doi: 10.15389/agrobiology.2023.1.87eng

UDC: 631.52:581.557:575

The authors are grateful to Ya.V. Pukhalsky for help in measuring nitrogenase activity.
Supported financially by the Russian Science Foundation under grant No. 19-16-00081П



O.P. Onischuk1, O.N. Kurchak1, A.K. Kimeklis1, T.S. Aksenova1,
E.E. Andronov1, 2, N.A. Provorov1

1All-Russian Research Institute for Agricultural Microbiology, 3, sh. Podbel’skogo, St. Petersburg, 196608 Russia, e-mail (✉ corresponding author),,,,,;
2Saint-Petersburg State University, 7-9, Universitetskaya nab., St. Petersburg, 199034 Russia, e-mail

Onishchuk O.P.
Aksenova T.S.
Kurchak O.N.
Andronov E.E.
Kimeklis A.K.
Provorov N.A.

Final revision received December 28, 2022
Accepted January 17, 2023

Nodule bacteria of the species Rhizobium leguminosarum are differentiated into two biovars (bv.) that form N2-fixing symbioses with leguminous plants of the galegoid complex, tribes Fabeae (genera Lathyrus, Lens, Pisum, Vavilovia, Vicia, symbiont — R. leguminosarum bv. viciae) and Trifolieae (genus Trifolium, symbiont — R. leguminosarum bv. trifolii) (J. Sprent et al., 2017). It was previously assumed that cross-inoculation between these biovars is impossible or rare, while data on the control of host specificity of R. leguminosarum were limited by interactions between pea (P. sativum) lines with different alleles of Sym2 gene and bv. viciae strains that differ in the presence of nodX gene (T.A. Lie, 1978). The aim of our work was to analyze the variability of R. leguminosarum bv. viciae strains from ancestral (A) and evolutionarily advanced (D) genomic groups in terms of host specificity and N2-fixing activity, aimed at the functional characterization of ancestral genome elements, which were previously identified by the comparative genetic analysis of strains isolated from representatives of the Fabeae tribe that differ in phylogenetic affiliation. In accordance with the previously proposed genotyping technique, strains were assigned to group A if they contained the nodX and fixW genes, did not contain a chromosomal copy of the fixNOPQ operon, and the nodT gene was outside the nod cluster. In the absence of at least one of these features, the strains were assigned to group D (E. Chirak et al., 2019). Group A strains were isolated from the relict legume Vavilovia formosa and from wild-growing Afghan lines of P. sativum, group D strains were isolated from cultivated European lines of P. sativum, from Vicia sativa and V. alpestris. In experiments on the analysis of cross-inoculation of two R. leguminosarum biovars we used bv. viciae strains isolated from nodules of Vavilovia formosa, Vicia sativa, V. subrotunda, European lines of Pisum sativum, Afghan lines of P. sativum, as well as bv. trifolii strains from clover (Trifolium pratense, T. ambiguum, T. montanum) nodules. In microvegetative experiments, plants inoculated with rhizobia were grown under gnotobiotic conditions on vermiculite. N2-fixing activity was determined using the acetylene method based on the use of C2H2 as a substrate for nitrogenase. Based on the results obtained, the following symbiotic phenotypes were identified: Fix+ — N2-fixing (large, pink) nodules; Fix- — non-fixing N2 (small, white, but morphologically normal) nodules; Fix+/-— nodules not fixing N2, but similar to Fix+ nodules (large, pink); Ndv- — non-fixing N2, tumor-like nodules; Nod- — nodules were absent. It turned out that 9 out of 11 strains of the ancestral group formed on clover nodules of Fix- phenotype, and 2 strains formed nodules of Ndv- phenotype. Among 8 strains of the evolutionarily advanced group, the Fix- and Ndv- phenotypes were detected in 4 and 2 strains, respectively, and 2 strains did not form nodules on clover (Nod-), indicating a narrowing of the host specificity of rhizobia during coevolution of bv. viciae with host plants. Therefore, we have shown for the first time that during the transition of bv. viciae strains to symbiosis with evolutionarily young representatives of the tribe Fabeae (transition from the A- to the D-group), bacteria lose the ability to form symbiosis with a heterologous host (Trifolium). Among 6 strains of clover rhizobia, 4 strains showed the ability to inoculate vetch forming Fix- nodules. In experiments to control the absence of contamination, DNA was isolated from nodules using the NucleoSpinÔ Soil (Macherey-Nagel GmbH & Co. KG, Germany), the nodA gene fragment was amplified using universal primers for R. leguminosarum biovars (ndARL302_F YTDGGMATCGC-HCACT/ndARL518_R RDACGAGBACRTCTTCRGT). The data obtained showed that under the conditions of sterile microvegetation experiments there is no contamination and the majority of strains are able for cross-inoculation: bv. viciae strains form nodules on clover and bv. trifolii form nodules on vetch. However, this ability is limited by the formation of non-fixing N2 nodules in heterologous hosts, including morphologically abnormal (tumor-like) nodules. The study of symbioses formed by 9 species of leguminous plants of tribe Fabeae (Pisum sativum, Vicia sativa, V. villosa, V. alpestris, Vavilovia formosa, Lens culinaris, L. nigricans, Lathyrus pratensis, L. sylvestris) with 6 R. leguminosarum bv. viciae strains, demonstrated a pronounced specificity of N2-fixing symbiosis formation, which depends mostly on the bacteria origin. Strains isolated from the same legume species (V. formosa or P. sativum) are more similar in host specificity than strains from different hosts. A hypothetical scheme of R. leguminosarum evolution is proposed, according to which: a) divergent evolution of bv. viciae is determined by host plant speciation in tribe Fabeae; b) the closest relative of a common ancestor of R. leguminosarum is represented by bv. trifolii, which display an evolutionary primitive sym gene organization and possibly originated from the ancestral bv. viciae strains that changed their host specificity. The rhizobia isolated from V. formosa may be considered as the close relatives of these ancestral strains since they exceed the pea and vetch isolates in the ability to form morphologically normal nodules with the heterologous host, clover. The data obtained show the possibility of constructing rhizobia strains with an increased symbiotic activity by editing the ancestral components of their genomes.

Keywords: nodule bacteria, leguminous plants, symbiotic N2-fixation, host specificity, biodiversity, Rhizobium leguminosarum, genomic groups, evolution of symbiosis.



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