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.

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.

Genome analysis and biogeographic distribution of the earliest divergent Frankia clade in the southern hemisphere.

Coriariaceae are a small plant family of 14-17 species and subspecies that currently have a global but disjunct distribution. All species can form root nodules in symbiosis with diazotrophic Frankia cluster-2 strains, which form the earliest divergent symbiotic clade within this bacterial genus. Studies on Frankia cluster-2 mostly have focused on strains occurring in the northern hemisphere. Except for one strain from Papua New Guinea, namely Candidatus Frankia meridionalis Cppng1, no complete genome of Frankia associated with Coriaria occurring in the southern hemisphere has been published thus far, yet the majority of the Coriariaceae species occur here. We present field sampling data of novel Frankia cluster-2 strains, representing two novel species, which are associated with Coriaria arborea and Coriaria sarmentosa in New Zealand, and with Coriaria ruscifolia in Patagonia (Argentina), in addition to identifying Ca. F. meridionalis present in New Zealand. The novel Frankia species were found to be closely related to both Ca. F. meridionalis, and a Frankia species occurring in the Philippines, Taiwan, and Japan. Our data suggest that the different Frankia cluster-2 species diverged early after becoming symbiotic circa 100 million years ago.

Frankia nepalensis sp. nov., a non-infective non-nitrogen-fixing isolate from root nodules of Coriaria nepalensis Wall.

Strains CN4T, CN6, CN7 and CNm7 were isolated from root nodules of Coriaria nepalensis from Murree in Pakistan. They do not form root nodules on C. nepalensis nor on Alnus glutinosa although they deformed root hairs of Alnus. The colonies are bright red-pigmented, the strains form hyphae and sporangia but no N2-fixing vesicles and do not fix nitrogen in vitro. The peptidoglycan of strain CN4T contains meso-diaminopimelic acid; whole cell sugars consist of ribose, mannose, glucose, galactose and rhamnose. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and two unknown lipids represent the major polar lipids; MK-9(H4) and MK-9(H6) are the predominant menaquinones (>15 %), and iso-C16 : 0 and C17 : 1ω8c are the major fatty acids (>15 %). The results of comparative 16S rRNA gene sequence analyses indicated that strain CN4T is most closely related to Frankia saprophytica CN 3T. An MLSA phylogeny using amino acids sequences of AtpD, DnaA, FtsZ, Pgk and RpoB, assigned the strain to cluster 4 non-nodulating species, close to F. saprophytica CN 3T , Frankia asymbiotica M16386T and Frankia inefficax EuI1cT with 0.04 substitutions per site, while that value was 0.075 with other strains. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between CN4T and all species of the genus Frankia with validly published names were below the defined threshold for prokaryotic species demarcation, with dDDH and ANI values at or below 27.8 and 83.7 %, respectively. The four strains CN4T, CN6, CN7 and CNm7 had dDDH (98.6-99.6 %) and ANI values that grouped them as representing a single species. CN4T has a 10.76 Mb genome. CN4T was different from its close phylogenetic neighbours with validly published names in being red-pigmented, in having several lantibiotic-coding clusters, a carbon monoxide dehydrogenase cluster and a clustered regularly interspaced short palindromic repeats (CRISPR) cluster. The results of phenotypic, physiological and phylogenomic analyses confirmed the assignment of strain CN4T (=DSM 114740T = LMG 32595T) to a novel species, with CN4T as type strain, for which the name Frankia nepalensis sp. nov. is proposed.

Identification and evolution of nsLTPs in the root nodule nitrogen fixation clade and molecular response of Frankia to AgLTP24.

Non-specific lipid transfer proteins (nsLTPs) are antimicrobial peptides, involved in several plant biological processes including root nodule nitrogen fixation (RNF). Nodulating plants belonging to the RNF clade establish symbiosis with the nitrogen-fixing bacteria rhizobia (legumes symbiosis model) and Frankia (actinorhizal symbiosis model) leading to root nodule formation. nsLTPs are involved in processes active in early step of symbiosis and functional nodule in both models. In legumes, nsLTPs have been shown to regulate symbiont entry, promote root cortex infection, membrane biosynthesis, and improve symbiosis efficiency. More recently, a nsLTP, AgLTP24 has been described in the context of actinorhizal symbiosis between Alnus glutinosa and Frankia alni ACN14a. AgLTP24 is secreted at an early step of symbiosis on the deformed root hairs and targets the symbiont in the nitrogen-fixing vesicles in functional nodules. nsLTPs are involved in RNF, but their functions and evolutionary history are still largely unknown. Numerous putative nsLTPs were found up-regulated in functional nodules compared to non-infected roots in different lineages within the RNF clade. Here, results highlight that nodulating plants that are co-evolving with their nitrogen-fixing symbionts appear to have independently specialized nsLTPs for this interaction, suggesting a possible convergence of function, which opens perspectives to investigate nsLTPs functions in RNF.

