Next Article in Journal
Design and Test of Duckbill Welding Robot for Cotton Seeder
Previous Article in Journal
Distribution of Genetic Diversity in Beta patula Aiton Populations from Madeira Archipelago, Portugal
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agriculture
Volume 13
Issue 1
10.3390/agriculture13010030
agriculture-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Symbiotic Performance and Characterization of Pigeonpea (Cajanus cajan L. Millsp.) Rhizobia Occurring in South African Soils

by
Francina L. Bopape
1,2,
Chrizelle W. Beukes
3,
Kopotsa Katlego
4,
Ahmed I. Hassen
1,2,
Emma T. Steenkamp
4 and
Eastonce T. Gwata
2,*
1
Agricultural Research Council, Plant Health and Protection (ARC-PHP), Private Bag X134, Queenswood 0121, South Africa
2
Department of Plant and Soil Sciences, Faculty of Science, Engineering and Agriculture, University of Venda, Private Bag X5050, Thohoyandou 0950, South Africa
3
The James Hutton Institute, Invergowrie, Dundee DD2 5DA, UK
4
Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria 0002, South Africa
*
Author to whom correspondence should be addressed.
Agriculture 2023, 13(1), 30; https://doi.org/10.3390/agriculture13010030
Submission received: 19 September 2022 / Revised: 26 November 2022 / Accepted: 19 December 2022 / Published: 22 December 2022
(This article belongs to the Section Agricultural Soils)
Browse Figures
Review Reports Versions Notes

Abstract

:
Pigeonpea (Cajanus cajan (L.) Millsp.) is an important grain legume, which, like several other legumes, depends on the process of biological nitrogen fixation for its nitrogen (N2) requirement by forming a symbiotic association with rhizobia. Compared to other tropical legumes, however, the productivity of pigeonpea in South Africa is low, despite the extensive interests in developing it for wider markets. To assist this process, the objectives of the current study were to (i) characterize putative indigenous rhizobial strains that were previously derived from local soils with no previous history of legume cultivation and (ii) confirm their nodulation abilities on a local landrace and a genetically improved (exotic) genotype of pigeonpea. DNA-based analyses using the 16S rRNA and recA genes showed that the strains predominantly represented Rhizobium and Bradyrhizobium, although we also recovered Phyllobacterium and Paraburkholderia. These rhizobia nodulated both the local landrace and the improved pigeonpea genotype that were included for comparative purposes. In many cases, rhizobia performed similarly on the two genotypes, although the locally sourced landrace mostly performed better in terms of nodulation and plant biomass. While the current study generated vital information regarding the diversity of indigenous rhizobia associating with pigeonpea, further screening (including field inoculation trials) would be necessary to identify possible elite nitrogen fixing rhizobial strains for development as inoculants to enhance South African pigeonpea production.

1. Introduction

Biological nitrogen fixation is one of the most important processes necessary for plant growth and yield improvements. This property is associated with two of the six subfamilies (i.e., Papilionoideae and Caesalpinioideae) of the Leguminosae/Fabaceae [1]. Specifically, these legumes make use of symbiotic nitrogen fixation (SNF), which relies on the association with a group of soil bacteria capable of inducing the formation of novel plant structures called nodules on either the stem or roots of the host, within which the fixation process happens [2]. Bacteria with the ability to induce such nitrogen-fixing nodules are referred to as rhizobia [3].
Like most other legumes, pigeonpea (Cajanus cajan) also derives its nitrogen mainly through the process of SNF [4,5]. Pigeonpea is widely cultivated for human consumption as a source of protein and food grain particularly in low-income communities. It serves as a valuable forage and green manure crop, and its cultivation contributes to soil fertility through SNF [6,7,8,9]. Due to its consumption by both humans and livestock, pigeonpea can be traded in both informal and formal markets, thereby generating household income [10]. However, the productivity of pigeonpea is generally low, particularly in smallholder cropping systems [11,12,13]. For example, in Tanzania an average yield of only 0.4 t/ha was reported for the growing season of 2002 to 2003 [12].
In terms of its agronomic and economic value, pigeonpea is a low-input crop with no chemical fertilizer requirements, as nitrogen is supplied through the natural symbiosis with rhizobia [14]. The legume is generally compatible with rhizobia that form part of the cowpea miscellany group, which commonly also nodulate legumes such as cowpea (Vigna unguiculata), siratro (Macropitilium atropurpureum), lima bean (Phaseolus lunatus), and peanut (Arachis hypogaea) [15,16,17,18]. This broad host-range group is indigenous to African soils and represent slow-growing rhizobia from the genus Bradyrhizobium [19]. Various studies from Africa have also shown that the interactions between these rhizobia and their legume hosts are promiscuous, especially in terms of their symbiosis with peanut, cowpea, and cowpea relatives [19,20,21,22]. However, despite the general lack of host-specificity in the cowpea miscellany group, all its members are not equally able to nodulate different legumes. For example, in one of the first studies to characterize this trait, some symbionts of groundnut and Vigna species could also nodulate pigeonpea, while others could not [18].
Pigeonpea is among the most drought-tolerant legumes and regarded as having great potential for cultivation in the water-stressed and semi-arid regions of Africa [23]. Although its origins are still debatable, the species is thought to have been introduced from India to East Africa, from where it was moved to other parts of Africa [23]. This includes South Africa, where perennial varieties of pigeonpea are predominantly grown as single plants in home gardens in the Kwazulu-Natal, Limpopo and Mpumalanga provinces [24]. However, in recognition of its potential for cultivation in the drought prone regions of South Africa, local stakeholders are using various initiatives to increase awareness about the benefits of pigeonpea cultivation and to promote it as a commercial crop in South Africa [25].
An important factor in pigeonpea cultivation is the availability of compatible rhizobia to allow efficient SNF. It rarely requires rhizobial inoculants due to the legume’s inherent promiscuity making it compatible with resident soil rhizobia [11,23], and thus, commercial inoculants are not available for this crop. In other words, the limited instances of pigeonpea cultivation in South Africa are achieved without the application of inoculum, as the legume associates with compatible rhizobia already present in the soil [26,27]. There is a dearth of reliable information regarding the identity and diversity of the rhizobial partners of pigeonpea, especially from South Africa, where the crop is cultivated on a non-commercial scale. Rhizobia are polyphyletic and consist of species which belong to 18 genera of the alpha-proteobacteria (alpha-rhizobia) and beta-proteobacteria (beta-rhizobia) [28,29]. However, previous studies elsewhere indicated that pigeonpea was associated with rhizobia from several genera including Bradyrhizobium spp. [30], Mesorhizobium [31], Rhizobium spp. [32,33] Sinorhizobium/Ensifer [34,35]. Although the Paraburkholderia genus consists of mostly plant-beneficial bacteria, including strains that are capable of SNF [36], some of the strains from this genus were associated mainly with mimosoid legumes [37,38]. However, little is known regarding the host range of rhizobia native to South African soils, nor whether they would sustain SNF with pigeonpea. This is particularly relevant given that not all cowpea miscellany group rhizobia are equally capable of nodulating pigeonpea. It would thus be important to determine whether South African soils contain rhizobia suitable for pigeonpea nodulation and nitrogen fixation. Such knowledge would be invaluable for improving production in the current growing areas as well as for the expansion of production into new areas in the country.
Therefore, the goal of this study was to characterize individual indigenous rhizobial strains that were previously derived from local soils with no previous history of legume cultivation and confirm their nodulation abilities on a locally sourced landrace and a genetically improved genotype of pigeonpea. This was achieved by using putative pigeonpea-nodulating rhizobia that were isolated using trapping experiments with South African soils and diverse genotypes of the legume in a previous study [26].

