Genetic diversity of microsymbionts from legumes Oxytropis putoranica M. Ivanova, Oxytropis mertensiana Turcz., Astragalus norvegicus Grauer, Astragalus tugarinovii Basil. growing on the Putorana Plateau in Arctic Russia
Irina G. Kuznetsova1, Anna L. Sazanova1, Denis S. Karlov1, Polina V. Guro1, Irina A. Alekhina2, Nina Yu. Tikhomirova1, Edgar A. Sekste1, Oleg S. Yuzikhin1, Igor N. Pospelov3, Elena B. Pospelova4, Andrey A. Belimov1, Vera I. Safronova1
1 All-Russia Research Institute for Agricultural Microbiology, 3 Podbelskogo Shosse, St. Petersburg-Pushkin-8, 196608, Russia
2 Arctic and Antarctic Research Institute, 38 Beringa St., St. Petersburg, 1999397, Russia
3 A.N. Severtsov Institute of Ecology and Evolution, 33 Leninsky Prospekt, Moscow, 119071, Russia
4 Reserves of Taimyr, 24 Kirov St., Norilsk, 663305, Russia
Corresponding author: Irina G. Kuznetsova (ig.kuznetsova@arriam.ru)
Academic editor: A. Matsyura | Received 20 January 2025 | Accepted 13 March 2025 | Published 2 April 2025 |
http://zoobank.org/9EAFB589-89E4-40AD-83AC-0D390BD0BC8C
Citation: Kuznetsova IG, Sazanova AL, Karlov DS, Guro PV, Alekhina IA, Tikhomirova NYu, Sekste EA, Yuzikhin OS, Pospelov IN, Pospelova EB, Belimov AA, Safronova VI (2025) Genetic diversity of microsymbionts from legumes Oxytropis putoranica M. Ivanova, Oxytropis mertensiana Turcz., Astragalus norvegicus Grauer, Astragalus tugarinovii Basil. growing on the Putorana Plateau in Arctic Russia. Acta Biologica Sibirica 11: 359–383. https://doi.org/10.5281/zenodo.15112972
Abstract
There is a significant potential for the introduction of legumes into the Arctic regions of Russia. The ability of legumes to form a nitrogen-fixing symbiosis with nodule bacteria is one of their most important characteristics. The article studies the genetic diversity of the 24 bacterial strains isolated from root nodules from wild populations of legumes Oxytropis putoranica M. Ivanova, O. mertensiana Turcz., Astragalus norvegicus Grauer, and A. tugarinovii Basil. collected on the Putorana Plateau (Krasnoyarsk region, Arctic Russia). The microbial strains were isolated using yeast broth made with a standard method using YMA mannitol. Genomic DNA was isolated from pure cultures and the primary identification of the strains was carried out by PCR followed by sequencing of the 16S rRNA marker gene fragment. To clarify the identity of the species, ITS sequencing of the region was performed. The isolates were assigned to six genera and to five families of the order Hyphomicrobiales: Pararhizobium and Neorhizobium (family Rhizobiaceae), Phyllobacterium (Phyllobacteraceae), Microvirga (Methylobacteriaceae), Bosea (Boseaceae) and Tardiphaga (Bradyrhizobiaceae). Isolates from O. putoranica nodules were identified as Neorhizobium galegae, Bosea sp., Bosea vaviloviae, and Tardiphaga robiniae. The isolated nodules of O. mertensiana were identified as Pararhizobium herbae, Tardiphaga robiniae, Microvirga ossetica, and Microvirga sp. Microsymbionts of A. norvegicus were assigned to Bosea psychrotolerans and to Pararhizobium herbae and Tardiphaga robiniae species, while isolates from A. tugarinovii were identified as Phyllobacterium zundukense, Bosea sp., and Tardiphaga robiniae. Symbiotic the nodA gene was detected in strains P. herbae P14/2-4 and P20/1-1, P. zundukense P17/1-7 and P17/3-2, while the nodC gene was not detected in any of the strains. The sterile test tube experiment confirmed the inability of strains P. herbae P14/2-4 and P20/1-1 to form nodules in host plants O. mertensiana and A. norvegicus, as well as in other wild arctic (A. tugarinovii, O. putoranica) and for- age legumes (Trifolium repens, Vicia cracca, and Lathyrus pratensis). The results obtained expand the understanding of the taxonomic status and biodiversity of local microsymbionts of wild legumes that grow on the Putorana Plateau. The study of the symbiotic efficiency of Arctic rhizobia will allow us to identify the most promising strains for the development of effective biofertilizers for the cultivation of forage and pasture legumes under the extreme soil and climatic conditions of the Russian Arctic. In turn, the creation of highly adapted legume-rhizobial systems based on valuable genetic resources of Arctic rhizobia strains will expand the range of legume species promising for use in the creation of multi-component agrophytocenoses necessary for the sustainable development of animal husbandry in Arctic regions of Russia.
