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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).

Sea buckthorn (Hippophae rhamnoides), a horticulturally multipurpose species in the family Elaeagnaceae, can build associations with Frankia actinomycetes to enable symbiotic nitrogen-fixing. Currently, no high-quality reference genome is available for an actinorhizal plant, which greatly hinders the study of actinorhizal symbiotic nodulation. Here, by combining short-read, long-read and Hi-C sequencing technologies, we generated a chromosome-level reference genome of H. rhamnoides (scaffold N50: 65 Mb, and genome size: 730 Mb) and predicted 30 812 protein-coding genes mainly on 12 pseudochromosomes. Hippophae rhamnoides was found to share a high proportion of symbiotic nodulation genes with Medicago truncatula, implying a shared molecular mechanism between actinorhizal and rhizobial symbioses. Phylogenetic analysis clustered the three paralogous NODULE INCEPTION (NIN) genes of H. rhamnoides with those of other nodulating species, forming the NIN group that most likely evolved from the ancestral NLP group. The genome of H. rhamnoides will help us to decipher the underlying genetic programming of actinorhizal symbiosis, and our high-quality genome and transcriptomic resources will make H. rhamnoides a new excellent model plant for actinorhizal symbiosis research.

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.

Publisher Copyright: © 2022 by the authors. Climate change and the accelerated rate of population growth are imposing a progressive degradation of natural ecosystems worldwide. In this context, the use of pioneer trees represents a powerful approach to reverse the situation. Among others, N2-fixing actinorhizal trees constitute important elements of plant communities and have been successfully used in land reclamation at a global scale. In this study, we have analyzed the transcriptome of the photosynthetic organs of Casuarina glauca (branchlets) to unravel the molecular mechanisms underlying salt stress tolerance. For that, C. glauca plants supplied either with chemical nitrogen (KNO3+) or nodulated by Frankia (NOD+) were exposed to a gradient of salt concentrations (200, 400, and 600 mM NaCl) and RNA-Seq was performed. An average of ca. 25 million clean reads was obtained for each group of plants, corresponding to 86,202 unigenes. The patterns of differentially expressed genes (DEGs) clearly separate two groups: (i) control- and 200 mM NaCl-treated plants, and (ii) 400 and 600 mM NaCl-treated plants. Additionally, although the number of total transcripts was relatively high in both plant groups, the percentage of significant DEGs was very low, ranging from 6 (200 mM NaCl/NOD+) to 314 (600 mM NaCl/KNO3+), mostly involving down-regulation. The vast majority of up-regulated genes was related to regulatory processes, reinforcing the hypothesis that some ecotypes of C. glauca have a strong stress-responsive system with an extensive set of constitutive defense mechanisms, complemented by a tight mechanism of transcriptional and post-transcriptional regulation. The results suggest that the robustness of the stress response system in C. glauca is regulated by a limited number of genes that tightly regulate detoxification and protein/enzyme stability, highlighting the complexity of the molecular interactions leading to salinity tolerance in this species. publishersversion published

Ginseng is an important cash crop. The long-term continuous cropping of ginseng causes the imbalance of soil environment and the exacerbation of soil-borne diseases, which affects the healthy development of ginseng industry. In this study, ginseng continuous cropping soil was treated with microbial inocula using broad-spectrum biocontrol microbial strain Frankia F1. Wheat straw, rice straw and corn straw were the best carrier materials for microbial inoculum. After treatment with microbial inoculum prepared with corn straw biochar, the soil pH value, organic matter, total nitrogen, available nitrogen, available phosphorus, and available potassium were increased by 11.18%, 55.43%, 33.07%, 26.70%, 16.40%, and 9.10%, the activities of soil urease, catalase and sucrase increased by 52.73%, 16.80% and 43.80%, respectively. A Metagenomics showed that after the application of microbial inoculum prepared with corn straw biochar, soil microbial OTUs, Chao1 index, Shannon index, and Simpson index increased by 19.86%, 16.05%, 28.83%, and 3.16%, respectively. Three classes (Alphaproteobacteria, Gammaproteobacteria and Sphingobacteria) were the dominant bacteria in ginseng soil, and their abundance increased by 7.87%, 9.81% and 1.24%, respectively, after treatment with microbial inoculum with corn straw biochar. Results indicated that the most effective treatment in ginseng soil would be the combined application of corn straw biochar and Frankia F1.

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 .

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.