2. Materials and Methods

2.1. Pigeonpea Genotypes and Rhizobial Strains

Two piegonepea genotypes were used in the study to confirm their nodulation abilities with each of the putative rhizobial strains. The genotypes consisted of a locally sourced landrace (PP1-3018) and an improved genotype (PP5-3021) (Table 1). A set of 39 randomly selected putative rhizobial strains was used in the study for inoculating the seed of the pigeonpea genotypes during planting as described below (Table 2). All the putative rhizobial strains were obtained originally from soil samples (that were collected from diverse locations across South Africa) and used for inoculating and trapping experiments involving five distinct pigeonpea genotypes in a glasshouse [26].

2.2. Glasshouse Trial Establishment

The seeds of each pigeonpea genotype were pretreated with 70% ethanol for 30 s, and surface sterilized with 3.5% sodium hypochlorite for 3 min. After rinsing 5 times with sterile distilled water, the seeds were imbibed in sterile distilled water for 3–4 h at room temperature. The seeds were then germinated overnight at 28 °C on water agar medium containing 20 g/L bacteriological agar (Merck, Midrand, South Africa). Three seeds of each genotype were planted separately in a 2.0 L Leonard jar containing sterilized sand and nitrogen-free Hoagland’s plant growth solution [39]. The soil was saturated with sterile distilled water, followed by planting the seeds and inoculating each genotype separately with each of the rhizobial strains [26]. The Leonard jars were then placed in a temperature-controlled glasshouse with a day length set at 14 h and temperature set at 28 °C, and night length set at 10h and temperature set at 20 °C.

2.3. Experimental Design, Measurements and Statistical Analysis

A randomized complete block design with three replications was used for the study. After six weeks of growth, prior to harvesting, the color of leaves and root nodules were assessed for evidence of nitrogen fixation. Plants with yellow, chlorotic leaves were expected to either lack nodules or have ineffective nodules with white interiors suggesting absence of nitrogen fixation [40]. In contrast, the plants with dark green leaves as well as nodules with pink to red interiors indicated successful nitrogen fixation. The latter is indicative of the presence of leghemoglobin, a protein produced by the host essential to the function of the oxygen-sensitive nitrogenase [41]. Upon harvesting the plants, four nitrogen fixation variables, namely, number of root nodules (NN), nodule fresh weight (NFW), root fresh weight (RFW), and shoot fresh weight (SFW), were measured for each plant after which the nodule dry weight (NDW), root dry weight (RDW), and shoot dry weight (SDW) were determined following oven-drying to a constant weight at 70 °C. The quantitative data sets for each of the seven nitrogen fixation variables for each pigeonpea genotype were analyzed using the standard analysis of variance procedure followed by mean separation at the 5.0% probability level of the least significance difference (LSD) test in the Statistical Analysis Software (SAS version 9.1, Cary, NC, USA) package [42].

2.4. DNA-Based Identification of Rhizobial Strains

Representatives of the rhizobial strains that were previously isolated from pigeonpea were identified using DNA sequences for both the 16S ribosomal RNA (rRNA) subunit gene and the recA house keeping gene [43,44,45]. To achieve this, the respective bacteria were used to inoculate tubes containing 5 mL of sterile tryptone yeast extract broth (TYB), which consisted of 5 g/L tryptone (Oxoid, Midrand, South Africa), 3 g/L yeast extract (Biolab, Midrand, South Africa), and 15 g/L agar (Merck, Midrand, South Africa). The inoculated tubes were incubated for 24 to 48 h at 28 °C and 150 rpm on a rotary shaker. Bacterial cells were then harvested by centrifugation and subjected to DNA extraction using the WIZARD genomic DNA purification kit (Promega, Madison, WI, USA).
The extracted DNA was used to PCR amplify the relevant regions. For 16S rRNA, the primers 27F (5′ AGA GTT TGA TCC TGG CTC AG 3′) and 1485R (5′ TAC CTT GTT ACG ACT TCA CCC CA 3′) were employed [46]. Each 50 μL-reaction 5 ng/μL DNA, 1.5 mM MgCl2, 800 μM dNTPs, 0.5 μM of each primer and 0.025 U/μL SuperTherm Taq polymerase (Promega, Madison, WI, USA) together with its reaction buffer. PCR amplification was carried out in an Eppendorf Master Cycler Gradient apparatus (Applied Biosystems, San Francisco, CA, USA). The cycling conditions entailed and initial denaturation at 94 °C was for 4 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 60 °C for 30 s, and extension at 72 °C for 1 min, after which a final extension step was performed at 72 °C for 1 min.
For the recA PCR, four different sets of primers were used. The primers recA63F (‘5 ATC GAG CGG TCG TTC GGC AAG GG 3′) and recA504R (‘5 TTG CGC AGC GCC TGG CTC AT 3′) were used to amplify a portion of recA in strains representing Bradyrhizobium, Paraburkholderia, and Phyllobacterium, as well as some strains of Rhizobium [46]. For Rhizobium strains, we also used primers recA6F (‘5 CGK CTS GTA GAG GAY AAA TCG GTG GA 3′) and recA555R (‘5-CGR ATC TGG TTG ATG AAG ATC ACC AT 3′). In both these cases, the reaction mixtures were constituted the same as for the 16S rRNA PCRs (except that the relevant recA primers were used) and optimum cycling conditions were applied [46]. For those strains in which the initial two sets of recA primers did not work, we used a third set of primers, TSrecAf5 (‘5 CAC TGC MYT GCG TAT YGT CGA AGG 3′) and TsrecAr3 (‘5 GAT CTT CAT SCG GAT CTG GTT GATG 3′) [46]. For these reactions, we employed FastStart High-Fidelity Taq and its MgCl2 and buffer (Roche Diagnostics, Midrand, South Africa) and optimum cycling conditions were applied.
All PCR products were purified with Exonuclease 1 and FASTAP alkaline phosphatase (Thermo Scientific, Johannesburg, South Africa). The PCR products were sequenced in both directions (using the original PCR primers) with an ABI PRISM BigDye Terminator v3.1 Cycle Sequencing Kit on an ABI3100 Automated Capillary DNA sequencer (Applied Biosystems, San Francisco, CA, USA). All raw sequence files were inspected and edited using Chromas Lite version 2.0 (Technelysium, Brisbane, Australia) and BioEdit version 5.0.9 [47]. The 16S rRNA sequence for each strain was compared to all publicly available sequences in the GenBank database of the National Centre for Biotechnology Information using the BLASTN software [48]. A separate dataset was constructed for representatives for each of the genera recovered in this study. The type strain information necessary to construct the datasets was obtained from a list of prokaryotic names with standing in nomenclature [49] and included multiple-sequence alignment files together with the pigeonpea rhizobial sequences generated in this study. All sequences generated during this study were submitted to National Center for Biotechnology Information using the Genbank submission portal National Institute of Health and were assigned the following accession numbers: 16S rRNA OK376606-OK384775, OK392633-OK393635, OP010913-OP010918, and OP013027-OP013030.
All 16S rRNA datasets were aligned with the online server of MAFFT (multiple alignment using fast Fourier transformation [50]), taking secondary structure into account by using the Q-INS-I strategy [51]. The recA datasets were aligned manually in BioEdit, based upon the inferred amino acid sequences. Sequences which were too short for meaningful comparison (for type strains as well as pigeonpea strains) were excluded from the alignments. To determine the appropriate evolutionary models for the alignments, jModelTest v2.1.7 was used [52,53]. For those datasets that did not use the general time reversible (GTR) model, maximum-likelihood analyses were performed in PhyML v3.1 while the analysis of those datasets using this model was performed with PhyML v.3.0 on the Montpellier bioinformatics platform [54]. Branch support was evaluated using bootstrap analysis [55] performed using 1000 pseudoreplicates.

