Diverse Rhizobium strains isolated from root nodules of Trifolium alexandrinum in Egypt and symbiovars

https://doi.org/10.1016/j.syapm.2020.126156Get rights and content

Abstract

Berseem clover (T. alexandrinum) is the main forage legume crop used as animal feed in Egypt. Here, eighty rhizobial isolates were isolated from root nodules of berseem clover grown in different regions in Egypt and were grouped by RFLP-16S rRNA ribotyping. Representative isolates were characterized using phylogenetic analyses of the 16S rRNA, rpoB, glnA, pgi, and nodC genes. We also investigated the performance of these isolates using phenotypic tests and nitrogen fixation efficiency assays. The majority of strains (<90%) were closely related to Rhizobium aegyptiacum and Rhizobium aethiopicum and of the remaining strains, six belonged to the Rhizobium leguminosarum genospecies complex and only one strain was assigned to Agrobacterium fabacearum. Despite their heterogeneous chromosomal background, most of the strains shared nodC gene alleles corresponding to symbiovar trifolii. Some of the strains closely affiliated to R. aegyptiacum and R. aethiopicum had superior nodulation and nitrogen fixation capabilities in berseem clover, compared to the commercial inoculant (Okadein®) and N-added treatments. R. leguminosarum strain NGB-CR 17 that harbored a nodC allele typical of symbiovar viciae, was also able to form an effective symbiosis with clover. Two strains with nodC alleles of symbiovar trifolii, R. aegyptiacum strains NGB-CR 129 and 136, were capable of forming effective nodules in Phaseolus vulgaris in axenic greenhouse conditions. This adds the symbiovar trifolii which is well-established in the Egyptian soils to the list of symbiovars that form nodules in P. vulgaris.

Introduction

Forage legumes have been the basis of livestock feed for centuries [40]. They have a high nutritional content and can provide a sustainable strategy for promoting food and protein global security. In contrast with forage cereals, legumes offer rich-protein and high-quality forage sources [25]. Egyptian berseem clover (T. alexandrinum) is a widely grown legume crop used as fodder and for soil fertility maintenance in many countries. It originated in Egypt and eventually became widespread in cropping systems in west and south Asia [35]. It is cultivated as a winter crop in areas such as eastern Australia, South Africa, southern Europe and the southeast USA [9], [33], [49]. Whereas in some European countries, the Midwest of USA, and Afghanistan, berseem clover has been introduced as a summer crop [6], [13], [53]. Currently, berseem clover is a global fodder crop that is cultivated on millions of hectares in the world. India has the greatest producing area (2 million ha), while Egypt comes second (1.18 million ha) with a slightly higher area than Pakistan (0.71 million ha) [35].

Clover, like other legume crops, establishes a symbiotic relationship with soil rhizobia forming root nodules, in which rhizobia fix atmospheric nitrogen [20]. Rhizobia were the first biofertilizers produced and now are applied across 400 million ha of agricultural land per year to improve legume production [19]. Their application saves billions of dollars as an alternative to synthetic nitrogen fertilizers [21] that could otherwise contaminate soil and water [57]. The annual nitrogen fixation inputs from pasture and fodder legumes is estimated to be 12–25 million tonnes [19]. Although berseem clover is one of the most efficient nitrogen-fixing legumes, meeting a large proportion of their nitrogen needs through symbiotic nitrogen fixation (SNF), a comprehensive summary on how much nitrogen is taken by the crop and how much nitrogen is supplied by SNF is lacking. The amounts of nitrogen fixed by berseem were reported in some cases to be higher than 250 kg N ha−1, which corresponds an average to 64% of berseem nitrogen derived from nitrogen fixation (%Ndfa) [17]. Nevertheless, a lower amount of nitrogen fixed (205 kg N ha−1) has been reported for berseem clover grown in Egypt under field conditions [34].

Bacterial genomes including rhizobia consist of two components that are comparable to the operating system and applications of smartphones [61]. The chromosome (the primary genome) is commonly shared by all members of a species and provides a stable basis for taxonomic identification [26]. In contrast, the accessory genome contains groups of genes that confer particular functions [41], and may have independent phylogenies that differ from those of the core genomes [30]. In rhizobial species, genes for plant nodulation (nod) and nitrogen fixation (nif and fix) are accessory genes that naturally spread from strain to strain via horizontal gene transfer [26]. This occurs whether these genes are found on plasmids or on chromosomal islands [11], which largely explains the evolution of rhizobial symbioses [29]. The concept of a symbiovar is a key to understand the diversity of rhizobia, whereby symbiovars define the host specificity [43]. The symbiovar unites a group of strains that share a genetic module conferring a distinct symbiotic phenotype [26]. There are over twenty different symbiovars reported in different genera of nodule-forming rhizobia [42].

