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Frankia actinobacteria occur in northwest Patagonian steppe soils and form nitrogen-fixing actinorhizal symbiotic root nodules with Ochetophila trinervis (Rhamnaceae). Soil disturbances affect soilborne Frankia, reducing the likelihood of establishing symbiosis, as found for Pinus ponderosa plantations in the region. The effect on local actinorhizal symbionts of other kinds of disturbance such as the ash that was deposited by the eruption of the Puyehue-Cordon Caulle (PCC) volcanic complex was unknown. We studied the initial succession steps for the Frankia colonization process and the soil change that might affect the establishment of symbiosis as a result of the combined action of volcanic ash deposition and P. ponderosa afforestation by comparing pre- and post-eruption time points. We collected soil and ash samples at three depths to use in plant bioassays with O. trinervis. These samples were also used to determine chemical properties (soil and ashes) and elemental composition (soil). Infective Frankia gradually colonized the ash deposited in steppe soils via soil legacy and immigration. The higher nodulation frequency found in plants inoculated with buried soil and deep ash than in plants inoculated with surface ash suggests that the colonization process was mainly due to soil legacy, but also, though at a slower rate, to immigration of Frankia from elsewhere. Nevertheless, the colonization process seemed to be hindered by the pine plantation establishment processes. C, N and P increased over time, favoring the establishment of Frankia and therefore the nodulation capacity of ash. The chemical and elemental characteristics of soils under natural vegetation seemed to be closer to pre-eruptive conditions than those of soils under the afforestation, and to favor nodulation capacity. The combination of ash deposition and the pine plantation appeared to have the strongest effect in impairing the restoration of previous conditions for the establishment of symbiosis with Frankia.

Shepherdia ×utahensis ‘Torrey’ (hybrid buffaloberry) is an actinorhizal plant that can form symbiotic nodules with the actinobacterial genus Frankia. However, little research has been conducted to investigate the presence of Frankia in their nodules and the effects on plant growth. In this study, plants were grown in a Metro-Mix® 820 substrate and inoculated with soils collected from Mohave County, AZ, or in a low organic-matter substrate inoculated with soils from North Logan, UT. The presence of Frankia was quantified using PolF/PolR primers to amplify their nitrogenase (nifH) gene sequences. In the Metro-Mix 820 substrate, plants irrigated with nitrogen (N)-free Hoagland’s solution at pH 6.5 formed nodules at week 12 after experiment initiation, whereas those receiving the same solution with 2 mm ammonium nitrate (NH4NO3) appeared healthy, but no nodules formed. In the low organic-matter substrate, nodules formed in 5 weeks when plants were irrigated with N-free Hoagland’s solution at pH 7.5. Four 300-bp fragments of query sequences (SU1, SU2, SU3, and SU4) were obtained from nodules. When compared with nifH gene sequences reported in the literature using the Basic Local Alignment Search Tool (BLAST), more than 90% similarity to the nifH of Frankia spp. was obtained. The Frankia strains in the nodules shared nifH sequences similar to those of the same host-specific group of Shepherdia. Furthermore, Frankia strains with similar nifH genes have been reported in nodules of Shepherdia argentea (silver buffaloberry). Additionally, Frankia strains belonging to cluster 3 infective strains consisting of Elaeagnaceae and Rhamnaceae infective Frankia showed high similarity to the query sequences. This research demonstrates that nodulation of S. ×utahensis is inhibited at 2 mm NH4NO3. Apart from N, nodule formation may be associated with the substrate type and pH of the nutrient solution. Based on nifH gene sequence amplification, Frankia strains in the root nodules may have the potential to fix atmospheric nitrogen (N2). These Frankia strains have signature gene sequence characteristics of Elaeagnaceae-infective Frankia, suggesting that S. ×utahensis shares Frankia strains similar to its parents.

Casuarina species are evergreen angiosperm trees native to Australia, South East Asia and the Pacific archipelagos. The genus comprises 14 confirmed species that are able to grow on a wide range of soils. Casuarina spp. are able to enter into an actinorhizal root nodule symbiosis with Frankia casuarinae, nitrogen-fixing soil actinobacteria; the symbiosis renders them independent of soil nitrogen sources. Casuarina spp. can also form symbiotic associations with ectomycorrhizal fungi (EMF) and/or arbuscular mycorrhizal fungi (AMF) for better phosphate supply. Under severe deficiency of phosphate and iron, several Casuarina spp. can form cluster roots. These trees are highly tolerant to salinity, drought, flooding and heavy metal pollution and are extensively used for the rehabilitation of degraded sites. Casuarina spp. plantations also play a significant role in prevention of soil erosion, coastal reclamation, dune stabilization, and as windbreaks in agricultural lands. Due to their multipurpose applications in agriculture and forestry, they have been widely introduced outside their native habitat especially in tropical, arid and semiarid countries across the globe. Therefore, the global distribution of Casuarina spp. has been significantly affected by anthropogenic influences. While in most of these countries, Casuarina spp. have become well-integrated into the regional farming systems, they have become invasive in other regions.

The symbiosis between plants and nitrogen‐fixing bacteria is widespread among legumes and actinorhizal plants within the nitrogen‐fixing root nodule (NFN) clade. However, there are major differences, as well as similarities, in the symbioses between actinorhizal plants and Frankia and those of legumes and their associated rhizobia. This review provides an overview of NFN symbioses. We outline the evolution and biogeography of actinorhizal plants and legumes and compare and contrast their microsymbionts and symbiotic processes. Within the NFN clade, a far greater number of nodulated legumes exists, compared with actinorhizal plants, and legumes have a much wider biogeographical distribution. There are genetic and physiological differences between free‐living diazotrophic Frankia and the phylogenetically diverse rhizobia, most strains of which are unable to fix N2 ex planta. Actinorhizal nodules are modified lateral roots with a central vascular system, whereas legume nodules are stem‐like organs with peripheral vascular systems. Most legumes contain their microsymbionts within symbiosomes, rather than the infection threads found in actinorhizal nodule cells. Legumes have greater control of their microsymbionts, and those within the Inverted Repeat Lacking Clade impose terminal differentiation on their bacteroids. Legumes also have effective processes for autoregulation of nodulation and downregulation of N2 fixation in response to high levels of soil N. These features of the legume‐rhizobia symbiosis have led to increased efficiencies in N2 fixation. Synthesis. We suggest that these characteristic features of the legume‐rhizobia symbiosis, specifically legumes' greater flexibility in the choice of microsymbiont partner and the evolution of increased efficiencies in N2 fixation, are factors that can explain why the majority of species within the Leguminosae have retained the ability to nodulate and how this has contributed to their evolutionary success.

