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Rhizobial isolates in active layer samples of permafrost soil of Spitsbergen, Arctic
Denis Karlov1, Anna Sazanova1, Irina Kuznetsova1, Nina Tikhomirova1, Zhanna Popova1, Yuriy Osledkin1, Nikita Demidov2, Andrey Belimov1, and Vera Safronova1
1All-Russia Research Institute for Agricultural Microbiology,
Shosse Podbel'skogo, 3, Saint Petersburg, 190608, Russian Federation
2Arctic and Antarctic Research Institute,
ul. Beringa, 38, Saint Petersburg, 199397, Russian Federation
Abstract
Citation: Karlov, D., Sazanova, A., Kuznetsova, I., Tikhomirova, N., Popova, Zh., Osledkin, Yu., Demidov, N., Belimov, A., and Safronova, V. 2021. Rhizobial isolates in active layer samples of permafrost soil of Spitsbergen, Arctic. Bio. Comm. 66(1): 73-82. https://doi.org/10.21638/spbu03.2021.109
Authors' information: Denis Karlov, PhD, Junior Researcher, orcid.org/0000-0002-9030-8820; Anna Sazanova, PhD, Senior Researcher, orcid.org/0000-0003-0379-6975; Irina Kuznetsova, Engineer-Researcher, orcid.org/0000-0003-0260-7677; Nina Tikhomirova, Researcher, orcid.org/0000-0002-8510-2123; Zhanna Popova, PhD, Senior Researcher, orcid.org/0000-0003-1570-613X; Yuriy Osledkin, PhD, Leading Researcher, orcid.org/0000-0001-8865-7434; Nikita Demidov, PhD, Researcher, orcid. org/0000-0002-3462-7747; Andrey Belimov, Dr. of Sci. in Biology, Head of Laboratory, orcid.org/0000-0002-9936-8678; Vera Safronova, PhD, Head of Laboratory, orcid. org/0000-0003-4510-1772
Manuscript Editor: Kirill Antonets, Department of Cytology and Histology, Faculty of Biology, Saint Petersburg State University, Saint Petersburg, Russia
Received: September 2, 2020;
Revised: November 26, 2020;
Accepted: December 21, 2020.
Copyright: © 2021 Karlov et al. This is an open-access article distributed under the terms of the License Agreement with Saint Petersburg State University, which permits to the authors unrestricted distribution, and self-archiving free of charge.
Funding: The work was performed with Russian Science Foundation support (grant No. 20-76-10042). Sampling, chemical analysis of samples and isolation of strains were supported by the Russian Science Foundation (grant No. 19-77-1006). The long-term storage of strains was supported by the Program for the Development and Inventory of Bioresource Collections. The research was performed using equipment of the Core Centrum "Genomic Technologies, Proteomics and Cell Biology" in ARRIAM.
Competing interests: The authors have declared that no competing interests exist.
Twenty-nine strains were isolated from two samples of the permafrost active layer of the Spitsbergen archipelago. The estimated number of bacteria ranged from 4.0-104 to 1.7-107 CFU-g-1. As a result of sequencing of the 16S rRNA (rrs) genes, the isolates were assigned to 13 genera belonging to the phyla Actino-bacteria, Proteobacteria (classes a, p, and y), Bacteroidetes, and Firmicutes. Six isolates belonged to the rhizobial genus Mesorhizobium (order Rhizobiales). A plant nodulation assay with seedlings of legume plants Astragalus norvegicus, A.frigidus, A.subpolaris and Oxytropis sordida, originated from Khibiny (Murmansk region, Russia) and inoculated with Mesorhizobium isolates, showed the inability of these strains to form nodules on plant roots. Symbiotic (sym) genes nodC and nodD were not detected in Mesorhizobium strains either.
Keywords: legume plants, root nodule bacteria, 16S rRNA genes, permafrost soil, Spitsbergen
Introduction
Root nodule bacteria (rhizobia) belong to one of the most important groups of soil microorganisms that form nitrogen-fixing symbiosis with legumes, which allows them to enrich nitrogen-depleted soils, making it available to other plants. (Tikhonovich and Provorov, 2009).