Plant–soil feedbacks regulate plant productivity and diversity, but potential mechanisms underpinning such feedbacks, such as the allocation of recent plant assimilate, remain largely untested especially for plants forming tripartite symbioses. We tested how soils from under alder Alnus glutinosa and beneath other species of the same and different families affected alder growth and nutrition, and colonization of roots by nitrogen-fixing Frankia bacteria and ectomycorrhizal fungi. We also measured how the soil environment affected carbon capture and allocation by pulse labelling seedlings with 13CO 2. We then tested for linkages between foliar nutrient stoichiometry and carbon capture and allocation and soil origin using statistical modelling approaches. Performance of alder and nitrogen nutrition were best on home and birch Betula pendula soils (both Betulaceae), whereas performance on Douglas fir Pseudotsuga menziesii (Pinaceae) soil was poor. Plants growing in P. menziesii soil were virtually devoid of Frankia and ectomycorrhizas, and the natural abundance 15N signatures of leaves were more enriched indicating distinct nitrogen acquisition pathways. Seedlings in these soils also had smaller 13C fixation and root allocation rates, leading to smaller 13C respiration rates by microbes. Statistical models showed that the best predictors of foliar N concentration were 13C allocation rates to fine roots and net CO 2 exchange from the mesocosms. The best predictors for foliar phosphorus concentration were net CO 2 exchange from the mesocosms and soil origin; seedlings in home soils tended to have greater foliar phosphorus compared to birch soils while seedlings from Douglas fir soils were no different from the other treatments. Foliar phosphorus concentration was not correlated with plant available or total soil phosphorus for any of the soils. Home soils also resulted in distinct ectomycorrhizal communities on seedlings roots, which could be responsible for greater foliar phosphorus concentration. Our findings show how the association of alder with nitrogen-fixing Frankia relieved nitrogen limitation in the seedling triggering a performance feedback loop. We propose that relief of nitrogen limitation likely increases plant phosphorus demand, which may promote the formation of ectomycorrhizas in nutrient-deficient soils. The formation of tripartite symbioses therefore generates positive plant–soil feedbacks, which enables plants to acquire mineral nutrients otherwise inaccessible in trade for carbon. A free Plain Language Summary can be found within the Supporting Information of this article.

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 LD

Nitrogen occurs as inert and inaccessible dinitrogen gaseous form (N

A series of new metallophores, referred to as frankobactins, were extracted from cultures of the symbiotic and nitrogen-fixing actinobacterium Frankia sp. CH37. Structure elucidation revealed a 2-hydroxyphenyl-substituted oxazoline core and a chain composed of five proteinogenic and nonproteinogenic amino acids, suggesting nonribosomal peptide synthesis as the biosynthetic origin. By whole-genome sequencing, bioinformatic analysis, and comparison with other Frankia strains, the genetic locus responsible for the biosynthesis was detected. Spectrophotometric titration of frankobactin with Fe(III) and Cu(II) and mass spectrometry established the 1:1 (metal:frankobactin) coordination. Uptake experiments suggested that frankobactin A1 (1) did not serve to recruit iron, but to detoxify Cu(II). As frankobactin A1 prevents the cellular entry of Cu(II), it could play a crucial role in the symbiosis of Frankia sp. and its host in the reclamation of copper-contaminated soil.

The phoD-harbouring bacterial community is responsible for much of the hydrolysis of organic phosphorus (Po) in soils and is therefore significant for the improvement of soil phosphorus (P) availability. However, the distribution of phoD-harbouring bacterial community structure and how it regulates the soil P fractions in steppe soils remain largely unknown. It is necessary to assess these relationships to maintain sustainable development on the steppe. We sampled soils from three steppe types across Inner Mongolia, China. Illumina high-throughput sequencing was used to quantify the bacterial phoD gene. The dominant phoD-harbouring genera were Amycolatopsis (5–11%), Bacillus (6–13%), Bradyrhizobium (3–8%) and Pseudomonas (3–5%) across all steppe soils. The relative abundances of phoD-harbouring Amycolatopsis and Bacillus increased significantly as available P (AP) decreased, while the relative abundances of Bradyrhizobium, Pseudomonas and Methylobacterium were significantly positively correlated with AP content. Redundancy analysis showed that the soil stoichiometric ratio of carbon (C), nitrogen (N) and P had a strong effect on the phoD-harbouring bacterial community structure. Correlation analyses further indicated that only phoD-harbouring Dietzia and Sphingomonas had a significant correlation with alkaline phosphatase activity and that they increased P availability by mineralizing Po. phoD-harbouring Frankia and Methylobacterium were positively correlated with labile-Po and negatively correlated with non-labile inorganic P. Moreover, phoD-harbouring Bacillus and Bradyrhizobium promoted the conversion of the non-labile Po pool into the labile Po pool, which can be attributed to microbial immobilization. Not all bacteria carrying the phoD gene promote soil P availability through mineralization or are induced not only in a P-repressible manner. Members of the phoD-harbouring bacterial community employ flexible P use strategies and can be strongly activated by their nutrient preferences and environmental conditions.