3. Results

3.1. Glasshouse Nodulation Test Experiment

Bacterial strains from the current study were tested for nodulation on an indigenous pigeonpea landrace (PP1-3018) and an improved genotype (PP5-3021). Effective nodulation was evident from the plants’ healthy appearance and green color, while those in which nodulation was ineffective or lacking were small, with yellow to light green leaves (Figure 1). Inspection of roots also revealed that the healthy plants had numerous nodules with pink interiors, while those of the less healthy plants lacked nodules or harboured ineffective, small nodules, lacking leghemoglobin. The uninoculated control plants were also stunted with yellow leaves, and their roots did not have nodules. In all cases, inoculation of the two genotypes and subsequent DNA sequence analysis confirmed that the inocula used in the various treatments were indeed responsible for the observed phenotypes.
Of the set of 39 strains that were used in the study, 32 nodulated the indigenous landrace effectively, while 34 effectively nodulated the improved genotype. Of the 32 which nodulated the indigenous genotype, 1–32 nodules/plant were produced on average, depending on the bacterial inoculum used. For those nodulating the improved genotype, this ranged from 1–27 nodules/plant, depending on the bacterial inoculum used. The highest NN was induced by the rhizobial strain 10ap3 (identified as Rhizobium sp 10) (Figure 2). In certain cases, strains capable of inducing most nodules and yielding the highest biomass on the indigenous genotype, were also able to do so on the improved genotype. Significant examples are strains 10ap3 (identified as Rhizobium sp 10) and 16a2p1 (identified as Rhizobium sp 12) (Figure 2).
The results also showed that the Bradyrhizobium strain 13bp3 (coded R30) produced NDW with the landrace genotype, but not with the improved genotype (Figure 3). In contrast, two Rhizobium sp (22ap1 and 29ap1) produced no detectable NDW with both pigeonpea genotypes (Figure 3). The SDW was similar between the landrace and the improved genotype with some of the rhizobial strains, for instance Phyllobacterium sp 31ap4 (code R23) and Paraburkholderia sp 30a2p3 (coded R8) (Figure 4). Two Bradyrhizobium strains (7a2p3 and 33ap4) and one Rhizobium strain (39a3p3) induced significantly high RDW in the improved genotype while inoculation with the Rhizobium sp 30bp3 resulted in a significantly high RDW in the landrace genotype (Figure 5). The respective fresh weights (of the nodules, shoots, and roots) showed some variable responses among the two pigeonpea genotypes but were considered less reliable indicators of nitrogen fixation (Supplementary Tables S1 and S2).

3.2. DNA-Based Identification of Rhizobial Strains

To identify the bacteria at species level, we utilized the phylogenies inferred from the 16S rRNA (diagram not shown) and recA alignments that were generated for the Rhizobium (Figure 6) and Bradyrhizobium (Figure 7). For this purpose, phylogenetic distances among known species were compared to those among the strains from this study. This approach was used in combination with the species delineation method which assumes that members of a species would consistently group together in different gene trees [21,34]. The strains that belong to the same genus or species mostly formed the same grouping either on the 16S rRNA or the recA phylogeny. For instance, in the Rhizobium genus, the Rhizobium sp 5b2p1, 10ap3, and 30bp3 formed the same cluster with R. lusitanum, R. multihospitium and R. tropici in the recA tree (Figure 6). In addition, the Bradyrhizobium strain 33ap4 is a close relative of B. ferriligni and B. elkanii (Figure 7). From the phylogenetic standpoint, these approaches revealed that all the strains isolated in the current study are likely new.