In this study, we describe the genomes of two novel candidate species of non-nitrogen fixing Frankia that were isolated from the root nodules of Coriaria nepalensis and Alnus glutinosa, genospecies CN and Ag, respectively. Comparative genomic analyses revealed that both genospecies lack genes essential for nitrogen-fixation and possess genes involved in the degradation of plant cell walls. Additionally, we found distinct biosynthetic gene clusters in each genospecies. The availability of these genomes will contribute to the study of the taxonomy and evolution of actinorhizal symbioses.

Frankia is a mycelium-forming actinobacterial genus that is found in actinorhizal plants as a nitrogen-fixing facultative endo-symbiont. Frankia has been a commonly uncultured endophytic actinobacteria with very slow growth germination. In the present study, the root nodules from Casuarina spp. were collected at hyper-saline and terrestrial habitats in Tamil Nadu, South India. The root nodules were surface-sterilized, dried, homogenized, and inoculated on the DPM medium with sodium propionate as a carbon source and biotin as a vitamin source. A total of 11 Frankia isolates were obtained from the specified habitats in a solid and liquid medium. The larger number of isolates was recorded in the hyper-saline habitats compared to terrestrial habitats. Finally, 2 predominant growth occurrences of isolates were selected; from each habitat, unique isolates were molecularly characterized and identified as Frankia sp. DDNSF-01 and Frankia casuarinae DDNSF-02. The physico-chemical property analysis of Casuarina rhizosphere soil of hyper-saline habitat showed higher pH, salinity and micro-nutrients like Fe−, Mn−, Cu−, and Zn− than terrestrial habitats, which could be the major factors responsible for the distribution dynamics of Frankia in hyper-saline habitats. Similarly, the biologically active metabolites of Frankia spp. were extracted by the ethyl acetate extraction method and tested by MIC assay, which was performed against emerging multi-host pathogens like Pseudomonas sp. and Candida sp. Likewise, the antibiofilm activity assay was done against the above pathogens. The biofilm inhibition of live/dead cells was confirmed by CLSM microscopic analysis. The F. casuarinae was inhibited the high level of biofilm formation of both pathogens when compared with Frankia sp. The present findings conclude the F. casuarinae is efficient in the control of emerging multi-host pathogens in actinorhizal plants.

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.

Rhizobium nitrogen-fixing nodule symbiosis occurs in two taxonomic lineages: legumes (Fabaceae) and the genus Parasponia (Cannabaceae). Both symbioses are initiated upon the perception of rhizobium-secreted lipochitooligosaccharides (LCOs), called Nod factors. Studies in the model legumes Lotus japonicus and Medicago truncatula showed that rhizobium LCOs are perceived by a heteromeric receptor complex of distinct Lys motif (LysM)-type transmembrane receptors named NOD FACTOR RECEPTOR1 (LjNFR1) and LjNFR5 (L. japonicus) and LYSM DOMAIN CONTAINING RECEPTOR KINASE3 (MtLYK3)-NOD FACTOR PERCEPTION (MtNFP; M. truncatula). Recent phylogenomic comparative analyses indicated that the nodulation traits of legumes, Parasponia spp., as well as so-called actinorhizal plants that establish a symbiosis with diazotrophic Frankia spp. bacteria share an evolutionary origin about 110 million years ago. However, the evolutionary trajectory of LysM-type LCO receptors remains elusive. By conducting phylogenetic analysis, transcomplementation studies, and CRISPR-Cas9 mutagenesis in Parasponia andersonii, we obtained insight into the origin of LCO receptors essential for nodulation. We identified four LysM-type receptors controlling nodulation in P. andersonii: PanLYK1, PanLYK3, PanNFP1, and PanNFP2. These genes evolved from ancient duplication events predating and coinciding with the origin of nodulation. Phylogenetic and functional analyses associated the occurrence of a functional NFP2-orthologous receptor to LCO-driven nodulation. Legumes and Parasponia spp. use orthologous LysM-type receptors to perceive rhizobium LCOs, suggesting a shared evolutionary origin of LCO-driven nodulation. Furthermore, we found that both PanLYK1 and PanLYK3 are essential for intracellular arbuscule formation of mutualistic endomycorrhizal fungi. PanLYK3 also acts as a chitin oligomer receptor essential for innate immune signaling, demonstrating functional analogy to CHITIN ELECITOR RECEPTOR KINASE-type receptors.

Actinorhizal plants host mutualistic symbionts of the nitrogen-fixing actinobacterial genus Frankia within nodule structures formed on their roots. Several plant-growth-promoting bacteria have also been isolated from actinorhizal root nodules, but little is known about them. We were interested investigating the in planta microbial community composition of actinorhizal root nodules using culture-independent techniques. To address this knowledge gap, 16S rRNA gene amplicon and shotgun metagenomic sequencing was performed on DNA from the nodules of Casuarina glauca. DNA was extracted from C. glauca nodules collected in three different sampling sites in Tunisia, along a gradient of aridity ranging from humid to arid. Sequencing libraries were prepared using Illumina NextEra technology and the Illumina HiSeq 2500 platform. Genome bins extracted from the metagenome were taxonomically and functionally profiled. Community structure based off preliminary 16S rRNA gene amplicon data was analyzed via the QIIME pipeline. Reconstructed genomes were comprised of members of Frankia, Micromonospora, Bacillus, Paenibacillus, Phyllobacterium, and Afipia. Frankia dominated the nodule community at the humid sampling site, while the absolute and relative prevalence of Frankia decreased at the semi-arid and arid sampling locations. Actinorhizal plants harbor similar non-Frankia plant-growth-promoting-bacteria as legumes and other plants. The data suggests that the prevalence of Frankia in the nodule community is influenced by environmental factors, with being less abundant under more arid environments.

Frankia and actinorhizal plants exchange signals in the rhizosphere leading to specific mutual recognition of partners and nitrogen-fixing nodule organogenesis. Frankia soli strain NRRL B-16219, from the Elaeagnus specificity group, colonizes the root tissues of its actinorhizal host through direct intercellular penetration of root epidermis cells and cortex. Here, we studied the early proteogenomic response of strain NRRL B-16219 to treatment with root exudates from compatible Elaeagnus angustifolia, and incompatible Ceanothus thyrsiflorus and Coriaria myrtifolia, host plants grown in nitrogen depleted hydroponic medium. Next-generation proteomics was used to identify the main Frankia proteins differentially expressed in response to the root exudates. No products of the nod genes present in B-16219 were detected. Proteins specifically upregulated in presence of E. angustifolia root exudates include those connected to nitrogen fixation and assimilation (glutamate synthetase, hydrogenase and squalene synthesis), respiration (oxidative phosphorylation and citric acid cycle pathways), oxidative stress (catalase, superoxide dismutase, and peroxidase), proteolysis (proteasome, protease, and peptidase) and plant cell wall degrading proteins involved in the depolymerization of celluloses (endoglucanase, glycosyltransferase, beta-mannanases, glycoside hydrolase and glycosyl hydrolase). Proteomic data obtained in this study will help link signaling molecules/factors to their biosynthetic pathways once those factors have been fully characterized.