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.

Root and rust rot are common fungal diseases that decrease both the yield and quality of ginseng, resulting in severe economic losses for the ginseng industry. In this study, we explored the bacter...

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.

Shepherdia ×utahensis ‘Torrey’ (‘Torrey’ hybrid buffaloberry) is an actinorhizal plant that can fix atmospheric nitrogen (N2) in symbiotic root nodules with Frankia. Actinorhizal plants with N2-fixing capacity are valuable in sustainable nursery production and urban landscape use. However, whether nodule formation occurs in S. ×utahensis ‘Torrey’ and its interaction with nitrogen (N) fertilization remain largely unknown. Increased mineral N in fertilizer or nutrient solution might inhibit nodulation and lead to excessive N leaching. In this study, S. ×utahensis ‘Torrey’ plants inoculated with soils containing Frankia were irrigated with an N-free nutrient solution with or without added 2 mm ammonium nitrate (NH4NO3) or with 0.0 to 8.4 g·L−1 controlled-release fertilizer (CRF; 15N–3.9P–10K) to study nodulation and plant morphological and physiological responses. The performance of inoculated plants treated with various amounts of CRF was compared with uninoculated plants treated with the manufacturer’s prescribed rate. Plant growth, gas exchange parameters, and shoot N content increased quadratically or linearly along with increasing CRF application rates (all P < 0.01). No parameters increased significantly at CRF doses greater than 2.1 g·L−1. Furthermore, the number of nodules per plant decreased quadratically (P = 0.0001) with increasing CRF application rates and nodule formation were completely inhibited at 2.9 g·L−1 CRF or by NH4NO3 at 2 mm. According to our results, nodulation of S. ×utahensis ‘Torrey’ was sensitive to N in the nutrient solution or in increasing CRF levels. Furthermore, plant growth, number of shoots, leaf area, leaf dry weight, stem dry weight, root dry weight, and N content of shoots of inoculated S. ×utahensis ‘Torrey’ plants treated with 2.1 g·L−1 CRF were similar to those of uninoculated plants treated with the manufacturer’s prescribed rate. Our results show that S. ×utahensis ‘Torrey’ plants inoculated with soil containing Frankia need less CRF than the prescribed rate to maintain plant quality, promote nodulation for N2 fixation, and reduce N leaching.

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.

International audience; Since their introduction at the beginning of the 19th century, Casuarina species have become important components of the Algerian landscape, yet the numbers of species present in Algeria and their identity, distribution, and symbiotic status are still not well understood. A survey conducted on 2,304 trees in 96 Casuarina stands across Algeria indicates that they are represented exclusively by five species: Casuarina cristata Miq., C. cunninghamiana Miq., C. equisetifolia L., C. glauca Sieber ex Spreng. and C. junghuhniana Miq. Two species, C. cunninghamiana and C. glauca, were the most common – the former occurring more often in the relatively humid and temperate areas of the country while the latter is most frequent in the drier and warmer areas. Production of seedlings of C. cunninghamiana and C. glauca in 12 nurseries resulted in low rates of spontaneous mycorrhizal infection frequency and nodulation, suggesting that inoculation of seedlings with selected mycorrhizal fungi and/or Frankia strains in the nursery would be necessary to improve their establishment in the field. This study is the first survey of the geographical distribution of Casuarina species in Algeria and should lead to more efficient utilization of this widely used tree genus in Algeria.; Malgré l’importance prise par les Casua-rina en Algérie depuis leur introductionau début du 19 e siècle, le nombre d’es-pèces aujourd’hui présentes dans lepays, leur identité, leur distribution ainsique leur statut symbiotique sont encorelargement méconnus. Une étude menéesur 2 304 arbres dans 96 peuplementsrépartis dans toute l’Algérie indiquequ’ils sont représentés exclusivementpar cinq espèces du genre Casuarina :Casuarina cristata Miq., C. cunningha-miana Miq., C. equisetifolia L., C. glaucaSieber ex Spreng. et C. junghuhnianaMiq. Deux espèces, C. cunninghamiana etC. glauca, sont largement prédominanteset différemment réparties, la premièreétant plus fréquente dans les zonesrelativement humides et tempérées dupays tandis que la seconde est présentedans les régions les plus arides et lesplus chaudes. La production de plantsde C. cunninghamiana et C. glauca dans12 pépinières situées dans le nord et lesud du pays indique qu’ils présententde faibles taux de mycorhization et denodulation spontanée. Cela suggèrequ’il est nécessaire d’inoculer les jeunesplants avec des souches sélectionnéesde champignons mycorhiziens et/oude Frankia au sein des pépinières afind’améliorer leur installation durable surle terrain. Cette étude constitue le pre-mier inventaire de la répartition géogra-phique des Casuarina en Algérie et peutconduire à une meilleure exploitationdes arbres de ce genre largement utiliséen Algérie