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

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

Frankia alni Carbonic Anhydrase Regulates Cytoplasmic pH of Nitrogen-Fixing Vesicles.

A phyloprofile of Frankia genomes was carried out to identify those genes present in symbiotic strains of clusters 1, 1c, 2 and 3 and absent in non-infective strains of cluster 4. At a threshold of 50% AA identity, 108 genes were retrieved. Among these were known symbiosis-associated genes such as nif (nitrogenase), and genes which are not know as symbiosis-associated genes such as can (carbonic anhydrase, CAN). The role of CAN, which supplies carbonate ions necessary for carboxylases and acidifies the cytoplasm, was thus analyzed by staining cells with pH-responsive dyes; assaying for CO2 levels in N-fixing propionate-fed cells (that require a propionate-CoA carboxylase to yield succinate-CoA), fumarate-fed cells and N-replete propionate-fed cells; conducting proteomics on N-fixing fumarate and propionate-fed cells and direct measurement of organic acids in nodules and in roots. The interiors of both in vitro and nodular vesicles were found to be at a lower pH than that of hyphae. CO2 levels in N2-fixing propionate-fed cultures were lower than in N-replete ones. Proteomics of propionate-fed cells showed carbamoyl-phosphate synthase (CPS) as the most overabundant enzyme relative to fumarate-fed cells. CPS combines carbonate and ammonium in the first step of the citrulline pathway, something which would help manage acidity and NH4+. Nodules were found to have sizeable amounts of pyruvate and acetate in addition to TCA intermediates. This points to CAN reducing the vesicles' pH to prevent the escape of NH3 and to control ammonium assimilation by GS and GOGAT, two enzymes that work in different ways in vesicles and hyphae. Genes with related functions (carboxylases, biotin operon and citrulline-aspartate ligase) appear to have undergone decay in non-symbiotic lineages.

Denitrification process of Casuarina root nodule endophyte Frankia.

To examine the characteristic of denitrification in Frankia, a symbiotic nitrogen-fixing microbe associated with non-leguminous plants, and its role as a N2O source or sink, Casuarina root nodule endophyte Frankia was isolated using sectioning method, which was then purely cultured to investigate the denitrification process under NO3- addition. The results showed that after addition of NO3- to the medium under anaerobic condition, the concentration of NO3- decreased with time, while the concentrations of NO2- and N2O initially increased and then decreased over time. Key denitrification genes and nitrogenase gene were detected at 26 h, 54 h and 98 h during incubation. Abundances of these genes significantly differed among each other, and their dynamics were asynchronous. Redundancy analysis of the effect of NO3-, NO2-, N2O concentrations on abundances of denitrification genes and nitrogenase gene indicated that 81.9% of the total variation in gene abundances could be explained by the first two axes. Frankia had a denitrifying activity under anaerobic condition, with denitrification genes, including nitrous oxide reductase gene (nosZ), being identified. Our results suggested that Frankia possessed a complete denitrification pathway and the ability of N2O reduction under anaerobic condition.

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.

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.

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.

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).

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')-ß-d-glucopyranoside, 3-pentyl α-l-rhamnopyranosyl-(1''→6')-ß-d-glucopyranoside, and 3-pentyl 6'-(3-hydroxy3-methylglutaryl)-ß-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.

Novel species of Frankia, Frankia gtarii sp. nov. and Frankia tisai sp. nov., isolated from a root nodule of Alnus glutinosa.