Russia's northern territories will increasingly be used for agricultural land as a result of global climate warming. In the Russian Arctic zone, there has an expansion of the areas occupied by more productive plant communities, the so-called "greening" of the tundra. This is mainly associated with an increase in temperature, the growing season duration and the thawing depth change of permafrost soils. These processes activate soil microorganisms, which contributes to a more intensive accumulation of organic matter in the forming phytocenoses (Belo-novskaya et al., 2016). As a result, free-living rhizobial bacteria can form highly adaptive legume-rhizobial symbioses, penetrating new landscapes, such as the Arctic tundra. Legumes are known to play an important role in pasture phytocenoses and are the main source of protein for herbivorous farm animals (Parakhin and Petrova, 2009; Pryadilshchikova, Kalabashkin and Konovalova, 2018).
From the permafrost soil of the Arctic, isolates were obtained related to the genera whose representatives can form nitrogen-fixing symbiosis with legumes. Strains of the genus Burkholderia were isolated from the permafrost of the Canadian Arctic (Wilhelm, Niederberger, Greer and Whyte, 2011). Isolates belonging to the genera Bradyrhizobium, Rhizobium, Devosia, and Methylobacterium, as well as the species Mesorhizobium gobiense, were obtained from the soils of northeastern Greenland (Ganzert, Bajerski and Wagner, 2014). It should be mentioned that the type strain Mesorhizobium gobiense 83330T was isolated from the nodule of Oxytro-
pis glabra growing in the desert soils of China. This strain, in addition to the host plant, is also able to form nodules on Glycyrrhiza uralensis, Lotus corniculatus, and Robinia pseudoacacia plants, as shown by cross-nodulating tests (Han et al., 2008). Of the Late Pleistocene (20-35 thousand years ago) permafrost soil of the Kolyma lowland, a dominant phylotype was identified, which showed 99.3 % similarity to Bradyrhizobium canariense (Kudryashova et al., 2013). The ability of strains of this species to form effective symbiosis with various legumes endemic from the tribe Genisteae growing in the Canary Islands has been shown (Vinuesa et al., 2005).
Isolates belonging to Rhizobium sp. (Singh et al., 2017) and Burkholderia sp. (Hansen, Herbert, Mikkelsen and Jensen, 2007) were isolated from permafrost soil of Spitsbergen. The metagenomic studies of the active permafrost layer in this archipelago revealed DNA and RNA of symbiotic nitrogen-fixing bacteria of the genera Rhizobium and Bradyrhizobium (Schostag et al., 2015). The above works were devoted to the description and study of the bacterial biodiversity, including root nodule bacteria, in the permafrost of Spitsbergen. However, their ability to form nitrogen-fixing symbioses with plants was not studied.
Currently, native species of legume plants do not grow on Spitsbergen. The presence of a randomly introduced species of Trifolium repens L. in the area of the village of Barentsburg (https://svalbardflora.no/index. php?id=638) and Pyramid (Svenning, Rosnes, Lund and Junttila, 2001) was noted. The clover genotypes from Spitsbergen have been shown to have a higher dry matter mass as well as greater frost resistance compared to the two varieties (Norstar and AberHerald) from central Norway (Svenning, Rosnes, Lund and Junttila, 2001). The same authors selected soil samples in various areas of Spitsbergen, including in the area of the village Pyramid. Clover plants planted in this soil formed nodules and their microsymbionts were identified as Rhizobium leguminosarum bv. trifolii (Fagerli and Svenning, 2005). Clover plants grown in other soil samples of Spitsbergen did not form nodules, which suggests the introduction of rhizobia together with clover seeds. However, attempts to use other species of leguminous plants, including Arctic ones, as a selective factor for the search for root-nodule bacteria have not been described.
It is known that the genus Mesorhizobium belongs to an extensive group of rhizobia, isolated from soils and root nodules of legume plants growing around the world (Helene, Dall'Agnol, Delamuta and Hungria, 2019). Mesorhizobium strains form nitrogen-fixing symbiosis with a wide range of host plants from the genera Acacia, Alhagi, Amorpha, Biserrula, Caragana, Chamaecrista, Leucaena, Lotus, Phaseolus, Prosopis, Robinia and Sophora, including various species of Astragalus (Willems, 2014). The broad distribution of Mesorhizobium strains, allied with the ability to establish symbiotic relationships with many
genera of legumes, makes it promising for use in agriculture (Helene, Dall'Agnol, Delamuta and Hungria, 2019).
Arctic rhizobia are of interest for studying the evolutionary development of nitrogen-fixing bacteria and their adaptation to low temperatures, and also make it possible to analyze the functional relations between rhizobia and leguminous plants in isolated indigenous populations of the North (Caudry-Reznicket, Prevost and Schulman, 1986). However, the biodiversity of root nodule bacteria in the Arctic territories and their symbiotic interactions with legumes are practically not studied in Russia.
