPKSIII2, and AsPKSIII3) predicted from next-generation sequencing analysis of A. sieboldiana seedling RNA were amplified and cloned. Phylogenetic tree analysis classified AsPKSIII2 and AsPKSIII3 into the chalcone synthase (CHS) group, whereas AsPKSIII1 was not classified into this group. We attempted to produce polyketides by adding cinnamic acid analogs to the culture medium of Escherichia coli, in which the respective PKSIII gene and the acetyl-CoA carboxylase (ACC) and 4-coumarate: CoA ligase (4CL) genes were simultaneously recombined. AsPKSIII1 is an enzyme that condensed only one molecule of malonyl-CoA to cinnamoyl-CoAs. In contrast, AsPKSIII2 and AsPKSIII3 produced chalcones as shown in a phylogenetic tree analysis, but also produced triketide pyrone. The ratio of these products differed between the two enzymes. We determined the gene and amino acid sequences as well as the substrate specificities of the two enzymes involved in flavonoid production and one enzyme potentially involved in diarylheptanoid production in A. sieboldiana.

High Frankia abundance and low diversity of microbial community are associated with nodulation specificity and stability of sea buckthorn root nodule.

Actinorhizal symbioses are gaining attention due to the importance of symbiotic nitrogen fixation in sustainable agriculture. Sea buckthorn (Hippophae L.) is an important actinorhizal plant, yet research on the microbial community and nitrogen cycling in its nodules is limited. In addition, the influence of environmental differences on the microbial community of sea buckthorn nodules and whether there is a single nitrogen-fixing actinomycete species in the nodules are still unknown. We investigated the diversity, community composition, network associations and nitrogen cycling pathways of the microbial communities in the root nodule (RN), nodule surface soil (NS), and bulk soil (BS) of Mongolian sea buckthorn distributed under three distinct ecological conditions in northern China using 16S rRNA gene and metagenomic sequencing. Combined with the data of environmental factors, the effects of environmental differences on different sample types were analyzed. The results showed that plants exerted a clear selective filtering effect on microbiota, resulting in a significant reduction in microbial community diversity and network complexity from BS to NS to RN. Proteobacteria was the most abundant phylum in the microbiomes of BS and NS. While RN was primarily dominated by Actinobacteria, with Frankia sp. EAN1pec serving as the most dominant species. Correlation analysis indicated that the host determined the microbial community composition in RN, independent of the ecological and geographical environmental changes of the sea buckthorn plantations. Nitrogen cycle pathway analyses showed that RN microbial community primarily functions in nitrogen fixation, and Frankia sp. EAN1pec was a major contributor to nitrogen fixation genes in RN. This study provides valuable insights into the effects of eco-geographical environment on the microbial communities of sea buckthorn RN. These findings further prove that the nodulation specificity and stability of sea buckthorn root and Frankia sp. EAN1pec may be the result of their long-term co-evolution.

Element contents and their seasonal dynamics in leaves of alder Alnus glutinosa (L.) Gaertn

Alnus glutinosa is an actinorhizal plant that fixes N via actinomycetes. Compared to other trees, A. glutinosa is more resistant to environmental stress and able to uptake soil nutrients more easily. Alnus glutinosa grows well not only in natural stands but also in degraded environment or soil in need of restoration. Changes in the contents of selected macro-, micro-, and non-nutrient elements in the leaves of A. glutinosa during the vegetation season were monitored in the Ore Mountains (Czech Republic), an area affected by extreme air pollution in the past. Decreased foliar content of N, P, K, and Cu, and increased content of Ca, Mn, Zn, and Al were observed; the content of other elements (S, Mg, Pb, and Cd) varied during the growing season or remained constant. From the viewpoint of nutrition, the content of N, S, Ca, and Mg macroelements was adequate; concentrations of P and K were low. Excessive amounts of Mn and Zn were measured, and the level of Cu was good. Non-nutrient elements Pb and Cd were present at the background level, and the level of Al was high. N/P, N/Ca, N/Mg, and Ca/Mg ratios were balanced, S/N value showed the lack of S, and N/K ratio indicated low content of K, which caused also suboptimal K/Ca and K/Mg values. The P/Al ratio varied from balanced to lower values. The content of individual elements and monitored changes were influenced by the amount of elements in the soil, moisture conditions, foliage phenology, and altitude.