The status of four Frankia strains isolated from a root nodule of Alnus glutinosa was established in a polyphasic study. Taxogenomics and phenotypic features show that the isolates belong to the genus Frankia. All four strains form extensively branched substrate mycelia, multilocular sporangia, vesicles, lack aerial hyphae, but contain meso-diaminopimelic acid as the diamino acid of the peptidoglycan, galactose, glucose, mannose, ribose, xylose and traces of rhamnose as cell wall sugars, iso-C16:0 as the predominant fatty acid, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol as the major polar lipids, have comparable genome sizes to other cluster 1, Alnus-infective strains with structural and accessory genes associated with nitrogen fixation. The genome sizes of the isolates range from 7.0 to 7.7 Mbp and the digital DNA G + C contents from 71.3 to 71.5 %. The four sequenced genomes are rich in biosynthetic gene clusters predicted to express for novel specialized metabolites, notably antibiotics. 16S rRNA gene and whole genome sequence analyses show that the isolates fall into two lineages that are closely related to the type strains of Frankia alni and Frankia torreyi. All of these taxa are separated by combinations of phenotypic properties and by digital DNA:DNA hybridization scores which indicate that they belong to different genomic species. Based on these results, it is proposed that isolates Agncl-4T and Agncl-10, and Agncl-8T and Agncl-18, be recognised as Frankia gtarii sp. nov. and Frankia tisai sp. nov. respectively, with isolates Agncl-4T (=DSM 107976T = CECT 9711T) and Agncl-8T (=DSM 107980T = CECT 9715T) as the respective type strains.

Neighbours in nodules: the interactions between Frankia sp. ACN10a and non-Frankia nodular endophytes of alder.

In the present study, we report the in vitro interactions between Frankia sp. ACN10a and non-Frankia nodular endophytes (NFNE) isolated from alder. The supernatant of NFNE grown in nitrogen-replete medium had neutral or negative effects on Frankia growth; none had a stimulatory effect. Inhibitory effects were observed for supernatants of some NFNE, notably Micromonospora, Pseudomonas, Serratia and Stenotrophomonas isolates. However, some NFNE-Frankia coculture supernatants could stimulate Frankia growth when used as a culture medium supplement. This was observed for supernatants of Frankia cocultured with Microvirga and Streptomyces isolates. In nitrogen-limited conditions, cocultures of Frankia with some NFNE, including some rhizobia and Cytobacillus, resulted in higher total biomass than Frankia-only cultures, suggesting cooperation, while other NFNE were strongly antagonistic. Microscopic observation of cocultures also revealed compromised Frankia membrane integrity, and some differentiation into stress resistance-associated morphotypes such as sporangia and reproductive torulose hyphae (RTH). Furthermore, the coculture of Frankia with Serratia sp. isolates resulted in higher concentrations of the auxinic plant hormone indole-3-acetic acid and related indolic compounds in the culture supernatant. This study sheds new light on the breadth of microbial interactions that occur amongst bacteria that inhabit the understudied ecological niche of the alder nodule.

A Nonspecific Lipid Transfer Protein with Potential Functions in Infection and Nodulation.

The response of Alnus glutinosa to Frankia alni ACN14a is driven by several sequential physiological events from calcium spiking and root-hair deformation to the development of the nodule. Early stages of actinorhizal symbiosis were monitored at the transcriptional level to observe plant host responses to Frankia alni. Forty-two genes were significantly upregulated in inoculated compared with noninoculated roots. Most of these genes encode proteins involved in biological processes induced during microbial infection, such as oxidative stress or response to stimuli, but a large number of them are not differentially modulated or downregulated later in the process of nodulation. In contrast, several of them remained upregulated in mature nodules, and this included the gene most upregulated, which encodes a nonspecific lipid transfer protein (nsLTP). Classified as an antimicrobial peptide, this nsLTP was immunolocalized on the deformed root-hair surfaces that are points of contact for Frankia spp. during infection. Later in nodules, it binds to the surface of F. alni ACN14a vesicles, which are the specialized cells for nitrogen fixation. This nsLTP, named AgLTP24, was biologically produced in a heterologous host and purified for assay on F. alni ACN14a to identify physiological effects. Thus, the activation of the plant immunity response occurs upon first contact, while the recognition of F. alni ACN14a genes switches off part of the defense system during nodulation. AgLTP24 constitutes a part of the defense system that is maintained all along the symbiosis, with potential functions such as the formation of infection threads or nodule primordia to the control of F. alni proliferation. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Cyclopeptide Alkaloids from Discaria chacaye (Rhamnaceae) as Result of Symbiosis with Frankia (Actinomycetales).

Cyclopeptide alkaloids with different biological activities are present in plants of the family Rhamnaceae. Plants of this family grow in a symbiotic relationship with aerobic Gram-positive actinomycetes belonging to the genus Frankia. This goal of this research was a study of the comparative profile of alkaloids present in Discaria chacaye and to establish a connection between the presence or absence of Frankia sp. and the alkaloids. In addition, insecticidal activities of the alkaloidal extract were examined. A total of 24 alkaloids were identified, of which 12 have a benzylisoquinoline skeleton, 9 were cyclopeptides, 2 isoquinolines, and 1 aporphine. The presence of cyclopeptide alkaloids is associated with Frankia nodules in the plant root. The alkaloid extracts showed insecticidal activity with mortality dose-dependence and LD50 values between 44 to 71 µg/mL.