The purpose of this work was to study the biodiversity of bacterial isolates in samples of the active layer of permafrost soil of Spitsbergen (including rhizobia, capable of forming a nitrogen-fixing symbiosis with legumes), as well as to study the ability of the obtained rhizobial isolates to form root nodules with Arctic legume plants.
Materials and methods
Seeds of legumes Astragalus norvegicus, A.frigidus, A. subpolaris and Oxytropis sordida were collected in Kh-ibiny (Kola Peninsula, Russia) in 2018. The coordinates of the collection sites are from 67°43'8.7" to 67°48'6.9" N and from 33°35'39.1" to 33°36'24.1" E.
Two samples of frozen soil were collected in 2018 by aseptic drilling of borehole 10 (56 m above sea level (a.s.l.), 77.99332° N, 14.66114° E) on the slope of pingo Fili in Grondalen valley in the vicinity of the Russian mine Barentsburg on West Spitsbergen (Demidov et al, 2019). Frozen conditions of cores were maintained during drilling, sampling and transportation to the laboratory. Modern mean annual air temperatures equals -2.2 °C, permafrost temperature at zero amplitude depth amounts to -3.56 °C and the active layer thickness amounts to approximately 1.5 m in this part of West Spitsbergen.
The uppermost part of borehole 10 from 0 to 2.5 m below surface (b.s.) includes the modern top soil at 0 to 0.1 m b.s. with living shrub material and a buried soil formation at 0.25 to 0.4 m b.s. with decomposed similar shrub material. Both modern and buried soil were sampled and used in this study (Table 1). The minerogenic material was characterised by fine sand and loam, including gravel. The cryostructures were wavy lenticular with
Table 1. Cryolithological description of cores from borehole 10 used in this study
№ sample Depth, m Cryolithological description
1 0.0-0.1 Modern top soil with living shrub material, fine sand and loam with wavy lenticular cryostructures including gravel.
2 0.3-0.39 Buried soil with decomposed shrub material, fine sand and loam with wavy lenticular cryostructures including gravel.
ice lenses up to 2 cm thick. Toward 2.5 m b.s. the clay content increased as the gravel content decreased. The ion content of dry residue was about 55 mg/l, with predominantly HCO3- and Ca2+. The pH was neutral (6.8).
Isolation of pure cultures of microorganisms
To isolate microorganisms, we used the original and 10-fold diluted modified yeast extract mannitol agar (YMA, Vincent, 1970) supplemented with 0.5 % succinate (YMSA, Safronova et al., 2015) for the cultivation of root nodule bacteria; Tryptic Soy Broth (TSB, Sigma, USA) supplemented with 20 g/L agar for het-erotrophs; R2A agar medium (Oxoid, UK) for oligo-trophs (Vishnivetskaya et al., 2000); meat peptone agar (GMF, NICF, Russia) for mesophilic microorganisms; Ashby's medium for nitrogen-fixing (oligonitrophils) microorganisms (Egorov, 1976). The cultivation was carried out at a temperature of 22 °C for two weeks, and results were recorded starting from the second day. To determine the number of colony-forming units (CFU), 1 g of soil was suspended in 10 ml of sterile water, followed by 10-fold dilutions and plating on solid nutrient media.
All strains were deposited in the Russian Collection of Agricultural Microorganisms (RCAM, WDCM 966) and stored at -80 °C in the automated Tube Store (Li-conic Instruments, Lichtenstein) as described previously (Safronova and Tikhonovich, 2012). Information on isolates is available in the online RCAM database (http:// www.arriam.spb.ru.).
Identification of the isolates and bioinformatic analysis
Identification of the isolates was determined by amplification, purification of the PCR product, and sequencing of the 16S rRNA gene, as described previously (Safronova et al., 2015, 2019). The DNA fragment was sequenced using an ABI PRISM 3500xl genetic analyzer (Applied Biosystems, USA).
The search for homologous sequences and related type strains was performed using the NCBI GenBank database (https://www.ncbi.nlm.nih.gov) and the BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The phylogenetic tree was constructed using the MEGA7 program and the neighbour-joining method (Tamura et al., 2011). The evolutionary distances were computed using the maximum composite likelihood model.
The rrs sequences were deposited to the NCBI GenBank database under accession numbers: MT912817-MT912845.
