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 the source of nitrogen is host dependent. The export of assimilated ammonium to the host suggests that 2-oxoglutarate is drawn from the tricarboxylic acid (TCA) cycle at a high rate. This specialized metabolism obviates the need for the reductive branch of the TCA cycle. We found that several genes encoding enzymes of 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 hypothesized that this could explain the low saprotrophic potential of Frankia cluster-2.

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.

Underneath our feet, in the soil, is a whole hidden world. It consists of microorganisms, small animals, and plant roots. As it is all concealed from us, there is a lot about them, how they interact, and their environment, which is still unknown. However, it has been revealed that these interactions are crucial for sustainable crop production and increasing yields in a changing environment. Chickpeas, soybeans, and peanuts are all examples of legumes, which have been part of the human diet since the earliest civilisations (Arranz-Otaegui & Roe 2023). In addition to their high-protein seeds and delicious flavour, legumes enrich the soil and help neighbouring and sequentially grown crops perform better. This is because legumes can engage in beneficial relationships –we call it symbiosis- with two groups of microbes: rhizobia and arbuscular mycorrhizal fungi (AMF). Rhizobia are a group of bacteria that can turn inaccessible atmospheric dinitrogen into the usable form of ammonia. This means that, unlike most other plants, legumes can tap into the atmospheric nitrogen supply, something most other plants cannot do.1 During this symbiosis, the rhizobia live inside specialised plant organs, nodules, found on the legume root system. Here, they receive carbon-rich molecules the plant produces via photosynthesis (Mathesius 2022). AMF are also associated with plant roots, but this symbiosis is much more common. More than 70% of all plant species can engage with AMF! While they cannot fix atmospheric nitrogen, they can scavenge other nutrients, such as phosphorus (P), from the soil that is out of reach for the roots. In turn, they receive carbon-rich molecules from the plant (Brundrett & Tedersoo, 2018). Associating with these beneficial microbes means plants must coordinate their resources since root growth also requires resources. For many plants, the roots are vital in the overall health and survival of the plant. They are responsible for anchorage, uptake of nutrients and water, and connecting with diverse microorganisms of the soil, such as bacteria and fungi. In addition, roots need to be able to adapt to different environments, such as the increasing amounts of extreme drought and rainfalls. Consequently, we can see that roots show a wide range of morphologies depending on their environment, including root length, diameter, and branching. Even within a species, roots adapt their root architecture depending on environmental cues (Cope et al., unpublished). We try to understand the interactions between root morphologies, nutrient uptake, and microbial interactions better, as this would allow us to select and breed crops more suited to the extreme environments that arise due to climate change (Cope et al., unpublished, Yang et al., 2024). It would also allow reducing the use of artificial nutrients and instead promote healthier soil. Ultimately, what happens belowground significantly affects plant growth aboveground and, consequentially, crop yield. For this reason, Pang and colleagues (Pang et al., 2023) investigated the coordination of AMF colonisation on two chickpea cultivars. These two cultivars differed in the thickness of their roots. The authors were interested in how different P levels and sources, with or without AMF, affected the roots. They looked at different root morphologies, carboxylate exudation, and root nodule formation. They found that, regardless of the source of P, AMF significantly affected the root morphology. The morphological change in response to AMF was more pronounced for the one genotype, which released greater amounts of carboxylates. The genotype with thinner roots had a significantly higher percentage of root length colonised by AMF than those with thicker roots. This colonisation could be due to the mobilisation of more P via carboxylates. This would increase the availability of P and, in turn, enhance AMF colonisation. The available level of P also played an important role in the level of AMF colonisation. At moderate P availability, AMF colonisation was found to be the highest. However, at very low or very high P availability, the AMF colonisation became very low. The two genotypes, as well as the P source, affected AMF colonisation. AMF play a more important role in P acquisition under low soluble P than under less soluble P. The effect of AMF on nodulation depended on the genotype, P source, and P availability. However, co-inoculation with AMF and rhizobia enhanced nodulation.