Background Recent studies highlighted the biosynthetic potential of nocardiae to produce diverse novel natural products comparable to that of Streptomyces, thereby making them an attractive source of new drug leads. Many of the 119 Nocardia validly named species were isolated from natural habitats but little is known about the diversity and the potential of the endophytic nocardiae of root nodule of actinorhizal plants. Results The taxonomic status of an actinobacterium strain, designated ncl2T, was established in a genome-based polyphasic study. The strain was Gram-stain-positive, produced substrate and aerial hyphae that fragmented into coccoid and rod-like elements and showed chemotaxonomic properties that were also typical of the genus Nocardia. It formed a distinct branch in the Nocardia 16S rRNA gene tree and was most closely related to the type strains of Nocardia nova (98.6%), Nocardia jiangxiensis (98.4%), Nocardia miyuensis (97.8%) and Nocardia vaccinii (97.7%). A comparison of the draft genome sequence generated for the isolate with the whole genome sequences of its closest phylogenetic neighbours showed that it was most closely related to the N. jiangxiensis, N. miyuensis and N. vaccinii strains, a result underpinned by average nucleotide identity and digital DNA-DNA hybridization data. Corresponding taxogenomic data, including those from a pan-genome sequence analysis showed that strain ncl2T was most closely related to N. vaccinii DSM 43285T. A combination of genomic, genotypic and phenotypic data distinguished these strains from one another. Consequently, it is proposed that strain ncl2T (= DSM 110931T = CECT 30122T) represents a new species within the genus Nocardia, namely Nocardia alni sp. nov. The genomes of the N. alni and N. vaccinii strains contained 36 and 29 natural product-biosynthetic gene clusters, respectively, many of which were predicted to encode for a broad range of novel specialised products, notably antibiotics. Genome mining of the N. alni strain and the type strains of its closest phylogenetic neighbours revealed the presence of genes associated with direct and indirect mechanisms that promote plant growth. The core genomes of these strains mainly consisted of genes involved in amino acid transport and metabolism, energy production and conversion and transcription. Conclusions Our genome-based taxonomic study showed that isolate ncl2T formed a new centre of evolutionary variation within the genus Nocardia. This novel endophytic strain contained natural product biosynthetic gene clusters predicted to synthesize novel specialised products, notably antibiotics and genes associated with the expression of plant growth promoting compounds.

Root nodule symbiosis evolved ca. 100 Mya between a nitrogen-fixing bacterium and the common ancestor to the Fabales, Fagales, Rosales, and Cucurbitales plant orders. Over time the majority of the lineages derived from this ancestor lost their symbiotic capability. While extant symbiotic members found in the Fabales order (legumes) all engage in symbiosis with rhizobia, extant symbiotic members of the latter three plant orders are referred to as actinorhizal plants. These engage in symbiosis with Frankia. Frankia is a genus of soil actinobacteria, which can be split into four phylogenetically distinct clades. The earliest divergent symbiotic clade, Frankia cluster-2, encompasses strains that have a broad host range and could not be cultured in vitro thus far with two exceptions. Based on Frankia enriched meta-genomes from whole nodules collected at different locations across the globe, it is clear there is very little diversity of Frankia cluster-2 in continental Eurasia, spanning from France to Japan. These strains are also closely related to strains found in North America. However, very little is known about strains occurring in the islands in the Pacific Ocean and the southern hemisphere. In short, this thesis aimed to investigate the biodiversity of the earliest divergent symbiotic Frankia clade and to understand how Frankia spread across the globe (Study 1 and Study 2). From nodules collected in study 1, a novel Streptomyces species was identified and declared (Study 3). The thesis also aimed to study genetic changes within Frankia cluster-2 which might be associated with their endosymbiotic lifestyle and low saprotrophic potential (Study 4 and Study 5).

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 inFrankiacluster-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 forFrankia 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 toFrankiacluster-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 withinFrankiacluster-2 and predict routes of dispersal from Gondwana, we sequenced and analysed the MAGs ofFrankiacluster-2 fromCoriaria japonicaandCoriaria intermediagrowing in Japan, Taiwan and the Philippines. Phylogenetic analyses indicate there is a clear split withinFrankiacluster-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.