A tale of two lineages: how the strains of the earliest divergent symbiotic Frankia clade spread over the world.

It is currently assumed that around 100 million years ago, the common ancestor to the Fabales, Fagales, Rosales and Cucurbitales in Gondwana, developed a root nodule symbiosis with a nitrogen-fixing bacterium. The symbiotic trait evolved first in Frankia cluster-2; thus, strains belonging to this cluster are the best extant representatives of this original symbiont. Most cluster-2 strains could not be cultured to date, except for Frankia coriariae, and therefore many aspects of the symbiosis are still elusive. Based on phylogenetics of cluster-2 metagenome-assembled genomes (MAGs), it has been shown that the genomes of strains originating in Eurasia are highly conserved. These MAGs are more closely related to Frankia cluster-2 in North America than to the single genome available thus far from the southern hemisphere, i.e., from Papua New Guinea.To unravel more biodiversity within Frankia cluster-2 and predict routes of dispersal from Gondwana, we sequenced and analysed the MAGs of Frankia cluster-2 from Coriaria japonica and Coriaria intermedia growing in Japan, Taiwan and the Philippines. Phylogenetic analyses indicate there is a clear split within Frankia cluster-2, separating a continental from an island lineage. Presumably, these lineages already diverged in Gondwana.Based on fossil data on the host plants, we propose that these two lineages dispersed via at least two routes. While the continental lineage reached Eurasia together with their host plants via the Indian subcontinent, the island lineage spread towards Japan with an unknown host plant.

Cyclic diarylheptanoids as potential signal compounds during actinorhizal symbiosis between Alnus sieboldiana and Frankia.

The nitrogen-fixing actinomycete Frankia coexists with actinorhizal plants via nodules and supplies nitrogen compounds to the plants. Although communication has been suggested to exist through chemical substances in this nodule symbiosis, the details underlying this mechanism remain elusive. The biphenyl-type diarylheptanoids (BP-CDHs), alnusonol, and alnusdione, previously isolated from the actinorhizal plant A. sieboldiana branch wood, are secondary metabolites that accumulate in a limited number of plant species. However, since relatively widely distributed in actinorhizal plants, we investigated whether adding A. sieboldiana root extracts and these BP-CDHs could affect plant seedlings inoculated with Frankia. The results showed that the addition of root extract or alnusonol significantly increased the number of nodules and lobes more than two times compared with that upon Frankia supplementation only. We also proved that the extracted components of this plant affected nodule symbiosis. Finally, we confirmed through LC-MS that the root extract component contained BP-CDH, alnusonol. The above-described results indicate that BP-CDHs, at leaset alnusonol, might function as signal compounds from the plant side of the actinorhizal symbiosis between A. sieboldiana and Frankia.

Paradigms of actinorhizal symbiosis under the regime of global climatic changes: New insights and perspectives.

Nitrogen occurs as inert and inaccessible dinitrogen gaseous form (N2 ) in the atmosphere. Biological nitrogen fixation is a chief process that makes this dinitrogen (N2 ) accessible and bioavailable in the form of ammonium (NH4 + ) ions. The key organisms to fix nitrogen are certain prokaryotes, called diazotrophs either in the free-living form or establishing significant mutual relationships with a variety of plants. On such examples is ~95-100 MY old incomparable symbiosis between dicotyledonous trees and a unique actinobacterial diazotroph in diverse ecosystems. In this association, the root of the certain dicotyledonous tree (~25 genera and 225 species) belonging to three different taxonomic orders, Fagales, Cucurbitales, and Rosales (FaCuRo) known as actinorhizal trees can host a diazotroph, Frankia of order Frankiales. Frankia is gram-positive, branched, filamentous, sporulating, and free-living soil actinobacterium. It resides in the specialized, multilobed, and coralloid organs (lateral roots but without caps), the root nodules of actinorhizal tress. This review aims to provide systematic information on the distribution and the phylogenetic diversity of hosts from FaCuRo and their micro-endosymbionts (Frankia spp.), colonization mechanisms, and signaling pathways. We also aim to provide details on developmental and physiological imperatives for gene regulation and functional genomics of symbiosis, phenomenal restoration ecology, influences of contemporary global climatic changes, and anthropogenic impacts on plant-Frankia interactions for the functioning of ecosystems and the biosphere.