The aim of this study was to investigate the effects of different plant parts and the age of plants at harvest as well as N2 fixing bacterial strains on the N concentration in symbiotic plant parts, especially on the δ15N signature of the actinorhizal plants and legumes. The 15N natural abundance method was used. Two actinorhizal plants were studied: Alnus incana (L.) infected with the Frankia strains ArI3 or “lsF” (local source of Frankia) and Hippophaë rhamnoides (L.) infected with the Frankia strains T1 or E15b. Two legume species were studied: Hedysarum coronarium (L.), infected with a soil suspension, and Robinia pseudoacacia (L.), infected with a crushed nodule suspension. It was particularly interesting that in A. incana, the two Frankia strains affected not only N concentration and δ15N signature of leaves and roots, but also had an impact on plant growth at first harvest. In Hippophaë rhamnoides plants inoculated with the Frankia strains T1 and E15b, N concentrations and δ15N values did not differ at any harvest time. However, plants nodulated by the Frankia strain T1 showed a higher nitrogen fixation rate and higher plant dry matter at all harvesting times. Based on our results for the quantification of N2 fixation with the “B” value, that is the δ15N value of the N2 fixing plants relying only on N2 fixation, plant parts, ages and strains should be carefully considered.

Plants capable of stimulating the N2 fixation in the root nodules with endosymbiotic associations of actinobacteria Frankia are known as actinorhizal plants. During the signaling, molecules communicate to develop the new root organs known as actinorhizal nodules. The Frankia are capable of interacting with 225 species belonging to 8 families and 25 genera of nonleguminous plants. Among the 8 families, the Casuarinaceae are most important due to their potential effect in agriculture and other environmental applications. Casuarina is ideal for growing in polluted dry lands and rain-fed areas. It plays a role in different applications such as pulp wood, fuel wood, timber production, medical use, windbreak, sand dune stabilization, improving the soil’s physical properties and maintaining soil organic matter, and fixing atmosphere N2 with the help of Frankia. The recent reports point out that the abundance of relative Frankia populations was more in actinorhizal plants. Actinorhizal interactions display several primitive features and thus provide the ideal opportunity to determine the minimal molecular toolkit desirable to construct a nodule and to understand the evolution of root nodule symbioses. Actinorhizal nodules shares a lot of features with rhizobial nodulation, yet our understanding of the molecular mechanisms implicated in actinorhizal nodulation remain very inadequate. Therefore, the overall bacterial community (bacteriome) diversity within Casuarina root nodules documented is very fewer. In this chapter, we provide an outline on root nodule bacteriome and microbiome networks from actinorhizal Casuarina plant in different diversities along with next-generation sequencing of 16S rRNA metagenomic analysis.

Casuarina equisetifolia L. (Casuarinaceae), an actinorhizal plant, exhibits mutualistic symbiosis with Frankia and promotes nitrogen fixation in root nodules. While the exchange of metabolites between host plant and microsymbiont is well understood in legume symbioses, the situation in the symbiosis between nitrogen-fixing Frankia and actinorhizal plants is less clear. In this study, a metabolomic approach was applied to root nodules of mature C. equisetifolia trees, leading to the identification of an undescribed taraxerane-type triterpenoid ester, 3-O-dihydrocoumaroyl β-taraxerol, along with twelve known compounds. An abundant component was tyramine with a content of 2.76 ± 0.315 mg/g FW in mature nodules. Tyramine specifically and abundantly accumulated in mature nitrogen-fixing nodules compared to senescent nodules, stems, leaves, and seeds. In addition, the potential function of tyramine was preliminarily examined and discussed.

Some plants fix atmospheric nitrogen by hosting symbiotic diazotrophic rhizobia or Frankia bacteria in root organs known as nodules. Such nodule symbiosis occurs in 10 plant lineages in four taxonomic orders: Fabales, Fagales, Cucurbitales, and Rosales, which are collectively known as the nitrogen-fixing clade. Nodules are divided into two types based on differences in ontogeny and histology: legume-type and actinorhizal-type nodules. The evolutionary relationship between these nodule types has been a long-standing enigma for molecular and evolutionary biologists. Recent phylogenomic studies on nodulating and nonnodulating species in the nitrogen-fixing clade indicated that the nodulation trait has a shared evolutionary origin in all 10 lineages. However, this hypothesis faces a conundrum in that legume-type and actinorhizal-type nodules have been regarded as fundamentally different. Here, we analyzed the actinorhizal-type nodules formed by Parasponia andersonii (Rosales) and Alnus glutinosa (Fagales) and found that their ontogeny is more similar to that of legume-type nodules (Fabales) than generally assumed. We also show that in Medicago truncatula, a homeotic mutation in the co-transcriptional regulator gene NODULE ROOT1 (MtNOOT1) converts legume-type nodules into actinorhizal-type nodules. These experimental findings suggest that the two nodule types have a shared evolutionary origin.

Nitrogen-fixing Actinobacteria of the genus Frankia can be subdivided into four phylogenetically distinct clades, members of clusters one to three engage in nitrogen-fixing root nodule symbioses with actinorhizal plants. Mur enzymes are responsible for the biosynthesis of the peptidoglycan layer of bacteria. The four Mur ligases, MurC, MurD, MurE, and MurF, catalyse the addition of a short polypeptide to UDP-N-acetylmuramic acid. Frankia strains of cluster-2 and cluster-3 contain two copies of murC, while the strains of cluster-1 and cluster-4 contain only one. Phylogenetically, the protein encoded by the murC gene shared only by cluster-2 and cluster-3, termed MurC1, groups with MurC proteins of other Actinobacteria. The protein encoded by the murC gene found in all Frankia strains, MurC2, shows a higher similarity to the MurC proteins of plants than of Actinobacteria. MurC2 could have been either acquired via horizontal gene transfer or via gene duplication and convergent evolution, while murC1 was subsequently lost in the cluster-1 and cluster-4 strains. In the nodules induced by the cluster-2 strains, the expression levels of murC2 were significantly higher than those of murC1. Thus, there is clear sequence divergence between both types of Frankia MurC, and Frankia murC1 is in the process of being replaced by murC2, indicating selection in favour of murC2. Nevertheless, protein modelling showed no major structural differences between the MurCs from any phylogenetic group examined.

