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AGRICULTURAL BIOLOGY, ISSN 2412-0324 ffngfeh ed. Online)
2015, V. 50, № 3, pp. 345-352
(SEL’SKOKHOZYAISTVENNAYA BIOLOGIYA) ISSN 0131-6397 (Russian ed. Print)
v_____________________________________' ISSN 2313-4836 (Russian ed. Online)
UDC 631.461.51:577.2 doi: 10.15389/agrobiology.2015.3.345rus
doi: 10.15389/agrobiology.2015.3.345eng
GENETIC DIVERSITY AMONG MICROSYMBIONTS OF Lathyrus, Vicia, Oxytropis AND Astragalus LEGUME SPECIES FROM BAIKAL REGION
I.G. KUZNETSOVA1, A.L. SAZANOVA1, V.I. SAFRONOVA1, A.G. PINAEV1, A.V. VERKHOZINA2, N.Yu. TIKHOMIROVA1, Yu.S. OSLEDKIN1, A.A. BELIMOV1
{All-Russian Research Institute for Agricultural Microbiology, Federal Agency of Scientific Organizations, 3, sh. Podbel’skogo, St. Petersburg, 196608 Russia, e-mail [email protected];
2Siberian Institute of Plant Physiology and Biochemistry, Federal Agency of Scientific Organizations, 132,
ul. Lermontova, Irkutsk, 664033 Russia
Acknowledgements:
Authors are thankful to SIPPB SB RAS employees L.E. Makarova for her help in organizing the expedition to the Chivyrkuy Gulf Coast and A.A. Kiseleva for plant identification.
Supported in part by Russian Science Foundation (Agreement number 14-26-00094) (sequencing of the isolates from Vicia baicalensis)
Received March 30, 2015
Abstract
Rhizobia are Gram-negative soil microorganisms that form intracellular nitrogen-fixing symbiosis with leguminous plants. Investigations of symbiotic systems with the participation of endemic or relict species have a particular importance for understanding of the evolution of plant-microbe interactions. The purpose of our work was to create a representative collection of microsymbionts of endemic Baikal legumes, as well as to estimate their biodiversity. The study of taxonomic positions of 69 isolates from root nodules Lathyrus humilis, Vicia baicalensis, Astragalus mongholicus and Oxytropis sylvatica was conducted. For primary identification of these isolates the methods of ITS-RFLP analysis was used that divided strains into 33 groups with identical DNA-profile. Then the taxonomy positions of isolates were determined by the 16S rRNA gene (rrs) and ITS region sequencing. Phylogenetic analysis revealed the considerable genetic diversity among microsymbionts of plants studied. Rhizobial isolates belonged to 5 genera: Rhizobium (family Rhizobiaceae), Mesorhizobium and Phyllobacterium (family Phyilobacteriaceae), Bosea and Tardiphaga (family Bradyrhizobiaceae). In addition, non-rhizobial isolates belonging to the genera Herbiconiux, Leifsonia, Burkholderia and Stenotrophomonas were obtained. It is known that some species of these genera may be present in the nodules of legumes, but also be inhabitants of rhizosphere or phyllosphere of different plants. The presence of atypical rhizobial microsymbionts in the studied plants was noted, which may indicate the active formation of relationships between partners in the legume-rhizobial systems of Baikal region.
Keywords: legumes of Baikal region, taxonomy of rhizobia, ribosomal genes sequencing.
Nodule bacteria (Rhizobia) belong to a large genetically diverse group of Gram-negative soil microorganisms that form intracellular nitrogen-fixing symbiosis with legumes. One of the urgent tasks of modern biotechnology is the study of mechanisms of interaction of legumes with rhizobia, required for evidence-based selection of highly effective plant-microbe systems [1].
