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There have been frequent reports of more than one strain of the nitrogen-fixing symbiont, Frankia, in the same root nodule of plants in the genus Alnus, but quantitative assessments of their relative contributions have not been made to date. Neither has the diversity of other microbes, having potential functional roles in symbiosis, been systematically evaluated. Alnus rubra root nodule microbiota were studied using Illumina short read sequencing and kmer-based read classification. Single end 76 bp sequencing was done to a median depth of 96 million reads per sample. Reads were assigned to taxa using KrakenUniq, with taxon abundances being estimated using its companion program Bracken. This was the first high resolution study of Alnus root nodules using next generation sequencing (NGS), quantifying multiple Cluster 1 A Frankia strains in single nodules, and in some cases, a Cluster 4 strain. Root nodules were found to contain diverse bacteria, including several genera containing species known to have growth-promoting effects. Evidence was found for partitioning of some bacterial strains in older versus younger lobes.

Uptake hydrogenase (Hup) recycles H2 formed by nitrogenase during nitrogen fixation, thereby preserving energy. Among root nodule bacteria, most rhizobial strains examined are Hup−, while only one Hup− Frankia inoculum had been identified. Previous analyses had led to the identification of two different [NiFe] hydrogenase syntons. We analysed the distribution of different types of [NiFe] hydrogenase in the genomes of different Frankia species. Our results show that Frankia strains can contain four different [NiFe] hydrogenase syntons representing groups 1f, 1h, 2a, and 3b according to Søndergaard, Pedersen, and Greening (HydDB: a web tool for hydrogenase classification and analysis. Sci Rep 2016;6:34212. https://doi.org/10.1038/srep34212.); no more than three types were found in any individual genome. The phylogeny of the structural proteins of groups 1f, 1h, and 2a follows Frankia phylogeny; the phylogeny of the accessory proteins does not consistently. An analysis of different [NiFe] hydrogenase types in Actinomycetia shows that under the most parsimonious assumption, all four types were present in the ancestral Frankia strain. Based on Hup activities analysed and the losses of syntons in different lineages of genome reduction, we can conclude that groups 1f and 2a are involved in recycling H2 formed by nitrogenase while group 1 h and group 3b are not. [ABSTRACT FROM AUTHOR]

Nearly 50 years after the ground-breaking isolation of the primary Comptonia peregrina microsymbiont under axenic conditions, efforts to isolate a substantial number of Protofrankia and Frankia strains continue with enduring challenges and complexities. This study aimed to streamline genomic insights through comparative and predictive tools to extract traits crucial for isolating specific Frankia in axenic conditions. Pangenome analysis unveiled significant genetic diversity, suggesting untapped potential for cultivation strategies. Shared metabolic strategies in cellular components, central metabolic pathways, and resource acquisition traits offered promising avenues for cultivation. Ecological trait extraction indicated that most uncultured strains exhibit no apparent barriers to axenic growth. Despite ongoing challenges, potential caveats, and errors that could bias predictive analyses, this study provides a nuanced perspective. It highlights potential breakthroughs and guides refined cultivation strategies for these yet-uncultured strains. We advocate for tailored media formulations enriched with simple carbon sources in aerobic environments, with atmospheric nitrogen optionally sufficient to minimize contamination risks. Temperature adjustments should align with strain preferences--28-29°C for Frankia and 32-35°C for Protofrankia--while maintaining an alkaline pH. Given potential extended incubation periods (predicted doubling times ranging from 3.26 to 9.60 days, possibly up to 21.98 days), patience and rigorous contamination monitoring are crucial for optimizing cultivation conditions. [ABSTRACT FROM AUTHOR]