International audience; Symbiosis established between actinorhizal plants and Frankia spp., which are nitrogen-fixing actinobacteria, promotes nodule organogenesis, the site of metabolic exchange. The present study aimed to identify amino acid markers involved in Frankia-Alnus interactions by comparing nodules and associated roots from field and greenhouse samples. Our results revealed a high level of citrulline in all samples, followed by arginine (Arg), aspartate (Asp), glutamate (Glu), γ-amino-n-butyric acid (GABA), and alanine (Ala). Interestingly, the field metabolome approach highlighted more contrasted amino acid patterns between nodules and roots compared with greenhouse samples. Indeed, 12 amino acids had a mean relative abundance significantly different between field nodule and root samples, against only four amino acids in greenhouse samples, underlining the importance of developing “ecometabolome” approaches. In order to monitor the effects on Frankia cells (respiration and nitrogen fixation activities) of amino acid with an abundance pattern evocative of a role in symbiosis, in-vitro assays were performed by supplementing them in nitrogen-free cultures. Amino acids had three types of effects: i) those used by Frankia as nitrogen source (Glu, Gln, Asp), ii) amino acids stimulating both nitrogen fixation and respiration (e.g., Cit, GABA, Ala, valine, Asn), and iii) amino acids triggering a toxic effect (Arg, histidine). In this paper, a N-metabolic model was proposed to discuss how the host plant and bacteria modulate amino acids contents in nodules, leading to a fine regulation sustaining high bacterial nitrogen fixation.

Members of the genus Frankia are gram-positive nitrogen-fixing actinobacteria that can live in a free-living form in the rhizosphere or in symbiotic association with non-leguminous actinorhizal plants belonging to 25 genera of angiosperms. Since the isolation of the first Frankia in 1978, several hundred isolates have been obtained worldwide from root nodules and soils and classified in four clusters. Although it is still difficult to study this valuable microorganism, the development of genomic and molecular tools both in Frankia and in model actinorhizal plants including Alnus glutinosa and Casuarina glauca, has advanced our understanding of this microorganism and its ability to develop a root endosymbiosis.

Actinobacteria from the genus Frankia are Gram+, aerobic, heterotrophic and filamentous bacteria found living independently in soil or as facultative symbionts that form N2-fixing root nodules on diverse and globally distributed angiosperms called actinorhizal species. This symbiotic association has important ecological and economic roles such as soil stabilization, land reclamation, crop protection and timber and fuel wood production. Some Frankia strains are resistant to abiotic stresses including salinity, heavy metal, pH, and temperature and have developed complexed mechanisms to adapt to these conditions. Under environmentally stressed conditions, actinorhizal plants that had associations with adapted Frankia strains show improved plant performance. Recent “Omics” studies including genomic, transcriptomic and proteomic developed on Frankia have provided different clues for a better understanding of these adaptation mechanisms. In this chapter, we review recent advances in Frankia genome studies and in molecular aspects of Frankia tolerance to environmental stresses.

Phytohormones play a crucial role in regulating plant developmental processes. Among them, ethylene and jasmonate are known to be involved in plant defense responses to a wide range of biotic stresses as their levels increase with pathogen infection. In addition, these two phytohormones have been shown to inhibit plant nodulation in legumes. Here, exogenous salicylic acid (SA), jasmonate acid (JA), and ethephon (ET) were applied to the root system of Casuarina glauca plants before Frankia inoculation, in order to analyze their effects on the establishment of actinorhizal symbiosis. This protocol further describes how to identify putative ortholog genes involved in ethylene and jasmonate biosynthesis and/or signaling pathways in plant, using the Arabidopsis Information Resource (TAIR), Legume Information System (LIS), and Genevestigator databases. The expression of these genes in response to the bacterium Frankia was analyzed using the gene atlas for Casuarina-Frankia symbiosis (SESAM web site).

The high-altitude Himalayan regions are unique micro-climatic and geographic separations that have precise agro-technical needs, which is quite different from other agro-climacteric zones of India for vegetable production. In such marginal degraded regions mainly drought stress causes significant harmful effect on survival, biomass production and yields of vegetable crops up to 70%. Since drought tolerance is quantitative and multigenic in nature, a massive challenge exists to solve this problem. The use of N-fixing growth promoting rhizobacteria (PGPR) in place of agrochemicals (pesticides and fertilizers) is a promising approach to increase the plant productivity by naturally increasing nitrogen content of soil and also not leads to deterioration of the soil micro-flora and environment. In this attempt, this chapter highlights the importance of promising indigenous Frankia sp. strains (potential N-fixing PGPR) isolated from the root nodules of Casuarina plant of Kumaon region of Uttarakhand, India. Also, the effect of bio-inoculation of isolated Frankia sp. strains has been demonstrated to improve PEG-mediated drought stress tolerance in tomato (Solanum lycopersicum L. cv. Pusa ruby) for improving sustainable hill agriculture in rain fed conditions. This successful model may be useful for improving the productivity of other crops grown in drought prone regions of Himalaya.

Legumes and actinorhizal plants are capable of forming root nodules symbiosis with rhizobia and Frankia bacteria. All these nodulating species belong to the nitrogen fixation clade. Most likely, nodulation evolved once in the last common ancestor of this clade. NIN (NODULE INCEPTION) is a transcription factor that is essential for nodulation in all studied species. Therefore, it seems probable that it was recruited at the start when nodulation evolved. NIN is the founding member of the NIN-like protein (NLP) family. It arose by duplication, and this occurred before nodulation evolved. Therefore, several plant species outside the nitrogen fixation clade have NLP(s), which is orthologous to NIN. In this review, we discuss how NIN has diverged from the ancestral NLP, what minimal changes would have been essential for it to become a key transcription controlling nodulation, and which adaptations might have evolved later.