Investigations of symbiotic systems with the participation of endemic or relict species have a particular importance for understanding of the evolution of plant-microbe interactions. These unique objects include Baikal legumes, such as low-growing vetchling (Lathyrus humilis), a Late Pleistocene relict of the South Siberian-Severouralsk area [2]; Mongolian milk vetch (Astragalus mong-holicus), a rare medicinal plant [3, 4]; Baikal endemics Baikal pea (Vicia baicalensis) and forest oxytrope (Oxytropis sylvatica) [5].
Few data on Astragalus microsymbionts show a great diversity of these microorganisms belonging to different genera of order Rhizobiales: Rhizobium, Sinorhizobium, Bradyrhizobium, and Mesorhizobium [6-9]. Vetch-
ling and pea species are nodulated with bacteria Rhizobium leguminosarum bv. viciae [10-12], and rhizobia and mesorhizobia strains are described among oxy-trope microsymbionts [7, 13]. However, microsymbionts of Baikal plants belonging to these genera have not been studied.
The purpose of this study was to create a representative collection of microsymbiont strains of endemic Baikal legumes (low-growing vetchling, Mongolian milk vetch, Baikal pea, and forest oxytrope) and to estimate their taxonomic position using the method of 16S rDNA and ITS-region sequencing.
Technique. A total of 69 strains isolated as described [14] from root nodules of low-growing vetchling Lathyrus humilis, Baikal pea Vicia baicalensis (two populations of each species) as well as Mongolian milk vetch Astragalus mongholicus and forest oxytrope Oxytropis sylvatica (one population of each species) which grow on the Chivyrkuy coast Bay (Baikal) were studied. Strains were grown on yeast mannitol agar (YMA) at 28 °С [7].
For primary identification of strain intraspecific diversity, RFLP 16S and 23S rRNA (ITS-region) sequencing analysis was performed. Amplified DNA was digested with MspI, and the resulting DNA fragments were separated by electrophoresis in the standard mode [15]. Species affiliation of strains was determined by 16S rRNA gene (rrs) sequencing. The taxonomy position of Bosea nodule bacteria was specified using the more variable ITS-region sequencing method.
To amplify 16S rDNA (about 1500 bp), the fD1 (5' -AGAGTTTGATCC-TGGCTCAG-3') and rD1 (5'-AAGGAGGTG-ATCCAGCC-3') primers were used, to amplify the ITS-region (800 bp), the FGPS1490-72 (5'-TGCGGC-TGGATCCCCTCCTT-3') and FGPL-132 (5'-CCGGGTTTCCCCATTCGG-3') primers were used. The resulting PCR product was gel purified [15] for subsequent sequencing.
The search for homologous sequences was performed using the GenBank database (BLAST program). The phylogenetic tree was constructed using the MEGA v. 4.0.2 program (Neighbor-Joining method). Sequence pairs were compared using the number of different nucleotides.
Results. The strains used in the study are summarized in the Table.
Taxonomic structure of microsymbiont strains isolated from root nodules in endemic Baikal legumes (according to rrs and ITS-region sequencing)
Legume microsymbiont No.
Microsymbiont species low-growing vetchling Lathyrus humilis Mongolian milk vetch Astragalus mongholicus Baikal pea Vicia baicalensis Forest oxytrope Oxytropis sylvatica
Rhizobium sp. 2/5(1), 2/12М - - -
Rhizobium leguminosarum bv. trifolii Mesorhizobium 1/10К, 2/10К 11/2, 11/3К, 11/4М, 11/7К, 11/12М, 11/19, 11/20, 11/21, 11/22, 11/23, 11/24К, 11/24М, 11/25, 11/33, 11/34К, 11/34М, 11/35, 11/36, 11/37К, 11/37М, 11/38, 11/39, 11/42К, 11/42М, 11/43К, 11/44К, 11/44М, 11/45К, 11/46, 11/47К, 11/47М, 11/48К, 11/48М, 13/3, 13/4, 13/6М, 13/7, 13/8К, 13/9М, 13/11 12/13М, 12/16К
metaHidurans - 3/14С1, 3/14С2 - -
Mesorhizobium ciceri 2/13К - - -
Tardiphaga robiniae 1/11М 3/6М, 3/11С, 3/21(2) - 12/11(1)
Bosea sp. 3/5М, 3/31К 12/22М
Bosea vaviloviae - 3/25 - -
Herbiconiux sp. 1/3М, 1/5М, 1/14М, 1/15М -
Burkholderia sp. - 3/8К 13/5
Leifsonia sp. - 3/23М, 3/27К
Stenotrophomonas sp. - 3/17 11/7М
Phyllobacterium sp. - 13/12М
Note. Dashes mean the absence of isolates.