Keywords
Putorana Plateau, arctic legumes, Astragalus, Oxytropis, arctic agriculture, legume-rhizobial symbiosis, ribosomal RNA genes
Introduction
The territory of the northern regions is an important resource for the development of agriculture (Unc et al. 2021; Naydenov 2020). The total area of the Arctic sector of Russia is approximately 3 million km2, which is 18% of the total territory of the Russian Federation, including 2.2 million km2 of land (Pestsov 2021). In the northwestern part of the central Siberian Plateau, on an area of 250 thousand km2, there is the Putorana mountain plateau. The plateau was formed by massive eruptions of a supervolcano about 247–252 million years ago. The solidified lava formed layers of basalt rock of varying composition. The plateau is covered by continuous permafrost. However, permafrost conditions change from west to east. In the Norilsk region, permafrost is partly discontinuous with through and interpermafrost taliks (Pospelov and Pospelova 2021). The climate is subarctic, strongly continental, but milder in some sheltered lake valleys. Winter is long and cold (average temperature -40 °C), and spring, summer, and autumn fall into three months: June, July, and August. Summer is rainy with an average temperature of +11...+13 °C. The plateau lies on the border between taiga and tundra. The vegetation is represented by larch-spruce forests in the valley part, sparse forests, and shrub tundra on the upper slopes and on the surface of the plateau. The highest parts of the plateau, near the watershed, are dominated by rock and lichen tundra. The main part of the plateau is characterized by a uniform geomorphological structure combining plateau-like surfaces of mountain ranges and deeply incised valleys with medium and steep slopes. In terms of soil-geographic zonation, the plateau corresponds to a separate Anabar-Putorana province, which is part of the East Siberian permafrost-taiga region. The Putorana province is characterized by taiga peaty-humic, high-humic nongleyed soils with high acidity of the upper horizons, ochre podburs, tundra podburs and stony placers. Many on the soils of the Putorana plateau are specific and do not have analogues among the soils described above in other regions. The specificity of soil formation is mainly determined by the combination of mountainous relief, cold continental climate with excess moisture, and the composition of soil-forming rocks. The soils accumulate large amounts of organic matter. In general, data on the study of the soil cover of the Putorana Plateau are limited. The literature contains descriptions mainly of soils of individual mountain-tundra and forest-tundra regions (Senkov 2014; The unified state register… 2025). Lake Duluk is located in the eastern half of the plateau. The tundra in this part is shrub-sedge-moss and the surface of the plateau is a cold mountain desert. Most of the plateau is covered by the Putorana Nature Reserve, one of the largest in Russia (Pospelov, Pospelova 2021; Merganič et al. 2021). It was established to "preserve and study the natural course of natural processes and phenomena, the genetic fund of flora and fauna, individual species and communities of plants and animals, typical and unique ecological systems of the Putorana Plateau" (The statute…2025). However, many valuable ecosystems remain poorly studied due to their inaccessibility (Merganič et al. 2021).
The traditional form of economic use of the territory of the subarctic and arctic sector of Russia is reindeer husbandry. The basis for successful reindeer husbandry in the territory is, first of all, the availability of the forage base (Mizin et al. 2018). One of the most important protein-rich components of natural pastures and pastures are legumes, which are widespread in the temperate and arctic zones of the northern hemisphere. Two thirds of the species represented in the Russian Arctic belong to the tribe Galegeae (Bornn) Torr.et Gray, subtribe Astragalinae (Adans.) Benth. (Yurtsev 1986; Kamelin 2017). On the Putorana plateau, legumes are mainly represented by the genera Oxytropis DC., Astragalus L. and Hedysarum L., which are included in the diet of animals and birds, including reindeer, snow rams, ground squirrels, pikas, brant geese, and graylag geese (Larin 1951; Yurtsev 1986; Rosenfeld 2009; Kamelin 2017). Legume species such as Astragalus alpinus L. subsp. arcticus (Bunge) Hult., Oxytropis adamsiana (Trautv.) Jurtz., Oxytropis nigrescens (Pall.) Fisch., Hedysarum arcticum B. Fedtsch are ubiquitous. A significant proportion of northern oxytropes and astragals are cryophytes endemic to the Arctic or certain parts of it. For example, the rare Taimyr-Putorana endemic Oxytropis putoranica M. Ivanova grows mainly on open gravel surfaces, screes, and along the dry beds of running streams. Oxytropis mertensiana Turcz. occurs only in the eastern part of Putorana in the highlands, in moist tundra. Astragalus norvegicus Grauer grows in lakeside meadows, river thickets, and damp open woodland. Astragalus tugarinovii Basil. prefers carbonate and non-turf soils. In the south-east of the Taimyr Peninsula, it is widespread on slopes and steppe meadows, common in the Golets tundra. However, on the Putorana plateau it was found only in the highlands of the dry mud tundra, at Lake Baselak and on a rocky outcrop in the western part of Lake Sobachye (Larin 1951; Pospelov and Pospelova 2021).
The geographical and ecological distribution of northern legumes and their role in ecosystems are largely determined by biocenotic and symbiotic relationships. Arctic soils differ from European soils in their low fertility and the presence of permafrost and seasonal frost (Larin 1951). One of the main limiting factors is the insufficient supply of nitrogen compounds readily available to plants in the soil (Beermann et al. 2015). To overcome this problem, legumes establish symbiotic relationships with nodule bacteria (rhizobia). These microorganisms are able to penetrate root hairs and form nodules in which atmospheric nitrogen is fixed. Therefore, rhizobia are important participants in the mutualistic plant-microbe symbiosis. They influence legume productivity and yield by providing additional means of survival in nitrogen deficiency and by contributing to an increase in soil fertility, which favours the introduction of new flora into local native communities (Provorov, Tikhonovich 2016). Therefore, rhizobia are important participants in mutualist plant-microbe symbiosis, influencing legume productivity and yield of legumes by providing plants with additional means of survival under nitrogen deficiency. Plants of the genera Oxytropis and Astragalus are nodulated by a wide range of rhizobial species. However, the dominant microsymbionts are members of the genus Mesorhizobium (Laguerre et al. 1997). In addition, members of the genera Rhizobium, Sinorhizobium, Bradyrhizobium, Bosea, and Tardiphaga can be found in Oxytropis and Astragalus nodules (Laguerre et al. 1997; Kuznetsova et al. 2015; Ampomah et al. 2017; Wdowiak and Malek 2020; Safronova et al. 2020). The low host specificity of Oxytropis and Astragalus species may indicate that they play an important role in the genetic diversification of microsymbionts, which may influence the adaptive capacity of legumes to different environmental extremes (Chen et al. 2015). There is no information in the literature on the rhizobia that inhabit the nodules of legumes that grow on the Putorana plateau. We propose that these legumes do not have an established species composition of nodule bacteria and that their nodules may contain taxonomically distinct groups of bacteria. Thus, arctic legume-rhizobial systems are good models for studying the formation of evolutionary relationships between legumes and rhizobia in harsh soil and climatic conditions of the north, which will facilitate the identification of adaptive microbial strains that can be used to obtain highly effective biofertilizers for growing legumes in various regions of the Russian Arctic.