Shepherdia ×utahensis ‘Torrey’ (Elaeagnaceae) is a hybrid of two native actinorhizal plants in the Intermountain West, S. argentea (silver buffaloberry) and S. rotundifolia (roundleaf buffaloberry). Due to actinorhizal symbiosis, atmospheric nitrogen (N2) can be converted to ammonium, a bioavailable form. Actinorhizal plants have great value in sustainable nursery production and urban landscape use. However, nitrogen fertilizer negatively affects the nodulation of actinorhizal plants. As a newly developed hybrid, both the symbiont identity and nodule formation of S. ×utahensis ‘Torrey’ remain largely unknown. Therefore, experiments were conducted to investigate the nodule formation of S. ×utahensis ‘Torrey’ inoculated with field soils from the Greenville Research Farm at Utah State University, North Logan, UT, or Mohave County, AZ. Further, S. ×utahensis ‘Torrey’ inoculated with field soils from the Greenville Research Farm were topdressed with controlled-released fertilizer (CRF) at eight application rates or irrigated with nutrient solutions at two nitrogen levels to study the impacts of nitrogen levels on nodule formation of S. ×utahensis ‘Torrey’. Nodules from S. ×utahensis ‘Torrey’ were used to identify the symbiont using nifH sequence and phylogenetic analysis. The results of our study showed that plants in a low organic substrate (e.g., perlite) and irrigated with a nutrient solution at pH 7.5 developed nodules 7 weeks earlier than in a commercial substrate (e.g., Metro-Mix® 820) irrigated with a nutrient solution at pH 6.5. Nodulated plants need less nitrogenous fertilizer to maintain plant growth and quality compared with uninoculated plants. However, S. ×utahensis ‘Torrey’ plants had fewer nodules when the nitrogen level of the fertilizer increased, and nodulation was completely inhibited when applying 2.9 g·L-1 CRF or 2 mM ammonium nitrate to plants. Phylogenetic analysis suggested that the symbiont of S. ×utahensis ‘Torrey’ could also induce nodules on plants in the Elaeagnaceae and Rhamnaceae to fix atmospheric nitrogen. According to the results in this research, to induce nodules earlier, S. ×utahensis ‘Torrey’ plants should be grown in a substrate with low organic-matter content and irrigated with a nitrogen-free nutrient solution at a relatively high pH when they are inoculated with Frankia-contained soil. Fertilizer lower than the manufacturer’s recommended rate may be applied to S. ×utahensis ‘Torrey’ plants, if inoculated with field soils, to promote nodule formation for nitrogen fixation and reduce nitrogen runoff.

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

Above-ground herbivory has a direct impact on plant life cycles, particularly at more sensitive stages, due to reduction of vegetative biomass. However, this effect may not be negative if it results in net biomass compensation. As sapling stage could be the best stage for native species to be outplanted, understanding the impact of aboveground herbivory on tree saplings is necessary for restoration purposes. We studied the effect of herbivory on saplings of Ochetophila trinervis (Rhamnaceae), a native woody species from North-west Patagonia, which forms an actinorhizal symbiosis with the N2-fixing actinobacteria Frankia. This tree species has the potential to be used for recovering degraded lands. Nevertheless, there is a perplexing contradiction between the high seed output of O. trinervis and the scarcity of saplings in the field. For 4 months, 1-year-old O. trinervis saplings were exposed to aboveground herbivory by generating different protection degrees (unprotected, protected against some kind of walking herbivores—protected saplings; and protected against all kind of walking herbivores—excluded saplings). The impact of herbivores over sapling survival was minimal (92 ± 3%, mean ± SE) and it was similar among saplings exposed to different protection degrees. The highest frequency of foliar damage in excluded saplings suggests the attack of flying herbivores. The increased emergence of new sprouts and root length growth in saplings highly damaged by herbivores (about three fold and two fold higher than in excluded saplings, respectively), evidenced the capacity of O. trinervis to develop a compensatory growth. The results contradict the assumption that herbivory explains the low density of saplings despite high seed production. Given the high-sapling survival and biomass compensation of O. trinervis after herbivory, we suggest that this species might be appropriate for restoration of degraded areas in the region.

Restoration of Alnus formosana (Burk.) Makino on landslide areas is important for agroforestry, forestry and soil erosion control in Taiwan. To ensure successful reforestation, A. formosana seedlings have to develop strong root system for nutrient and water acquisition as well as anchorage. Inoculating of A. formosana with symbiotic nitrogen-fixing actinobacteria Frankia may help mitigate drought and nutrient deficiencies on landslide sites. However, the effects of Frankia inoculation on growth, root architecture and mechanical properties of A. formosana seedlings are not well understood. In this research, a Frankia strain AF1 was isolated from actinorhizal nodules of local A. formosana and recognized as Frankia species, and its influences on growth performance and root mechanical properties of A. formosana seedlings were examined and analyzed. The results showed that the inoculated seedlings had significantly larger height and root biomass, longer root length, and more root tip number than that of the non-inoculated controls. Consistently, the inoculated seedlings had statistically significant higher uprooting resistance, root tensile resistance force and tensile strength than the controls. The results reveal that this native Frankia strain can promote growth performance, root system architecture, anchorage ability and root tensile strength of A. formosana. Key words: Actinorhiza, Betulaceae, inoculation, root morphology, tensile strength, uprooting resistance.

Frankia sp. NRRL B-16219 was directly isolated from a soil sample obtained from the rhizosphere of Ceanothus jepsonii growing in the USA. Its host plant range includes members of Elaeagnaceae species. Phylogenetically, strain NRRL B-16219 is closely related to “Frankia discariae” with a 16S rRNA gene similarity of 99.78%. Because of the lack of genetic tools for Frankia, our understanding of the bacterial signals involved during the plant infection process and the development of actinorhizal root nodules is very limited. Since the first three Frankia genomes were sequenced, additional genome sequences covering more diverse strains have helped provide insight into the depth of the pangenome and attempts to identify bacterial signaling molecules like the rhizobial canonical nod genes. The genome sequence of Frankia sp. strain NRRL B-16219 was generated and assembled into 289 contigs containing 8,032,739 bp with 71.7% GC content. Annotation of the genome identified 6211 protein-coding genes, 561 pseudogenes, 1758 hypothetical proteins and 53 RNA genes including 4 rRNA genes. The NRRL B-16219 draft genome contained genes homologous to the rhizobial common nodulation genes clustered in two areas. The first cluster contains nodACIJH genes whereas the second has nodAB and nodH genes in the upstream region. Phylogenetic analysis shows that Frankia nod genes are more deeply rooted than their sister groups from rhizobia. PCR-sequencing suggested the widespread occurrence of highly homologous nodA and nodB genes in microsymbionts of field collected Ceanothus americanus. Electronic supplementary material The online version of this article (10.1186/s40793-017-0261-3) contains supplementary material, which is available to authorized users.

