The genetic diversity of microsymbionts from Thermopsis lanceolata growing in Mongolia

Cover Page
  • Authors: Karlov D.S.1, Sazanova A.L.1, Kuznetsova I.G.1, Safronova V.I.1, Tikhomirova N.Y.1, Popova Z.P.1, Osledkin Y.S.1, Verkhozina A.V.2, Belimov A.A.1
  • Affiliations:
    1. All-Russia Research Institute for Agricultural Microbiology
    2. Siberian Institute of Plant Physiology and Biochemistry (SIPPB)
  • Issue: Vol 17, No 1 (2019)
  • Pages: 43-51
  • Section: 1. Genetic basis of ecosystems evolution
  • URL: https://journals.eco-vector.com/ecolgenet/article/view/10336
  • DOI: https://doi.org/10.17816/ecogen17143-51

Abstract


For the first time, bacteria were isolated and identified from the root nodules of a wild-growing medicinal legume plant Thermopsis lanceolata, originated from Mongolia. The taxonomic position of 14 isolates obtained was determined using of sequencing of the 16S rRNA (rrs) and atpD genes. It was shown a significant biodiversity of the isolates from T. lanceolata, which belonged to three genera of the order Rhizobiales: Phyllobacterium (family Phyllobacteriaceae), Rhizobium (family Rhizobiaceae) and Bosea (family Bradyrhizobiaceae). Six isolates belonged to the species Phyllobacterium zundukense and Phyllobacterium trifolii (100 и 99,9% rrs similarity with the type strains P. zundukense Tri-48T and P. trifolii PETP02T, respectivelly), three isolates were identified as Rhizobium anhuiense (99,8% rrs similarity with the type strain R. anhuiense CCBAU 23252T). Two slow-growing isolates of the genus Bosea Tla-534 and Tla-545 may potentially belong to new species, since their rrs-similarity to the closest type strains B. massiliensis LMG 26221T, B. lathyri LMG 26379T and B. vaviloviae Vaf18T was 98,5-99,0%. Non-rhizobial strains were not isolated. The isolation and future investigation of the rhizobial microsymbionts of the valuable medicinal legume Thermopsis lanceolata is one of the necessary prerequisites for its industrial cultivation.


Legume-rhizobium symbiosis is a unique and widespread phenomenon among leguminous plants. Nodule bacteria (Rhizobia), which are the integral elements of such symbiotic activity, are used to explore such interactions and reveal the mechanisms of plant–microbe interactions. Leguminous plants have high morphophysiological and ecological diversity and contribute a great deal to nitrogen balance in numerous land ecosystems and agrocenosis. This is achieved by broadening the range of biochemical function in both symbiotic partners and obtaining novel adaptive features of the plants [1]. Therefore, studying legume-rhizobium symbiosis has significant ecological and practical value.

Thermopsis lanceolata, R. Br. is a perennial wild leguminous plant growing in West and East Siberia, Baikal region, Middle Asia, North of Mongolia, and China [2]. The plant contains numerous alkaloids and has various pharmacological applications. One of the major biologically active alkaloids is cytosine, which is mostly present in the seeds [3] and is used in nicotine addiction treatment (Tabex) [4] and as an expectorant drug (Thermopsol and Codelac Broncho). It is also widely applied in veterinary medicine (Cytitonum) [5]. Current literature does not report any studies on extraction of nodule bacteria from T. lanceolata. In addition, to effectively introduce the plants, it is necessary to collect its microsymbionts for the production of growth-promoting agents. The first stage of microsymbiont strain studying, which is their identification, requires the use of modern molecular genetic methods, such as sequencing of 16S rDNA and “housekeeping” genes.

Therefore, the goal of the present study is to prepare a collection of rhizobial microsymbionts of the wild-growing medical leguminous plant, T. lanceolata, growing in North Mongolia, and to determine the taxonomic status of the strains by sequencing 16S rDNA and the “housekeeping” gene atpD.