4. Discussion

This study characterized the rhizobial strains which were originally sampled from diverse locations (virgin or grasslands transects) that had no history of pigeonpea cultivation across South Africa. In these regions, some studies on edible legumes such as cowpea, dry bean, and soybean were conduted previously, but little is known about their symbiotic efficiency with pigeonpea [24]. We thus aimed to characterize pigeonpea-nodulating bacteria from soils that were previously collected from different regions of South Africa, and then evaluate their ability to nodulate and promote the growth of pigeonpea using an indigenous and an improved genotype (Figure 3). The results of the nodulation test indicated that some rhizobial strains that were evaluated (for instance, Bradyrhizobium strain 13bp3) could nodulate with the indigenous genotype (Figure 3) but could not nodulate with the improved genotype, thus suggesting that some soils had strains with low nodulation and nitrogen fixation efficiencies as compared to soils from other locations such as those with strains 23ap5, 34a2p5, and 22ap1. Similarly, some rhizobial strains (35bp1, 14a1p5 and 22ap1) failed to nodulate the improved genotype suggesting that there was host genotype x rhizobial strain incompatibility between some strains and the improved pigeonpea genotype. Nevertheless, there were nodulation compatibilities with similar rhizobial strains, such as 32b2p5, 10ap3, and 16a2p1, in both genotypes.
Our results revealed that diverse bacteria from both the α-proteobacteria and β-proteobacteria may occupy the root nodules of pigeonpea. We further assembled confirmed rhizobia, belonging to distinct genera, namely, Rhizobium, Bradyrhizobium, and Phyllobacterium, and one strain from the genus Paraburkholderia in the β-proteobacteria. In addition to the rhizobia, other non-rhizobial bacteria were also isolated and partially identified with 16S rRNA. These mostly represented Bacillus, Pseudomonas, Burkholderia, and Paenibacillus, which have no known history of nodulation [56]. However, these genera are known to contain endophytes and plant growth promoting rhizobacteria (PGPR) [57]. Another study identified endophytes in nodules of groundnut improving plant growth [58]. In addition, PGPR, Burkholderia sp. Nafp2/4-1b (SARCC-3049) could improve the growth of maize, while coinoculation of Burkholderia and rhizobia on Medicago sativa increased the number of nodules and plant biomass of Medicago sativa [59]. Future research should thus seek to investigate their potential for promoting the growth of pigeonpea, as previous work has shown that they can benefit legume growth in various ways.
Rhizobium and Bradyrhizobium appear to be common symbionts of pigeonpea (Figure 2) across the sample areas in South Africa. This was consistent with the observation that pigeonpea-nodulating rhizobial strains native to African soils belong to the broader B. japonicum and B. elkanii groups [60]. In addition, slow-growing Bradyrhizobium sp of the cowpea miscellany group and fast-growing Rhizobium sp nodulated pigeonpea [61]. Similarly, other studies identified Bradyrhizobim species as a commonly nodulating pigeonpea on the coast of West Africa in Côte d’ Ivoire, while Rhizobium species commonly nodulated pigeonpea in semi-arid regions of India [33,62].
Our study also showed for the first time that members of Phyllobacterium and Paraburkholderia can nodulate pigeonpea (Figure 3). In previous studies, endophytic strains of the genus Paraburkholderia that exhibited antimicrobial potential against Fusarium udum were isolated from the leaves, roots, and stems of pigeonpea [63]. Nonetheless, some strains from this genus that could fix N were associated mainly with mimosoid legumes [37,38]. Similarly, there are no reports of symbiotic strains of Phyllobacterium that were isolated from pigeonpea. Therefore, this legume is thus compatible with diverse symbionts, and some of these less commonly encountered bacteria might have potential in agriculture. Moreover, the availability of diverse inoculants would contribute to the expansion of pigeonpea to new areas. The indigenous genotype of pigeonpea performed better than the improved genotype. Our findings correlate with those from other studies, which also found that pigeonpea can grow well in natural environments without artificial inoculation [64]. The indigenous pigeonpea genotype interacted more effectively with indigenous soil rhizobia in comparison to the imported genotype. Therefore, indigenous rhizobia and unimproved pigeonpea genotypes may be are better suited to one another when grown in these environments. However, field trails will be necessary before proposing these strains for commercial inoculum production. They could also be tested on more genotypes, with the emphasis on climate change, e.g., by testing at higher temperatures and under semi-arid conditions.
In conclusion, this study revealed that pigeonpea is compatible with diverse groups of rhizobia occurring in South Africa which include both α- and β-proteobacteria as their symbiotic partners. Furthermore, Rhizobium and Bradyrhizobium from South African soils are the common symbionts of pigeonpea. In addition, the indigenous pigeonpea genotypes appear to be more compatible with the indigenous strains than the improved genotypes. For future studies, several strains from this study can be used for co-inoculating pigeonpea seed to determine if a significant improvement in the crop productivity will be achieved.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture13010030/s1, Table S1: The nodule fresh weight of the improved genotype and landrace of pigeonpea; Table S2: The shoot fresh weight of the improved genotype and landrace of pigeonpea.

Author Contributions

Conceptualization, E.T.G. and F.L.B., methodology, F.L.B., C.W.B., K.K., E.T.S. and E.T.G., analysis of data, F.L.B., C.W.B., A.I.H. and E.T.G., writing—original draft preparation, F.L.B. and E.T.G., writing—review and editing, E.T.G., C.W.B. and E.T.S. All authors have read and agreed to the published version of the manuscript.