Climate change and the accelerated rate of population growth are imposing a progressive degradation of natural ecosystems worldwide. In this context, the use of pioneer trees represents a powerful approach to reverse the situation. Among others, N2-fixing actinorhizal trees constitute important elements of plant communities and have been successfully used in land reclamation at a global scale. In this study, we have analyzed the transcriptome of the photosynthetic organs of Casuarina glauca (branchlets) to unravel the molecular mechanisms underlying salt stress tolerance. For that, C. glauca plants supplied either with chemical nitrogen (KNO3 +) or nodulated by Frankia (NOD+) were exposed to a gradient of salt concentrations (200, 400, and 600 mM NaCl) and RNA-Seq was performed. An average of ca. 25 million clean reads was obtained for each group of plants, corresponding to 86,202 unigenes. The patterns of differentially expressed genes (DEGs) clearly separate two groups: (i) control- and 200 mM NaCl-treated plants, and (ii) 400 and 600 mM NaCltreated plants. Additionally, although the number of total transcripts was relatively high in both plant groups, the percentage of significant DEGs was very low, ranging from 6 (200 mM NaCl/NOD+) to 314 (600 mM NaCl/KNO3 +), mostly involving down-regulation. The vast majority of up-regulated genes was related to regulatory processes, reinforcing the hypothesis that some ecotypes of C. glauca have a strong stress-responsive system with an extensive set of constitutive defense mechanisms, complemented by a tight mechanism of transcriptional and post-transcriptional regulation. The results suggest that the robustness of the stress response system in C. glauca is regulated by a limited number of genes that tightly regulate detoxification and protein/enzyme stability, highlighting the complexity of the molecular interactions leading to salinity tolerance in this species info:eu-repo/semantics/publishedVersion

Background Alpine alder vegetation acts upon the nearby grass and dwarf shrub vegetation by the nitrogen supply from the symbiotic bacteria Frankia alni of Alnus viridis. This has been studied in two transects concerning plant distribution, plant diversity, nitrate concentration in soil and photosynthetic performance of specific marker plants. Results Away from the alder stand, a band of some meters was dominated by Calamagrostis varia which then was followed by alpine dwarf shrub vegetation. Nitrate in the soil showed a concentration decrease away from the alder stand leading to values near the detection limit in the dwarf shrub zone. Within these three zones, plant species were distributed according to their N-index, given in the ecological literature. Three dominant species, Calamagrostis varia, Rhododendron ferrugineum and Vaccinium myrtillus were examined at sites of different N-availability in the horizontal transect for their photosynthetic performance, by measuring the prompt fluorescence, the OJIP named polyphasic rise of chlorophyll-a fluorescence. All three plant species showed signs of stress in the fluorescence rise kinetics at decreased nitrate availability. These are similar to other known stress effects such as faster reduction of the primary acceptor or an electron supply limitation on the donor site of photosystem II. Conclusion Prompt chlorophyll-a fluorescence data of the examined leaves in a natural vegetation system showed the effects of a decrease in the essential nutrient nitrogen and in a manner parallel to changes in plant diversity. The selected marker plants behaved differently towards decreasing nitrogen concentrations in soil.

The physicochemical profiling of e-waste was done by AAS, XRD, and SEM with EDX that reveals the composition of valuable metals like Au−, Ag−, Zn−, and Cu−. Thus, the present findings provide the important resource of precious metal in the e-waste (PCBs) as pollutant in the environment and human impact. The native isolates Frankia sp. DDNSF-01 and Frankia casuarinae DDNSF-02 were subjected for bioleaching of gold and precious elements bearing e-waste under one and two-step leaching methods. The two-step bioleaching protocol was found effectual in expression with high level of metal leaching within the culture medium of Frankia metabolites and organic acids which were obtained by the leaching of metals. The presence of organic acids functional groups was determined by FT-IR spectrum and by low pH of growth medium. The AAS analysis quantified the initial concentration of metals and their significant increase brought by the leaching. The initial concentration of precious metal elements was found in the e-waste like Au−, Ag−, Cu−, and Zn− (0.04, 0.05, 0.35, 0.1 mg/g) respectively. Frankia sp. leached out precious metals like Au−, Ag−, Cu−, and Zn− (0.2, 0.14, 0.68, 0.2 mg/g) respectively. Frankia casuarinae leached out precious metals like Au−, Ag−, Cu−, and Zn− (0.25, 0.15, 0.55, and 0.2 mg/g) respectively. The precious metal gold was highly leached out by F. casuarinae than Frankia sp.. The valuable metal copper was significantly increased by Frankia sp. than F. casuarinae. Higher gold recovery, at 75%, was obtained for F. casuarinae and copper 94% recovery by Frankia sp. bioleaching of the biooxidised e-waste at 0.2% low pulp densities. The results were confirmed by XRD and SEM with EDX analysis.