MATERIALS AND METHODS

The study materials were 14 bacterial isolates extracted from the root nodules of a leguminous plant, T. lanceolata, which were collected from the mountain-taiga region of Mongolia (vicinity of the lake Khubsugul, right bank of the river Eg-Gol, village Alag-Erdene), according to the standard method [6]. Bacteria were cultured on modified mannitol-yeast agar YMSA with 0.5% succinate [7]. One isolate was selected from each nodule. Species affiliation of the isolates was determined by amplifying and sequencing the 16S rRNA genes as previously described [7]. To specify the taxonomic status of the isolates, amplification and sequencing of atpD, which codes for β-subunit of the ATP-synthesis complex, were performed using atpD-273F/atpD-771R [8] and atpD352F/atpD871R [9] primers. The obtained PCR-product was extracted from gel and purified as previously described [10] for further sequencing on ABI PRISM 3500xl genetic analyzer (Applied Biosystems, USA). Search for homologous sequences was performed using the NCBI GenBank database (https://www.ncbi.nlm.nih.gov) and BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). MEGA7 (MEGA, PA, USA) and neighbor-joining method [11] were used for the construction of phylogenetic trees. Nucleic acid sequences obtained were deposited in the GenBank database under the accession numbers MH779890–MH779903 and MK135051–MK135064.

All obtained isolates were deposited in the Russian Collection of Agricultural Microorganisms (RCAM, WDCM 966) and placed at the Station for Low-Temperature Automated Storage of Biological Samples (Liconic Instruments, Liechtenstein) [12]. Information on isolates is available in the Internet database of RCAM (http://www.arriam.spb.ru.).

RESULTS AND DISCUSSION

The vicinity of Lake Khusbugul, where plants were collected for further extraction of bacteria from the root nodules, has a distinctly continental climate and the soils exist under aridization and cryodization conditions [13]. The soil at collection site can be characterized as gray humus lithozem [14], and the soil pH is acidic and neutral [15]. As a result of analysis of nodules collected from different T. lanceolata plants within a population, 14 bacterial isolates were obtained: two of them formed colonies on the 5–6th day; three of them on the 3rd day; and nine of them on the 4–5th day.

Analysis of 16S rRNA (rrs) gene sequences allowed to refer of obtained isolates to three genera in the order Rhizobiales: Phyllobacterium (fam. Phyllobacteriaceae), Rhizobium (fam. Rhizobiaceae), and Bosea (fam. Bradyrhizobiaceae). Figure 1 shows that the strains belonging to genera Phyllobacterium and Rhizobium form two separate groups. Strains of Phyllobacterium, however, are divided in three clusters. Rrs-cluster I included isolates Tla-531, Tla-536, Tla-538, Tla-546, and Tla-549, in addition to type strains P. trifolii PETP02T and P. loti S658T at 100% support level (see Fig. 1). Similarity of isolates based on gene rrs with specified type strains was 99.9% and 100% accordingly, whereas the gene sequences of the isolates were identical (Table 1). However, a phylogenetic tree constructed based on the analysis of gene atpD revealed that the isolates were split into two statistically reliable clusters (Fig. 2). Cluster Ia was formed at 100% support level and consisted of isolates Tla-531, Tla-546, Tla-549, and a type strain P. trifolii PETP02T, whose similarity based on atpD gene was 100% (see Table 1). Therefore, based on the results of atpD sequencing, isolates Tla-531, Tla-546, and Tla-549 were identified as P. trifolii. It should be noted that bacteria of this type werefirst isolated from the nodules of Trifolium pretense [16]. Previously, it was reported that the strain P. trifolii PETP02T could establish symbiotic interactions with Trifolium repens and Lupinus albus [16, 17]. In addition, bacteria that are closely related to species P. trifolii, with 99.9% similarity based on rrs were isolated from nodules of Onobrychis viciifolia [18], which indicates that there is a wide range of host plants for the group of nodule bacteria under study. Cluster Ib was formed by isolates Tla-536 and Tla-538, identified as Phyllobacterium sp., and were most similar to P. loti and P. trifolii (see Fig. 1, 2; Table 1).

 

Fig. 1. Phylogenetic tree generated by the neighbour-joining method using partial 16S rRNA gene sequences of the isolated strains from Thermopsis lanceolata nodules and representatives of closely related to Phyllobacterium and Rhizobium species. The isolated strains in bold. Type species are indicated by the letter T. I–III clusters that formed by Phyllobacterium isolates obtained in the work. Bootstrap values more than 50% are given

 

Fig. 2. Phylogenetic tree generated by the neighbour-joining method using atpD gene sequences of the isolated strains from Thermopsis lanceolata nodules and representatives of closely related to Phyllobacterium and Rhizobium species. The isolated strains in bold. Type species are indicated by the letter T. Iа, Ib, II и III – clusters that formed by Phyllobacterium isolates obtained in the work. Bootstrap values more than 50% are given