Table (continued)
49
2/10K 11/21 11/3K
Ehizobium leguminosarum bv trifolii ATCC14480T 11/37M 11/12M 13/7M 13/3 13/4 11/7K 11/34M 13/8K 13/6M 12/13M 12/16K 11/4M 11/23 11/24K 1/10K ---2/12M
---Ehizobium leguminosarum bv viciae ATCCI0004T
I—Ehizobium leucaenae LMG9517T
1 I----Rhizobium tropici CIAT899T
<Ёг Rhizobium freirei PRF81T Ehizobium leguminosarum bv phaseoli ATCC14482T Rhizobium etli CFN42T
■Ehizobium mesoamericanum CCGE501T Ehizobium grahamii CCGE502T
t Ehizobium alamii GBV016T —Rhizobium sullae IS123T ' -Ehizobium gallicum R602spT
991----Rhizobium mongolense USDA1844T
Ehizobium giardinii E1152T 2/5(1)
Rhizobium selenitireducens BIT Rhizobium vignae CCBAU05176T
Rhizobium undicola LMG11875T 56rMesorhizobium ciceri NBRC100389T mAj 2/13K
■““J---Mesorhizobium australicum WSM2073T
Mesorhizobium loti ATCC33669T
Mesorhizobium alhagi CCNWXJ12-Mesorhizobium opportunistum WSM2075T Mesorhizobium amorphae ACCC19665T Mesorhizobium huakuii IF015243T Mesorhizobium plurifarium LMG11892T ,
Mesorhizobium mediterraneum LMG17148T ' 3/14C1
\Mesorhizobium metallidurans STM2683T 3/14C2 -
2T
III
0.005
Fig. 1. Rr-phylogram demonstrating the taxonomic position of genera Rhizobium and Mesorhizobium isolates. The tested strains are marked in bold. Letter «Т» marks typical strains; I-III — significantly different clusters.
Isolates were divided into two groups by their growth rate. In 9 strains, visible colonies were formed from the day 5 to 6 of growth on YMA medium, other strains formed colonies from the day 3 to 4. Based on the results of ITS-region RFLP-analysis, studied isolates were divided into 33 groups with identical DNA profile. One species of each group was selected for rrs sequencing. Rrs sequencing analysis showed that 23 strains belonged to genera Rhizo-
bium and Mesorhizobium and formed three significantly different clusters with the level of support of more than 95 % (Fig. 1).