The use of wastewater for irrigation in agroforestry is cost-effective for water management. It is well established that rhizospheric microorganisms such as N2-fixing bacteria are able to modulate rhizobioaugmention and to boost phyoremediation process. To date, no study has been conducted to evaluate biological effects of rhizobioaugmentation in Casuarina glauca trees induced by their symbiont N-fixing actinobacteria of the genus Frankia. The objective of the present study was to evaluate the main effects of rhizobioaugmentation on the biological activity in the C. glauca’s rhizosphere and on C. glauca growth in soils irrigated with industrial wastewater. Two Frankia strains (BMG5.22 and BMG5.23) were used in a single or dual inoculations of C. glauca seedlings irrigated with industrial wastewater. Soil enzymes activity related to carbon, phosphorus, sulfur and nitrogen cycling were measured. Results revealed that the BMG5.22 Frankia strain increases significantly the size (dry weight) of C. glauca shoots and roots while dual inoculation increased significantly the root length. Surprisingly, β-glucosidase (BG), cellobiohydrolase (CBH), β-N-acetylglucosaminidase (NAGase), aryl sulfatase (AS), acid phosphatase (AP), alkaline phosphatase (AlP), glycine aminopeptidase (GAP), leucine aminopeptidase (LAP), and peroxidase (PER) activity in the rhizosphere decreased significantly in soils treated with the two strains of symbionts. This suggests no positive correlations between enzymatic activity and C. glauca growth.

Plant interactions with single symbionts of nitrogen fixing bacteria or mycorrhizal fungi are well studied. However, less is known about how plants interact with multiple symbiont species. The aim of this study was to assess whether multiple symbionts had a synergistic effect on host plant performance compared with a single symbiont and whether increasing the number of non-specific fungal species would increase plant performance due to niche complementarity. Green alder (Alnus viridis ssp. crispa) were inoculated with non-specific ectomycorrhizal fungi (Lactarius torminosus, Lactarius theiogalus, Hebeloma crustuliniforme) alone or in combination, with and without Frankia, and grown in nutrient-poor soil. Frankia significantly increased plant growth by 48.5%, doubled the proportion of plants forming ectomycorrhizae, as well as increased root extracellular phosphatase activity by 8.8% compared to non-Frankia treatments. However, increasing number of fungal species decreased nodulated plant biomass. Plants that formed a Hartig net had reduced nodule number and nodule biomass allocation, as well as total nodule activity compared with plants with no Hartig net. Our results indicated that Frankia inoculation provided benefit for both host plants and ectomycorrhizal fungi in the nutrient-poor soil, while ectomycorrhizal fungi did not. These negative effects of mycorrhizal fungi are likely dependent on soil nutrient P availability.

Actinobacterial strain CjT was directly isolated from soil beneath Ceanothus jepsonii growing in the USA. The strain formed cell structures typical of the genus Frankia including extensive hyphae, vesicles and sporangia, and it effectively nodulated members of the actinorhizal Colletieae, Elaeagnaceae and Myricaceae. The whole-cell hydrolysate of strain CjT was rich in meso-diaminopimelic acid and galactose, glucose, mannose, xylose, ribose and a trace of rhamnose. Tbe polar lipid profile contained phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol and glycophospholipid. The menaquinone was predominantly MK-9(H4). The fatty acid profile predominantly consisted of C17 : 1ω8c, iso-C16 : 0, C15:0, C16 : 0 and C17 : 0. A multilocus sequence analysis phylogeny based on atp1, ftsZ, dnaK, gyrA and secA gene sequences positioned the strain within Elaeagnaceae- and Colletieae -nodulating species together with Frankia elaeagni DSM 46783T, Frankia discariae DSM 46785T and Frankia irregularis DSM 45899T. Pairwise 16S rRNA gene sequence similarities showed that strain CjT was most closely related to F. discariae DSM 46785T (99.78 %) while their digital DNA–DNA hybridization value was 41.1 %. Based on the overall analyses, strain CjT (=DSM 100623T=CECT 9041T) warrants classification as the type strain of a novel species, for which the name Frankia soli sp. nov. is proposed.

Frankia sp. strain B2 was isolated from Casuarina cunninghamiana nodules. Here, we report the 5.3-Mbp draft genome sequence of Frankia sp. strain B2 with a G+C content of 70.1 % and 4,663 candidate protein-encoding genes. Analysis of the genome revealed the presence of high numbers of secondary metabolic biosynthetic gene clusters.

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.

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.