Sterile test-tube experiment
Seeds of leguminous plants Astragalus norvegicus, A. frigidus, A. subpolaris and Oxytropis sordida were surface sterilized by treatment with H2SO4 for 5 min. The treated seeds were rinsed carefully with sterile water and germinated on filter paper in Petri dishes at +25 °C in the dark for 3 days. Seedlings 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, H3BO3 0.005, (NH4)2MoO4 0.005, ZnSO4 x 7 H2O 0.005, MnSO4 0.002 (Novikova and Safronova, 1992). Seedlings were inoculated with Mesorhizobium isolates 1Y-1, 1Y-3, 1Y-5, 1Y-8, 1G-2 and 1G-4 in the approximate amount of 106 cells per tube as well as with two soil extracts from different horizons of the active layer of permafrost soil of borehole 10 (Spitsbergen). The uninoculated plants were used as negative control. The plant nodulation assay was carried out in duplicate. Plants were cultivated for 35 days in the growth chamber with 50 % relative humidity and four levels of illumination and temperature: night (dark, 18 oC, 8 h), morning (200 ^mol m-2 s-1, 20 oC, 2 h), day (400 ^mol m-2 s-1, 23 oC, 12 h), evening (200 ^mol m-2 s-1, 20 oC, 2 h). Illumination was performed by L 36W/77 FLUORA lamps (Osram, Germany).
Amplification of nodC and nodD genes in rhizobial isolates
To reveal the potential ability of Mesorhizobium isolates to nodulate, the symbiotic nodC and nodD genes were amplified. For nodC genes, primers were used: nodCF (5'- AYGTHGTYGAYGACGGTTC — 3') / nodCI (5'-CGYGACAGCCANTCKCTATTG — 3') (Laguerre et al., 2001) and the PCR protocol as described earlier (Wei, Young and Bontemps, 2009). For nodD genes, primers were used: (5'- GCGAACGYWTTCTGACACC- 3') / (5'- TCSGTAAATSCSGGAAG — 3') and the PCR protocol as described earlier (Ji et al., 2015). The strains Mesorhizobium japonicum Opo-235 and Opo-242, as well as the strain M. kowhaii Ach-343 (Safronova et al., 2018; 2019) were used as reference strains.
Multi-substrate testing
The enzymatic activities of the isolates were studied using the GENIII MicroPlate microassay system (BioLog, USA), which analyzes the ability of bacteria to metabolize 71 carbon sources and resistance to 23 chemicals. For the test analysis, isolates belonging to the bacterial genera presented in the GEN III database were selected. The analysis was performed according to the manufacturer's recommendations, with the exception of the extended incubation period for strains 2G-2 and 2R-3 (2 days).
Results and discussion
Isolation of pure cultures of microorganisms
The number of microorganisms varied within 1.4-1061.7-107 CFU-g-1 (sample 1) and 4.0-10M.1-105 CFU^g1 (sample 2) depending on the medium and the dilution rate. The data obtained are comparable with the results of previous studies on the number of microorganisms in the active layer of the permafrost soil of Spitsbergen (Singh et al., 2017; Trubitsyn et al., 2019). For sample 1, the maximum number of CFU/g was detected on YMSA, 1/10 R2A and 1/10 TSA; for sample 2 — on YMSA medium.
Due to the use of various nutrient media, it was possible to expand the range of isolated bacteria. Representatives of four bacterial taxa (Streptomyces filde-sensis, Arthrobacter humicola, Mesorhizobium sp. and Pseudomonas sp.) were isolated on two or more media. The other strains were isolated on one medium: on
YMSA — representatives of the genera Rhodococcus, Ko-curia, Citricoccus, Sphingomonas, Mucilaginibacter and Planococcus; on R2A — Streptomyces, Streptacidiphilus and Arthrobacter; on GMF — Arthrobacter; on TSA — Luteibacter; on Ashby — Caballeronia.