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 S & oslash;ndergaard, 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. Different types of [NiFe] hydrogenases in the genus Frankia and their roles in nitrogen fixation deduced from losses in lineages of genome erosion.

AimsRoot system architecture (RSA) plays an important role in the plant’s ability to sustain yield under abiotic stresses such as drought. Preceding crops (precrops) can affect the yield of the proceeding crop, partially by affecting the RSA. This experiment aims to explore the interactions between precrop identity, crop genotype and drought at early growth stages.MethodsRhizotrons, sized 60 × 80 × 3.5 cm, were used to assess the early root growth of two winter wheat (Triticum aestivum L.) genotypes, using precrop-treated soil around the seedlings and differing water regimes. The rhizotrons were automatically imaged 3 times a week to track root development.ResultsPrecrop-treated soil affected the RSA and changes caused by the reduced water treatment (RWT) were different depending on the precrop. Largest of these was the 36% reduction in root depth after wheat, but 44% after OSR. This indicates that effects caused by the precrop can be simulated, at least partially, by transferring precrop-treated soils to controlled environments. The genotypes had differential RSA and reacted differently to the RWT, with Julius maintaining an 8.8-13.1% deeper root system compared to Brons in the RWT. In addition, the combined environmental treatment affected the genotypes differently.ConclusionOur results could help explain discrepancies found from using precrops to enhance yield as they indicate differences in the preceding crop effect when experiencing drought stress. Further, these differences are affected by genotypic interactions, which can be used to select and adapt crop genotypes for specific crop rotations, depending on the year. Additionally, we have shown a viable method of stimulating a partial precrop effect at the seedling stage in a controlled greenhouse setting using field soil around the germinated seed.

Background Increasing nitrogen (N) use efficiency (NUE) can enhance cereal yields, improve grain quality and reduce agrochemical inputs. However, several challenges are associated with achieving enhanced NUE, e.g., a frequently poor synchronization between N supply and demand; low grain yields per unit of N input; and inherent trade-offs between grain yield and quality. Scope To address these challenges, we focus on the temporal perspective of NUE related processes at different growth stages, and propose the utilization of beneficial microbes, e.g. arbuscular mycorrhizal fungi (AMF) for their roles in affecting aspects of NUE particularly at the later development stages. Conclusion Some evidence suggests that AMF can directly promote N uptake and use in cereals, by regulating N transporters, but the indirect effects of AMF on NUE related processes at different growth stages are poorly known. Here we explore AMF and their potential roles in promoting NUE related processes that enhance crop P and N uptake post-anthesis, when nutrient supply often is low. In order to fully exploit the opportunities for beneficial root-fungal symbiosis, we propose approaches for plant breeding and crop management, and consider the potential of plant-microbe interactions post-anthesis to promote NUE and the sustainable production of especially cereals.

Roots play a pivotal role in the adaption of a plant to its environment, with different root traits adapting the plant to different stresses. The environment affects the Root System Architecture (RSA), but the genetic factors determine to what extent, and whether stress brought about by extreme environmental conditions is detrimental to a specific crop. This study aimed to identify differences in winter wheat RSA caused by cultivation region and practice, in the form of preceding crop (precrop), and to identify if modern cultivars used in Sweden differ in their reaction to these environments. This was undertaken using high-throughput phenotyping to assess the RSA. Clear differences in the RSA were observed between the Swedish cultivation regions, precrop treatments, and interaction of these conditions with each other and the genetics. Julius showed a large difference between cultivars, with 9.3–17.1% fewer and 12–20% narrower seminal roots. Standardized yield decreased when grown after wheat, 23% less compared to oilseed rape (OSR), and when grown in the Southern region, 14% less than the Central region. Additionally, correlations were shown between the root number, angle, and grain yield, with different root types being correlated depending on the precrop. Cultivars on the Swedish market show differences that can be adapted to the region-precrop combinations. The differences in precrop effect on RSA between regions show global implications and a need for further assessment. Correlations between RSA and yield, based on root-type × precrop, indicate different needs of the RSA depending on the management practices and show the potential for improving crop yield through targeting genotypic and environmental conditions in a holistic manner. Understanding this RSA variance, and the mechanisms of conditional response, will allow targeted cultivar breeding for specific environments, increasing plant health and food security.