 

Table 1

The similarity of 16S rRNA and atpD genes between the isolated strains from Thermopsis lanceolata nodules and the type strains of closely related to the Bosea and Rhizobium species

Type strains

Loci

Isolates similarity (%)

Cluster I

Cluster II

Cluster III

Tla-531

Tla-536

Tla-538

Tla-546

Tla-549

Tla-540

Tla-537

Tla-543

Tla-544

P. trifolii PETP02T

16S рРНК

99.9

99.9

99.9

99.9

99.9

98.3

98.9

98.9

98.9

 

atpD

100

93.3

93.4

100

100

89.5

89.0

89.0

89.0

P. loti S658T

16S рРНК

100

100

100

100

100

98.4

99.0

99.0

99.0

 

atpD

96.7

92.6

92.6

96.7

96.7

89.3

87.7

87.7

87.7

P. bourgognense STM 201T

16S рРНК

99.1

99.1

99.2

99.2

99.2

98.8

99.4

99.3

99.3

 

atpD

88.9

89.6

89.6

88.9

88.9

89.6

91.8

91.8

91.8

P. brassicacearum STM 196T

16S рРНК

98.7

98.7

98.7

98.7

98.7

99.3

99.3

99.3

99.3

 

atpD

96.9

94.2

94.2

96.9

96.9

88.9

87.9

87.9

87.9

P. endophyticum PEPV15T

16S рРНК

98.1

98.1

98.7

98.7

98.7

99.2

99.5

99.4

99.4

 

atpD

90.8

90.8

90.1

90.8

90.8

89.8

89.6

89.6

89.6

P. zundukense Tri-48T

16S рРНК

99.0

99.0

99.0

99.0

99.0

99.3

100

99.9

99.9

 

atpD

89.0

89.8

89.8

89.0

89.0

89.3

100

100

100

P. sophorae CCBAU03422T

16S рРНК

98.3

98.3

98.5

98.5

98.5

99.1

99.0

99.2

99.2

 

atpD

88.9

89.4

89.4

88.9

88.9

87.3

91.5

91.5

91.5

 

Rrs-cluster II was formed at a low support level of 54% by strains P. sophorae CCBAU03422T, P. brassicacearum STM 196T, and isolate Tla-540, which, based on an atpD-dendrogram, was not grouped with any other strains (see Fig. 1, 2). Similarity of the isolate with the closest species, P. brassicacearum and P. zundukense, was 99.3%, based on rrs (see Table 1). Based on the analysis of the data obtained, isolate Tla-540 was identified as Phyllobacterium sp.

Rrs-cluster III combined isolates Tla-537, Tla-543, Tla-544, and type strain P. zundukense Tri-48T at a support level of 88% (see Fig. 1). On the atpD-dendrogram (see Fig. 2), the strains also formed a statistically reliable cluster (support level 100%). With regard to the high degree of homology based on rrs (99.9%–100%) and atpD (100%), isolates Tla-537, Tla-543, and Tla-544 were identified as P. zundukense (see Table 1), and have recently been described as microsymbionts of a relict leguminous plant, Oxytropis triphylla, growing in the Baikal region [19]. In addition, it has been demonstrated that strains of P. zundukense isolated from nodules of O. triphylla do not potentially carry out individual symbiosis as they do not have common nodABC genes required for plants nodulation [19]. Currently, genus Phyllobacterium is represented by only 11 species, most of which are isolated from the root nodules of leguminous plants [16, 19–23]. However, nodACD and nifH genes, which are required for effective symbiosis with host plants, have been found only in two species (P. trifolii and P. sophorae). Phyllobacterium trifolii and P. sophorae are able to form nodules independently on host plants [16, 23, and 24].

Fast-growing isolates, Tla-541, Tla-550, and Tla-552, belonged to genus Rhizobium and demonstrated similar levels of rrs-homology (99.8%) with three type strains, R. leguminosarum LMG 14904T, R. anhuiense CCBAU23252T, and R. laguerreae FB206T, with which they formed a common cluster with a support level of 98% (see Fig. 1, Table 2). On atpD-dendrogram, isolates Tla-541, Tla-550, and Tla-552 were clustered only with type strain R. anhuiense CCBAU23252T at a relatively high support level of 83% (see Fig. 2). Considering the significant similarity in atpD gene between strain R. anhuiense CCBAU23252T and isolates Tla-541, Tla-550, and Tla-552 (96.8%–97.0%), the isolates were identified as Rhizobium anhuiense (see Table 2). Strains of the species were isolated from nodules of Vicia faba and Pisum sativum growing in China [25]. R. anhuiense are also microsymbionts of Lathyrus japonicus [26]. It has been demonstrated that L. japonicus has a high capacity for nitrogen fixation, particularly under low temperature conditions associated with arctic and subarctic regions, where the plant is considered a prospective forage crop [27]. In general, genus Rhizobium represents the most representative group in the family Rhizobiaceae, with all species being able to fix atmospheric nitrogen and form stable symbioses with leguminous plants [28].