Thus, the aim of our work was to create a collection of the wild microsymbionts of arctic legumes O. putoranica, O. mertensiana, A. norvegicus, and A. tugarinovii collected on the Putorana Plateau, to study of the genetic diversity of microorganisms of the order Hyphomicrobiales by sequencing rrs gene sequences and the region between 16S and 23S rDNA (ITS region), to study the ability of the obtained strains to form effective symbiosis with leguminous plants by searching symbiotic nodA and nodC genes and under conditions of sterile test-tube experiment.
Materials and methods
Collection of nodules and isolation of bacterial strains
Root nodules were collected from wild populations of legumes Oxytropis putoranica (2 populations), Oxytropis mertensiana, Astragalus norvegicus and Astragalus tugarinovii growing in the vicinity of Lake Duluk (69.544246 N, 94.141467 E) during the 2022 expedition to the Putorana Plateau (Krasnoyarsk region) (Figs 1, 2).
Figure 1. Putorana Plateau and Lake Duluk. Map from the site https://nakarte.me (ESRI Sputnik), 2024.
The selection of nodules was carried out from 3-5 individual plants so that the total number of nodules for each plant species was at least 20. Due to the different growing conditions and phenotypes of legumes, various tools were used to collect root nodules: garden shovels, chisels and hammers. Roots with nodules were carefully removed from the soil and placed in separate paper bags. The bags were stored in a ventilated, cool and dry place in the shade until the roots were completely dry. Five nodules of each plant species were selected in the laboratory for further work. Individual nodules were sterilized for 1 min in 96% ethanol, and strains were isolated from homogenized material on yeast–mannitol agar (YMA) at 28°C using the standard technique (Novikova, Safronova 1992). To obtain pure bacterial cultures, visible colonies were picked and sequentially cloned twice in Petri dishes with YMA medium using the streak plate technique.
DNA extraction and PCR protocols
DNA was isolated using KIT (Thermo Scientific, EU) according to the manufacturer’s instructions. The strains were identified by sequencing of 16S rDNA and ITS region. Primary identification of the strains was performed by PCR amplification of 16S marker fragments of the rRNA gene (rrs) fragments (1341–1404 bp) with subsequent sequencing of the amplicons. Amplification was carried out with the primers fD1 5′-AGAGTTTGATCCTGGCTCAG-3′ and rD1 5′-AAGGAGGT- GATCCAGCC-3′. PCR was performed on a T100 automated amplifier (Bio-Rad, United States). PCR was carried out in 50 μL reaction mixtures containing 150 μM dNTP (Promega, United States), 5 pmol of each primer, 1 U Taq polymerase (Helicon, Russia) and 50–100 ng purified DNA template. The PCR conditions for 16S rDNA amplification were as follows: 95 °C 3.5 min; 94 °C, 1 min 10 s; 56°C, 40 s; 72°C, 2 min 10 s and final elongation 72 °C, 6 min 10 s. To amplify the ITS-region we used primers FGPS FGPS1490-72 5'-TGCGGCTGGGGATCCCCTCCTT-3' and FGPL132'-38 5'-CCGGGGTTTCCCCATTCGG-3'. The PCR condition for ITS region amplification: 95°C, 3.5 min; 94°C, 1 min; 50°C, 1 min; and 72 °C, 2 min with final elongation 72°C, 6 min 10 s (Normand et al. 1996).
Amplification of the nodC and nodA genes in isolates
To determine the nodulation capacity, a 666 bp fragment of the nodA gene was sequenced using the primers nodA-1 5′-GCRGTGGAARNTRNNCTGGGAAA-3 and nodA-2 5′-GNCCGTCRTCRAASGTCARGTA-3′ (Haukka et al. 1998); and a 930 bp fragment of the nodC gene using the primers nodCF 5′-AYGTHGTYGAYGACG- GTTC-3′ and nodCR 5′-CGYGACAGCCANTCKCTATTG-3′ (Laguerre et al. 2021). PCR fragments amplified:95°C, 2 min; 94°C, 30 s; 50°C (primer pair nodA-1, nodA-2) or 53°C (primer pair nodСF, nodСR) for 1 min and 72°C, 1 min with final elongation 72°C, 3 min. The strain Rhizobium leguminosarum bv. trifolii RCAM1365 was used as a positive control.
Visualization and purification of the PCR product
Electrophoresis was performed on 1% agarose gel (Invitrogen, United States) in 0.5% TAE. The GeneRuler 1 Kb Plus DNA Ladder TM and Lambda DNA/HindIII marker (Fermentas, Lithuania) were used for the determination of the size and approximate quantification of the DNA fragments. The PCR product was purified using a Cleanup S-Cap Kit (Eurogen, Russia) according to the manufacturer’s instructions.
Figure 2. Plants Oxytropis putoranica, Oxytropis mertensiana, Astragalus norvegicus and Astragalus tugarinovii found during the expedition.
Sequencing and data processing
The sequencing of the prepared PCR products was performed on the ABI PRISM 3500×l genetic analyzer (Life Technologies, United State) in the Core Centrum “Genomic Technologies, Proteomics and Cell Biology’, All-Russia Research Institute for Agricultural Microbiology. The DNA sequences obtained were analyzed using the ChromasLite 2.6.4 program. Sequences of closely related strains were searched for in the GenBank database (https://www.ncbi.nlm.nih.gov) and the BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic trees were construct- ed using the MEGA -X, XI software package (Tamura et al. 2011). Evolutionary distances were computed using the maximum composite likelihood method. Bootstrap analysis with 1000 replications was performed to estimate the support of clusters. The nucleotide sequences were deposited in the GenBank database with accession numbers PQ870313 – PQ870336 for the rrs gene, PQ871498 – PQ871503 for the ITS region.