In agriculture, plant growth enrichment via plant growth stimulating microbes has been recognized as an emergency, it is used as an alternatives to chemical pesticides and growth stimulants. The phytopathogens cause various diseases such as blister bark; stem cankers, and pink and brown rot diseases besides affect the growth frequency of Casuarina spp. toward biotic and abiotic stresses. Bio-control and plant growth-promoting potential of native Frankia isolates from Casuarina spp. in Tamil Nadu, India, was not much explored. Hence, in the present study, we are investigating the plant growth improvement activity and phytopathogen control in Casuarina spp. The Frankia sp. DDNSF-01 and Frankia casuarinae DDNSF-02 were isolated and identified from the root nodules of Casuarina spp. Additionally, it is recognized for plant growth promoter activity and in vitro antimicrobial activity against phytopathogens including Pseudomonas sp. and Colletotrichum sp. The plant growth regulators including IAA, siderophore, ammonia production, and phosphate solubilization were found out. Therefore, the formation of the most significant plant growth-promoting phytohormone IAA was confirmed by UV, FT-IR, TLC, HPLC, HPTLC, and NMR spectrum. Bioactive metabolites including methyl 4-hydroxybenzoate, dodecanoic acid, and some novel flavonoids were identified. Therefore, various growth regulators such as L-aspartic acid, 1H-indole-3-carboxaldehyde were confirmed by GC-MS spectra. The present findings conclude Frankia spp. as efficient plant growth enhancement mediator and also inhibit the phytopathogens.

A stable and efficient plasmid transfer system was developed for nitrogen-fixing symbiotic actinobacteria of the genus Frankia, a key first step in developing a genetic system. Four derivatives of the broad-host-range cloning vector pBBR1MCS were successfully introduced into different Frankia strains by a filter mating with Escherichia coli strain BW29427. Initially, plasmid pHKT1 that expresses green fluorescent protein (GFP) was introduced into Frankia casuarinae strain CcI3 at a frequency of 4.0 × 10−3, resulting in transformants that were tetracycline resistant and exhibited GFP fluorescence. The presence of the plasmid was confirmed by molecular approaches, including visualization on agarose gel and PCR. Several other pBBR1MCS plasmids were also introduced into F. casuarinae strain CcI3 and other Frankia strains at frequencies ranging from 10−2 to 10−4, and the presence of the plasmids was confirmed by PCR. The plasmids were stably maintained for over 2 years and through passage in a plant host. As a proof of concept, a salt tolerance candidate gene from the highly salt-tolerant Frankia sp. strain CcI6 was cloned into pBBR1MCS-3. The resulting construct was introduced into the salt-sensitive F. casuarinae strain CcI3. Endpoint reverse transcriptase PCR (RT-PCR) showed that the gene was expressed in F. casuarinae strain CcI3. The expression provided an increased level of salt tolerance for the transformant. These results represent stable plasmid transfer and exogenous gene expression in Frankia spp., overcoming a major hurdle in the field. This step in the development of genetic tools in Frankia spp. will open up new avenues for research on actinorhizal symbiosis. IMPORTANCE The absence of genetic tools for Frankia research has been a major hindrance to the associated field of actinorhizal symbiosis and the use of the nitrogen-fixing actinobacteria. This study reports on the introduction of plasmids into Frankia spp. and their functional expression of green fluorescent protein and a cloned gene. As the first step in developing genetic tools, this technique opens up the field to a wide array of approaches in an organism with great importance to and potential in the environment.

International audience; Actinorhizal root nodules result from the interaction between a nitrogen-fixing actinomycete from the genus Frankia and roots of dicotyledonous trees and shrubs belonging to 25 genera within 8 plant families. Most actinorhizal plants can reach high rates of nitrogen fixation comparable to those found in root nodule symbiosis of the legumes. As a consequence, these trees are able to grow in poor and disturbed soils and are important elements in plant communities worldwide. While the basic knowledge of these symbiotic associations is still poorly understood, actinorhizal symbioses emerged recently as original systems to explore developmental strategies to form nitrogen-fixing nodules. Many tools have been developed in recent years to explore the interaction between Frankia and actinorhizal plants including molecular biology, biochemistry, and genomics. However, technical difficulties are often encountered to explore these symbiotic interactions, mainly linked to the woody nature of the plant species and to the lack of genetic tools for their bacterial symbionts. In this chapter, we report an inventory of the main recent molecular tools and techniques developed for studying actinorhizae.

Frankia spp. are widespread nitrogen-fixing soil bacteria, which often live in symbiosis with a broad range of hosts. Metal homeostasis plays a crucial role in the success of the symbiosis regarding the acquisition of essential trace metals and detoxification of potentially toxic elements. We have hypothesised that Frankia releases many organic ligands with a broad spectrum of affinity for essential and toxic metals. We coined the term ‘ligandosphere’ to describe the entirety of excreted metal complexing agents and ligands derived from the dissolved organic matter. Using metal isotope-coded profiling (MICP); metallophores of physiological important and toxic trace metals were identified by the addition of stable metal isotope pairs such as 54Fe/58Fe, 63Cu/65Cu, 66Zn/68Zn or 95Mo/98Mo. Liquid chromatography coupled to a mass spectrometer revealed strong variations of the metallophore profile in between the 14 test-strains. In total, about 83 organic ligands were identified as binding to one of the tested metals. The predicted sum formula of the major Fe binding ligands and MS/MS experiments suggested that several metallophore candidates have a similar molecular backbone. Growth experiments with a hyper-producer of metallophores revealed a positive relationship between metallophore production and the concentration of Cu in the growth medium. The present study provides the first comprehensive overview of the complexity of Frankia’s ligandosphere. It opens a path to a deeper understanding of mechanisms that regulate metal homeostasis in frankiae. Deciphering these mechanisms is important since the fitness of actinorhizal plants and their potential in ecological restoration relies heavily on their symbiosis with frankiae.

International audience; Plants able to establish a nitrogen-fixing root nodule symbiosis with the actinobacterium Frankia are called actinorhizal. These interactions lead to the formation of new root organs, called actinorhizal nodules, where the bacteria are hosted intracellularly and fix atmospheric nitrogen thus providing the plant with an almost unlimited source of nitrogen for its nutrition. Like other symbiotic interactions, actinorhizal nodulation involves elaborate signalling between both partners of the symbiosis, leading to specific recognition between the plant and its compatible microbial partner, its accommodation inside plant cells and the development of functional root nodules. Actinorhizal nodulation shares many features with rhizobial nodulation but our knowledge on the molecular mechanisms involved in actinorhizal nodulation remains very scarce. However recent technical achievements for several actinorhizal species are allowing major discoveries in this field. In this review, we provide an outline on signalling molecules involved at different stages of actinorhizal nodule formation and the corresponding signalling pathways and gene networks.