In root nodule symbioses (RNS) between nitrogen (N)‐fixing bacteria and plants, bacterial symbionts cycle between nodule‐inhabiting and soil‐inhabiting niches that exert differential selection pressures on bacterial traits. Little is known about how the resulting evolutionary tension between host plants and symbiotic bacteria structures naturally occurring bacterial assemblages in soils. We used DNA cloning to examine soil‐dwelling assemblages of the actinorhizal symbiont Frankia in sites with long‐term stable assemblages in Alnus incana ssp. tenuifolia nodules. We compared: (1) phylogenetic diversity of Frankia in soil versus nodules, (2) change in Frankia assemblages in soil versus nodules in response to environmental variation: both across succession, and in response to long‐term fertilization with N and phosphorus, and (3) soil assemblages in the presence and absence of host plants. Phylogenetic diversity was much greater in soil‐dwelling than nodule‐dwelling assemblages and fell into two large clades not previously observed. The presence of host plants was associated with enhanced representation of genotypes specific to A. tenuifolia, and decreased representation of genotypes specific to a second Alnus species. The relative proportion of symbiotic sequence groups across a primary chronosequence was similar in both soil and nodule assemblages. Contrary to expectations, both N and P enhanced symbiotic genotypes relative to non‐symbiotic ones. Our results provide a rare set of field observations against which predictions from theoretical and experimental work in the evolutionary ecology of RNS can be compared. [ABSTRACT FROM AUTHOR]

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

Nearly 50 years after the ground-breaking isolation of the primary Comptonia peregrina microsymbiont under axenic conditions, efforts to isolate a substantial number of Protofrankia and Frankia strains continue with enduring challenges and complexities. This study aimed to streamline genomic insights through comparative and predictive tools to extract traits crucial for isolating specific Frankia in axenic conditions. Pangenome analysis unveiled significant genetic diversity, suggesting untapped potential for cultivation strategies. Shared metabolic strategies in cellular components, central metabolic pathways, and resource acquisition traits offered promising avenues for cultivation. Ecological trait extraction indicated that most uncultured strains exhibit no apparent barriers to axenic growth. Despite ongoing challenges, potential caveats, and errors that could bias predictive analyses, this study provides a nuanced perspective. It highlights potential breakthroughs and guides refined cultivation strategies for these yet-uncultured strains. We advocate for tailored media formulations enriched with simple carbon sources in aerobic environments, with atmospheric nitrogen optionally sufficient to minimize contamination risks. Temperature adjustments should align with strain preferences—28–29°C for Frankia and 32–35°C for Protofrankia—while maintaining an alkaline pH. Given potential extended incubation periods (predicted doubling times ranging from 3.26 to 9.60 days, possibly up to 21.98 days), patience and rigorous contamination monitoring are crucial for optimizing cultivation conditions.

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.

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

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

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. [Graphic: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The analysis of transmembrane proteins in Frankia genomes provided insight into the host-microbe interaction strategy of this nitrogen-fixing Actinobacteria. The transmembrane proteins (TM) with their extracellular part exposed on the surface of the cell and the peripheral membrane proteins play a crucial role in the interaction of Frankia with the infected host plant and are termed as 'surfactome' for this study. Our analysis identified two categories of transmembrane proteins: signaling-TM (STM) and non-signaling TM (NSTM) proteins. The STM category was further subdivided into Lipo, Sec, and TAT. The codon and amino acid usage analysis revealed the importance of compositional constraints, translational efficiency, tRNA adaptation index, and RSCU over Frankia. The COG analysis proved the role of surfactome in signal transduction. It was evident that STMs were evolving faster than NSTMs. Moreover, the signal part of the STM proteins was found to be more evolved than the mature part. This rapid evolution enables the signaling proteins to interact with diverse external stimuli more effectively, which may be essential for the host-microbe association between Frankia and Actinorhizal plants. In conclusion, the comprehensive analysis of surfactome among Frankia genomes provided valuable perceptions into the host-microbe interaction strategy of this nitrogen-fixing actinobacteria. The results suggest that the composition and evolution of these proteins are essential for successful host-microbe associations. Considering the uniqueness of these proteins we coined the term "surfactome" for them and called for further research on the mechanisms of plant–microbe interactions. [ABSTRACT FROM AUTHOR]