 

Table 2

The similarity of 16S rRNA and atpD genes between the isolated strains from Thermopsis lanceolata nodules and the type strains of closely related to the Bosea and Rhizobium species

Type strains

Loci

Isolates  similarity (%)

 

Type strains

Loci

Isolates  similarity (%)

Tla-534

Tla-545

Tla-541

Tla-550

Tla-552

B. massiliensis LMG 26221T

16S rRNA

99.0

98.6

R. leguminosarum LMG 14904T

16S rRNA

99.8

99.8

99.8

 

atpD

92.4

95.6

 

atpD

93.5

93.7

93.7

B. lathyri LMG 26379T

16S rRNA

99.0

98.5

R. anhuiense CCBAU23252T

16S rRNA

99.8

99.8

99.8

 

atpD

91.9

94.0

 

atpD

96.8

97.0

97.0

B. vaviloviae Vaf18T

16S rRNA

98.9

98.5

R. laguerreae FB206T

16S rRNA

99.8

99.8

99.8

 

atpD

91.0

92.2

 

atpD

95.7

95.9

95.9

 

Slow-growing isolates, Tla-534 and Tla-545, did not form statistically reliable groups with any other strains on either rrs- or atpD-dendrograms (Fig. 3, 4). Based on the homology levels of rrs (see Table 2), isolates Tla-534 and Tla-545 demonstrated maximum similarity with type strains of genus Bosea: B. massiliensis LMG 26221T, B. lathyri LMG 26379T, and B. vaviloviae Vaf18T (similarity 98.5–99.0%). Based on the obtained data, both isolates were identified as Bosea sp. With regard to the obtained results, one could assume that isolates Tla-534 and Tla-545 are novel species in genus Bosea. The genus consists of nine species, among which four species, B. lupini, B. lathyri, B. robiniae, and B. vaviloviae, were isolated from nodules of leguminous plants of genus Lupinus, Lathyrus, Robinia, and Vavilovia, respectively. However, the capacity of the strains to independently form symbioses has not yet been studied [7, 29, 30].

 

Fig. 3. Phylogenetic tree generated by the neighbour-joining method using partial 16S rRNA gene sequences of the isolated strains from Thermopsis lanceolata nodules and representatives of closely related to Bosea species. The isolated strains in bold. Type species are indicated by the letter T. Bootstrap values more than 50% are given

 

Fig. 4. Phylogenetic tree generated by the neighbour-joining method using atpD gene sequences of the isolated strains from Thermopsis lanceolata nodules and representatives of closely related to Bosea species. The isolated strains in bold. Type species are indicated by the letter T. Bootstrap values more than 30% are given

 

We obtained bacterial isolates from the root nodules of a leguminous plant, T. lanceolata, for the first time. Nodule bacteria, P. trifolii and и R. anhuiense, were identified, which form nodules on the plant, as well as bacteria P. zundukense, with the capacity to independently carry out symbiosis, which has not been previously reported. Isolates belonged to genus Bosea could be representatives of novel species of slow-growing nodule bacteria. It should be noted that to clarify the taxonomic status of nine out of 14 obtained isolates, analysis of gene atpD was used, which facilitated the description of novel species, P. zundukense and B. vaviloviae [7, 19]. Formation and study of collections of microsymbionts of Thermopsis lanceolata could facilitate the enhanced and effective industrial production of this valuable medical plant.

The work was performed as part of scientific project of FASO of Russia (Title No. 0664-2018-0001). Sequencing of atpD gene was conducted with RSF support (grant No. 16-16-00080). The long-term storage of strains was supported by the Program for the Development and Inventory of Bioresource Collections.