The effect of host plants on the abundance and distribution of introduced and indigenous Frankia populations was assessed in soils and root nodules of four alder species, Alnus glutinosa,Alnus cordata,Alnus rubra and Alnus viridis. Plants were grown in microcosms with either a sandy soil without detectable frankiae, with or without inoculation of a mixture of Frankia isolates, or a silty clay loam soil with indigenous Frankia. The presence of frankiae in soils increased plant height and root nodule formation, with significant increases in the presence of indigenous frankiae. Abundance in soils increased significantly for both introduced and indigenous Frankia populations independent of alder species, with generally largest increases in cluster 1b frankiae. Root nodules formed by introduced frankiae did not reflect the diversity of strains inoculated, with nodules generally only formed by strain ArI3 representing cluster 1a/d. All indigenous Frankia populations detected in soil were also found in A. glutinosa nodules, while A. cordata or A. rubra nodules contained different subsets of frankiae with unique abundances dependent on plant species. These results demonstrate the intrageneric differences of host plants in the selection of specific Frankia populations in soils for root nodule formation.

Microbes live in a complex communal ecosystem. The structural complexity of microbial community reflects diversity, functionality as well as habitat type. Delineation of ecologically important microbial populations along with exploration of their roles in environmental adaptation or host-microbe interaction has a crucial role in modern microbiology. In this scenario, reverse ecology (the use of genomics to study ecology) plays a pivotal role. Some studies have reported the presence of other non-Frankia genus from the same root nodule from where the Frankia was isolated. Since co-existance of two different genus in one small niche should maintain a strict direct interaction, it will be interesting to utilize the concept of reverse ecology in this scenario.Here, we exploited an ‘R’ package, the RevEcoR, to resolve the issue of co-existing microbes which are proven to be a crucial tool for identifying the nature of their relationship (competition or complementation) persisting among them. Our target organism here is Frankia, a nitrogen-fixing actinobacterium popular for its genetic and host specificity nature. According to their plant host, Frankia has already been subdivided into four clusters CI, CII, CIII and CIV. Our results revealed a strong competing nature of CI Frankia. The competition index between CI and CIII was greater than other studied Frankia clusters. The other interesting result was the co-occurrence of C-II and C-IV groups. It was revealed that these two groups follow the theory of resource partitioning in their lifestyle. Metabolic analysis along with their differential transporter machinery validated our hypothesis of resource partitioning among C-II and C-IV group.

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.

SummaryAlnus glutinosa response to Frankia alni is driven by several sequential physiological modifications that include calcium spiking, root hair deformation, penetration, induction of primordium, formation and growth of nodule. Here, we have conducted a transcriptomic study to analyse plant responses to Frankia alni at early stages of symbiosis establishment.Forty-two genes were significantly activated by either with a Frankia culture supernatant or with living cells separated from the roots by a dialysis membrane permitted to identify plant genes which expression changes upon early contact with Frankia. Most of these genes encode biological processes, including oxidative stress and response to stimuli. The most upregulated gene is the non-specific lipid transfer protein (nsLTP) encoding gene with a fold change of 141. Physiological experiments showed that nsLTP increases Frankia nitrogen fixation at sub-lethal concentration. Immunohistochemistry experiments conducted at an early infection stage indicated that nsLTP protein is localized at the deformed root hair region after Frankia inoculation and later in nodules, precisely around bacterial vesicles. Taken together, these results suggest that nsLTP acts at early and late stages of symbiosis, probably by increasing nitrogen uptake by Frankia.

Halogenation often improves the bioactive properties of natural products and is used in pharmaceutical research for the generation of new potential drug leads. High regio- and stereospecificity, simple reaction conditions and straightforward downstream processing are the main advantages of halogenation using enzymatic biocatalysts compared to chemical synthetic approaches. The identification of new promiscuous halogenases for the modification of various natural products is of great interest in modern drug discovery. In this paper, we report the identification of a new promiscuous FAD-dependent halogenase, DklH, from Frankia alni ACN14a. The identified halogenase readily modifies various flavonoid compounds, including those with well-studied biological activities. This halogenase has been demonstrated to modify not only flavones and isoflavones, but also flavonols, flavanones and flavanonols. The structural requirements for DklH substrate recognition were determined using a feeding approach. The homology model of DklH and the mechanism of substrate recognition are also proposed in this paper.

Community assembly of diverse mutualistic symbionts in host plants have recently received much attention. On the other hand, for decades, researchers have also focused on a role of genetic variation for shaping ecological communities. However, it remains unclear how local interaction networks between a host and symbionts are shaped from the pool of genetically diverse microorganisms in the natural rhizosphere. In this study, we comprehensively analyzed local and regional genetic communities of Frankia in host individuals and in surrounding soils using metabarcoding to unravel community assembly in shaping interaction networks between plants and root nodule symbionts, both of which are genetically diverse, in natural ecosystems. We found that Frankia communities in rhizosphere soil were consisted of tremendously diverse strains. The diverse Frankia communities in rhizosphere soils were heterogeneously structured by the presence of host individuals and soil nutritional status. Furthermore, quantification of the filtering forces by community analysis demonstrated that the filtering forces that shape symbiotic networks would depend on genetic variation in the host plant. This study suggests that filtering mechanisms and host genetics would have a profound role in shaping plant-bacterium symbiotic networks in natural environments.