Frankia strains induce the formation of nitrogen-fixing nodules on roots of actinorhizal plants. Phylogenetically, Frankia strains can be grouped in four clusters. The earliest divergent cluster, cluster-2, has a particularly wide host range. The analysis of cluster-2 strains has been hampered by the fact that with two exceptions, they could never be cultured. In this study, 12 Frankia-enriched metagenomes of Frankia cluster-2 strains or strain assemblages were sequenced based on seven inoculum sources. Sequences obtained via DNA isolated from whole nodules were compared with those of DNA isolated from fractionated preparations enhanced in the Frankia symbiotic structures. The results show that cluster-2 inocula represent groups of strains, and that strains not represented in symbiotic structures, that is, unable to perform symbiotic nitrogen fixation, may still be able to colonize nodules. Transposase gene abundance was compared in the different Frankia-enriched metagenomes with the result that North American strains contain more transposase genes than Eurasian strains. An analysis of the evolution and distribution of the host plants indicated that bursts of transposition may have coincided with niche competition with other cluster-2 Frankia strains. The first genome of an inoculum from the Southern Hemisphere, obtained from nodules of Coriaria papuana in Papua New Guinea, represents a novel species, postulated as Candidatus Frankia meridionalis. All Frankia-enriched metagenomes obtained in this study contained homologs of the canonical nod genes nodABC; the North American genomes also contained the sulfotransferase gene nodH, while the genome from the Southern Hemisphere only contained nodC and a truncated copy of nodB.

Frankia strains induce the formation of nitrogen-fixing nodules on roots of actinorhizal plants. Phylogenetically, Frankia strains can be grouped in four clusters. The earliest divergent cluster, cluster-2, has a particularly wide host range. The analysis of cluster-2 strains has been hampered by the fact that with two exceptions, they could never be cultured. In this study, 12 Frankia-enriched metagenomes of Frankia cluster-2 strains or strain assemblages were sequenced based on seven inoculum sources. Sequences obtained via DNA isolated from whole nodules were compared with those of DNA isolated from fractionated preparations enhanced in the Frankia symbiotic structures. The results show that cluster-2 inocula represent groups of strains, and that strains not represented in symbiotic structures, that is, unable to perform symbiotic nitrogen fixation, may still be able to colonize nodules. Transposase gene abundance was compared in the different Frankia-enriched metagenomes with the result that North American strains contain more transposase genes than Eurasian strains. An analysis of the evolution and distribution of the host plants indicated that bursts of transposition may have coincided with niche competition with other cluster-2 Frankia strains. The first genome of an inoculum from the Southern Hemisphere, obtained from nodules of Coriaria papuana in Papua New Guinea, represents a novel species, postulated as Candidatus Frankia meridionalis. All Frankia-enriched metagenomes obtained in this study contained homologs of the canonical nod genes nodABC; the North American genomes also contained the sulfotransferase gene nodH, while the genome from the Southern Hemisphere only contained nodC and a truncated copy of nodB.

Background: Cadmium (Cd2+) is one of the highly toxic heavy metals and is considered as a carcinogenic agent. Our aim was to confirm the ability of Frankia alni ACN14a to resist Cd2+ and to determine the genes involved in the resistance mechanism. Results: F. alni ACN14a and Frankia casuarinae CcI3 hyphae showed up to 10 and 22 times Cd2+ accumulation when exposed to 1 mM Cd2+, respectively. Scanning electron microscopy (SEM) exhibited a stable Cd2+ precipitate on the cell surface, and the increase in Cd2+ weight level reached 16.45% when evaluated with SEM-EDAX analysis. The following two potential Cd2+ operons were identified: 1. cadCA operon, which encodes a copper-transporting P-type ATPase A (cadA, FRAAL0989) and an ArsR family regulator (cadC, FRAAL0988), with 37- and 70-fold increase in their expression by qRT-PCR, respectively and 2. cadB/DX, which encodes a putative cobalt-zinc-cadmium resistance protein (cadD, FRAAL3628) and heavy metal-associated domain protein (cadX, FRAAL3626), with 22- and 16-fold upregulation when exposed to Cd2+ stress. Conclusions: Cd2+ tolerance by F. alni ACN14a involved efflux of Cd2+ outside the cells and binding it to the membrane surface. Our results indicate the existence of two cadmium-resistance mechanisms in Frankia strains, which support the idea of using them as a bioremediation agent.How to cite: Rehan M, Alhusays A, Serag AM, et al. The cadCA and cadB/DX operons are possibly induced in cadmium resistance mechanism by Frankia alni ACN14a. Electron J Biotechnol 2022;60. https://doi.org/10.1016/j.ejbt.2022.09.006.