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

Genus Frankia is comprised primarily of nitrogen-fixing actinobacteria that form root nodule symbioses with a group of hosts known as the actinorhizal plants. These plants are evolutionarily closely related to the legumes that are nodulated by the rhizobia. Both host groups utilize homologs of nodulation genes for root-nodule symbiosis, derived from common plant ancestors. The corresponding endosymbionts, Frankia and the rhizobia, however, are distantly related groups of bacteria, leading to questions about their symbiotic mechanisms and evolutionary history. To date, a stable system of electrotransformation has been lacking in Frankia despite numerous attempts by research groups worldwide. We have identified type IV methyl-directed restriction systems, highly-expressed in a range of actinobacteria, as a likely barrier to Frankia transformation. Here we report the successful electrotransformation of the model strain F. alni ACN14a with an unmethylated, broad host-range replicating plasmid, expressing chloramphenicol-resistance for selection and GFP as a marker of gene expression. This system circumvented the type IV restriction barrier and allowed the stable maintenance of the plasmid. During nitrogen limitation, Frankia differentiates into two cell types: the vegetative hyphae and nitrogen-fixing vesicles. When the expression of egfp under the control of the nif gene cluster promoter was localized using fluorescence imaging, the expression of nitrogen fixation in nitrogen-limited culture was localized in Frankia vesicles but not in hyphae. The ability to separate gene expression patterns between Frankia hyphae and vesicles will enable deeper comparisons of molecular signaling and metabolic exchange between Frankia-actinorhizal and rhizobia-legume symbioses to be made, and may broaden potential applications in agriculture. Further downstream applications are possible, including gene knock-outs and complementation, to open up a range of experiments in Frankia and its symbioses. Additionally, in the transcriptome of F. alni ACN14a, type IV restriction enzymes were highly expressed in nitrogen-replete culture but their expression strongly decreased during symbiosis. The down-regulation of type IV restriction enzymes in symbiosis suggests that horizontal gene transfer may occur more frequently inside the nodule, with possible new implications for the evolution of Frankia.

International audience; Actinobacteria from genus Frankia are able to form symbiotic associations with actinorhizal plants including alders. Among them, Sp+ strains are characterized by their ability to differentiate numerous sporangia inside host plant cells (unlike "Sp-" strains unable of in-planta sporulation). Here, we report the first genome sequences of three unisolated Sp+ strains: AgTrS, AiOr and AvVan obtained from Alnus glutinosa, A. incana and A. alnobetula (previously known as viridis), respectively (with genome completeness estimated at more than 98%). They represent new Frankia species based on Average Nucleotide Identity (ANI) calculations, and the smallest Alnus-infective Frankia genomes so far sequenced (~5 Mbp), with 5,178, 6,192 and 5,751 candidate protein-encoding genes for AgTrS, AiOr and AvVan, respectively.

Genus Frankia is comprised primarily of nitrogen-fixing actinobacteria that form root nodule symbioses with a group of hosts known as the actinorhizal plants. These plants are evolutionarily closely related to the legumes, which are nodulated by the rhizobia. Both host groups utilize homologs of nodulation genes for root-nodule symbiosis, derived from common plant ancestors. However the corresponding endosymbionts, Frankia and the rhizobia, are distantly related groups of bacteria, leading to questions of their symbiotic mechanisms and evolutionary history. To date, a stable system of genetic transformation has been lacking in Frankia. Here, we report the successful electrotransformation of Frankia alni ACN14a, by means of replicating plasmids expressing chloramphenicol-resistance for selection, and the use of GFP as a marker of gene expression. We have identified type IV methyl-directed restriction systems, highly-expressed in a range of actinobacteria, as a likely barrier to Frankia transformation and circumvented this barrier by using unmethylated plasmids, which allowed the transformation of F. alni as well as the maintenance of the plasmid. During nitrogen limitation, Frankia differentiates into two cell types: the vegetative hyphae and nitrogen-fixing vesicles. When the plasmid transformation system was used with expression of egfp under the control of the nif gene cluster promoter, it was possible to demonstrate by fluorescence imaging the expression of nitrogen fixation in vesicles but not hyphae in nitrogen-limited culture. Importance To date, the study of Frankia-actinorhizal symbioses has been complicated by the lack of genetic tools for manipulation of Frankia, especially stable genetic transformation. The transformation system reported here, particularly coupled with marker genes, can be used to differentiate patterns of gene expression between Frankia hyphae and vesicles in symbiosis or in free-living conditions. This will enable deeper comparisons between Frankia-actinorhizal symbioses and rhizobia-legume symbioses in terms of molecular signaling and metabolic exchange that will broaden understanding of the evolution of these symbioses and potentially make possible their application in agriculture. The development of transformation methods will allow further down-stream applications including gene knock-outs and complementation that will, in turn, open up a much broader range of experiments into Frankia and its symbioses.

International audience; Since the recovery of the first transgenic plant in the early 1980s, plant transformation technologies have enabled advances in many aspects of basic plant science, including nitrogen-fixing root endosymbioses. Using the biological vectors Agrobacterium tumefaciens and A. rhizogenes, gene constructs have been successfully introduced in the actinorhizal tree species Casuarina glauca, thereby paving the way for functional analysis of the key genes involved in the symbiotic process with the actinobacterium Frankia. In recent years, not only studies of gene promoters in transgenic Casuarinaceae, but also the use of RNA interference to down-regulate genes of interest, have provided new insights into the early stages of the interaction between the root system and the actinobacterium. Opportunities offered by recent developments in genome editing technologies based on the engineered nucleases ZFNs (zinc-finger nucleases), TALENs (transcription activator-like effector nucleases) and RNA-guided CRISPR-Cas (clustered regularly interspaced short palindromic repeats-associated protein/Cas) will be briefly presented.