An actinobacterial strain, CMB- FB, was isolated from surface- sterilized root nodules of a Coriaria intermedia plant growing along Halsema Highway in the province of Benguet (Luzon, Philippines). The 16S rRNA gene sequence of CMB- FB showed high sequence similarity to those of the type strains of Streptomyces rishiriensis (99.4%), Streptomyces humidus (99.1 %), Streptomyces cacaoi subsp. asoensis (99.0 %), and Streptomyces phaeofaciens (98.6 %). The major menaquinones of CMB- FB were composed of MK- 9(H4), MK-9(H6) and MK- 9(H8), and there was a minor contribution of MK- 9(H10). The polar lipid profile consisted of phosphatidylethanolamine, unidentified aminolipids and phospholipids, a glycophospholipid and four unidentified lipids. The diagnostic diamino acid of the peptidoglycan was meso- diaminopimelic acid. The major fatty acids were iso- C16:0, anteiso-C15: 0 and anteiso-C17:0. The results of physiological analysis indicated that CMB- FB was mesophilic. The results of phylogenetic, genome- genome distance calculation and average nucleotide identity analysis indicated that the isolated strain represents the type strain of a novel species. On the basis of these results, strain CMB- FB (=DSM 112754T=LMG 32457T) is proposed as the type strain of the novel species Streptomyces coriariae sp. nov.

Microorganisms, such as bacteria and fungi, can live together with plants in a tight relationship where they help each other to provide nutrients. Some groups of bacteria can turn nitrogen, an essential element, from a gas in the atmosphere into a form that plants can use. In return, plants provide carbon to the bacteria. This special relationship is called symbiosis. Researchers try to understand why some plants interact with these helpful bacteria and others do not. They do this by looking at how the symbiotic relationship was formed and how it evolved over a long period of time of several million years. If we can use symbiotic microorganisms to provide plants with nutrients instead of using fertilizers, we could reduce the negative effects caused by greenhouse gasses associated with their production.

Summary Frankia cluster-2 are diazotrophs that engage in root nodule symbiosis with host plants of the Cucurbitales and the Rosales. They are rarely found in the soil in the absence of their hosts. Previous studies have shown that an assimilated nitrogen source, presumable arginine, is exported to the host in nodules of Datisca glomerata (Cucurbitales), but not in the nodules of Ceanothus thyrsiflorus (Rosales). To investigate if an assimilated nitrogen form is commonly exported by cluster-2 strains, and which nitrogen source would then be exported to C. thyrsiflorus , gene expression levels, metabolite profiles and enzyme activities were analysed. We found that the export of assimilated nitrogen in symbiosis is a common feature for Frankia cluster-2 strains, but which source is host-plant dependent. We also identified several gene losses. The ammonium assimilation via the GS/GOGAT cycle for export to the host, entails a high demand of 2-oxoglutarate from the TCA cycle. This specialised metabolism seems to have led to genome reduction: we show that Frankia cluster-2 strains have lost the glyoxylate shunt and succinate semialdehyde dehydrogenase, leading to a linearization of the TCA cycle. This could explain the low saprotrophic potential of Frankia cluster-2.

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.

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.

We describe a new Frankia species, for three non-isolated strains obtained from Alnus glutinosa in France and Sweden, respectively. These strains can nodulate several Alnus species (A. glutinosa, A. incana, A. alnobetula), they form hyphae, vesicles and sporangia in the root nodule cortex but have resisted all attempts at isolation in pure culture. Their genomes have been sequenced, they are significantly smaller than those of other Alnus-infective species (5 Mb instead of 7.5 Mb) and are very closely related to one another (ANI of 100%). The name Candidatus Frankia nodulisporulans is proposed. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and draft genome sequences reported in this study for AgTrS, AgUmASt1 and AgUmASH1 are MT023539/LR778176/LR778180 and NZ_CADCWS000000000.1/CADDZU010000001/CADDZW010000001, respectively.

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.