To examine the characteristic of denitrification in Frankia, a symbiotic nitrogen-fixing microbe associated with non-leguminous plants, and its role as a N2 O source or sink, Casuarina root nodule endophyte Frankia was isolated using sectioning method, which was then purely cultured to investigate the denitrification process under NO3-addition. The results showed that after addition of NO3-to the medium under anaerobic condition, the concentration of NO3-decreased with time, while the concentrations of NO2-and N2 O initially increased and then decreased over time. Key denitrification genes and nitrogenase gene were detected at 26 h, 54 h and 98 h during incubation. Abundances of these genes significantly delivered among each other, and their dynamics were asynchronous. Redundancy analysis of the effect of NO3-, NO2-, N2O concentrations on abundances of denitrification genes and nitrogenase gene indicated that 81.9% of' the total variation in gene abundances could be explained by the first two axes. Frankia had a denitrifying activity under anaerobic condition, with denitrification genes, including nitrous oxide reductase gene (nosZ), being identified. Our results suggested that Frankia possessed a complete denitrification pathway and the ability of N2O reduction under anaerobic condition. [ABSTRACT FROM AUTHOR]

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

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.

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). [ABSTRACT FROM AUTHOR]

As the oldest species of Betulaceae, Nepalese alder (Alnus nepalensis) shows a high capacity for nitrogen fixation, rapid growth rate, and strong adaptability to stress environments, and it plays an important role in maintaining the structure and function of forest and agroforestry ecosystems. We explored its geographic distribution and the corresponding environmental drivers through collecting specimen records and published literature for Nepalese alder over the world during the past 40 years. The research trends, the growth limiting factors, the physiological characteristics, and ecological functions were all summarized as well. In terms of geographical distribution and limiting factors, Nepalese alder is mainly distributed in southern mountainous areas of the Himalayas and southwest China. Since it presented a clear northern limit of distribution and an upper limit of elevation, temperature is assumed to be the main environmental limiting factor. According to historical development, the research history of Nepalese alder could be divided into three main periods: the initial development (before 2001), the fast development (2002–2015), and the high-quality development (2016–2022), with the two key points in 2002 and 2015 relating to the conversion of cropland to a forest project that the government conducted and the application from theory to practice, respectively. As can be seen from the ecological functions, Nepalese alder could form symbiotic nodules with Frankia, which plays an important role in improving soil physical and chemical properties and facilitating vegetation secondary succession. Overall, the present review provides a reference for further studies on ecological adaptability and sustainable utilization of Nepalese alder under climate change, and also for regional ecosystem service, forestry production practice, and vegetation restoration. [ABSTRACT FROM AUTHOR]

Based on analysis of the nucleotide sequences of the ITS regions, LSU gene fragments of rDNA, and cultural and morphological characteristics, species affiliation of six strains of mycelial fungi isolated from root nodules of actinorhizal plants in the subtropical zone (Corriaria myrtifolia, Myrica cerifera, and Eleagnus pungeus) was determined. The micromycetes studied were identified as Pezicula radicicola. Members of this species have not previously been revealed in the root nodules of actinorhizal plants. The possible functions and ecological role of Pezicula radicicola as mycosymbionts of actinorhizal plants are discussed. [ABSTRACT FROM AUTHOR]