64
57
56
81
40
98 L
^^Bradyrhizobium rifense CTAW71T Л Bradyrhizobium cytisi CTAW11T С Щ—Bradyrhizobium yuanmingense 100594T '-Bradyrhizobium arachidis CCBAU051107T jBradyrhizobium huanghuaihaiense CCBAU23303T 'Bradyrhizobium iriomotense NBRC102520T — Bradyrhizobium daqingense CCBAU15774T Bradyrhizobium betae LMG21987T
Bradyrhizobium japonicum USDA6T Bradyrhizobium canariense BTA-1T —Nitrobacter vulgaris DSM10236T Nitrobacter alkalicus AN IT Nitrobacter winogradskyi Nb-255T ----Nitrobacter hamburgensis X14T
Rhodopseudomonas rhenobacensis DSM12706T Bhodopseudomonas palustris 22| ATCC17001T
'■Rhodopseudomonas faecalis JCM11668T
----Metalliresistens boonkerdii 106595T N
1/11M 12/11(1)
3/21(2) V rv
100 3/11C
—Tardiphaga robiniae LMG26467 3/6M
rBradyrhizobium retamae LMG 27393T
Bradyrhizobium lablabi CCBAU23086T Bradyrhizobium pachyrhizi PAC48T 1—Bradyrhizobium jicamae PAC68T Bosea lathyri LMG26379T 3/5M 12/22 3/31 3/25
Bosea vaviloviae Vaf- 18T
44
67
99
45
100
_99
> V
951 Bosea minatitlanensis AMX51T
1 >61 'Bosea robiniae LMG26381T '—Bosea thiooxidans DSM9653T ““ ftftBosea massiUensis 63287T
__ 'Bosea lupini LMG26383T
59 j Bosea eneae 34614T 9VBosea vestrisii 34635T
0.01
Fig. 2. Ris-phylogram demonstrating the taxonomic position of isolates within the Bradyihi-zobiaceae family. Tested strains are marked in bold. Letter «T» marks typical strains; IV, V — significantly different clusters.
Cluster I included 19 strains isolated from low-growing vetchling, baikal pea and forest oxytrope (3, 14 and 2 strains, respectively) and two typical strains R. leguminosarum bv. trifolii ATCC14480Т and R. leguminosarum bv. viciae ATCC10004Т. Based on the results of rrs sequencing (see Table), isolates 1/10K, 2/10K, 11/3K, 11/4M, 11/7K, 11/12M, 11/21, 11/23, 11/24K, 11/34M, 11/37M, 12/13M, 12/16K, 13/3, 13/4, 13/6M, 13/7M, and 13/8K were identified as R. leguminosarum bv. trifolii (index of similarity to typical strain ATCC14480Т was 99.7-99.9 %).
Cluster II was formed by strain 2/5(1) isolated from low-growing vetchling and typical strain Ш52Т Rhizobium giardinii which has been described as microsymbiont strain [16]. However, since the similarity between these strains in gene rrs was 99.0 % only, strain 2/5(1) was identified as
Rhizobium sp. (see Table).
Three isolates were classified as Mesorhizobium (see Fig. 1, Table). Strains 3/14С1 and 3/14С2 (cluster III) isolated from Mongolian milk vetch were identified as Mesorhizobium metallidurans and M ciceri (rrs homology to typical strains STM2683Т and NBRC100389Т was 100 and 99.6 %, respectively).
Figure 2 shows the rs-phylogram demonstrating the taxonomic position of 9 slow-growing Rhizobium isolates within the Bradyrhizobiaceae family. Strains 1/11M, 3/6M, 3/11C, 3/21(2), and 12/11(1) isolated from various plants formed cluster IV with typical strain Tardiphaga robiniae LMG26467Т and were assigned to this species (rrs index of similarity of 99.8-99.9 %). Cluster V with the level of statistical support of 99.0 % was formed by four strains 3/5M, 3/31К, 12/22М, and 3/25 and two typical Bosea (B. lathyri and B. vaviloviae) strains. The rrs similarity of isolates to typical strains B. lathyri LMG26379T and B. vaviloviae Vaf-18T was 98.4-99.4 and 99.4-100 %, respectively. It should be noted that species B. vaviloviae was described as recently as 2015 for three microsymbiont strains of a relic legume Vavilovia formosa which grows in North Ossetia [17]. Therefore, Baikal isolates belonging to genus Bosea and having a high index of similarity to species B. vaviloviae are of great interest for further study.
8fl~Brndyrhizobiutn daqingense CCBAU15774T
4
93.