Phylogenetic analysis
A total of 29 bacterial isolates were obtained, grouped into 13 genera and 16 species (Table 2). Sequence analysis of the rrs gene of the obtained isolates showed that they belong to the following bacterial phyla: Actino-bacteria, Proteobacteria (classes a, p and y), Bacteroide-tes and Firmicutes. The most numerous (both in the number of isolated strains and in the number of genera) was the phylum Actinobacteria, represented by the genera Arthrobacter (6 strains), Streptomyces (5 strains), as well as Streptacidiphilus, Rhodococcus, Kocuria, and Citricoccus (1 strain each). The phylum Proteobacte-ria was represented by the genera Mesorhizobium and
Table 2. The similarity between the isolates obtained in the work and the closest type strains based on the 16S rRNA gene sequencing
Isolate number* Closest type strain Isolation source Similarity, % Phylogenetic group Accession number
1R-6,1G-1, 1A-1, 1T-1 Streptomyces fildesensis GW25-5T Soil of Antarctica 99.3699.63 DQ408297
1Y-4 Rhodococcus qingshengii JCM 15477T Carbendazim-contaminated soil 99.36 DQ090961
1R-3 Streptomyces paucisporeus 1413T Forest soil 98.10 AY876943
1R-4 Streptacidiphilus durhamensis FSCA67T Forest soil 98.13 NR_125637
2A-2, 2G-1, 2R-2, 2Y-4 Arthrobacter humicola KV-653T Soil 99.93-100 Actinobacteria AB279890
2G-2 Arthrobacter humicola KV-653T Soil 98.27 AB279890
2R-3 Arthrobacter oryzae KV-651T Soil 98.56 AB279889
2Y-1 Kocuria rosea DSM 20447T Soil 100 X87756
2Y-2 Citricoccus nitrophenolicus PNP1T Citricoccus alkalitolerans YIM 70010T Sludge from a wastewater treatment plant Desert soil 100 100 GU797177 AY376164
1Y-1, 1Y-3, 1Y-5, 1Y-8, 1G-2, 1G-4 Mesorhizobium sp. CCANP61 Mesorhizobium qingshengii CCBAU 33460T Mesorhizobium shangrilense CCBAU 65327T Nodules Cicer canariense Nodules Astragalus sinicus Nodules Caragana bicolor 100 99.92 99.85 Alphaproteobacteria HF931055 NR_109565 NR_116163
1Y-7 Sphingomonas panacis DCY99T Rhizosphere Panax ginseng 99.05 NR_146850
1A-2, 1A-4 Caballeronia udeis Hg2T Caballeronia sordidicola S5-BT PAH** — contaminated soil fungus Phanerochaete sordida 98.81 98.60 Beta proteobacteria NR_125500 NR_104563
1T-2 Luteibacter anthropi CCUG 25036T Human blood 99.28 NR_116911
2A-1, 2R-1 Pseudomonas frederiksbergensis JAJ28T Soil 99.02 Gammaproteobacteria AJ249382
1Y-6 Mucilaginibacter dorajii DR-f4T Rhizosphere Platycodon grandi-florus 98.15 Bacteroidetes GU139697
2Y-3 Planomicrobium soli XN13T Soil 99.47 Firmicutes NR_134133
* Strains isolated on media: R — R2A; G — GMF; A — Ashby; T — TSA; Y — YMSA
**PAH — polycyclic aromatic hydrocarbon
51
76
96
65
1Y-1 (MT912825) 1Y-3 (MT912826) 1Y-5 (MT912827) 1Y-8 (MT912828) 1G-2 (MT912830) 1G-4 (MT912831)
Mesorhizobium sp. CCANP61 (HF931055) Mesorhizobium shangrilense CCBAU 65327T (NR_116163)
-Mesorhizobium australicum WSM20731 (NR_115281)
63
Mesorhizobium sp. S23442 (D84631)
Mesorhizobium qingshengii CCBAU 33460T (NR109565)
92
52
Mesorhizobium cantuariense ICMP 19515T (NR 137373) Mesorhizobium ciceri UPM-Ca7T (NR 025953) -Mesorhizobium loti NZP 2213T (NR 025837)
77
Mesorhizobium newzealandense ICMP 19545T (NR 148639) — Mesorhizobium sangaii SCAU7T (NR_ 108202)
60
67
Mesorhizobium waitakense ICMP 19523T (NR 148637) Mesorhizobium sophorae ICMP 19535T (NR 148638)
82
-Mesorhizobium albiziae CCBAU 61158T (NR 043549)
941Mesorhizobium gobiense CCBAU 83330T (NR 044052)
58
80
Mesorhizobium tianshanense A-1BST (NR 024880) -Mesorhizobium caraganae CCBAU 11299T (NR 044118)
92
la
lb
Mesorhizobium mediterraneum UPM-Ca36T (NR_118684) -Mesorhizobium robiniae CCNWYC 115T (NR_116467)
71
94
Mesorhizobium abyssinicae AC98cT (NR_108621) -Mesorhizobium acaciae RITF7411 (NR 137366)
L Mesorhizobium amorphae ACCC 19665T (NR 024879) -Mesorhizobium huakuii IFO 15243T (NR 043390)
■Mesorhizobium jarvisii ATCC 33669T (KM192335)
-Mesorhizobium opportunistum WSM20751 (NR_115280)
Mesorhizobium erdmanii USDA 3471T (NR_135857) Mesorhizobium japonicum Opo-242 (MF661788) -Mesorhizobium camelthorni A38 (JX298859)
80
87
0.0020
Fig. 1. Phylogenetic tree generated by the neighbour-joining method using partial 16S rRNA gene sequences of the strains isolated from active layer of permafrost soil and representatives of closely related Mesorhizobium species. The isolated strains are in bold. Type strains are indicated by the letter T. Subclusters la and Ib are formed by Mesorhizobium isolates obtained in this work and representatives of closely related species. Bootstrap values of more than 50% are given.