Clovers usually form effective symbiosis with R. leguminosarum, which is one of the most exploited species of root-nodule bacteria in world agriculture [20]. The description of three symbiovars in R. leguminosarum (sv. viciae, sv. trifolii, sv. phaseoli) was first reported by Jordan [23]. To date, R. leguminosarum can be divided into distinct genospecies (gsA, gsB, gsC, gsD, gsE, gsF, and gsG), that are not symbiovar specific [[47],25], personal communication Peter Young]. In 2013, strain K3.22, belonging to Rhizobium pisi (Pisum sativum nodulating rhizobial species), was identified as a clover-microsymbiont [30]. Strain K3.22 was distinguished from R. leguminosarum because its chromosomal gene sequences had identical similarities to R. pisi DSM 30132T [30]. However, the K3.22 plasmid symbiosis nod genes demonstrated high sequence identity to those from R. leguminosarum sv. trifolii [30]. Later, a new species isolated from Egypt, R. aegyptiacum, was added to the list of N2-fixing rhizobia that can form effective symbiosis with berseem clover [46]. Based on 16S rRNA gene analysis, strains from R. aegyptiacum were firstly classified into the species Rhizobium etli (the traditional microsymbiont of P. vulgaris [47]. However, the DNA-DNA hybridization analysis supported the new species status [46].

To give more insights on the genetic diversity and symbiotic effectiveness of berseem clover microsymbionts in Egyptian soils, 80 rhizobial isolates were obtained from the root nodules of clover plants grown in different geographical regions and diverse soils. The taxonomy and phylogenetic relationships of isolated bacteria were analyzed using restriction fragment length polymorphism of the 16S rRNA gene (16S rRNA-RFLP) and sequencing of 16S rRNA, glnA, rpoB, pgi, and nodC genes. The symbiotic performance of all isolates was screened in axenic conditions and subsequently the symbiotic effectiveness of highly-efficient rhizobial isolates was screened in pot experiment with field soils in controlled greenhouse conditions.

Section snippets

Bacterial isolation and sampling sites

Nodules were collected from field-grown berseem clover in 80 different sites that represented different agro/climatic soil conditions in Egypt (Fig. 1). Rhizobium isolates were obtained from surface-sterilized berseem clover root nodules as described by Vincent [58]. Root nodules were surface-sterilized by washing for 30 s with 95% ethanol, immersed in 10% sodium hypochlorite for 90 s, and finally were washed six times using sterile distilled water. One hundred μl aliquot of the last nodule

Bacterial isolates, phenotypic characterization and nodulation assay

A total of 80 rhizobial isolates were isolated from root nodules of field grown berseem clover under different Egyptian soil conditions. All isolates were able to nodulate berseem clover cv. Serw 1 and formed pink nodules within 2 weeks after inoculation in sterilized Leonard jar assemblies in the greenhouse. After 50 days, different nodulation patterns were induced by bacterial inoculations, resulting in nodules number ranging from 14 to 49 nodules per plant with dry mass of nodules from 6.3

Discussion

Berseem clover is the main forage legume crop that is used as animal feed in Egypt [24]. In this study, 80 bacterial isolate were purified from root nodules of berseem clover grown in various pedoclimatic conditions in Egypt. All isolates were authenticated as clover nitrogen-fixing rhizobia by nodulating their original host in axenic greenhouse conditions. A preliminary analysis of genetic relationship using PCR-RFLP of 16S rRNA gene selected 12 out of the 80 isolates that were further

Conclusion

This study shows the genetic diversity among local rhizobia nodulating T. alexandrinum in Egyptian soils with different agro-climatic conditions. All isolated strains belonged to R. aegyptiacum, R. aethiopicum, and A. fabacearum containing the trifolii symbiovar. Remarkably, R. leguminosarum strains identified in this study probably constitute new genospecies of R. leguminosarum species complex. Here we reported the ability of T. alexandrinum to establish effective symbiosis with strains

Funding

The authors received no specific funding for this work.