Storage of strains
Pure microsymbiont cultures (after sequential double cloning) were placed in the UNU ‘Station for low-temperature automated storage of biological samples’ at -80 °C (Liconic Instruments, Liechtenstein) for long-term storage. Information on the strains is available in the RCAM Internet database (https://arriam.ru/kollekciya-kultur1/).
Sterile test tube experiment
The seeds of wild arctic leguminous plants A. norvegicus, A. tugarinovii, O. putoranica, O. mertensiana and the forage legumes Trifolium repens, Lathyrus pratensis, Vicia cracca were surface sterilized by treatment with 98% H2SO4 for 5–10 min (depending on the size of the seeds). The treated seeds were carefully rinsed with sterile water and germinated on filter paper in Petri dishes at +25 °C in the dark for 3–5 days. The seeds were transferred in 50 ml glass tubes (2 seedlings per tube) containing 3 g of sterile vermiculite. Each glass tube was supplemented with 6 ml of the nutrient solution (g/l): K2HPO4 – 1.0, KH2PO4 – 0.25, MgSO4 – 1.0, Ca3(PO4)2 – 0.2, FeSO4 – 0.02, as well as microelements according to M.V. Fedorov in a volume of 1 ml of the following composition: H3BO3 – 0.005, (NH4)2MoO4 – 0.005, ZnSO4 × 7 H2O – 0.005, MnSO4 – 0.002 g/l. The seedlings were inoculated with P. herbae P14/2-4 and P20/1-1 in an approximate amount of 106 cells per tube. Uninoculated plants were used as negative control. The plant nodulation assay was carried out in duplicate. The plants were grown for 35 days in the growth chamber with 50 % relative humidity and four levels of illumination and temperature: night (dark, 18 °C, 8 h), morning (200 μmol m–2s–1, 20 °C, 2h), day (400 μmol m–2s–1, 23 °C, 12h), evening (200 μmol m–2s–1, 20 °C, 2h). Illumination was performed with L36W / 77 FLUORA lamps (Osram, Germany).
Results
Molecular phylogenetic identification of strains
As a result of this work, 24 bacterial isolates were obtained from nodules collected from the roots of O. putoranica, O. mertensiana, A. norvegicus, and A. tugarinovii. Four of them were fast-growing and formed colonies on the third day, while the rest appeared on the fourth or fifth day. BLAST analysis of the rrs gene sequences allowed all the isolates obtained to be assigned to six genera of the order Hyphomicrobiales (formerly Rhizobiales): Pararhizobium and Neorhizobium (family Rhizobiaceae), Phyllobacterium (Phyllobacteriaceae), Microvirga (Methylobacteriaceae), Bosea (Boseaceae), and Tardiphaga (Bradyrhizobiaceae) (Table 1).
Table 1. BLAST results of 16S rRNA gene fragments comparison for strains of nodules of O. putoranica, O. mertensiana, A. norvegicus, and A. tugarinovii plants collected near Lake Duluk (Putorana Plateau, Krasnoyarsk region)
Strain ID | Species | Source of isolation | Closely related type strain(s) identified by BLAST | Similarity, % |
P13/1-1 | Tardiphaga robiniae | Tardiphaga robiniae R-45977 | 99.71 | |
P13/1-2 | B. vaviloviae | Bosea vaviloviae Vaf-18 | 100.00 | |
P13/2-2 | B. vaviloviae | Bosea vaviloviae Vaf-18 | 100.00 | |
P13/2-3 | Tardiphaga robiniae | Tardiphaga robiniae R-45977 | 99.78 | |
P18/1-2 | Tardiphaga robiniae | Tardiphaga robiniae R-45977 | 99.93 | |
P18/3-2 | Neorhizobium galegae | O. putoranica | Neorhizobium galegae NBRC 14965, Neorhizobium vignae CCBAU 05176 | 99.48, 99.77 |
P18/3-6 | Tardiphaga robiniae | Tardiphaga robiniae R-45977 | 99.85 | |
P18/5-1 | Neorhizobium galegae | Neorhizobium galegae NBRC 14965, Neorhizobium vignae CCBAU 05176 | 99.41, 99.66 | |
P18/5-2 | Bosea sp. | B. lathyri R-46060, B. caraganae RCAM04680, B. psychrotolerans 1131 | 99.14 | |
P14/2-2 | Bosea psychrotolerans | Bosea psychrotolerans 1131 | 99.93 | |
P14/2-3 | Tardiphaga robiniae | Tardiphaga robiniae R-45977 | 99.86 | |
P14/2-4 | Pararhizobium herbae | A. norvegicus | Pararhizobium herbae CCBAU 83011 | 100.00 |
P14/3-3 | Bosea psychrotolerans | Bosea psychrotolerans 1131 | 99.48 | |
P14/5-1 | Bosea psychrotolerans | Bosea psychrotolerans 1131 | 99.33 | |
P20/1-1 | Pararhizobium herbae | Pararhizobium herbae CCBAU 83011 | 100.00 | |
P20/1-2 | Tardiphaga robiniae | O. mertensiana | Tardiphaga robiniae R-45977 | 99.86 |
P20/2-1 | Tardiphaga robiniae | Tardiphaga robiniae R-45977 | 99.86 | |
P20/4-1 | Microvirga ossetica | Microvirga ossetica V5/3M | 99.86 | |
P20/5-1 | Microvirga sp. | Microvirga ossetica V5/3M | 99.18 | |
P17/1-6 | Bosea sp. | Bosea caraganae RCAM04680 | 99.34 | |
P17/1-7 | P. zundukense | A. tugarinovii | Phyllobacterium zundukense Tri-48 | 99.93 |
P17/2-3 | Tardiphaga robiniae | Tardiphaga robiniae R-45977 | 99.93 | |
P17/3-2 | P. zundukense | Phyllobacterium zundukense Tri-48 | 99.93 | |
P17/3-3 | Tardiphaga robiniae | Tardiphaga robiniae R-45977 | 99.93 |
Four fast growing isolates P18/3-2 and P18/5-1 from O. putoranica nodules, P14/2-4 from A. norvegicus nodule and P20/1-1 from O. mertensiana nodule were assigned to the family Rhizobiaceae based on the results of the sequencing of the rrs gene fragment. These isolates were grouped into two groups on the phylogenetic tree (Fig. 3).