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.

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.

Certain bacteria and archaea are responsible for biological nitrogen fixation. Metabolic pathways usually are common between archaea and bacteria. Diazotrophs are categorized into two main groups namely: root-nodule bacteria and plant growth-promoting rhizobacteria. Diazotrophs include free living bacteria, such as Azospirillum, Cupriavidus, and some sulfate reducing bacteria, and symbiotic diazotrophs such Rhizobium and Frankia. Three types of nitrogenase are iron and molybdenum (Fe/Mo), iron and vanadium (Fe/V) or iron only (Fe). The Mo-nitrogenase have a higher specific activity which is expressed better when Molybdenum is available. The best hosts for Rhizobium legumiosarum are Pisum, Vicia, Lathyrus and Lens; Trifolium for Rhizobium trifolii; Phaseolus vulgaris, Prunus angustifolia for Rhizobium phaseoli; Medicago, Melilotus and Trigonella for Rhizobium meliloti; Lupinus and Ornithopus for Lupini, and Glycine max for Rhizobium japonicum. Termites have significant key role in soil ecology, transporting and mixing soil. Termite gut microbes supply the enzymes required to degrade plant polymers, synthesize amino acids, recycle nitrogenous waste and fix atmospheric nitrogen. The positive effects of Arbuscular mycorrhizal (AM) fungi such as growth promotion, increased root length, leaf area, stem diameter, transplant performance and tolerance to stresses have been reported previously.

The aims of this study were to evaluate the effect of macronutrients on nitrogen fixation in mycorrhizal and non-mycorrhizal grey alder (Alnus incana (L.) Moench), and to evaluate the effect of ectomycorrhizal mycelium on the transfer of symbolically fixed nitrogen from grey alder to Scots pine (Pinus sylvestris L). One alder and one pine were grown together in pots with root systems separated by a 20μm mesh nylon filter which allowed hyphae but not roots to penetrate. Half the plants of both species were inoculated with Paxillus involutus (Ft.) Ft. and all alders were inoculated with Frankia. Nutrient solutions were added with macronutrient (N, K, P, Ca, Mg and S) concentrations varied according to a two-level fractional factorial design. The plants were harvested after two growing periods in a growth chamber. Nitrogen fixation by alder and transfer of symbiotically fixed N from alder to pine was measured by

A series of new metallophores, referred to as frankobactins, were extracted from cultures of the symbiotic and nitrogen-fixing actinobacterium

Frankia bacteria are nitrogen fixing species from the Actinobacterium phylum which live on the root nodules of plants. They have been hypothesised to have significant potential for natural product biosynthesis. The cytochrome P450 monooxygenase complement of Frankia sp. EuI1c (Frankia inefficax sp.), which comprises 68 members, was analysed. Several members belonged to previously uncharacterised bacterial P450 families. There was an unusually high number of CYP189 family members (21) suggesting that this family has undergone gene duplication events which are classified as "blooms". The likely electron transfer partners for the P450 enzymes were also identified and analysed. These consisted of predominantly [3Fe-4S] cluster containing ferredoxins (eight), a single [2Fe-2S] ferredoxin and a couple of ferredoxin reductases. Three of these CYP family members were produced and purified, using Escherichia coli as a host, and their substrate range was characterised. CYP1027H1 and CYP150A20 bound a broad range of norisoprenoids and terpenoids. CYP1074A2 was highly specific for certain steroids including testosterone, progesterone, stanolone and 4-androstene-3,17-dione. It is likely that steroids are the physiological substrates of CYP1074A2. These results also give an indication that terpenoids are the likely substrates of CYP1027H1 and CYP150A2. The large number of P450s belonging to distinct families as well as the associated electron transfer partners found in different Frankia strains highlights the importance of this family of enzymes has in the secondary metabolism of these bacteria.

Root nodule endosymbiosis with nitrogen-fixing bacteria provides plants with unlimited access to fixed nitrogen, but at a significant energetic cost. Nodulation is generally considered to have originated in parallel in different lineages, but this hypothesis downplays the genetic complexity of nodulation and requires independent recruitment of many common features across lineages. Recent phylogenomic studies revealed that genes that function in establishing or maintaining nitrogen-fixing nodules are independently lost in non-nodulating relatives of nitrogen-fixing plants. In our opinion, these data are best explained by a scenario of a single gain followed by massively parallel loss of nitrogen-fixing root nodules triggered by events at geological scale.