Denis S. Karlov

All-Russia Research Institute for Agricultural Microbiology

Author for correspondence.
Email: makondo07@gmail.com
SPIN-code: 8355-8091
ResearcherId: E-2552-2014

Russian Federation, 3, Podbelsky highway, Pushkin, Saint-Petersburg, 196608

PhD, Junior Researcher, Russian Collection of Agricultural Microorganisms

Anna L. Sazanova

All-Russia Research Institute for Agricultural Microbiology

Email: anna_sazanova@mail.ru

Russian Federation, 3, Podbelsky highway, Pushkin, Saint-Petersburg, 196608

PhD, Senior Researcher, Russian Collection of Agricultural Microorganisms

Irina G. Kuznetsova

All-Russia Research Institute for Agricultural Microbiology

Email: kuznetsova_rina@mail.ru

Russian Federation, 3, Podbelsky highway, Pushkin, Saint-Petersburg, 196608

Engineer-Researcher, Russian Collection of Agricultural Microorganisms

Vera I. Safronova

All-Russia Research Institute for Agricultural Microbiology

Email: v.safronova@rambler.ru

Russian Federation, 3, Podbelsky highway, Pushkin, Saint-Petersburg, 196608

PhD, Head, Russian Collection of Agricultural Microorganisms

Nina Y. Tikhomirova

All-Russia Research Institute for Agricultural Microbiology

Email: arriam2008@yandex.ru

Russian Federation, 3, Podbelsky highway, Pushkin, Saint-Petersburg, 196608

Researcher, Russian Collection of Agricultural Microorganisms

Zhanna P. Popova

All-Russia Research Institute for Agricultural Microbiology

Email: elestd@yandex.ru

Russian Federation, 3, Podbelsky highway, Pushkin, Saint-Petersburg, 196608

PhD, Senior Researcher, Russian Collection of Agricultural Microorganisms

Yuriy S. Osledkin

All-Russia Research Institute for Agricultural Microbiology

Email: arriam2008@yandex.ru

Russian Federation, 3, Podbelsky highway, Pushkin, Saint-Petersburg, 196608

PhD, Leading Researcher, Russian Collection of Agricultural Microorganisms

Alla V. Verkhozina

Siberian Institute of Plant Physiology and Biochemistry (SIPPB)

Email: allaverh@list.ru

Russian Federation, 132, Lermontova street, Irkutsk, 664033

PhD, Head of The Herbarium Group, Department of Terrestrial Ecosystems Resistance

Andrey A. Belimov

All-Russia Research Institute for Agricultural Microbiology

Email: belimov@rambler.ru

Russian Federation, 3, Podbelsky highway, Pushkin, Saint-Petersburg, 196608

DrSci, Head of Laboratory of Rhizosphere Microflora

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Supplementary files

Supplementary Files Action
1. Fig. 1. Phylogenetic tree generated by the neighbour-joining method using partial 16S rRNA gene sequences of the isolated strains from Thermopsis lanceolata nodules and representatives of closely related to Phyllobacterium and Rhizobium species. The isolated strains in bold. Type species are indicated by the letter T. I–III clusters that formed by Phyllobacterium isolates obtained in the work. Bootstrap values more than 50% are given View (205KB) Indexing metadata
2. Fig. 2. Phylogenetic tree generated by the neighbour-joining method using atpD gene sequences of the isolated strains from Thermopsis lanceolata nodules and representatives of closely related to Phyllobacterium and Rhizobium species. The isolated strains in bold. Type species are indicated by the letter T. Iа, Ib, II и III – clusters that formed by Phyllobacterium isolates obtained in the work. Bootstrap values more than 50% are given View (163KB) Indexing metadata
3. Fig. 3. Phylogenetic tree generated by the neighbour-joining method using partial 16S rRNA gene sequences of the isolated strains from Thermopsis lanceolata nodules and representatives of closely related to Bosea species. The isolated strains in bold. Type species are indicated by the letter T. Bootstrap values more than 50% are given View (164KB) Indexing metadata
4. Fig. 4. Phylogenetic tree generated by the neighbour-joining method using atpD gene sequences of the isolated strains from Thermopsis lanceolata nodules and representatives of closely related to Bosea species. The isolated strains in bold. Type species are indicated by the letter T. Bootstrap values more than 30% are given View (228KB) Indexing metadata

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Copyright (c) 2019 Karlov D.S., Sazanova A.L., Kuznetsova I.G., Safronova V.I., Tikhomirova N.Y., Popova Z.P., Osledkin Y.S., Verkhozina A.V., Belimov A.A.

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