Figure 3. Unrooted phylogenetic tree constructed on the basis of a comparative analysis of the nucleotide sequences of 16S rRNA gene fragment of strains obtained from nodules of O. putoranica, O. mertensiana, A. norvegicus and representatives of the related species Rhizobium, Neorhizobium, and Pararhizobium. Isolates are shown in bold. Clusters I–II are statistically different clusters. Support levels above 50% are indicated.
Cluster I included isolates P18/3-2, P18/5-1 and type strains Neorhizobium vignae CCBAU 05176 and Neorhizobium galegae NBRC 14965 with a low level of support (Fig. 3). The level of similarity of rrs between isolates P18/3-2, P18/5-1 and the type strain N. vignae CCBAU 05176 was 99.77 and 99.66%, respectively, whereas the level of rrs similarity between isolates P18/3-2, P18/5-1 and the type strain of N. galegae NBRC 14965 was 99.48 and 99.41%, respectively (Table 1).
Cluster II was formed by isolates P14/2-4, P20/1-1 and the type strains Pararhizobium polonicum F5.1 and Pararhizobium herbae CCBAU 83011 with a relatively low (77%) level of support. The isolates had a 100% rrs similarity to the type strain P. herbae CCBAU 83011, while the level of rrs similarity between isolates and the type strain P. polonicum F5.1 was 99.61% (Table1).
For isolates P18/3-2, P18/5-1, P14/2-4 and P20/1-1, the ITS fragment was sequenced and a phylogenetic tree constructed. These isolates were grouped into two groups (Fig. 4).
Figure 4. Unrooted phylogenetic tree constructed on the basis of a comparative analysis of the nucleotide sequences of the fragment of the ITS region of four strains, as well as representatives of related species of Rhizobium, Neorhizobium, Pseudorhizobium and Pararhizobium. Isolates are shown in bold. Clusters I–II are statistically different clusters. Support levels above 50% are indicated.
Cluster I included isolates P18/3-2, P18/5-1 and the type strain N. galegae HAMBI 540 at 100% support level (Fig. 4). The level of ITS region similarity between isolates P18/3-2, P18/5-1 and this type strain was 96.16 and 95.97%, respectively (Table 2).
Table 2. Similarity levels (%) of the ITS region of strains P18/3-2, P18/5-1, P14/2-4, and P20/1-1, isolated from root nodules of O. putoranica, O. mertensiana, A. norvegicus and closely related type strains, belonging to the genera Neorhizobium, Pararhizobium, Pseudorhizobium and Rhizobium
Closely related type strains | Strain ID | |||
P18/3-2 | P18/5-1 | P14/2-4 | P20/1-1 | |
N. galegae HAMBI 540 | 96.16 | 95.97 | - | - |
N. vignae CCBAU 05176 | 86.36 | 86.06 | - | - |
P. banfieldiae NT-26 | 83.01 | 82.24 | - | - |
R. lentis BLR27 | 79.04 | 77.89 | - | - |
P. gei ZFJT.2T | - | - | 87.77 | 87.80 |
P. giardinii USDA 2914 | - | - | 84.74 | 84.93 |
P. herbae DSM26427 | - | - | 98.91 | 99.00 |
P. polonicum F5.1 | - | - | 89.64 | 89.64 |
Cluster II included isolates P14/2-4, P20/1-1, and the type strain P. herbae DSM26427 at 100% support level (Fig. 4). The ITS regions of isolates P14/2-4 and P20/1-1 showed 98.91 and 99.00% similarity to this type strain, respectively (Table 2). In order for a strain to be classified as a specific species, there must be at least 95.5% similarity in the ITS sequence between them (Ferraz et al. 2022). The results of the ITS sequencing confirmed that the isolates P14/2-4 and P20 / 1-1 were related to the species P. herbae, while P18/3-2 and P18/5-1 to the species N. galegae.
Phylogenetic analysis of the sequences of the fragments of the rrs gene of isolates P17/1-7 and P17/3-2 from A. tugarinovii nodules showed that they formed a cluster I with the type strain Phyllobacterium zundukense Tri-48 at a support level (Fig. 5) and had 99.93% similarity to it (Table 2). On the ITS tree, strains P17/1-7 and P17/3-2 formed a group I (97% support level) with the type strain P. zundukense Tri-48 (Fig. 6) and had 98.20 and 98.23% similarity in the ITS gene, respectively. Therefore, isolates P17/1-7 and P17/3-2 were identified as P. zundukense. The isolates P13/1-2, P13/2-2, and P18/5-2 from the O. putoranica nodules, P14/2-2, P14/3-3, and P14/5-1 from the A. norvegicus nodules and the P17/1-6 from the A. tugarinovii nodule were assigned to the Bosea genus according to the results of rrs gene fragment (Table 1, 3). These isolates were grouped into four groups on the phylogenetic tree (Fig. 7). Cluster I was formed by isolates P14/2-3, P14/3-3, P14/5-1 and the type strain
Bosea psychrotolerans 1131 with a rather high support level (Fig. 7). The isolate P14/2-2 showed 99.93 and 99.85%, the isolate P14/3-3 showed 99.48 and 99.14%, the isolate P14/5-1 showed 99.21 and 99.14% rrs similarity to the closest type strain B. psychrotolerans 1131 and B. vaviloviae Vaf-18, respectively (Table 3). Thus, these strains were assigned to the species B. psychrotolerans.