Symbiotic nitrogen fixation (SNF) represents the largest natural input of bioavailable nitrogen into the biosphere, impacting key processes spanning from local community dynamics to global patterns of nutrient limitation and primary productivity. While research on SNF historically focused largely on herbaceous and agricultural species, the past two decades have seen major advances in our understanding of SNF by tree species in forest and savanna communities. This has included important developments in the mathematical theory of SNF in forest ecosystems, experimental work on the regulators of tree SNF, broad observational analyses of tree N-fixer abundance patterns and increasingly process-based incorporation of tree SNF into ecosystem models. This review synthesizes recent work on the local and global patterns, environmental drivers and community and ecosystem effects of nitrogen-fixing trees in natural ecosystems. By better understanding the drivers and consequences of SNF in forests, this review aims to shed light on the future of this critical process and its role in forest functioning under changing climate, nutrient cycling and land use., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

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. [ABSTRACT FROM AUTHOR]

Coriaria myrtifolia occurs as natural flora of warm temperate climates of northern Algeria which commonly found in hedges, forest and ravine edges. This actinorhizal species was known to establish a mutualistic symbiosis with members of phylogenetic cluster 2 (including strains associated to Coriaria spp., Ceanothus, Datiscaceae, and Dryadoideae) within the genus Frankia. Attempts to isolate C. myrtifolia microsymbionts from native plants growing in 4 locations in Algeria permitted to only recover asymbiotic Frankia strains (unable to reestablish nodulation and to fix nitrogen) from phylogenetic cluster 4 and several non- Frankia actinobacteria including members of Micrococcus, Micromonospora, Nocardia, Plantactinospora, and Streptomyces genera. The biodiversity of Frankia microsymbionts of C. myrtifolia root nodules was assessed using PCR-amplification followed by partial nucleotide sequencing of gln A1 (glutamine synthetase type 1) gene. On the 12 different gln A1 gene sequences obtained in this study, 9 were detected for the first time, and were mainly closelyrelated to Mediterranean genotypes previously described in the Grand Maghreb countries (Morocco and Tunisia) and in Europe (France) but without clear separations from other cluster 2 genotypes. [ABSTRACT FROM AUTHOR]

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. Since the co-existence of two different genera 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-specific nature. According to their plant host, Frankia has already been sub-divided into four clusters C-I, C-II, C-III, and C-IV. Our results revealed a strong competing nature of CI Frankia. Among the clusters of Frankia studied, the competition index between C-I and C-III was the largest. 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 groups. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Plant Production is the property of Egyptian National Agricultural Library (ENAL) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of Plant Production is the property of Egyptian National Agricultural Library (ENAL) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The actinobacterial genus Frankia establishes nitrogen-fixing root nodule symbioses with specific hosts within the nitrogen-fixing plant clade. Of four genetically distinct subgroups of Frankia, cluster I, II, and III strains are capable of forming effective nitrogen-fixing symbiotic associations, while cluster IV strains generally do not. Cluster II Frankia strains have rarely been detected in soil devoid of host plants, unlike cluster I or III strains, suggesting a stronger association with their host. To investigate the degree of host influence, we characterized the cluster II Frankia strain distribution in rhizosphere soil in three locations in northern California. The presence/absence of cluster II Frankia strains at a given site correlated significantly with the presence/absence of host plants on the site, as determined by glutamine synthetase (glnA) gene sequence analysis, and by microbiome analysis (16S rRNA gene) of a subset of host/nonhost rhizosphere soils. However, the distribution of cluster II Frankia strains was not significantly affected by other potential determinants such as host-plant species, geographical location, climate, soil pH, or soil type. Rhizosphere soil microbiome analysis showed that cluster II Frankia strains occupied only a minute fraction of the microbiome even in the host-plant-present site and further revealed no statistically significant difference in the α-diversity or in the microbiome composition between the host-plant-present or -absent sites. Taken together, these data suggest that host plants provide a factor that is specific for cluster II Frankia strains, not a general growth-promoting factor. Further, the factor accumulates or is transported at the site level, i.e., beyond the host rhizosphere. Biological nitrogen fixation is a bacterial process that accounts for a major fraction of net new nitrogen input in terrestrial ecosystems. Transfer of fixed nitrogen to plant biomass is especially efficient via root nodule symbioses, which represent evolutionarily and ecologically specialized mutualistic associations. Frankia spp. (Actinobacteria), especially cluster II Frankia spp., have an extremely broad host range, yet comparatively little is known about the soil ecology of these organisms in relation to the host plants and their rhizosphere microbiomes. This study reveals a strong influence of the host plant on soil distribution of cluster II Frankia spp.