66
100
38
391
52
100
'—Bradyrhizobium huanghuaihaiense CCBAU23303T Bradyrhizobium yuanmingense CCBAU1007 IT ■Bradyrhizobium arachidis CCBAU051107T Bradyrhizobium Japonicum LMG6138T —Bradyrhizobium canariense BTA-1T I—Bradyrhizobium betae LMG21987T Y Bradyrhizobium cytisi CTAW11T jjj-Bradyrhizobium rifense CTAW71T ■Bradyrhizobium iriomotense NBRC102520T
---Bradyrhizobium pachyrhizi PAC48T
-Bradyrhizobium retamae Rol9T ■Bradyrhizobium jicamae PAC68T —Bradyrhizobium lablabi CCBAU23086T Nitrobacter hamburgensis DSM10229T
911-----Nitrobacter winogradskyi DSM10237T
-----Tardiphaga robiniae LMG26467T
—Rhodopseudomonas palustris ATCC17001T -----Bosea sp. ORS1414
92|_T
68f
43
99
-Bosea sp. STM358 — 12/12M
Bosea lathyri LMG26379T )^3/31K 3/5M -3/25
Bosea vaviloviae Vaf-17 Bosea vaviloviae Vaf- 18T Bosea vaviloviae Vaf-43
93 48 38 92 94
0.05
Fig. 3. ITS-phylogram demonstrating the taxonomic position of genera Bosea isolates isolated from Mongolian milk vetch (Astragalus mongholicus) and forest oxytrope (Oxytiopis sylvatica) nodules.
Tested strains are marked in bold. Letter «T» marks typical strains.
ITS-region sequencing was performed in strains 3/5M, 3/31К, 12/22М, and 3/25 as this method provides higher resolution in the identification of closely related strains than rrs sequencing [9]. The dendrogram shown in Figure 3 demonstrates the correlation between the results of rrs and ITS-region sequencing. All isolates could be clustered with typical strains B. lathyri LMG26379T and B. va-
viloviae Vaf-18T under the statistical support level of 99.0%. However, the maximum levels of ITS-region homology between these isolates and typical strains B. lathyri and B. vaviloviae were low (of 84.9 and 89.4 %, respectively). Isolate 3/25 was an exception, its ITS-region similarity to typical strain B. vaviloviae Vaf-18T was 97.8 %. Based on the results of rrs ITS-region sequencing, it was assigned to species B. vaviloviae. The taxonomic position of the other Bosea isolates is unclear as it is considered that nodule bacteria strains belonging to one species can not be less than 95 % homologous in ITS-region [9]. Analysis of rrs sequencing in other Baikal isolates revealed their belonging to genera Herbi-coniux (4 strains), Leifsonia (2 strains), Burkholderia (2 strains), Stenotro-phomonas (2 strains), and Phyllobacterium (1 strain). Reported in the literature, some species of these genera may be present in legume nodules [18, 19] and be inhabitants of rhizosphere or phyllosphere of different plants.
Thus, rhizobial strains belonging to species Rhlzobium spp., Mesorhizobium ciceri, Tardiphaga robiniae, and species of genus Herbiconiux (family Micro-bacteriaceae) can be found among the low-growing vetchling microsymbionts. Species of genera Mesorhizobium, Bosea, Tardiphaga, Leifsonia, Burkholderia and Stenotrophomonas were found among Mongolian milk vetch symbionts. Isolates of species R. leguminosarum bv. trifolii, T. robiniae and Bosea sp. were obtained from forest oxytrope nodules. A total of 40 strains belonging to species R. leguminosarum bv. trifolii were isolated from Baikal pea nodules, as well as one Phyllobacterium and one Burkholderia species. We would like to note that the presence of atypical rhizobial microsymbionts in legume nodules (M ciceri and T. robiniae in vetchling; Bosea spр. and T. robiniae in milk vetch and oxytrope; Phyllobacterium sp. in pea) may indicate the active formation of relationships between partners in the legume-rhizobial systems of Baikal region.
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