The genetic diversity and symbiotic efficiency of the nodule microsymbionts isolated from Oxytropis taimyrensis (Jurtz.) A. et D. Love, Astragalus frigidus (L.) A. Gray and Astragalus tugarinovii Basil. from Arctic Yakutia

Legumes have significant potential for introduction into the Arctic regions of Russia. One of the key characteristics of legumes is the ability to form nitrogen-fixing symbioses with nodule bacteria (rhizobia). However, insufficient attention has been paid to the study of the biodiversity and symbiotic efficiency of Arctic rhizobia in Russia. The present work describes for the first time 13 strains of the order Hyphomicrobiales (formerly Rhizobiales ) isolated from nodules of Oxytropis taimyrensis , Astragalus frigidus and A. tugarinovii growing in Arctic Yakutia. The ability of nine rhizobial strains Rhizobium sp. 7/1-1, 19-1/1, 20-1/1 and 33-1/1, R. giardinii 20/1-1, M. norvegicum 20/1-4 and Mesorhizobium sp. 9-4/1, 25-2/1 and 32-2/1 to nodulate the wild Arctic legumes Oxytropis adamsiana and Astragalus frigidus and the fodder legumes Trifolium repens and Medicago sativa was investigated under the conditions of a sterile test-tube experiment. The aim of the work was to isolate and study the genetic diversity of strains of the order Hyphomicrobiales isolated from nodules of the wild legumes Oxytropis taimyrensis (Jurtz.) A. et D. Love, Astragalus frigidus (L.) A. Gray and Astragalus tugarinovii Basil, collected in the Arctic zone of Yakutia, and to determine the ability of rhizobial strains to form nitrogen-fixing nodules on the roots of fodder and wild legumes Trifolium repens L., Medicago sativa L., Oxytropis adamsiana (Trautv.) Jurtzev and Astragalus frigidus (L.) A.Gray under the conditions of a sterile test-tube cross-nodulation experiment. Root nodules of wild populations of O. taimyrensis , A. frigidus , A. tugarinonovii were collected in 2021 near the lake Sevastyan-Kyule and on Tit-Ary during the Russian-German expedition to the Lena River Delta. Rhizobial strains were isolated from legume nodules by standard methods using mannitol-yeast YMA nutrient medium. Genomic DNA was isolated from pure cultures using the DNeasy Blood&Tissue Kit (QIAGEN, Germany) and Monarch® (New England Biolabs, USA). The primary identification of the strains was carried out by PCR followed by sequencing of the 16S rRNA marker gene ( rrs ) fragment (900-1400 bp). The ability of nine Arctic strains of the genera Rhizobium and Mesorhizobium to form nitrogen-fixing nodules on the roots of Trifolium repens L., Medicago sativa L., O. adamsiana and A. frigidus was studied under the conditions of sterile test-tube experiment. The strains studied were isolated in the present work and previously from nodules of the Arctic legumes Lathyrus palustris L., Vicia cracca L. and Hedysarum arcticum B. Fedtsch growing in the Lena River delta. Plants were grown in sterile 50 ml glass vessels containing 3 g vermiculite and 6 ml of Krasilnikov-Korenyako medium. Seedlings were inoculated with suspensions of individual strains at 106 c ells/vessel. Commercial strains of Rhizobium leguminosarum RCAM1365 and Sinorhizobium meliloti RCAM1750 from the Russian Collection of Agricultural Microorganisms (RCAM, ARRIAM, St. Petersburg) were used as positive controls. Non-inoculated plants were used as negative controls. At the end of cultivation, nodules were counted and crude plant biomass was determined. Nitrogen fixation activity was determined by the acetylene method using a GC-2014 gas chromatograph (Shimadzu, Japan). The obtained isolates were assigned to the genera Rhizobium (family Rhizobiaceae ), Mesorhizobium (family Phyllobacteriaceae ), Bosea (family Boseaceae ) and Tardiphaga (family Bradyrhizobiaceae ). The strains Rhizobium sp. 7/1-1, Tardiphaga robiniae 7/2-2 and 7/4-2 were isolated from A. tugarinovii , the strains Mesorhizobium sp. 25-2/1, 25A/5-1, Bosea sp. 25A/1-3, B. lathyri 25A/2-1, B. psychrotolerans 25A/2-2 and 25A/4-1 were isolated from A. frigidus , whereas the strains Mesorhizobium sp. 9-4/1, T. robiniae 9/1-5, 9/3-1 and 9/5-1 were isolated from nodules of O. taimyrensis . Under sterile test-tube experiments the strain R. giardinii 20/1-1 did not form nodules in any of the inoculation variants, whereas the other eight strains were able to form both ineffective and nitrogen-fixing nodules depending on the legume inoculation variant. With respect to the native Arctic species O. adamsiana and A. frigidus , strains isolated from native legumes ( O. taimyrensis , A. frigidus , H. arcticum ) were more active, whereas the cultivated plants M. sativa and T. repens were more responsive to inoculation with strains isolated from introduced plants L. palustris and V. cracca . The ability of O. adamsiana to form an effective symbiosis with M. norvegicum 20/1-4, Mesorhizobium sp. 9-4/1 and 25-2/1, isolated from representatives of the legume genera Hedysarum , Oxytropis and Astragalus , respectively, indicates a low host specificity of this plant species, which may presumably allow it to use the resource potential of a wide range of symbiotic microorganisms inhabiting the soil of various Arctic regions.