BackgroundThe ability to establish root nodule symbioses is restricted to four different plant orders. Soil actinobacteria of the genus Frankia can establish a symbiotic relationship with a diverse group of plants within eight different families from three different orders, the Cucurbitales, Fagales and Rosales. Phylogenetically, Frankia strains can be divided into four clusters, three of which (I, II, III) contain symbiotic strains. Members of Cluster II nodulate the broadest range of host plants with species from four families from two different orders, growing on six continents. Two Cluster II genomes were sequenced thus far, both from Asia.ResultsIn this paper we present the first Frankia cluster II genome from North America (California), Dg2, which represents a metagenome of two major and one minor strains. A phylogenetic analysis of the core genomes of 16 Frankia strains shows that Cluster II the ancestral group in the genus, also ancestral to the non-symbiotic Cluster IV. Dg2 contains the canonical nod genes nodABC for the production of lipochitooligosaccharide Nod factors, but also two copies of the sulfotransferase gene nodH. In rhizobial systems, sulfation of Nod factors affects their host specificity and their stability.ConclusionsA comparison with the nod gene region of the previously sequenced Dg1 genome from a Cluster II strain from Pakistan shows that the common ancestor of both strains should have contained nodABC and nodH. Phylogenetically, Dg2 NodH proteins are sister to rhizobial NodH proteins. A glnA-based phylogenetic analysis of all Cluster II strains sampled thus far supports the hypothesis that Cluster II Frankia strains came to North America with Datisca glomerata following the Madrean-Tethyan pattern.

Aims: Research objectives were to determine nodule-forming capacities and competition for nodule-formation of Frankia strains ArI3 and Ag45/Mut15, which were assessed 90 days after planting of host seedlings in containers within soil microcosms. Methods: Both strains were inoculated in a '4 × 4' factorial design with all combinations (n = 16) of cell numbers of the two strains (0, 102, 103, or 104 cells g−1 of soil) determined by qPCR. Results: In all cases, inoculation significantly increased plant height compared to non-inoculated controls. Root nodules were formed on all inoculated plants with strain Ag45/Mut15 alone forming few nodules, for all inoculum amounts whiles strain ArI3 alone formed about ten-times more nodules at the lowest inoculum amount than Ag45/Mut15, with notable increases in nodule numbers for each ten-fold increase in inoculum. Except for nodules formed after inoculation of strain Ag45/Mut15 alone, nodules in all combinations harbored strain ArI3 only. Both strains were detected in rhizosphere soils for all three inoculum amounts. In contrast to nodules and rhizosphere soil, however, strain ArI3 was not detected in any bulk soil samples after three months of incubation, while strain Ag45/Mut15 was detected with densities about ten-fold higher than the original inoculum. Conclusions: These analyses demonstrate that strain ArI3 is initially more infective and competitive for root nodule formation than strain Ag45/Mut15. Strain Ag45/Mut15, however, survived and increased in cell numbers in bulk soil after 90 days whereas strain ArI3 disappeared indicating a reversal of competitive advantage between these two strains in the soil matrix over time. [ABSTRACT FROM AUTHOR]

The potential interference of Frankia asymbiotica NRRL B-16386 (nitrogen-fixing but non-infective) during nodule establishment on Alnus glutinosa seedlings by Frankia torreyi CpI1 (infective and effective) was investigated. Basic plant growth characteristics (stem and root elongation, dry weight production and total chlorophyll) and nodulation were assessed. The analysis showed that NRRL B-16386 did not greatly affect strain CpI1 during root nodule establishment and seedling growth of A. glutinosa despite the increase in its quantity and decrease in the symbiotic strain in the inocula. An increase in plant growth was noted even when the level of the effective Frankia strain decreased. The asymbiotic strain appeared to have an inhibitory effect on seedling growth and to thrive on the surface of root nodules well established by the symbiotic strain. [ABSTRACT FROM AUTHOR]

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.