NIN-at the heart of NItrogen-fixing Nodule symbiosis.

Legumes and actinorhizal plants establish symbiotic relationships with nitrogen-fixing bacteria, resulting in the formation of nodules. Nodules create an ideal environment for nitrogenase to convert atmospheric nitrogen into biological available ammonia. NODULE INCEPTION (NIN) is an indispensable transcription factor for all aspects of nodule symbiosis. Moreover, NIN is consistently lost in non-nodulating species over evolutions. Here we focus on recent advances in the signaling mechanisms of NIN during nodulation and discuss the role of NIN in the evolution of nitrogen-fixing nodule symbiosis.

Comparative analysis of nitrogen content and its influence on actinorhizal nodule and rhizospheric microorganism diversity in three Alnus species.

Alnus spp. (alder) are typical nonleguminous nitrogen-fixing trees that have a symbiotic relationship with Frankia. To explore the differences in nitrogen-fixing microorganisms between three alders (A. cremastogyne, A. glutinosa, and A. formosana) with different chromosome ploidies, the community structure and compositional diversity of potential nitrogen-fixing microorganism in root nodules and rhizosphere soil were comparatively analyzed using 16S rRNA and nitrogenase (nifH) gene sequencing. The nitrogen contents in the root nodules and rhizosphere soil were also determined. The results showed that the contents of total nitrogen and nitrate nitrogen in the root nodules of the three alders are significantly higher than those in the rhizosphere soils, while the ammonium nitrogen content show the opposite trend. The family, genus, and species levels showed obviously differences between root nodules and rhizosphere soils, while there were no significant differences at the classification level between the three alders. At the phylum level, the dominant phyla from 16S rRNA and nifH gene data in the root nodules and rhizosphere soil of the three alders are phylum Actinomycetota and phylum Pseudomonadota, respectively. The LEfSe results showed that there are significant differences in the dominant groups in the root nodules and rhizosphere oil of the three alders. The relative abundances of dominant groups also showed obvious differences between the root nodules and rhizosphere soils of three alders. The relative abundances of Frankia and unclassified_Frankia in root nodules are obviously higher than those in rhizosphere soils, and their relative abundances in A. glutinosa root nodules are significantly higher than those in A. cremastogyne and A. formosana at the genus and species levels. The diversity of potential nitrogen-fixing microorganism from 16S rRNA and nifH gene data in the A. glutinosa root nodules and rhizosphere soils are all higher than those in A. cremastogyne and A. formosana. The results of functional prediction also showed that the OTUs for nitrogen fixation, nitrate respiration, and ureolysis in A. glutinosa root nodules are higher than those in the other two alders. Redundancy analysis revealed that the total nitrogen content mostly affects the Frankia community. Overall, there are significant differences in the community composition and structure of potential nitrogen-fixing microorganism in the root nodules and rhizosphere soils between the three alders. A. glutinosa showed a relatively stronger nitrogen fixation capacity than A. formosana and A. cremastogyne. The results help elucidates how the community structure and nitrogen-fixing ability of potential nitrogen-fixing microorganism differ between alder species and serve as a reference for applying Frankia to alder plantations.

A rare non-canonical splice site in Trema orientalis SYMRK does not affect its dual symbiotic functioning in endomycorrhiza and rhizobium nodulation