Ethics approval and consent to participate

Authors declare that they have consented to participate in the manuscript and publish it.

Acknowledgment

We specially thank professor Peter Young (University of York) for sharing unpublished data and productive discussions.

References (65)

  • K. Taha et al.

    Rhizobium laguerreae is the main nitrogen-fixing symbiont of cultivated lentil (Lens culinaris) in Morocco

    Syst. Appl. Microbiol.

    (2018)
  • S. Tounsi-Hammami et al.

    Genetic diversity of rhizobia associated with root nodules of white lupine (Lupinus albus L.) in Tunisian calcareous soils

    Syst. Appl. Microbiol.

    (2019)
  • D. Wibberg et al.

    Complete genome sequencing of Agrobacterium sp. H13-3, the former Rhizobium lupini H13-3, reveals a tripartite genome consisting of a circular and a linear chromosome and an accessory plasmid but lacking a tumor-inducing Ti-plasmid

    J. Biotechnol.

    (2011)
  • J.P.W. Young

    Bacteria are smartphones and mobile genes are apps

    Trends Microbiol.

    (2016)
  • S.H. Youseif et al.

    Phylogenetic multilocus sequence analysis of native rhizobia nodulating faba bean (Vicia faba L.) in Egypt

    Syst. Appl. Microbiol.

    (2014)
  • S.H. Youseif et al.

    Phenotypic characteristics and genetic diversity of rhizobia nodulating soybean in Egyptian soils

    Eur. J. Soil Biol.

    (2014)
  • S.F. Altschul et al.

    Gapped BLAST and PSI-BLAST: a new generation of protein database search programs

    Nucleic Acids Res.

    (1997)
  • M. Andrews et al.

    Horizontal transfer of symbiosis genes within and between rhizobial genera: occurrence and importance

    Genes

    (2018)
  • A.A. Aserse et al.

    Draft genome sequence of type strain HBR26T and description of Rhizobium aethiopicum sp. nov

    Stand. Genomic Sci.

    (2017)
  • B. Asfaw et al.

    Genetically diverse lentil and faba bean-nodulating rhizobia are present in soils across Central and Southern Ethiopia

    FEMS Microbiol. Ecol.

    (2019)
  • M.M. Bashir

    Fodder production in Afghanistan

  • S. Boivin et al.

    Host-specific competitiveness to form nodules in Rhizobium leguminosarum symbiovar viciae

    New Phytol.

    (2020)
  • W.J. Broughton et al.

    Plant nutrient solutions

  • J.A. Carpenter et al.

    Pastures

  • R. de Castro Pires et al.

    Soil characteristics determine the rhizobia in association with different species of Mimosa in central Brazil

    Plant Soil

    (2018)
  • M.I.A. Cavassim et al.

    Symbiosis genes show a unique pattern of introgression and selection within a Rhizobium leguminosarum species complex

    Microb. Genomics

    (2020)
  • Y.X. Chen et al.

    Faba bean (Vicia faba L.) nodulating rhizobia in Panxi, China, are diverse at species, plant growth promoting ability, and symbiosis related gene levels

    Front. Microbiol.

    (2018)
  • A. Clark

    Managing Cover Crops Profitably

    (2007)
  • J.R.M. Delamuta et al.

    Genetic diversity of Agrobacterium species isolated from nodules of common bean and soybean in Brazil, Mexico, Ecuador and Mozambique, and description of the new species Agrobacterium fabacearum sp. nov

    Int. J. Syst. Evol. Microbiol.

    (2020)
  • R.C. Efrose et al.

    Molecular diversity and phylogeny of indigenous Rhizobium leguminosarum strains associated with Trifolium repens plants in Romania

    Antonie van Leeuwenhoek, Int. J. Gen. Mol. Microbiol.

    (2018)
  • D. Giambalvo et al.

    Forage production, N uptake, N2 fixation, and N recovery of berseem clover grown in pure stand and in mixture with annual ryegrass under different managements

    Plant Soil

    (2011)
  • Q. Han et al.

    Variation in rhizosphere microbial communities and its association with the symbiotic efficiency of rhizobia in soybean

    ISME J

    (2020)
  • Cited by (11)

    View all citing articles on Scopus
    View full text