Abstract 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.

Frankia cluster-2 strains are diazotrophs that engage in root nodule symbiosis with actinorhizal plants of the Cucurbitales and the Rosales. Previous studies have shown that an assimilated nitrogen source, presumably arginine, is exported to the host in nodules of Datisca glomerata (Cucurbitales), while a different metabolite is exported in the nodules of Ceanothus thyrsiflorus (Rosales). To investigate if an assimilated nitrogen form is commonly exported to the host by cluster-2 strains, and which metabolite would be exported in Ceanothus, we analysed gene expression levels, metabolite profiles, and enzyme activities in nodules. We conclude that the export of assimilated nitrogen in symbiosis seems to be a common feature for Frankia cluster-2 strains, but which source is host-dependent. The export of assimilated ammonium to the host suggests that 2-oxoglutarate is drawn from the TCA cycle at a high rate. This specialised metabolism obviates the need for the reductive branch of the TCA cycle. We found several genes encoding enzymes of the central carbon and nitrogen metabolism were lacking in Frankia cluster-2 genomes: the glyoxylate shunt and succinate semialdehyde dehydrogenase. This led to a linearization of the TCA cycle, and we hypothesize this could explain the low saprotrophic potential of Frankia cluster-2., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Purpose: The short growing season and cold climate of the boreal forest can restrict soil nitrogen availability, limiting plant growth and ecosystem productivity. Vascular nitrogen-fixing plants should have an advantage in low nitrogen environments. Yet, their abundance in the boreal forest is low. How nitrogen fixation is affected when temperature differences occur between the soil and air, especially in the spring when soil temperatures remain cool, has not been documented in actinorhizal shrubs. Methods: A lab study was performed on Alnus alnobetula subsp. crispa (Aiton) Raus. For 13 weeks, soil was kept at either 10˚C, 14˚C or 16˚C, independently of shoot temperature, at 20˚C. Results: Soils at 14˚C and 10˚C inhibited whole-plant nitrogen fixation (by 53% and 68%) and photosynthesis (by 43% and 39%), respectively, compared to soils at 16˚C. Reductions in photosynthetic rate were mainly attributed to a reduction in the fixed nitrogen supply and subsequent reduction in chlorophyll formation. Photosynthesis was not reduced immediately, suggesting some utilization of a nitrogen source not supplied from current fixation. Reduced amounts of fixed nitrogen and photosynthates resulted in diminished biomass production and relative growth rate. Conclusion: The assumed advantages of being a nitrogen-fixing plant in a low nitrogen environment may be constrained by soil temperature to a larger extent than previously considered. This may restrict the abundance of nitrogen-fixing species in the boreal forest. [ABSTRACT FROM AUTHOR]

Frankia strains are nitrogen-fixing soil actinobacteria that can form root symbioses with actinorhizal plants. Phylogenetically, symbiotic frankiae can be divided into three clusters, and this division also corresponds to host specificity groups. The strains of cluster II which form symbioses with actinorhizal Rosales and Cucurbitales, thus displaying a broad host range, show suprisingly low genetic diversity and to date can not be cultured. The genome of the first representative of this cluster, Candidatus Frankia datiscae Dg1 (Dg1), a microsymbiont of Datisca glomerata, was recently sequenced. A phylogenetic analysis of 50 different housekeeping genes of Dg1 and three published Frankia genomes showed that cluster II is basal among the symbiotic Frankia clusters. Detailed analysis showed that nodules of D. glomerata, independent of the origin of the inoculum, contain several closely related cluster II Frankia operational taxonomic units. Actinorhizal plants and legumes both belong to the nitrogen-fixing plant clade, and bacterial signaling in both groups involves the common symbiotic pathway also used by arbuscular mycorrhizal fungi. However, so far, no molecules resembling rhizobial Nod factors could be isolated from Frankia cultures. Alone among Frankia genomes available to date, the genome of Dg1 contains the canonical nod genes nodA, nodB and nodC known from rhizobia, and these genes are arranged in two operons which are expressed in D. glomerata nodules. Furthermore, Frankia Dg1 nodC was able to partially complement a Rhizobium leguminosarum A34 nodC::Tn5 mutant. Phylogenetic analysis showed that Dg1 Nod proteins are positioned at the root of both α- and β-rhizobial NodABC proteins. NodA-like acyl transferases were found across the phylum Actinobacteria, but among Proteobacteria only in nodulators. Taken together, our evidence indicates an Actinobacterial origin of rhizobial Nod factors.