Table 3. Phenotypic properties of some strains obtained in the study
Phenotypic property 1Y-4 2A-2 2G-2 2R-3 2A-1 2R-1 2Y-1 Phenotypic property 1Y-4 2A-2 2G-2 2R-3 2A-1 2R-1 2Y-1
Growth at: Utilization of:
pH 6 + + + + + + - D-Fructose-6-phosphate - + - - - - -
pH 5 +
D-Aspartic Acid + + + +
1 % NaCl + + + + + +
Glycyl-L-Proline +
4% NaCl
L-Alanine + + +
8 % NaCl
L-Arginine + + + + +
Utilization of:
L-Aspartic Acid + + + + +
D-Maltose + +
L-Glutamic Acid + + + + +
D-Trehalose + +
L-Histidine + +
D-Cellobiose + +
L-Pyroglutamic Acid + + + +
Sucrose + + + - + + -
D-Turanose + + - + - - - L-Serine + - + - + + -
Stachyose + + + - - - - Pectin - + - - + + -
D-Raffinose + + - + - - - D-Galacturonic Acid - + - - + + -
a-D-Lactose + +
L-Galactonic Acid Lactone
ß-Methyl-D-Glucoside - - + - - - - + +
D-Gluconic Acid
N-Acetyl-D-Glucosamine + -
D-Glucuronic Acid + + - - + + -
N-Acetyl-D-Galactosamine - - + - + - - Glucuronamide + - - - + + -
Mucic Acid + + + +
N-Acetyl Neuraminic Acid + +
Quinic Acid + - + - + + -
a-D-Glucose - + - - + + - D-Saccharic Acid + + - - + + -
D-Mannose - + + + - - - p-Hydroxy-Phenylacetic Acid - - - - + + -
D-Fructose + + + +
D-Lactic Acid Methyl Ester +
D-Galactose - + - - + - -
3-Methyl Glucose + + - - - - - D-Lactic Acid - + + - + + -
D-Fucose - + - + - - - Citric Acid + + - - + + -
L-Fucose - + + - - - - a-Keto-Glutaric Acid - + + - + + -
L-Rhamnose +
D-Malic Acid
1 % Sodium Lactate + + + + - - - -
L-Malic Acid + - + - + + -
Fusidic Acid - - - - - - - Bromo-Succinic Acid - - + - - - -
D-Serine +
Tween 40
D-Sorbitol + + + + + - -
y-Amino-Butryric Acid
D-Mannitol - + - + + - - + + + +
D-Arabitol - - + - + + - a-Hydroxy-Butyric + + - - - - -
myo-Inositol + + + + Acid
(3-Hydroxy-D, L-Butyric Acid
Glycerol - - + + + + - + +
Phenotypic property 1Y-4 2A-2 2G-2 2R-3 2A-1 2R-1 2Y-1
Utilization of:
a-Keto-Butyric Acid + + - + - - -
Acetoacetic Acid + - - + + - +
Propionic Acid + + - - + - +
Acetic Acid + + + + + + +
Formic Acid + - - - - - -
Resistance to:
Troleandomycin - - - - + + -
Rifamycin SV - - - - + + -
Minocycline - - - - - - -
Lincomycin - - - - + + -
Guanidine HCl - - - - - - -
Niaproof4 - - - - + + -
Vancomycin - - - - + + -
Nalidixic Acid + + + + - - +
Aztreonam + + + - - - -
Lithium Chloride + - - - - - -
Potassium Tellurite + - - + + + -
Sodium Butyrate + - - - - - +
Tetrazollium Violet - - - - + + -
Tetrazollium Blue - - - - + + -
Sphingomonas (6 and 1 strain, respectively) related to class a-Proteobacteria, by the genera Caballeronia and Luteibacter (2 and 1 strain, respectively) related to class ß-Proteobacteria, as well as by the genus Pseudomonas (2 strains) related to class y-Proteobacteria. The phyla Bacteroidetes and Firmicutes were represented by the genera Mucilaginibacter and Planomicrobium, respectively (1 strain each).