BackgroundNitrogen-fixing nodules occur in ten related taxonomic lineages interspersed with lineages of non-nodulating plant species. Nodules result from an endosymbiosis between plants and diazotrophic bacteria; rhizobia in the case of legumes and Parasponia and Frankia in the case of actinorhizal species. Nodulating plants share a conserved set of symbiosis genes, whereas related non-nodulating sister species show pseudogenization of several key nodulation-specific genes. Signalling and cellular mechanisms critical for nodulation have been co-opted from the more ancient plant-fungal arbuscular endomycorrhizal symbiosis. Studies in legumes and actinorhizal plants uncovered a key component in symbiotic signalling, the LRR-type SYMBIOSIS RECEPTOR KINASE (SYMRK). SYMRK is essential for nodulation and arbuscular endomycorrhizal symbiosis. To our surprise, however, despite its arbuscular endomycorrhizal symbiosis capacities, we observed a seemingly critical mutation in a donor splice site in the SYMRK gene of Trema orientalis, the non-nodulating sister species of Parasponia. This led us to investigate the symbiotic functioning of SYMRK in the Trema-Parasponia lineage and to address the question of to what extent a single nucleotide polymorphism in a donor splice site affects the symbiotic functioning of SYMRK.ResultsWe show that SYMRK is essential for nodulation and endomycorrhization in Parasponia andersonii. Subsequently, it is revealed that the 5’-intron donor splice site of SYMRK intron 12 is variable and, in most dicotyledon species, doesn’t contain the canonical dinucleotide ‘GT’ signature but the much less common motif ‘GC’. Strikingly, in T. orientalis, this motif is converted into a rare non-canonical 5’-intron donor splice site ‘GA’. This SYMRK allele, however, is fully functional and spreads in the T. orientalis population of Malaysian Borneo. A further investigation into the occurrence of the non-canonical GA-AG splice sites confirmed that these are extremely rare.ConclusionSYMRK functioning is highly conserved in legumes, actinorhizal plants, and Parasponia. The gene possesses a non-common 5’-intron GC donor splice site in intron 12, which is converted into a GA in T. orientalis accessions of Malaysian Borneo. The discovery of this functional GA-AG splice site in SYMRK highlights a gap in our understanding of splice donor sites.

Significance of nitrogen-fixing actinorhizal symbioses for restoration of depleted, degraded, and contaminated soil.

Atmospheric nitrogen (N2)-fixing legume trees are frequently used for the restoration of depleted, degraded, and contaminated soils. However, biological N2 fixation (BNF) can also be performed by so-called actinorhizal plants. Actinorhizal plants include a high diversity of woody species and therefore can be applied in a broad spectrum of environments. In contrast to N2-fixing legumes, the potential of actinorhizal plants for soil restoration remains largely unexplored. In this Opinion, we propose related basic research requirements for the characterization of environmental stress responses that determine the restoration potential of actinorhizal plants for depleted, degraded, and contaminated soils. We identify advantages and unexplored processes of actinorhizal plants and describe a mainly uncharted avenue of future research for this important group of plant species.

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

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

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).

NAD+ Synthetase is Required for Free-living and Symbiotic Nitrogen Fixation in the Actinobacterium Frankia casuarinae.

Frankia spp. are multicellular actinobacteria that fix atmospheric dinitrogen (N2) not only in the free-living state, but also in root-nodule symbioses with more than 200 plant species, called actinorhizal plants. To identify novel Frankia genes involved in N2 fixation, we previously isolated mutants of Frankia casuarinae that cannot fix N2. One of these genes, mutant N3H4, did not induce nodulation when inoculated into the host plant Casuarina glauca. Cell lineages that regained the ability to fix N2 as free-living cells were isolated from the mutant cell population. These restored strains also regained the ability to stimulate nodulation. A comparative ana-lysis of the genomes of mutant N3H4 and restored strains revealed that the mutant carried a mutation (Thr584Ile) in the glutamine-dependent NAD+ synthetase gene (Francci3_3146), while restored strains carried an additional suppressor mutation (Asp478Asn) in the same gene. Under nitrogen-depleted conditions, the concentration of NAD(H) was markedly lower in the mutant strain than in the wild type, whereas it was higher in restored strains. These results indicate that glutamine-dependent NAD+ synthetase plays critical roles in both free-living and symbiotic N2 fixation in Frankia.

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.