Table 3. Similarity levels (%) of the 16S rRNA gene fragments comparison for strains from nodules of O. putoranica, A. norvegicus, A. tugarinovii, and closely related type strains, belonging to genera Bosea
Strain ID | Closely related type strains | ||||
B. caraganae RCAM04680 | B. lathyri R-6060 | B. psychrotolerans 1131 | B. vaviloviae Vaf-18 | B. massiliensis 63287 | |
P13/1-2 | 98.71 | 99.07 | 99.78 | 100 | 98.35 |
P13/2-2 | 98.68 | 99.05 | 99.78 | 100 | 98.32 |
P14/2-2 | 98.67 | 99.19 | 99.93 | 99.85 | 98.16 |
P14/3-3 | 98.00 | 98.50 | 99.48 | 99.14 | 97.57 |
P14/5-1 | 98.00 | 98.50 | 99.21 | 99.14 | 97.56 |
P17/1-6 | 99.34 | 98.98 | 98.83 | 98.91 | 98.98 |
P18/5-2 | 99.14 | 99.14 | 99.14 | 99.07 | 97.92 |
Figure 5. Unrooted phylogenetic tree constructed on the basis of a comparative analysis of the nucleotide sequences of the 16S rRNA gene fragment of strains obtained from nodules of A. tugarinovii and representatives of the related species Phyllobacterium and Mesorhizobium. Isolates are shown in bold. Cluster I is a statistically different cluster. Support levels above 50% are indicated.
Figure 6. Unrooted phylogenetic tree constructed on the basis of a comparative analysis of the nucleotide sequences of the fragment of the ITS region of two strains, as well as representatives of related species of Phyllobacterium and Mesorhizobium. Isolates are shown in bold. Cluster I is a statistically different cluster. Support levels above 50% are indicated.
Cluster II included isolates P13/1-2, P13/2-2 and the type strain Bosea vaviloviae Vaf-18 at a support level (Fig. 7). The 16S rRNA sequence analysis showed that isolates P13/1-2 and P13/2-2 were 100 and 99.78% similar to the closest type strain B. vaviloviae Vaf-18 and B. psychrotolerans 1131, respectively (Table 3). Thus, isolates P13/1-2 and P13/2-2 were assigned to species B. vaviloviae.
Cluster III included the isolate P18/5-2 and the type strain Bosea lathyri R-46060 with a low level of support (50%, Fig. 7). The similarity between the isolate and the type strains B. lathyri R-46060, B. caraganae RCAM04680, B. psychrotolerans 1131 was 99.14% (Table 3). Therefore, isolate P18/5-2 was identified as Bosea sp.
Cluster IV was formed by isolate P17/1-6 and the type strain Bosea caraganae RCAM04680 with a low support level (Fig. 7). The level of rrs similarity between the isolate and the type strain B. caraganae RCAM04680 was 99.34% (Table 3). The isolate P17 / 1-6 was identified as Bosea sp. To clarify the species identity of isolates P18/5-2 and P17/1-6, sequencing and phylogenetic analysis of the ITS gene should be performed. Slow growing strains P20/4-1 and P20/5-1 were isolated from root nodules of O. mertensiana. Based on the results of the fragment of the sequencing of the rrs gene, the isolates were assigned to the genus Microvirga (Table 1). On the phylogenetic tree, they were grouped into cluster V with the type strain Microvirga ossetica V5/3M at a high support level (Fig. 7). The rrs similarity between isolates P20/4-1, P20/5-1 and the closest type strain of M. ossetica V5/3M was 99.86 and 99.17%, respectively. The rrs-similarity between isolates P20/4-1, P20 / 5-1, and the type strain of Microvirga zambiensis WSM3693 was 98.63 and 97.91%, respectively. The strain P20 / 4-1 was identified as M. ossetica, while strain P20/5-1 was identified as Microvirga sp. More molecular phylogenetic analysis is needed to clarify the species identity of strain P20/5-1.
Figure 7. Unrooted phylogenetic tree constructed on the basis of a comparative analysis of the nucleotide sequences of the 16S rRNA gene fragment of strains obtained from nodules of O. putoranica, O. mertensiana, A. norvegicus, and A. tugarinovii and representatives of the related species Bosea and Microvirga. Isolates are shown in bold. Clusters I-V are statistically different clusters. Support levels above 50% are indicated.
Slow growing strains P13/1-1, P13/2-3, P18/1-2, and P18/3-6 were isolated from O. putoranica nodules, P14/2-3 from the A. norvegicus nodule, P20/2 and P20/2-1 from O. mertensiana nodules, P17/2-3 and P17/3-3 from the A. tugarinovii nodules. The similarity rrs between the isolates and the closest type strain of Tardiphaga robiniae R-45977 and Tardiphaga zeae SS122 varied from 99.71 to 99.93% and from 99.34 to 99.56%, respectively. All of these isolates were classified as T. robiniae.
Analyzing the Genetic Diversity of Symbiotic Genes
The search for symbiotic (symbiotic) nodA and nodC genes was carried out in strains of N. galegae P18/3-2 and P18/5-1 of O. putoranica nodules, P. herbae P14/2-4 of A. norvegicus nodule, P. herbae P20/1-1, M. ossetica P20/4-1 and Microvirga sp. P20/5-1 strains from O. mertensiana nodules, Phyllobacterium zundukense P17/1-7, and P17/3-2 strains from A. tugarinovii nodules. The nodA gene was detected in strains P. herbae P14/2-4 and P20/1-1, P. zundukense P17/1-7 and P17/3-2, while the nodC gene was not detected in any of the strains. Both symbiotic genes were present in the positive control Rhizobium leguminosarum bv. trifolii RCAM1365.