From sample 1, 18 strains were isolated, while 11 strains were isolated from sample 2 (Table 2). There were no coincidences at the genus level between the isolates of both samples. In sample 1, the greatest number of strains belonged to the genera Mesorhizobium and Streptomyces (6 and 5 strains, respectively), while in sample 2 most of the strains were affiliated to the genus Arthrobacter (6 strains).
Isolates 1Y-1, 1Y-3, 1Y-5, 1Y-8, 1G-2, and 1G-4 showed 100 % similarity of the rrs gene to the strain Mesorhizobium sp. CCANP61 (family Phyllobacteriaceae) isolated from the root nodule of the legume plant Ci-
cer canariense, an endemic to the Canary Islands (Armas-Capote et al., 2014). The closest type strains to this group were Mesorhizobium qingshengii CCBAU 33460T and Mesorhizobium shangrilense CCBAU 65327T, which showed a high rate of rrs-similarity to the obtained isolates (99.92 and 99.85 %, respectively). On the phy-logenetic tree (Fig. 1) isolates 1Y-1, 1Y-3, 1Y-5, 1Y-8, 1G-2, 1G-4 and strain Mesorhizobium sp. CCANP61 formed the single subcluster Ia; Mesorhizobium shangrilense CCBAU 65327T and Mesorhizobium australicum WSM2073T formed subcluster Ib. The levels of support of these subclusters were low (45 and 65 %, respectively). Both subclusters were grouped together with a 51 % support level. The type strain Mesorhizobium qingshengii CCBAU 33460T previously isolated from the root nodule of the legume plant Astragalus sinicus did not form a cluster with the obtained isolates. It was shown that this strain contains the symbiotic genes nifH and nodC and effectively nodulates the host plant, which is traditionally used as a green manure in wintry fallow paddy fields (Zheng et al., 2013). The strain Mesorhizobium shangrilense CCBAU 65327T was isolated from the root nodule of Caragana bicolor growing in China. Strain CCBAU 65327T was able to nodulate a wide range of plant species of genera Caragana, Glycyrrhiza, Astragalus, Vigna and Phaseolus (Lu et al., 2009).
Isolates 2G-1, 2A-2, 2R-2, 2Y-4 showed a high level of rrs-similarity (99.93-100 %) with the type strain Ar-throbacter humicola KV-653T, isolated from a paddy soil sample in Saitama prefecture in Japan (Kageyama, Morisaki, Omura and Takahashi, 2008), while the isolate 2Y-1 was identified as Kocuria rosea (100 % rrs-similarity with the type strain DSM 20447T) (Stackebrandt, Koch, Gvozdiak and Schumann, 1995).
Isolate 2Y-2 showed a 100 % similarity with the type strains Citricoccus nitrophenolicus PNP1T and Citricoc-cus alkalitolerans YIM 700101- The strain PNP1T was isolated from sludge from a wastewater treatment plant at a chemical factory producing pesticides in Denmark. It has been shown that this strain can utilize the xenobiotic para-nitrophenol (pNP), which is a product of human activity and a common pollutant in the environment (Nielsen, Kjeldsen and Ingvorsen, 2011). The strain Cit-ricoccus alkalitolerans YIM 70010T was isolated from desert soil in Egypt (Li et al., 2005). The rest of the isolates showed a low level of rrs-similarity (98.1-99.47 %) to the closest type strains.
Multi-substrate testing of isolates
Seven isolates were tested for the ability to utilize the major classes of carbon sources and for resistance to different inhibiting chemicals (Table 3). Each strain had a unique metabolic profile. All strains grew at 1 % (w/v)
1Y-1 1Y-3 1Y-5 1Y-8 1G-2 1G-4 235 242 343
Fig. 2. Agarose gel electrophoresis of nodC PCR-products from rhizo-bial isolates Mesorhizobium and reference strains. 235 — M.japonicum Opo-235; 242 — M.japonicum Opo-242; 343 — M. kowhaii Ach-343
Fig. 3. Agarose gel electrophoresis of nodD PCR-products from rhizo-bial isolates Mesorhizobium and reference strains. 235 — M.japonicum Opo-235; 242 — M.japonicum Opo-242; 343 — M. kowhaii Ach-343
NaCl and pH 6 with the exception of 2G-2 and 2Y-1. Growth at pH 5 was only in strain 2R-1. Isolate Arthro-bacter sp. 2G-2 was able to utilize P-methyl-D-glucoside, N-acetyl-D-glucosamine, glycyl-L-proline, D-lactic acid methyl ester and bromo-succinic acid; isolate of Arthro-bacter sp. 2A-2 utilized L-rhamnose and D-fructose-6-phosphate and isolate Rhodococcus sp. 1Y-4 utilized fu-sidic and formic acids.