Funding

The University of Venda Research and Publications Committee, funded this research (Grant number: SARDF/18/PPR/02).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The study was sponsored by the University of Venda Research and Publications Committee, (Grant number: SARDF/18/PPR/02). We also thank the staff at the Nitrogen Fixation Unit of the ARC-Plant Health and Protection Institute for their assistance in completing the glasshouse trails included in this study.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Legume Phylogeny Working Group (LPWG). A new subfamily classification of the Leguminosae based on a taxonomically comprehensive phylogeny. Taxon 2017, 66, 44–77. [Google Scholar] [CrossRef] [Green Version]
  2. Wang, Q.; Liu, J.; Zhu, H. Genetic and Molecular mechanism underlying symbiotic specificity in legume-rhizobium interactions. Front. Plant Sci. 2018, 9, 313. [Google Scholar] [CrossRef] [PubMed]
  3. Zahran, H.H. Rhizobium-Legume symbiosis and nitrogen fixation under severe conditions and in an arid climate. Microbiol. Mol. Biol. Rev. 1999, 63, 968–989. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Egbe, O.M. Assessment of biological nitrogen fixing potentials of pigeonpea genotypes intercropped with sorghum for soil fertility improvement in Southern Guinea savanna of Nigeria. J. Trop. Agric. Food Environ. Ext. 2007, 6, 33–45. [Google Scholar] [CrossRef] [Green Version]
  5. Mhango, W.G.; Snapp, S.; Kanyama-Phiri, G.Y. Biological nitrogen fixation and yield of pigeonpea and groundnut: Quantifying response on smallholder farms in northern Malawi. Afr. J. Agric. Res. 2017, 12, 1385–1394. [Google Scholar]
  6. Amarteifio, J.O.; Muthali, D.C.; Karikari, S.K.; Morake, T.K. The composition of pigeonpea (Cajanus cajan (L.) Millsp.) grown in Botswana. Plant Foods Hum. Nutr. 2002, 57, 173–177. [Google Scholar] [CrossRef]
  7. Rao, S.C.; Coleman, S.W.; Mayeux, H.S. Forage production and nutritive value of selected pigeonpea ecotypes in the southern Great Plains. Crop Sci. 2002, 42, 1259–1263. [Google Scholar] [CrossRef]
  8. Mapfumo, P.; Giller, K.E.; Mpepereki, S.; Mafongoya, P.L. Dinitrogen fixation by pigeonpea of different maturity types on granitic sandy soils in Zimbabwe. Symbiosis 1999, 27, 305–318. [Google Scholar]
  9. Hillocks, R.J.; Minja, E.; Nahdy, M.S.; Subrahmanyam, P. Diseases and pests of pigeonpea in eastern Africa. Int. J. Pest Man. 2000, 46, 7–18. [Google Scholar] [CrossRef]
  10. Gwata, E.T.; Shimelis, H. Evaluation of pigeonpea germplasm for important agronomic traits in Southern Africa. In Crop Production, 26th ed.; Goyal, A., Asif, M., Eds.; Intech Open Limited: London, UK, 2013; pp. 1–15. [Google Scholar]
  11. Gwata, E.T.; Siambi, M. Genetic enhancement of pigeonpea for high latitude areas in Southern Africa. Afr. J. Biotechnol. 2009, 8, 4413–4417. [Google Scholar]
  12. Myaka, F.A.; Sakala, W.D.; Adu-Gyamfi, J.J.; Kamalongo, D.; Ngwira, A.; Odgaard, R.; Nielsen, N.E.; Høgh-Jensen, H. Yields and accumulations of N and P in farmer-managed maize-pigeonpea intercrops in semi-arid Africa. Plant Soil 2006, 285, 207–220. [Google Scholar] [CrossRef]
  13. Ayenan, M.A.T.; Ofori, K.; Ahoton, L.E.; Danguah, A. Pigeonpea [(Cajanus cajan (L.) Millsp)] production systems, farmers’ preferred traits and implications for variety development and introduction in Benin. J. Agric. Food Secur. 2017, 6, 48. [Google Scholar] [CrossRef]
  14. Abaidoo, R.C.; Dashiell, K.E.; Sanginga, N.; Keyser, H.H.; Singleton, P.W. Time course of dinitrogen fixation of promiscuous soybean genotypes measured by the isotope dilution method. Biol. Fert. Soils. 1999, 30, 187–192. [Google Scholar] [CrossRef]
  15. Thies, J.E.; Bohlool, B.B.; Singleton, P.W. Subgroups of the Cowpea Miscellany: Symbiotic Specificity within Bradyrhizobium spp. for Vigna unguiculata, Phaseolus lunatus, Arachis hypogaea and Macroptilium atropurpureum. Appl. Environ. Microbiol. 1991, 57, 1540–1545. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Saxena, K.B. Genetic improvement of pigeonpea—A review. Trop. Plant Biol. 2008, 1, 159–178. [Google Scholar] [CrossRef]
  17. Gronemeyer, J.L.; Reinhod-Huren, B. Diversity of Bradyrhizobia in subsahara Africa: A rich resource. Front. Microbiol. 2018, 9, 2194. [Google Scholar] [CrossRef]
  18. Pandya, S.; Uchil, P.; Subramanian, M.; Desai, A. Determination of Host specificity of Cowpea Miscellany Rhizobium spp. by nod ABC-lacZ fusion. Curr. Microbiol. 1998, 36, 361–364. [Google Scholar] [CrossRef]
  19. Mpepereki, S.; Wollum, A.G., II; Makonese, F. Diversity in symbiotic specificity of cowpea rhizobia indigenous to Zimbabwean soils. Plant Soil 1996, 186, 167–171. [Google Scholar] [CrossRef]
  20. Law, I.J.; Botha, W.J.; Majaule, U.C.; Phalane, F.L. Symbiotic and genomic diversity of “Cowpea” bradyrhizobia from soils in Botswana and South Africa. Biol. Fert. Soils 2007, 43, 653–663. [Google Scholar] [CrossRef]
  21. Steenkamp, E.T.; Stepkowski, T.; Prymusiak, A.; Botha, W.J.; Law, I.J. Cowpea and peanut in southern Africa are nodulated by diverse Bradyrhizobium strains harboring nodulation genes that belong to the large pantropical clade common in Africa. Mol. Phylogen. Evol. 2008, 48, 1131–1144. [Google Scholar] [CrossRef]
  22. Pule-Meulenberg, F.; Belane, A.K.; Krasova-Wade, T.; Dakora, F.D. Symbiotic functioning and bradyrhizobial biodiversity of cowpea (Vigna unguiculata L. Walp.) in Africa. BMC Microbiol. 2010, 10, 89. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. Odeny, D.A. The potential of pigeonpea (Cajanus cajan (L.) Millsp.) in Africa. Nat. Resour. Forum 2007, 31, 297–305. [Google Scholar] [CrossRef]
  24. Grain, S.A. Pigeonpea—A Potential Crop for Food and Health Security. 2020. Available online: https://www.grainsa.co.za/pigeon-pea-a-potential-crop-for-food-and-health-security (accessed on 12 February 2022).
  25. Mathews, C.; Saxena, K.B. Prospects for pigeonpea cultivation in drought-prone areas of South Africa. In Proceedings of the First International Edible Legume Conference, Durban, South Africa, 17–21 April 2005; pp. 1–11. [Google Scholar]