Sterile test tube experiment
To study the nodulating activity of strains P. herbae P14/2-4 and P20/1-1, a sterile test tube experiment with legume plants A. norvegicus, A. tugarinovii, O. putoranica, O. mertensiana, T. repens, L. pratensis, and V. cracca was performed. The result obtained showed the lack of ability of isolates to form nodules on the roots of these plant species.
Discussion
As a result, nodules of the wild arctic legumes O. putoranica, O. mertensiana, A. norvegicus, and A. tugarinovii that grow on the shore of Lake Duluk (Putorana plateau, Krasnoyarsk region) were collected. Twenty-four bacterial isolates belonging to six genera of the order Hyphomicrobiales: Pararhizobium and Neorhizobium (Rhizobiaceae), Phyllobacterium (Phyllobacteraceae), Microvirga (Methylobacteriaceae), Bosea (Boseaceae) and Tardiphaga (Bradyrhizobiaceae) were isolated and studied. Species affiliation was established for 18 isolates.
The microsymbionts of O. putoranica belonged to N. galegae, B. vaviloviae, Bosea sp., and T. robiniae, the microsymbionts of A. norvegicus were represented by P. herbae, Bosea sp. and T. robiniae. The microsymbionts of O. mertensiana were associated with P. herbae, T. robiniae, M. ossetica, and Microvirga sp. The microsymbionts of A. tugarinovii were related to P. zundukense, Bosea sp., and T. robiniae. The genus Neorhizobium was segregated from the genus Rhizobium based on multilocus sequence analysis (MLSA) of six housekeeping genes. The type strain N. galegae HAMBI 540 was shown to be able to form nitrogen-fixing root nodules on Galega orientalis Lam. (Lindström 1989; Mousavi et al. 2014). The four species N. galegae, N. vignae, N. huautlense, and N. alkalisoli are members of the "R. galegae complex" and are closely related phylogenetically. Some strains were described as nodule microsymbionts of various legumes (Wang et al. 1998; Ren, Chen et al. 2011). Other strains were found in the soil (Zhang et al. 2012; Soenens et al. 2019; Pan et al. 2022).
The genus Pararhizobium was separated from the genus Rhizobium based on MLSA analysis of housekeeping genes (Mousavi et al. 2015). The strain R. herbae CCBAU 83011 was first isolated from Nodules of Astragalus membranaceus (Fisch.) The bundle was growing in Xinjiang (China) and was renamed in 2015 Pararhizobium herbae (Ren, Wang et al. 2011; Mousavi et al. 2015). Representatives of the species were also found in the nodules of various leguminous plants of the genera Astragalus, Oxytropis, Vicia and Lathyrus (Kuznetsova et al. 2023), while other species of Pararhizobium were isolated, for example, from Antarctic water samples (Naqvi et al. 2017) and Alpine soils (Zhang et al. 2017).
The genus Phyllobacterium was first described by Knoesel in 1962 (Knösel 1962) for bacteria developing in leaf nodules of tropical ornamental plants. Phyllobacterium strains were isolated from root nodules of various legumes, including Astragalus (Mantelin et al. 2006) and Oxytropis (Safronova et al. 2018), from the Brassica rhizo- plane (Mantelin et al. 2006) or from leaf nodules of Ardisia (Mergaert et al. 2002). It is known that strains P. sophorae CCBAU 03422 and P. trifolii PETP02 possessed the core nodulation nodACD and nifH genes, encoding Fe protein nitrogenase, and were capable of forming an effective symbiosis with host plants (Valverde et al. 2005; Jiao et al. 2015). The genomes of P. zundukense strains of O. triphylla nodules lacked the common nodABC genes required for legume nodulation, although some other symbiotic nod and fix genes were detected (Safronova et al. 2018). Thus, Neorhizobium, Pararhizobium, and Phyllobacterium species can be both free-living and plant-associated bacteria, indicating their high ability to adapt to the environment, accompanied by the complexity of microbial genomes and deep specialization to host plants.
The genus Microvirga was first described in 2003 (Kanso, Patel 2003). Most species of Microvirga were described as isolates from soil, air, and water samples. However, strains of several species were isolated from root nodules of legumes and can form effective symbioses with host plants (Ardley et al. 2012; Radl et al. 2014). The type strain Microvirga ossetica V5/3M was isolated from root nodules of Vicia alpestris Steven growing in the north Ossetia region (Caucasus). However, it was unable to form nodules or swellings on the roots of V. alpestris and V. formosa plants in pot experiments (Safronova et al. 2017).
The genus Bosea is currently represented by 14 species. Members of the genus Bosea may be present in nodules of various legumes of the genera Lupinus, Lathyrus, Robinia, Vavilovia, Caragana, Spartocytisus, Vicia, Astragalus, Oxytropis, and Hedysarum (De Meyer and Willems 2012; Sazanova et al. 2019; Pulido-Suárez et al. 2022; Kuznetsova et al. 2024). Strains of B. vaviloviae were detected in the nodules of Vavilovia formosa (Steven) Fed (North Caucasus), A. schelichowii (Norilsk) Turcz. and narrowly endemic Kamchatka species of Oxytropis (Safronova et al. 2020; Kuznetsova et al. 2023). B. psychrotolerans, which is phylogenetically related to B. vaviloviae, was described in 2019 as a psychrotrophic alphaproteobacterial species isolated from Lake Michigan water (Albert et al. 2019). The nodule genes involved in the formation of nodules on legume roots were found to be present in the genomes of several Bosea species. However, the ability of these species to form nodules independently was not demonstrated (Sazanova et al. 2019).