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.

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 13CO2. 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 CO2 exchange from the mesocosms. The best predictors for foliar phosphorus concentration were net CO2 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. [ABSTRACT FROM AUTHOR]

Symbiotic nitrogen- (N) fixing trees can influence multiple biogeochemical cycles by fixing atmospheric N, which drives net primary productivity and soil carbon (C) and N accumulation, as well as by mobilizing soil phosphorus (P) and other nutrients to support growth and metabolism. The soil micronutrient molybdenum (Mo) is essential to N-fixation, yet surprisingly little is known of whether N-fixing trees alter soil Mo cycling, and if changes to soil Mo are coupled to soil C, N, and P. We compared how symbiotic N-fixing red alder and non-N-fixing Douglas-fir trees modified surface soil C, N, P, and Mo across variation in climate and other site factors in the Pacific Northwest. We found that after two decades, N-fixing trees drove coupled increases in surface soil C, N, total P, and organic P. Consistent with contributions of N-fixing trees to soil organic matter, increased soil C and N were accompanied by lower δ13C in all sites, and lower δ15N in sites where non-fixer plots exhibited elevated soil δ15N. However, N-fixing trees did not affect surface soil Mo concentrations or fractions, suggesting that different factors control the cycling of P versus Mo over decadal timescales. Random forest analysis revealed that surface soil P was most strongly influenced by factors related to soil C accumulation, whereas surface soil Mo was related primarily to environmental factors, including potential differences in atmospheric Mo deposition across sites. Ratios of surface soil P:Mo were higher in extractable pools than in total soil digests, reinforcing the idea of stronger biotic cycling of P than Mo. Overall, our multi-site, multi-decadal field study found surprisingly small effects of N-fixing trees on soil Mo, despite rapid increases in soil organic C, N, and P. We hypothesize that, rather than direct effects of N-fixing vegetation, abiotic or indirect biotic factors such as soil sorption of atmospheric Mo inputs can link C–N–P–Mo cycles in terrestrial ecosystems on longer timescales. [ABSTRACT FROM AUTHOR]

Aims: Revealing genetic diversity in a root nodulation symbiosis under field conditions is critical to understand the formation of ecological communities of organisms associated with hosts and the nitrogen cycle in natural ecosystems. However, our knowledge of the genetic diversity of bacterial mutualists on a local scale is still poor because of the assumption that the genetic diversity of mutualistic bacteria is constrained by their hosts. Methods: We thoroughly investigated the genetic diversity of Frankia in a local forest stand. We collected root nodules from 213 Alnus hirsuta seedlings covering the spatial range of the continuous population, which means that Alnus individuals occurred in a relatively homogeneous distribution in a continuous forest. Then, a phylogenetic and diversity analysis was performed for the nifD-K IGS region, including global Frankia sequences from Alnus hosts. Results: The genetic diversity of Frankia detected even on a local scale measured as high as that shown by previous studies conducted on local and regional scales. Moreover, a genetic structure analysis revealed a spatially mosaic-like distribution of genetic variation in Frankia despite the small spatial scale. Conclusions: The genetic diversity and composition of bacterial mutualists are heterogeneous on a local scale. Our findings demonstrate that genetically different bacterial symbionts simultaneously interact with a single host population and interaction partnerships spatially vary. The standing variation could produce dynamic ecological and evolutionary outcomes in a heterogeneous forest ecosystem. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]