The actinorhizal symbiosis is a mutualistic relationship between an actinobacterium from the genus Frankia and a wide variety of dicotyledonous plants representing 8 different families of angiosperms. Molecular phylogenetic approaches have identified four major Frankia lineages that have distinctive plant host ranges. Since the first published three Frankia genomes, an effort was undertaken to provide full genomic databases covering all four well established Frankia lineages and to provide depth of the number of strains covered. Here, we report on the updated status of these sequencing efforts. At present, there are 25 complete or draft Frankia genomes that have been sequenced and annotated, and several others are now in the pipeline being sequenced. An overview of the Frankia genomes will be presented focusing on their general genomic properties including size of the pan- and core-gene pool, size relationship and genome plasticity. Furthermore, a description of biosynthetic potential and a discussion about genes (nitrogenase, hopanoid biosynthesis, truncated hemoglobin, hydrogenase uptake gene clusters) involved in the symbiosis will be discussed. The absence of common nod genes within these Frankia genomes provides clues about the host-microbe recognition process for the actinorhizal symbiosis

Salinity is one of the most wide spread abiotic stresses affecting agricultural productivity, with an impact on more than 800 million hectares worldwide. A promising solution for the recovery of saline soils encompasses the use of actinorhizal plants, a group of perennial dicotyledonous angiosperms including species highly resilient to extreme environmental conditions. These plants are able to establish root-nodule symbiosis with N2-fixing actinobacteria of the genus Frankia. In this review, we discuss the main physiological and biochemical mechanisms underlying salt tolerance in the model Casuarina glauca supplemented with chemical nitrogen or obtaining it from symbiotic Frankia. In the first part, an overview of the impact of increasing NaCl concentrations in photosynthesis, antioxidative system and membrane integrity is presented. The second part addresses the effect of salt stress in the symbiosis between C. glauca and Frankia strain Thr. Preliminary results from analyses of the branchlets proteome and nodule metabolome are presented as well.

International audience; Nitrogen and phosphorus availability are frequent limiting factors in plant growth and development. Certain bacteria and fungi form root endosymbiotic relationships with plants enabling them to exploit atmospheric nitrogen and soil phosphorus. The relationships between bacteria and plants include nitrogen-fixing Gram-negative proteobacteria called rhizobia that are able to interact with most leguminous plants (Fabaceae) but also with the non-legume Parasponia (Cannabaceae), and actinobacteria Frankia, which are able to interact with about 260 species collectively called actinorhizal plants. Fungi involved in the relationship with plants include Glomeromycota that form an arbuscular mycorrhizal (AM) association intracellularly within the roots of more than 80% of land plants. Increasing numbers of reports suggest that the rhizobial association with legumes has recycled part of the ancestral program used by most plants to interact with AM fungi. This review focuses on the most recent progress made in plant genetic control of root nodulation that occurs in non-legume actinorhizal plant species.

Frankia Sp+ strains maintain their ability to sporulate in symbiosis with actinorhizal plants, producing abundant sporangia inside host plant cells, in contrast to Sp- strains, which are unable to perform in-planta sporulation. We herein examined the role of in-planta sporulation in Frankia infectivity and competitiveness for root infection. Fifteen strains belonging to different Sp+ and Sp- phylogenetic lineages were inoculated on seedlings of Alnus glutinosa (Ag) and A. incana (Ai). Strain competitiveness was investigated by performing Sp-/Sp+ co-inoculations. Plant inoculations were standardized using crushed nodules obtained under laboratory-controlled conditions (same plant species, age, and environmental factors). Specific oligonucleotide primers were developed to identify Frankia Sp+ and/or Sp- strains in the resulting nodules. Single inoculation experiments showed that (i) infectivity by Sp+ strains was significantly greater than that by Sp- strains, (ii) genetically divergent Sp+ strains exhibited different infective abilities, and (iii) Sp+ and Sp- strains showed different host preferences according to the origin (host species) of the inocula. Co-inoculations of Sp+ and Sp- strains revealed the greater competitiveness of Sp+ strains (98.3 to 100% of Sp+ nodules, with up to 15.6% nodules containing both Sp+ and Sp- strains). The results of the present study highlight differences in Sp+/Sp- strain ecological behaviors and provide new insights to strengthen the obligate symbiont hypothesis for Sp+ strains.

Biological nitrogen fixation is one of the most critical processes contributing to ecosystem productivity and stability on a global scale. In temperate climates of the northern hemisphere, plant-root associated bacteria of the genus Frankia are the major nitrogen fixers in forest environments. Trees belonging to the genus Alnus are the most widespread hosts of Frankia in the Pacific Northwest, and a myriad of biotic and abiotic factors can influence the robustness of this symbiosis. Host identity and bacterial strain are important features that can impact Alnus-Frankia association, but little is known about the interplay of intrageneric hosts that co-occur in natural settings. In this study we investigated the genetic diversity and host specificity of Frankia bacteria associated with sympatrically occurring populations of Alnus rubra (red alder) and Alnus rhombifolia (white alder) in Oregon. Based on sequence analysis of the nifH gene recovered from root nodules we found low overall bacterial diversity. One dominant Frankia genotype was associated with both host species, indicating a lack of strong host specificity in this system. Our results suggest that certain intrageneric plant hosts with overlapping distributions show cross-compatibility with symbiotic actinorhizal bacteria, and that low strain diversity of these bacteria can persist across mixed host populations.

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.

Large areas in the extra-Andean region in the forest - steppe ecotone in “Northwestern Argentinean Patagonia” have been replaced by plantations of the exotic conifer Pinus ponderosa which modify soils physical and chemical factors and alter the biodiversity. Considering that in the region occur saprophytic soilborne actinobacteria that play important role as the fixation of atmospheric nitrogen (N2) in symbiosis with native plant species and the production of bioactive molecules in plants rhizosphere, we aimed to study the effect of the plantation on the abundance of the N2 fixer Frankia and on the genus diversity of cultivable rhizospheric actinobacteria. The study was performed with soils of six paired sites with pine plantations and natural neighbor areas (including steppes or shrublands). Abundance of infective Frankia was estimated by evaluating the nodulation capacity of soils, through a plant bioassay using Ochetophila trinervis as trap plant. Isolation trials for saprophytic actinobacteria were performed by applying chemotactic and successive soils dilutions methods. We concluded that P. ponderosa afforestation affect soil actinobacteria. This was mainly evidenced by a decrease in the Frankia nodulation capacity in O. trinervis, which was related to plantation age, to lower soil carbon and nitrogen content, higher available phosphorus, and to a slight decrease in soils pH. Pine plantation influence on the cultivable saprophytic actinobacteria was less clear. The study highlights the importance of soils as source of Frankia and rhizospheric actinobacteria in relation to disturbance caused by pine plantation in natural environments with native actinorhizal plant species. Fil: Arancibia, Natalia B.. Universidad Nacional del Comahue. Centro Regional Universitario Bariloche; Argentina Fil: Solans, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; Argentina Fil: Mestre, María Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales. Universidad Nacional del Comahue. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales.; Argentina Fil: Chaia, Eugenia Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; Argentina