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