Two Broad Host Range Rhizobial Strains Isolated From Relict Legumes Have Various Complementary Effects on Symbiotic Parameters of Co-inoculated Plants.

Vera Safronova,Evgeny Andronov,Anna Tsyganova,Anna Kitaeva,Igor Tikhonovich,Elizaveta Chirak,Alexander Pinaev,Andrey Belimov,Anna Sazanova,Irina Kuznetsova,Viktor Tsyganov,Elena Seliverstova

doi:10.3389/fmicb.2019.00514

Abstract

Two bacterial strains Ach-343 and Opo-235 were isolated, respectively from nodules of Miocene-Pliocene relict legumes Astragalus chorinensis Bunge and Oxytropis popoviana Peschkova originated from Buryatia (Baikal Lake region, Russia). For identification of these strains the sequencing of 16S rRNA (rrs) gene was used. Strain Opo-235 belonged to the species Mesorhizobium japonicum, while the strain Ach-343 was identified as M. kowhaii (100 and 99.9% rrs similarity with the type strains MAFF 303099T and ICMP 19512T, respectively). Symbiotic genes of these strains as well as some genes that promote plant growth (acdS, gibberellin- and auxin-synthesis related genes) were searched throughout the whole genome sequences. The sets of plant growth-promoting genes found were almost identical in both strains, whereas the sets of symbiotic genes were different and complemented each other with several nod, nif, and fix genes. Effects of mono- and co-inoculation of Astragalus sericeocanus, Oxytropis caespitosa, Glycyrrhiza uralensis, Medicago sativa, and Trifolium pratense plants with the strains M. kowhaii Ach-343 and M. japonicum Opo-235 expressing fluorescent proteins mCherry (red) and EGFP (green) were studied in the gnotobiotic plant nodulation assay. It was shown that both strains had a wide range of host specificity, including species of different legume genera from two tribes (Galegeae and Trifolieae). The effects of co-microsymbionts on plants depended on the plant species and varied from decrease, no effect, to increase in the number of nodules, nitrogen-fixing activity and plant biomass. One of the reasons for this phenomenon may be the discovered complementarity in co-microsymbionts of symbiotic genes responsible for the specific modification of Nod-factors and nitrogenase activity. Localization and co-localization of the strains in nodules was confirmed by the confocal microscopy. Analysis of histological and ultrastructural organization of A. chorinensis and O. popoviana root nodules was performed. It can be concluded that the strains M. kowhaii Ach-343 and M. japonicum Opo-235 demonstrate lack of high symbiotic specificity that is characteristic for primitive legume-rhizobia systems. Further study of the root nodule bacteria having complementary sets of symbiotic genes will contribute to clarify the evolutionary paths of legume-rhizobia relationships and the mechanisms of effective integration between partners.

Highlights

It has been shown that primitive tropical legumes possess relatively low symbiotic specificity, whereas evolutionarily young legumes of temperate climate show higher specificity and can form symbiosis with a single species of root nodule bacteria (Lie et al, 1987; Tikhonovich and Provorov, 2009; Provorov and Vorobyev, 2011; Bakker et al, 2014)
The species Vavilovia formosa growing in Western Asia and Caucasus mountains as well as Oxytropis triphylla, O. popoviana, O. tragacanthoides, Hedysarum zundukii, Astragalus chorinensis, and Glycyrrhiza uralensis originated from Baikal Lake region are known as Miocene-Pliocene relics (Malyschev, 2006; Sinjushin et al, 2009; Turuta et al, 2015)
The whole genome sequence analysis of this pair of strains showed the presence in Opo-243 additional genes nodPQ, nolK, and noeL involved in the modification of Nod factors (NFs) and affecting the specificity of plant-rhizobia interactions

Summary

Introduction

It has been shown that primitive tropical legumes possess relatively low symbiotic specificity, whereas evolutionarily young legumes of temperate climate show higher specificity and can form symbiosis with a single species of root nodule bacteria (Lie et al, 1987; Tikhonovich and Provorov, 2009; Provorov and Vorobyev, 2011; Bakker et al, 2014). Information about strains isolated from root nodules of these plant species was recently published (Safronova et al, 2014, 2015a,b, 2017a,b). It was shown that the multi-strain treatments of two Australian wattle species Acacia salicina and A. stenophylla interacting with highly diverse communities of rhizobia had a negative effect on plant growth, probably due to strong competition between strains with different levels of nitrogen-fixing activity and symbiotic effectiveness (Barrett et al, 2015). Opo-243 isolated from the same nodule of a relict legume O. popoviana significantly accelerated the root nodule formation on the host plant after a combined inoculation (Safronova et al, 2018a). The whole genome sequence analysis of this pair of strains showed the presence in Opo-243 additional genes nodPQ, nolK, and noeL involved in the modification of Nod factors (NFs) and affecting the specificity of plant-rhizobia interactions. This report demonstrated that taxonomically different strains forming symbiotic systems with relics can be co-microsymbionts infecting the same nodule and promoting the nodulation process due to complementary sets of symbiotic genes

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