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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]

Abstract 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.

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.

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

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]

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]

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.

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.

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.

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]

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. [ABSTRACT FROM AUTHOR]

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.)

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]

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).

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.

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]

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.

Alnus spp. (alder) are typical nonleguminous nitrogen-fixing trees that have a symbiotic relationship with Frankia . To explore the differences in nitrogen-fixing microorganisms between three alders ( A. cremastogyne , A. glutinosa , and A. formosana ) with different chromosome ploidies, the community structure and compositional diversity of potential nitrogen-fixing microorganism in root nodules and rhizosphere soil were comparatively analyzed using 16S rRNA and nitrogenase ( nifH ) gene sequencing. The nitrogen contents in the root nodules and rhizosphere soil were also determined. The results showed that the contents of total nitrogen and nitrate nitrogen in the root nodules of the three alders are significantly higher than those in the rhizosphere soils, while the ammonium nitrogen content show the opposite trend. The family, genus, and species levels showed obviously differences between root nodules and rhizosphere soils, while there were no significant differences at the classification level between the three alders. At the phylum level, the dominant phyla from 16S rRNA and nifH gene data in the root nodules and rhizosphere soil of the three alders are phylum Actinomycetota and phylum Pseudomonadota, respectively. The LEfSe results showed that there are significant differences in the dominant groups in the root nodules and rhizosphere oil of the three alders. The relative abundances of dominant groups also showed obvious differences between the root nodules and rhizosphere soils of three alders. The relative abundances of Frankia and unclassified_ Frankia in root nodules are obviously higher than those in rhizosphere soils, and their relative abundances in A. glutinosa root nodules are significantly higher than those in A. cremastogyne and A. formosana at the genus and species levels. The diversity of potential nitrogen-fixing microorganism from 16S rRNA and nifH gene data in the A. glutinosa root nodules and rhizosphere soils are all higher than those in A. cremastogyne and A. formosana . The results of functional prediction also showed that the OTUs for nitrogen fixation, nitrate respiration, and ureolysis in A. glutinosa root nodules are higher than those in the other two alders. Redundancy analysis revealed that the total nitrogen content mostly affects the Frankia community. Overall, there are significant differences in the community composition and structure of potential nitrogen-fixing microorganism in the root nodules and rhizosphere soils between the three alders. A. glutinosa showed a relatively stronger nitrogen fixation capacity than A. formosana and A. cremastogyne . The results help elucidates how the community structure and nitrogen-fixing ability of potential nitrogen-fixing microorganism differ between alder species and serve as a reference for applying Frankia to alder plantations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yuan, Chen, Huang, Wang, Guo, Huang and Yang.)

Strains CN4 T , CN6, CN7 and CNm7 were isolated from root nodules of Coriaria nepalensis from Murree in Pakistan. They do not form root nodules on C. nepalensis nor on Alnus glutinosa although they deformed root hairs of Alnus . The colonies are bright red-pigmented, the strains form hyphae and sporangia but no N 2 -fixing vesicles and do not fix nitrogen in vitro . The peptidoglycan of strain CN4 T contains meso -diaminopimelic acid; whole cell sugars consist of ribose, mannose, glucose, galactose and rhamnose. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and two unknown lipids represent the major polar lipids; MK-9(H 4 ) and MK-9(H 6 ) are the predominant menaquinones (>15 %), and iso-C 16 : 0 and C 17 : 1 ω8 c are the major fatty acids (>15 %). The results of comparative 16S rRNA gene sequence analyses indicated that strain CN4 T is most closely related to Frankia saprophytica CN 3 T . An MLSA phylogeny using amino acids sequences of AtpD, DnaA, FtsZ, Pgk and RpoB, assigned the strain to cluster 4 non-nodulating species, close to F. saprophytica CN 3 T , Frankia asymbiotica M16386 T and Frankia inefficax EuI1c T with 0.04 substitutions per site, while that value was 0.075 with other strains. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between CN4 T and all species of the genus Frankia with validly published names were below the defined threshold for prokaryotic species demarcation, with dDDH and ANI values at or below 27.8 and 83.7 %, respectively. The four strains CN4 T , CN6, CN7 and CNm7 had dDDH (98.6-99.6 %) and ANI values that grouped them as representing a single species. CN4 T has a 10.76 Mb genome. CN4 T was different from its close phylogenetic neighbours with validly published names in being red-pigmented, in having several lantibiotic-coding clusters, a carbon monoxide dehydrogenase cluster and a clustered regularly interspaced short palindromic repeats (CRISPR) cluster. The results of phenotypic, physiological and phylogenomic analyses confirmed the assignment of strain CN4 T (=DSM 114740 T = LMG 32595 T ) to a novel species, with CN4 T as type strain, for which the name Frankia nepalensis sp. nov. is proposed.