  26. Bopape, F.L.; Gwata, E.T.; Hassen, A.I.; Zhou, M.M. Symbiotic efficiency of pigeonpea (Cajanus cajan) with different sources of nitrogen. Plant Gen. Resour. 2021, 19, 312–315. [Google Scholar] [CrossRef]
  27. Gwata, E.T. Potential impact of edible tropical legumes on crop productivity in the small-holder sector in sub-Saharan Africa. J. Food Agric. Environ. 2010, 8, 939–944. [Google Scholar]
  28. Beukes, C.W.; Palmer, M.; Manyaka, P.; Chan, W.Y.; Avontuur, J.R.; van Zyl, E.; Huntemann, M.; Clum, A.; Pillay, M.; Palaniappan, K.; et al. Genome data provides high support for generic boundaries in Burkholderia sensu lato. Front. Microbiol. 2017, 8, 1154. [Google Scholar] [CrossRef] [Green Version]
  29. Poole, P.; Ramachandran, V.; Terpolilli, J. Rhizobia: From saprophytes to endosymbionts. Nat. Rev. Microbiol. 2018, 16, 291–303. [Google Scholar] [CrossRef]
  30. Nautiyal, C.S.; Hegde, S.V.; van Berkum, P. Nodulation, nitrogen fixation, and hydrogen oxidation by pigeonpea Bradyrhizobium spp. in symbiotic association with pigeonpea, cowpea, and soybean. Appl. Environ. Microbiol. 1988, 54, 94–97. [Google Scholar] [CrossRef] [Green Version]
  31. Jain, D.; Kumari, A.; Saheewala, H.; Sanadhya, S.; Maheshwari, D.; Meena, R.H.; Singh, A.; Gera, R.; Mohanty, S.R. Biochemical, functional and molecular characterization of pigeonpea rhizobia isolated from semi-arid regions of India. Arch. Microbiol. 2020, 202, 1809–1816. [Google Scholar] [CrossRef]
  32. Rajendran, G.; Sing, F.; Desai, A.J.; Archana, G. Enhanced growth and nodulation of pigeonpea by co-inoculation of Bacillus strains with Rhizobium spp. Bioresour. Technol. 2008, 99, 4544–4550. [Google Scholar] [CrossRef]
  33. Singh, K.; Dhull, S.; Gera, R. Genetic diversity of diazotrophs nodulating pigeonpea in arid and semi-arid zones of Haryana, India. Int. J. Curr. Microbiol. App. Sci. 2018, 7, 3447–3467. [Google Scholar] [CrossRef]
  34. Gosai, J.; Anandhan, S.; Bhattacharjee, A.; Archana, G. Elucidation of quorum sensing components and their role in regulation of symbiotically important traits in Ensifer nodulating pigeonpea. Microbiol. Res. 2020, 231, 126354. [Google Scholar] [CrossRef] [PubMed]
  35. Dubey, R.C.; Maheshwari, D.K.; Kumar, H.; Choure, K. Assessment of diversity and plant growth promoting attributes of rhizobia isolated from Cajanus cajan L. Afric. J. Biotechnol. 2010, 9, 8619–8629. [Google Scholar]
  36. Bellés-Sancho, P.; Lardi, M.; Liu, Y.; Hug, S.; Pinto-Carbó, M.A.; Zamboni, N.; Pessi, G. Paraburkholderia phymatum homocitrate synthase NifV plays a key role for nitrogenase activity during symbiosis with papilionoids and in free-living growth conditions. Cells 2021, 10, 952. [Google Scholar] [CrossRef] [PubMed]
  37. Beukes, C.W.; Boshoff, F.S.; Phalane, F.L.; Hassen, A.I.; le Roux, M.M.; Stepkowski, T.; Venter, S.N.; Steenkamp, E.T. Both alpha and beta-rhizobia occupy the root nodules of Vachellia karroo in South Africa. Front. Microbiol. 2019, 10, 1195. [Google Scholar] [CrossRef]
  38. Silva, V.C.; Alves, P.A.C.; Rhem, M.F.K.; dos Santos, J.M.F.; James, E.K.; Gross, E. Brazilian species of Calliandra Benth. (tribe Ingeae) are nodulated by diverse strains of Paraburkholderia. Syst. Appl. Microbiol. 2018, 41, 241–250. [Google Scholar] [CrossRef]
  39. Somasegaran, P.; Hoben, H.J. Hand Book for Rhizobia; Springer: New York, NY, USA; Berlin/Heidelberg, Germany, 1994. [Google Scholar]
  40. Gwata, E.T.; Wofford, D.S. Potential of RAPD analysis of the promiscuous nodulation trait in soybean (Glycine max L.). Biol. Fert. Soils 2013, 49, 241–244. [Google Scholar] [CrossRef]
  41. Dakora, F.D. A functional relationship between leghaemoglobin and nitrogenase based on novel measurements of the two proteins in legume root nodules. Ann. Bot. 1995, 75, 49–54. [Google Scholar] [CrossRef] [Green Version]
  42. SAS. Statistical Analysis System User’s Guide: Statistical Version, 8th ed.; SAS Institute: Cary, NC, USA, 2003. [Google Scholar]
  43. De Castro Pires, R.; dos Reis Junior, F.B.; Zilli, J.E.; Fischer, D.; Hofmann, A.; James, E.K.; Simon, M.F. Soil characteristics determine the rhizobia in association with different species of Mimosa in central Brazil. Plant Soil 2018, 423, 411–428. [Google Scholar] [CrossRef] [Green Version]
  44. Dabo, M.; Sanjay, K.J.; Dakora, F.D. Phylogenetic evidence of allopatric speciation of bradyrhizobia nodulating cowpea (Vigna unguiculata L. walp) in South African and Mozambican soils. FEMS Microbiol. Ecol. 2019, 95, fiz067. [Google Scholar] [CrossRef] [Green Version]
  45. Lane, D.J. 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics; Stackebrandt, E., Goodfellow, M., Eds.; John Wiley and Sons: New York, NY, USA, 1991; pp. 115–175. [Google Scholar]
  46. Beukes, C.W.; Venter, S.N.; Law, I.J.; Phalane, F.L.; Steenkamp, E.T. South African Papilionoid legumes are nodulated by diverse Burkholderia with unique nodulation and nitrogen fixation loci. PLoS ONE. 2013, 8, 7. [Google Scholar] [CrossRef]
  47. Hall, T.A. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 1999, 41, 95–98. [Google Scholar]
  48. Altschul, S.F.; Gish, W.; Miller, W.; Myers, E.W.; Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 1990, 215, 403–410. [Google Scholar] [CrossRef] [PubMed]
  49. Parte, A.C.; Sardà Carbasse, J.; Meier-Kolthoff, J.P.; Reimer, L.C.; Göker, M. List of prokaryotic names with standing in nomenclature (LPSN) moves to the DSMZ. Int. J. Syst. Evol. Microbiol. 2020, 70, 5607–5612. [Google Scholar] [CrossRef] [PubMed]
  50. Katoh, K.; Toh, H. Improved accuracy of multiple ncRNA alignment by incorporating structural information into a MAFFT-based framework. BMC Bioinform. 2008, 9, 212. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  51. Guindon, S.; Gascuel, O. A simple, fast and accurate method to estimate large phylogenies by maximum-likelihood. Syst. Biol. 2003, 52, 696–704. [Google Scholar] [CrossRef] [Green Version]