The genus Tardiphaga is currently represented by two species, T. robiniae and
T. alba. The first T. robiniae was isolated from Robinia pseudoacacia L., growing in Flanders (Belgium) (De Meyer et al. 2012). T. robiniae strains have also been found in nodules of V. formosa, A. schelichowii, and in narrow local endemics of the genus Oxytropis (Safronova et al. 2020, Kuznetsova et al. 2023). Some symbiotic (sym) genes responsible for nodulation and nitrogen fixation may be present in the genome of T. robiniae strains. However, the ability of Tardiphaga representatives to form nodules independently was not identified (Safronova et al. 2020). The type strain T. alba SK50-23 was isolated from Thallium-loaded garden soil in Japan (Bao et al. 2024). Thus, despite the lack of the ability of Bosea and Tardiphaga strains to form symbiotic nodules, the frequency of their occurrence and the presence of individual sym genes probably indicate their ability to influence the efficiency of legume-rhizobium symbiosis.
A study of the genetic diversity of symbiotic genes was carried out to investigate the ability of strains of the genus Neorhizobium, Pararhizobium, Phyllobacterium, and Microvirga to form nodules. The strains Pararhizobium sp. P14 / -2, P20 / 1-1, and P. zundukense P17/1-7, P17/3-2 possessed the main symbiotic (symbiotic) gene nodA, but lacked the nodC gene, without which nodule formation is impossible, while other strains lacked both genes. The nodA and nodC genes are common genes and, together with the nodB gene, are responsible for the synthesis of the cortical part of the Nod factors. NodABC genes are located in plasmids in fast-growing rhizobia, are involved in horizontal transfer genes processes and can be easily lost by bacterial cells (Provorov, Tikhonovich 2016). The negative results of the amplification of the nodA and nodC gene in the studied isolates may be due to the absence of target genes or their peculiar structure. The absence of nodules in a sterile test tube experiment using strains P. herbae P14/2-4 and P20/1-1 and legumes A. norvegicus, O. mertensiana, A. tugarinovii, O. putoranica, T. repens, L. pratensis, and V. cracca confirmed the negative results of amplification of the nodC gene in these strains. Thus, based on the results of the nod-gene search and the test tube experiment, no strains capable of forming nodules on host plants were identified, which may be due to both the mediocre quality of the collected nodules and the suboptimal conditions for culturing Arctic bacteria. In the future, it is proposed to use additional nutrient medium and different temperature cultivation modes to identify nodulating strains.
It is important to note the presence of two or three strains belonging to different families of the order Hyphomicrobiales in one nodule. Thus, strains N. galegae P18/3-2 and P18/5-1 (Rhizobiaceae), T. robiniae P18/3-6 (Bradyrhizobiaceae), and Bosea sp. P18/5-2 (Boseaceae) were detected in nodules of O. putoranica. Strains P. herbae P14/2-4 (Rhizobiaceae), B. psychrotolerans P14/2-2 (Boseaceae), and T. robiniae P14/2-3 (Bradyrhizobiaceae) were isolated from a nodule of A. norvegicus. The strains P. herbae P20/1-1 (Rhizobiaceae) and T. robiniae P20/1-2 (Bradyrhizobiaceae) were isolated from a nodule of O. mertensiana. Strains P. zundukense P17/1- 7 and P17/3-2 (Phyllobacteriaceae), Bosea P17/1-6 (Boseaceae), and T. robiniae P17/3-3 (Bradyrhizobiaceae) were isolated from the nodules of A. tugarinovii. This confirms the hypothesis that legume symbiotic systems can be multicomponent and include rhizobial strains from different taxonomic groups. This creates conditions for effective exchange of genetic material between microsymbionts cohabiting in root nodules (Safronova et al. 2023).
Conclusions
This paper presents the first data on the genetic diversity of native arctic bacteria associated with nodules of the wild legumes O. putoranica, O. mertensiana, A. norvegicus, and A. tugarinovii growing in the vicinity of Lake Duluk (Putorana Plateau, Krasnoyarsk Krai). A total of 24 isolates belonging to six genera of the order Hyphomicrobiales were obtained: Pararhizobium and Neorhizobium, Phyllobacterium, Microvirga, Bosea, and Tardiphaga. The species N. galegae (fam. Rhizobiaceae), T. robiniae (fam. Bradyrhizobiaceae), B. vaviloviae and Bosea sp. (fam. Boseaceae) were described as microsymbionts of O. putoranica. The species P. herbae (fam. Rhizobiaceae), T. robiniae, M. ossetica, and Microvirga sp. (fam. Methylobacteriaceae) were described as microsymbionts of O. mertensiana. Representatives of P. herbae, B. psychrotolerans, and T. robiniae were isolated from the nodules of A. norvegicus, and representatives of P. zundukense (fam. Phyllobacteriaceae), Bosea sp. and T. robiniae were found in the nodules of A. tugarinovii. To clarify the species assignment of the strains, Microvirga sp. P20/5-1, Bosea sp. P17/1-6 and P18/5-2 require the sequencing and analysis of additional marker genes. In addition, strains of different taxonomic groups of the order of Hyphomicrobiales order are present in the nodules of all legume species, which can affect the increase in the potential of symbiotic complementarity for increasing the productivity of plant-microbe interactions due to the possibility of joint localization of taxonomically different co-microsymbionts in one nodule. Such symbiotic systems are promising models for studying the formation of the specificity of legume-rhizobial interaction and its effect on the productivity of symbiosis in extreme Arctic conditions. Therefore, the creation of plant-microbial systems based on the genetic resources of Arctic rhizobia will allow us to expand the range of leguminous plant species that are promising as a high-protein component in the creation of highly productive agrophytocenoses for the sustainable development of northern livestock farming in the context of climate change and radical restructuring of Arctic landscapes.
Acknowledgements
The research was conducted utilizing the equipment of the Collective Center "Genomic technologies, proteomics and cell biology" of the All-Russia Research Institute for Agricultural Microbiology. This work was supported by the Russian Foundation for Basic Research project No. 20-76-10042.
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