Strains Pseudomonas sp. 2A-1 and 2R-1 had nearly identical phenotypic profiles (Table 3).
The common assimilated substrates for isolates Ar-throbacter spp. 2A-2, 2G-2 and 2R-3 were D-mannose, D-gluconic acid and acetic acid. All strains grew at pH 6 and in the presence of Tween 40, and also were sensitive to nalidixic acid.
Isolates Pseudomonas sp. 2A-1 and Arthrobacter humi-cola 2A-2 had the largest range of utilized substrates (42 and 38 substrates, respectively), isolate Kocuria rosea 2Y-1 revealed the smallest spectrum (3 substrates). Isolate 2Y-1 assimilated acetoacetic acid, propionic acid and acetic acid.
Sterile test-tube experiment and the search for nodC and nodD genes in Mesorhizobium isolates
To study the nodulating activity of Mesorhizobium isolates 1Y-1, 1Y-3, 1Y-5, 1Y-8, 1G-2 and 1G-4, a sterile test-tube experiment with legume plants growing in the Murmansk region (Astragalus norvegicus, A. frigidus, A. subpolaris u Oxytropis sordida) was performed. The obtained result showed the lack of ability of isolates to form nodules on the roots of these plant species. The presence in Mesorhizobium-related isolates of symbiotic genes nodC and nodD, which belong to the common nodulation genes, was not revealed (Figs. 2 and 3).
Conclusions
In total, 29 isolates were isolated from samples of the active layer of Spitsbergen permafrost due to the use of a wide range of nutrient media. The isolates belonged to the phyla Actinobacteria, Proteobacteria (classes a, P
and y), Bacteroidetes and Firmicutes, and represented 13 genera and 16 species. Most of the isolated strains had a low level of rrs-similarity (less than 99.5 %) with the closest type strains, which indicates they may possibly belong to new species of microorganisms. A number of strains belong to the practically valuable groups of microorganisms that promote plant growth (genera Arthrobacter and Caballeronia) (Chung et al., 2010; Pa-laniappan et al., 2010) or biodegradation of xenobiotics (genus Citricoccus) (Nielsen, Kjeldsen and Ingvorsen, 2011). The multi-substrate analysis of the isolates revealed a significant variety of their phenotypic profiles. At the same time, some strains had a number of features (growth in the presence of antibiotics and heavy metal salts, acid and salt tolerance) that can be of practical use. Despite the fact that most of the isolates belong to non-symbiotic bacteria, six isolates belonging to root nodule bacteria Mesorhizobium were isolated. To clarify the species affiliation of the Mesorhizobium isolates, additional analysis of the "housekeeping" genes is required. Although representatives of this genus are known as symbiotic microorganisms, nodulating a wide range of legumes, the isolated Mesorhizobium strains did not form nodules on the roots of the Arctic species Astragalus norvegicus, A. frigidus, A. subpolaris or Oxytropis sordida. The presence of the common nodulation genes nodC and nodD was also not detected in the obtained isolates. The negative results of the plant nodulation assay and amplification of nod-genes in these isolates could be associated with either the actual absence of these genes or their different structure. For further study of the nodulation capacity of rhizobial isolates, it is necessary to obtain their whole genome sequences and analyze symbiotic genes. The data obtained make it possible to consider the collection of strains isolated from the active layer of permafrost soil of Spitsbergen as a promising object for further research.
The selection of frost-resistant and effective strains of nodule bacteria and soil microorganisms, and analysis of the whole genomic sequences of the obtained isolates, will allow us to evaluate their agricultural potential
for the formation of new pasture phytocenoses and for the restoration of previously disturbed lands in the Arctic regions of Russia.
Acknowledgements
The authors are grateful to L.Yu. Shipilina of the N. I.Vavilov All-Russian Institute of Plant Genetic Resources, for kindly collecting and providing seeds of legume plants originated from Murmansk region.
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