  52. Darriba, D.; Taboada, G.L.; Doallo, R.; Posada, D. jModelTest 2: More models, new heuristics and parallel computing. Nat. Methods 2012, 9, 772. [Google Scholar] [CrossRef]
  53. Tavaré, S. Some probabilistic and statistical problems in the analysis of DNA sequences. In Some Mathematical Questions in Biology—DNA Sequence Analysis; Miura, R.M., Ed.; American Mathematical Society: Providence, RI, USA, 1986; pp. 57–86. [Google Scholar]
  54. Guindon, S.; Dufayard, J.F.; Lefort, V.; Anisimova, M.; Hordijk, W.; Gascuel, O. New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0. Syst. Biol. 2010, 59, 307–321. [Google Scholar] [CrossRef] [Green Version]
  55. Felsenstein, J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 1985, 39, 783–791. [Google Scholar]
  56. Hassen, A.I.; Bopape, F.L.; Trytsman, M. Nodulation study and characterization of rhizobial microsymbionts of forage and pasture legumes in South Africa. World J. Agric. Res. 2014, 2, 93–100. [Google Scholar] [CrossRef] [Green Version]
  57. Dhole, A.; Shelat, H.; Vyas, R.; Jhala, Y.; Bhange, M. Endophytic occupation of legume root nodules by nifH-positive non-rhizobial bacteria and their efficacy in the groundnut (Arachis hypogaea). Ann. Microbiol. 2016, 66, 1397–1407. [Google Scholar] [CrossRef]
  58. Hassen, A.I.; Khambani, L.S.; Swanevelder, Z.H.; Mtshweni, N.P.; Bopape, F.L.; Van Vuuren, A.; Van Der Linde, E.J.; Morey, L. Elucidating key plant growth-promoting (PGPR) traits in Burkholderia sp. Nafp2/4-1b(=SARCC-3049) using gnotobiotic assays and whole-genome-sequence analysis. Lett. Microbiol. 2021, 73, 658–671. [Google Scholar] [CrossRef] [PubMed]
  59. Degefu, T.; Wolde-Meskel, E.; Adem, M.; Fikre, A.; Amede, T.; Ijiewo, C. Morphophysiological diversity of rhizobia nodulating pigeonpea (Cajanus cajan L. Millsp.) growing in Ethiopia. Afr. J. Biotechnol. 2018, 17, 167–177. [Google Scholar]
  60. Mapfumo, P.; Mpepereki, S.; Mafongoya, P.L. Pigeonpea rhizobia prevalence and crop response to inoculation in Zimbabwean smallholder-managed soils. Exper. Agric. 2000, 36, 423–434. [Google Scholar] [CrossRef]
  61. Fossou, R.K.; Ziegler, D.; Zeze, A.; Barja, F.; Perret, X. Two major clades of bradyrhizobia dominate symbiotic interactions with pigeonpea in fields of Côte d’Ivoire. Front. Microbiol. 2016, 7, 1793. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  62. Botha, W.J.; Bloem, J.F.; Law, I.J. Bradyrhizobium sp. (Lupinus) in the winter rainfall region of South Africa. Biol. Fert. Soils 2002, 36, 335–343. [Google Scholar] [CrossRef]
  63. Bind, M.; Nema, S. Isolation and molecular characterization of endophytic bacteria from pigeonpea along with antimicrobial evaluation against Fusarium udum. Appl. Microbiol. 2019, 5, 163. [Google Scholar] [CrossRef]
  64. Gwata, E.T.; Silim, S.N. Utilization of landraces for the genetic enhancement of pigeonpea in eastern and southern Africa. J. Food Agric. Environ. 2009, 7, 803–806. [Google Scholar]
Agriculture 13 00030 g001 550
Figure 1. Pigeonpea plants of the (a) local landrace genotype PP1-3018 and (b) the exotic improved genotype PP5-3021. Genotype PP1-3018 appears a much healthier with a dark green color, while some of the plants of genotype PP5-3021 appear less healthy with light green/yellow leaves; (c) most of the plants with yellow leaves, as well as the uninoculated controls, lacked nodules (left) while the healthy-looking plants had roots bearing numerous indeterminate nodules (right).
Figure 1. Pigeonpea plants of the (a) local landrace genotype PP1-3018 and (b) the exotic improved genotype PP5-3021. Genotype PP1-3018 appears a much healthier with a dark green color, while some of the plants of genotype PP5-3021 appear less healthy with light green/yellow leaves; (c) most of the plants with yellow leaves, as well as the uninoculated controls, lacked nodules (left) while the healthy-looking plants had roots bearing numerous indeterminate nodules (right).
Agriculture 13 00030 g001
Agriculture 13 00030 g002 550
Figure 2. The effects of inoculation with rhizobial strains on the number of nodules per plant of the landrace and improved pigeonpea genotypes. The identity of each rhizobial strain (R1–R40) is described in Table 2 and the results of the statistical analysis are reported in Tables S1 and S2 (depending on the genotype).
Figure 2. The effects of inoculation with rhizobial strains on the number of nodules per plant of the landrace and improved pigeonpea genotypes. The identity of each rhizobial strain (R1–R40) is described in Table 2 and the results of the statistical analysis are reported in Tables S1 and S2 (depending on the genotype).
Agriculture 13 00030 g002
Agriculture 13 00030 g003 550
Figure 3. The effects of inoculation with rhizobial strains on the nodule dry weight per plant of the landrace and improved pigeonpea genotypes. The identity of each rhizobial strain (R1–R40) is described in Table 2 and the results of the statistical analysis are reported in Tables S1 and S2 (depending on the genotype).
Figure 3. The effects of inoculation with rhizobial strains on the nodule dry weight per plant of the landrace and improved pigeonpea genotypes. The identity of each rhizobial strain (R1–R40) is described in Table 2 and the results of the statistical analysis are reported in Tables S1 and S2 (depending on the genotype).
Agriculture 13 00030 g003
Agriculture 13 00030 g004 550
Figure 4. The effects of inoculation with rhizobial strains on the shoot dry weight per plant of the landrace and improved pigeonpea genotypes. The identity of each rhizobial strain (R1–R40) is described in Table 2 and the results of the statistical analysis are reported in Tables S1 and S2 (depending on the genotype).
Figure 4. The effects of inoculation with rhizobial strains on the shoot dry weight per plant of the landrace and improved pigeonpea genotypes. The identity of each rhizobial strain (R1–R40) is described in Table 2 and the results of the statistical analysis are reported in Tables S1 and S2 (depending on the genotype).
Agriculture 13 00030 g004
Agriculture 13 00030 g005 550