Actinobacteria as biological nitrogen fixation (BNF) and symbiotic association with actinorhizal plants for restoration of salinized soils

Soil salinity is a significant environmental problem affecting agricultural productivity worldwide. Salinized soils are one of the highest exchangeable sodium salts present in zone of the roots. In this scenario, much focus was drawn towards the symbiotic association between the plant species and the nitrogen fixing actinobacterial species. Actinobacteria are well known for their ability to produce secondary metabolites with potential applications in bioremediation and plant growth promotion. This study investigated the efficiency of co-culturing with actinobacteria to enhance the growth of plants under 0.5% NaCl conditions. Pot trials were conducted with three different treatments, fertile soil without actinobacteria, 0.5% NaCl mixed soil with actinobacteria co-culturing, and 0.5% NaCl mixed soil without actinobacteria co-culturing. The results showed that the plants grown in the 0.5% NaCl mixed soil with actinobacteria co-culturing treatment exhibited significantly plant growth, compared to the other two treatments. The co-culturing of actinobacteria was found to improve the plant's ability to tolerate high salinity conditions and enhance their growth. In this study, actinobacteria were isolated from high saline soil samples using selective media, and their morphological and biochemical characteristics were examined.

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

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

Decrypting phytomicrobiome of the neurotoxic actinorhizal species, Coriaria myrtifolia, and dispersal boundary of Frankia cluster 2 in soil outward compatible host rhizosphere.

The actinorhizal plant, Coriaria myrtifolia, is a neurotoxic plant species endemic to the western Mediterranean area, which forms a nitrogen-fixing symbiosis with members of Frankia cluster 2. Contrarily to other Frankia clusters, the occurrence and mode of dispersal for infective cluster 2 units outside of the host plant rhizosphere remains controversial. The present study was designed to investigate the structure of the microbiomes of C. myrtifolia phytosphere, rhizosphere, and soil samples extending outward linearly up to 1 km. Results showed that the epiphyte and endophyte communities were not significantly different from each other for most of the plant tissues. The communities associated with the below-ground tissues (nodule and root) were significantly different from those found on the above-ground tissues (fruit, leaves, and stems) and had a higher community richness. Coriaria myrtifolia phytomicrobiomes were dominated by Cyanobacteria for leaf, stem, and fruit while Actinobacteria and Proteobacteria were dominant in the root and nodule organelles. The nodule, a special niche for nitrogen fixation, was mainly inhabited by Frankia but contained several non-Frankia bacteria. Beside Frankia cluster 2, the presence of clusters 1, 4, and large numbers of cluster 3 strains have been detected in nodules, roots, and rhizospheres of C. myrtifolia. Despite Frankia being found in all plots using plant trapping bioassays with C. myrtifolia seedlings, Frankia cluster 2 was not detected in soil metagenomes showing the limits of detection by this approach. This result also suggests that in the absence of appropriate host plant species, Frankia cluster 2 has a reduced number of infective units present in the soil outward from the rhizosphere.

Taxogenomic status of phylogenetically distant Frankia clusters warrants their elevation to the rank of genus: A description of Protofrankia gen. nov., Parafrankia gen. nov., and Pseudofrankia gen. nov. as three novel genera within the family Frankiaceae.

The genus Frankia is at present the sole genus in the family Frankiaceae and encompasses filamentous, sporangia-forming actinomycetes principally isolated from root nodules of taxonomically disparate dicotyledonous hosts named actinorhizal plants. Multiple independent phylogenetic analyses agree with the division of the genus Frankia into four well-supported clusters. Within these clusters, Frankia strains are well defined based on host infectivity range, mode of infection, morphology, and their behaviour in culture. In this study, phylogenomics, overall genome related indices (OGRI), together with available data sets for phenotypic and host-plant ranges available for the type strains of Frankia species, were considered. The robustness and the deep radiation observed in Frankia at the subgeneric level, fulfilling the primary principle of phylogenetic systematics, were strengthened by establishing genome criteria for new genus demarcation boundaries. Therefore, the taxonomic elevation of the Frankia clusters to the rank of the genus is proposed. The genus Frankia should be revised to encompass cluster 1 species only and three novel genera, Protofrankia gen. nov., Parafrankia gen. nov., and Pseudofrankia gen. nov., are proposed to accommodate clusters 2, 3, and 4 species, respectively. New combinations for validly named species are also provided.