Figure 5. The effects of inoculation with rhizobial strains on the root dry weight per plant of the landrace and improved pigeonpea genotypes. The identity of each rhizobial strain (R1–R40) is described in Table 2 and the results of the statistical analysis are reported in Tables S1 and S2 (depending on the genotype).
Figure 5. The effects of inoculation with rhizobial strains on the root dry weight per plant of the landrace and improved pigeonpea genotypes. The identity of each rhizobial strain (R1–R40) is described in Table 2 and the results of the statistical analysis are reported in Tables S1 and S2 (depending on the genotype).
Agriculture 13 00030 g005
Agriculture 13 00030 g006 550
Figure 6. A recA maximum-likelihood phylogeny of Rhizobium strains associated with pigeonpea. The strains in red originated from pigeonpea nodules. Bootstrap support for the groupings above 50% are indicated. Information regarding the type strain is provided while the GenBank accession number or locus tag for each species is indicated in brackets. Species names which appear in inverted commas (‘…’) are combinations which have not yet been validly published. The scale bar corresponds to the number of nucleotide changes per site. The model used is GTR + I + G with invariable proportion site value 0.4310 and gamma value 0.8190.
Figure 6. A recA maximum-likelihood phylogeny of Rhizobium strains associated with pigeonpea. The strains in red originated from pigeonpea nodules. Bootstrap support for the groupings above 50% are indicated. Information regarding the type strain is provided while the GenBank accession number or locus tag for each species is indicated in brackets. Species names which appear in inverted commas (‘…’) are combinations which have not yet been validly published. The scale bar corresponds to the number of nucleotide changes per site. The model used is GTR + I + G with invariable proportion site value 0.4310 and gamma value 0.8190.
Agriculture 13 00030 g006
Agriculture 13 00030 g007 550
Figure 7. A recA maximum-likelihood phylogeny of Bradyrhizobium strains associated with pigeonpea. The strains in red originated from pigeonpea nodules. Bootstrap Bootstrap support for the groupings above 50% are indicated. Information regarding the type strain is provided while the GenBank accession number or locus tag for each species is indicated in brackets. Species names which appear in inverted commas (‘…’) are combinations which have not yet been validly published. The scale bar corresponds to the number of nucleotide changes per site. The model used is GTR+ I+ G with invariable proportion site value 0.4986 and gamma value 0.6930.
Figure 7. A recA maximum-likelihood phylogeny of Bradyrhizobium strains associated with pigeonpea. The strains in red originated from pigeonpea nodules. Bootstrap Bootstrap support for the groupings above 50% are indicated. Information regarding the type strain is provided while the GenBank accession number or locus tag for each species is indicated in brackets. Species names which appear in inverted commas (‘…’) are combinations which have not yet been validly published. The scale bar corresponds to the number of nucleotide changes per site. The model used is GTR+ I+ G with invariable proportion site value 0.4986 and gamma value 0.6930.
Agriculture 13 00030 g007
Table
Table 1. Pigeonpea germplasm used in the study.
Table 1. Pigeonpea germplasm used in the study.
Genotype Seed ColourStatus (Source)
PP1-3018GreyUnimproved landrace (locally sourced)
PP5-3021White/CreamImproved germplasm, (International Crops Research Institute for the Semi-Arid Tropic)
Table
Table 2. Rhizobial strains used in the study.
Table 2. Rhizobial strains used in the study.
Rhizobial StrainRhizobial Species
CodeDesignated Name
R18b2p1Rhizobium sp.
R28a2p3Bradyrhizobium sp.
R332b2p5Rhizobium sp.
R432b1p5Rhizobium sp.
R56bp3Bradyrhizobium sp.
R67a2p3Bradyrhizobium sp.
R730bp3Rhizobium sp.
R830a2p3Paraburkholderia sp.
R911a2p3Bradyrhizobium sp.
R1010ap3Rhizobium sp.
R1139a3p3Rhizobium sp.
R1216a2p1Rhizobium sp.
R1315ap1Rhizobium sp.
R1418ap3Rhizobium sp.
R1535ap3Rhizobium sp.
R1635bp1Rhizobium sp.
R1737ap4Rhizobium sp.
R185b2p1Rhizobium sp.
R1927b2p5Bradyrhizobium sp.
R2031b1p5Rhizobium sp.
R2131b2p3Rhizobium sp.
R2231b1p3Rhizobium sp.
R2331ap4Phyllobacteriumsp.
R2438a1p5Rhizobium sp.
R2533ap4Bradyrhizobium sp.
R2617ap1Rhizobium sp.
R2717a1p3Rhizobium sp.
R2814a1p5Rhizobium sp.
R2913b1p4Rhizobium sp.
R3013bp3Bradyrhizobium sp.
R3123ap5Rhizobium sp.
R3219bp5Bradyrhizobium sp.
R3336ap5Rhizobium sp.
R3434a2p5Rhizobium sp.
R3522ap1Rhizobium sp.
R3629ap1Rhizobium sp.
R3729a1p2Rhizobium sp.
R3829a2p2Rhizobium sp.
R3926bp3Rhizobium sp.
R40Controlwater
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Bopape, F.L.; Beukes, C.W.; Katlego, K.; Hassen, A.I.; Steenkamp, E.T.; Gwata, E.T. Symbiotic Performance and Characterization of Pigeonpea (Cajanus cajan L. Millsp.) Rhizobia Occurring in South African Soils. Agriculture 2023, 13, 30. https://doi.org/10.3390/agriculture13010030

AMA Style

Bopape FL, Beukes CW, Katlego K, Hassen AI, Steenkamp ET, Gwata ET. Symbiotic Performance and Characterization of Pigeonpea (Cajanus cajan L. Millsp.) Rhizobia Occurring in South African Soils. Agriculture. 2023; 13(1):30. https://doi.org/10.3390/agriculture13010030

Chicago/Turabian Style

Bopape, Francina L., Chrizelle W. Beukes, Kopotsa Katlego, Ahmed I. Hassen, Emma T. Steenkamp, and Eastonce T. Gwata. 2023. "Symbiotic Performance and Characterization of Pigeonpea (Cajanus cajan L. Millsp.) Rhizobia Occurring in South African Soils" Agriculture 13, no. 1: 30. https://doi.org/10.3390/agriculture13010030

APA Style

Bopape, F. L., Beukes, C. W., Katlego, K., Hassen, A. I., Steenkamp, E. T., & Gwata, E. T. (2023). Symbiotic Performance and Characterization of Pigeonpea (Cajanus cajan L. Millsp.) Rhizobia Occurring in South African Soils. Agriculture, 13(1), 30. https://doi.org/10.3390/agriculture13010030

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop