Characterization of rhizobia and their symbiotic performance on selected important legumes 
under abiotic stress conditions 

by 

Langutani Sanger Khambani  

(2050271) 

 

 

Thesis 

Submitted in fulfilment of the requirements for the degree 

Philosophiae Doctor  

in  

Molecular and Cell Biology 

in the Faculty of Science, University of the Witwatersrand, Johannesburg, South Africa 

 

 

Supervisor: Prof Karl Rumbold 

Co-supervisor: Dr Ahmed Hassen 

 

January 2023 

 

 

 



i 
 

 

Declaration 

 

I, Langutani Sanger Khambani, 2050271 am a student registered for the degree of Doctor of 

Philosophy in the academic year 2023. 

I hereby declare the following: 

 I am aware that plagiarism (the use of someone else’s work without their permission and/or 

without acknowledging the original source) is wrong. 

 I confirm that the research proposal submitted for assessment for the above course is my own 

unaided work except where I have explicitly indicated otherwise. 

 I have followed the required conventions in referencing the thoughts and ideas of others. 

 I understand that the University of the Witwatersrand may take disciplinary action against me 

if there is a belief that this is not my own unaided work or that I have failed to acknowledge 

the source of the ideas or words in my writing. 

 

Signature:                      Date: 11 January 2023 

 

 

 

 

 

 

 



ii 
 

Acknowledgements 

 

To my supervisor Prof Karl Rumbold and co-supervisor Dr Ahmed Hassen , thank you for your 

guidance and professional support throughout the study. 

 

Agricultural Research Council (ARC), thank you for awarding me the bursary to pursue my 

doctoral degree under the Professional Development Programme (PDP) for two years.  

 

Special thanks to Mr Freddie Domba from the Agricultural Research Council for your 

unreserved assistance with the glasshouse trials. 

 

Namhla Ngxamani, thank you for your assistance when I conducted some of my laboratory 

experiments at Wits.  

 

To my family, your endless love and support kept me going, thank you. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



iii 
 

Characterization of rhizobia and their symbiotic performance on selected important legumes 

under abiotic stress conditions 

Abstract 

 

Introduction: Legumes are a source of proteins in human diet and a staple food for many 

cultures globally as an inexpensive meat alternative. They form a symbiotic relationship with 

rhizobia to form root nodules. Inside the nodules, the rhizobia fix atmospheric nitrogen (N2) by 

the biological nitrogen fixation (BNF) process. This provides a supply of nitrogen to plants. 

Lately, farmers are more open to the use of rhizobia inoculants due to the availability of effective 

and quality products in the market. These inoculants improve yields at a low cost when 

compared to chemical or artificial fertilizers. Symbiotic genes (nodA and nodC), the ACC 

deaminase enzyme (encoded by acdS gene) and exopolysaccharides (exoR gene) are essential for 

the rhizobium-legume association, particularly under abiotic stress conditions for effective 

nodulation. 

Aim: The current study aims at isolating stress tolerant SARCC (South African Rhizobium 

Culture Collection) isolates to be applied as inoculants under abiotic stress conditions. 

Materials and methods: Rhizobia spp. tolerance to various levels of temperature, 

acidity/alkalinity (pH5, pH7 and pH9), heavy metals (50mM, 100mM and 150mM 

concentrations of AlCl3.6H2O) and salinity (50mM, 100mM and 150mM concentrations of 

NaCl) stresses were investigated. Phylogenetic characterization of the isolates was determined 

using nucleotide sequence analysis of the 16S rRNA, exoR gene, acdS gene, the house keeping 

recA gene, as well as nodA and nodC symbiotic genes. A glasshouse nodulation efficacy test 

under normal and abiotic stress conditions was also conducted. 



iv 
 

Results: This study reports tolerance to abiotic stresses and the phylogenetic characterization of 

40 rhizobia strains previously isolated from the root nodules of Medicago sativa, Trifolium 

repens, Lupinus albus, Vigna unguiculata and Phaseolus vulgaris. The isolates exhibited 

significant variations in their tolerance to abiotic stresses. Using molecular approaches, some of 

the rhizobia isolates have been detected to possess certain beneficial traits involved in the 

reduction of abiotic stresses such as the acdS (9 isolates) and exoR (5 isolates) genes. The 

symbiotic genes nodC (7 isolates) and nodA (16 isolates) were detected as well. Phylogenetic 

analyses based on 16S rRNA gene, housekeeping genes (recA) revealed that the isolates belongs 

to five genera: Sinorhizobium, Bradyrhizobium, Rhizobium, Mesorhizobium and Aminobacter. 

Under glasshouse nodulation efficacy test, effective isolates provided the highest plant biomass 

and number of nodules under normal conditions, acidity/alkalinity and salinity abiotic stresses 

when compared to the un-inoculated controls. 

Conclusion: Amid the increasing threats of the global climate change and the rising abiotic 

stresses, these current results provide baseline information in the selection of rhizobia for use as 

inoculants under abiotic stressed conditions in South Africa. 

Key words: Rhizobia, abiotic stress, biological nitrogen fixation, nodules 

 

 

 

 

 

 



v 
 

Research outputs 

Publication forming part of PhD thesis:  

A manuscript has been submitted to Letters in Applied Microbiology journal: 

L.S. Khambani, A.I. Hassen, and K. Rumbold (2023). Characterization of Rhizobia spp. and 

detection of essential traits that promote nodulation of legumes under abiotically stressed 

conditions. Letters in Applied Microbiology journal (submitted). 

Conferences presentation 

Poster presentation 

Khambani, L.S., Hassen, A.I., Rumbold, K. & Steenkamp, E. (2018). Molecular characterization 

and nodulation efficacy test of rhizobium isolates on legumes of economic importance. Poster 

presented at the 45th Annual South African Association of Botany, AMA and SASSB Joint 

Congress, 8 -11 January 2019, University of Johannesburg, Kingsway Campus, South Africa. 

 

 

 

 

 

 

 

 

 

 



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Table of contents 

Chapter 1: Introduction         1 

1.1 Classification of rhizobia         1 

1.1.1 Alpha-rhizobia (α-rhizobia)        1 

1.1.2 Beta-rhizobia (β-rhizobia)        2 

1.1.3 Gamma-rhizobia (γ-rhizobia)        2 

1.2 Role of rhizobia          3 

1.3 Nodulation and nitrogen fixation genes        4 

1.3.1 Nodulation genes         4 

1.3.2 Nitrogen fixation genes         6 

1.4 Problem identification         7 

1.5 Aim and objective          8 

1.5.1 Aim           8 

1.5.2 Specific objective         8 

1.6 References           9 

Chapter 2 

2.1 Legumes           17 

Declaration  i 

Acknowledgement  ii 

Abstract 

Research outputs 

 iii 

iv 

Table of contents  v 

List of figures  viii 

List of tables  x 

List of abbreviations  xi 



vii 
 

2.2 Climate change and abiotic stress        19 

2.3 Nitrogen fixation          20 

2.4 Nodule formation          21 

2.5. Materials and methods         25 

2.5.1 Sampling          25 

2.5.2 Rhizobia isolates and growth media       26 

2.6 Glasshouse nodulation efficacy test       26 

2.6.1 Seeds germination         26 

2.6.2. Bacterial inoculum preparation        27 

2.6.3. Glasshouse nodulation efficacy test       27 

2.6.4. Statistical analysis         29 

2.7. Results           29 

2.8. Discussion          44 

2.9 Conclusion          52 

2.10 References           53 

Chapter 3 

3.1 Significance and impact of the study        73 

3.2 Abstract           74 

3.3. Introduction          75 

3.4 Materials and methods         78 

3.4.1 Sample collection and growth conditions      78 

3.4.2 In vitro assays for various abiotic stress tolerances     79 

3.4.3 Phylogenetic characterization of bacterial isolates     79 

3.5 Results and discussion         81 



viii 
 

3.6 Acknowledgement         99 

3.7 Conflicts of interest         99 

3.8 Funding information          99 

3.9 Author contribution         99 

3.10 References          100 

Chapter 4 

4. Tolerance of Rhizobia spp. to abiotic stress mechanisms     118 

4.1. 1-aminocyclopropane-1-carboxylate (ACC) deaminase     118 

4.2. Exopolysaccharides (EPS)        121 

4.3. Ultraviolet (UV) mutagenesis        123 

4.4. Materials and methods         124 

4.4.1. Screening for exopolysaccharide production      125 

4.4.2. ACC deaminase activity        125 

4.4.2.1 Growth of rhizobia         125 

4.4.2.2 Preparation of ACC solution and RMM      125 

4.4.2.3 The ACC deaminase activity        125 

4.4.3 Mutagenesis          126 

4.4.3.1 Ultraviolet (UVC) interval based mutagenesis     127 

4.4.3.2 Selection of potential mutants from UVC mutagenesis    127 

4.4.3.3 Molecular characterization to confirm acdS and exoR genes mutant   127 

4.5 Results            127 

4.6 Discussion           138 

4.7 Conclusion          142 



ix 
 

4.8 References           143 

Chapter 5: General conclusion        158 

 

List of figures 

Chapter 1 

Figure 1.1 Phylogenetic tree of Proteobacteria       3  

Figure 1.2 The synthesis of the nod factor core by common nodulation gene   5 

Figure 1.3.Oxygen controls nifA and fixK expression      6 

Chapter 2 

Figure 2.1 Variety of legume          18 

Figure 2.2 Shcematic overview of nodule formation      22 

Figure 2.3: Types of nodules         23 

Figure 2.4: Seeds germination        27 

Figure 2.5: Nodulation under glasshouse       29 

Figure 2.6: Effect of legume inoculation with Rhizobia      33 

Chapter 3:  

Figure 3.1 Maximum likelihood phylogenetic tree of 16S rRNA sequences   83 

Figure 3.2 Maximum likelihood phylogenetic tree of recA      86 

Figure 3.3 The Neighbor-Joining phylogenetic tree of nodA sequences   88 

Figure 3.4 Maximun likelihood phylogenetic tree of acdS     91 

Figure 3.5 Maximum likelihood phylogenetic tree of exoR sequences   92 

Chapter 4 

Figure 4.1 Ethylene synthesis pathway       119 

Figure 4.2 Rhizobium forming mucoid colonies on YMCR agar    128 



x 
 

Figure 4.3 Exopolysaccharide production by the rhizobium isolate     129 

Figure 4.4 Exopolysaccharide production by the rhizobium isolate    130 

Figure 4.5 Screening for ACC deaminase activity      131 

Figure 4.6 Screening for ACC deaminase activity       136 

Figure 4.7 1% agarose gel electrophoresis of PCR amplified acdS gene   137 

Figure 4.8 1% agarose gel electrophoresis of PCR amplified exoR gene   138 

List of Tables 

Chapter 2 

Table 2.1. Authentication test of rhizobia strains inoculated on Vigna inguiculata       34 

Table 2.2 Authentication test of rhizobia strains inoculated on of Medicago sativa       35 

Table 2.3 Authentication test of rhizobia strains inoculated on Phaseolus vulgaris        36 

Table 2.4 Authentication test of rhizobia strains inoculated on Trifolium repens        37 

Table 2.5 Authentication test of rhizobia strains inoculated on Lupinus albus        38 

Table 2.6 Effect of Acidity/alkalinity on nodulation and plant biomass of Vigna unguiculata   39 

Table 2.7 Effect of Acidity/alkalinity on nodulation and plant biomass of Medicago sativa      40 

Table 2.8 Effect of Acidity/alkalinity on nodulation and plant biomass of Phaseolus vulgaris  41 

Table 2.9 Effect of Acidity/alkalinity on nodulation and plant biomass of Trifolium repens      42 

Table 2.10 Effect of Acidity/alkalinity on nodulation and plant biomass of Lupinus         43 

 

Chapter 3        

Table 3.1 SARCC isolates exhibiting tolerance to various abiotic stress in-vitro        97 

Chapter 4 

Table 4.1 Screening of Rhizobia isolates for the utilization of ACC deaminase        132 
 



xi 
 

List of abbreviations 

SARCC  South African rhizobium culture collection 

PGP   Plant growth promoters 

UV   Ultraviolet 

ACC   1-Amino cyclopropane 1-carboxylic acid 

LPS   Lipopolysaccharides 

EPS   Exopolysaccharides  

DNA   Deoxyribonucleic acid    

BNF   Biological nitrogen fixation 

nodA   N-acyltransferase 

nodC   N-acetylglucosaminyltransferase 

recA   Recombinase A 

ARC-PHP  Agricultural research council-plant health protection 

RNA   Ribonucleic acid 

NCBI   National Center for Biotechnology Information 

nodD   Nodulation proten D 

LCOs   Lipo-Chitooligosaccharides 

YMCR   Yeast mannitol congo red 

YMB   Yeast mannitol broth 

RMM   Rhizobium minimal medium 

CRD   Completely Randomized design 

ANOVA   Analysis of variance 

LSD   Least significant difference 

bp   Base pair 

BLAST  Basic local alignment search 

ML   Maximum likelihood 

NJ   Neibhour Joining 



xii 
 

NCD   National communicable diseases 

PEG   Polyethylene glycol  

 



1 
 

CHAPTER 1 
 

INTRODUCTION 

 

1.1 Classification of Rhizobia 

Rhizobia are root nodulating bacteria found in the soil and form symbiotic relationships with 

legumes, this enables them to fix atmospheric nitrogen (Aminu et al., 2015). They are classified 

into two diverse groups which are represented by the α- and β-proteobacteria species in 18 

genera (Poole et al, 2018; De Lajudie et al., 2019). The α-proteobacteria are more diverse and 

most rhizobia species are Bradyrhizobium, Allorhizobium, Mesorhizobium, Azorhizobium, and 

Rhizobium and Sinorhizobium (Ensifer) (Maynaud, et al., 2012) and Microvirga genera, (Ardley 

et al., 2012). The β-proteobacteria genus was recently discovered and it comprises of 

Cupriavidus and Burkholderia species (Gyaneshwar et al., 2011) and Paraburkholderia tuberum 

(formerly Burkholderia tuberum) which was amongst beta-rhizobia species recognized first 

(Sawana et al., 2014). Thus, α and β-rhizobia distinguishes each class of the symbionts 

(Compant et al., 2008). Moreover, several studies have reported Pseudomonas sp.classified as γ-

Proteobacteria in black locust (Shiraishi et al., 2010). 

1.1.1 Alpha-rhizobia (α-rhizobia) 

The α-rhizobia are Hyphomicrobiales (formerly known as Rhizobiales) distributed in 17 genera 

of 7 famalies and these microsymbionts are common in legumes (Hordt et al., 2020). 

Mesorhizobium, Rhizobium, Bradyrhizobium and Sinorhizobium species are the most common 



2 
 

out of the 17 genera and the majority of rhizobia species. The α-rhizobia commonly found in 

common bean (Phaseolus vulgaris), mung bean (Vigna radiata), chickpea (Cicer arietinum), 

soybean (Glycine max), alfalfa (Medicago sativa), peanut (Arachis hypogaea), and pea (Pisum 

sativum) legumes (Chen et al., 2020). Ensifer (non-symbiotic) and Sinorhizobium (symbiotic) are 

currently comined and should be classified into two genera (pre-existing). This is based on their 

phenotypic and distinct genomic properties and their separation into two phylogenetically 

distinct clades (Fargozi et al., 2020). 

1.1.2 Beta-rhizobia (β-rhizobia) 

The β-rhizobia were isolated from tropical legumes, e.g Mimosa species, and other legumes. The 

β-rhizobia are less diverse and were discovered much later (Moulin et al., 2001; Liu et al., 2020). 

Moreover, they are currently classified into three genera i.e. Cupriavidus, Paraburkholderia and 

Trinickia belonging to the Burkholderiaceae family (Estrada-de los Santos et al., 2018). The 

Paraburkholderia and Trinickia genera were described based on the whole genome analyses of 

some former Burkholderia species (Dobritsa and Samadpour, 2016; Estrada-de los Santos et al., 

2018). 

1.1.3 Gamma-rhizobia (γ-rhizobia) 

The γ-rhizobia remain disputed and must be authenticated further. In Pseudomonas species 

(Shiraishi et al., 2010), γ-rhizobia were first described in black locust (Robinia pseudoacacia) 

root nodules isolates and Mesorhizobium species commonly nodulates black locust (Kang et al., 

2012). The nodC, nodA, nifD and nifH (symbiotic genes) sequences of these Pseudomonas 

species were similar to rhizobia species, indicating that these genes were possibily acquired via 

lateral transfer (Shiraishi et al., 2010). Moreover, Pseudomonas isolates were possibly 



3 
 

Mesorhizobium and β-rhizobia (mixed cultures). Subsequently, Pseudomonas in Trifolium 

legume root microbiome was abundant and bacteria without nodA or nodC genes are common in 

legumes root nodules (De Meyer and Willems 2012; (Hartman et al., 2017). 

 

Figure 1.1. The phylogenetic tree constructed from the 16S rRNA gene sequences of Proteobacteria. Rhizobia 

genera are indicated in bold. Image adapted from (Barreda and Fikri-Benbrahim, 2014). 

1.2 Role of Rhizobia 

Rhizobia species invade their legume hosts through root hairs, sites where the lateral root emerge 

or root epidermis and induces legume root nodules development. The rhizobia inside the root 

nodules of the host then fix atmospheric nitrogen (N2) in the presence of nitrogenase whereby 

nitrogen is converted to ammonium (NH4
+). The nitrogenase activity determines the amount of 

ammonium supply given to plants by rhizobia. High nitrogenase activity will produce a high 

amount of nitrogen that is utilized by the legume host plant and ultimately enters the earth’s food 

web to improve plant growth. In exchange, rhizobia acquire carbohydrates, proteins, and oxygen 



4 
 

produced by the legume plants to live and breed (Sprent et al., 2013; Aminu et al., 2015). The 

genus Rhizobium was the first group to be described in nitrogen fixing bacteria; it is the largest 

genus of rhizobia accommodating 112 species and has been used frequently as legumes nitrogen-

fixing bacteria (Mousavi, 2016; De Lajudie et al., 2019). 

1.3 Nodulation and nitrogen fixation genes  

The symbiotic nitrogen fixation process is dependant on the nitrogen fixing and nodulation 

genes, mostly the nod, nif and fix genes in the rhizobia genera. The accessory genes of the 

bacteria encode the Nod and Nif proteins and these genes are found in the transmissible genetic 

elements. Thus, they can be frequently transferred within the bacterial species genus and not 

frequently between the genera (Remigi et al., 2016). Rhizobia strains have the ability to infect 

compatible legume host and the growth conditions vary, this is based on the symbiotic 

development stage. These includes the growth conditions in the curled root hair pocket’s which 

are acidic and oxidative (Hawkins et al., 2017). Moreover, free-living and symbiotic growth 

stages require metabolic capabilities and a set of genes which are unique. Therefore, when 

developing strains to be used in the production of commercial inoculants, one must account for 

these requirements and other biological properties, including the industrial scale growth and the 

ability to survive during desiccation (O’Callaghan, 2016). 

1.3.1 Nodulation genes 

The nodA, nodB and nodC genes are essential in rhizobia species to synthesize the core structure 

of nod factor because they encode enzymes that synthesize the lipo-oligosaccharide core. The 

inactivation of these genes affects the ability of rhizobia spp. to evoke any kind of the plant’s 

symbiotic reaction. This is despite of the legume host species, their infection mode, location and 



5 
 

the type of the nodules they form (Long, 1989; Martinez et al., 1990). Moreover, the Nod genes 

enzymes have various functions. The nodC synthesizes the N-acetyl glucosamine which 

determines the Nod factor backbone synthesis whilst nodB is a chito-oligosaccharide deacetylase 

responsible for removing an N-acetyl group from the Nod factor and nodA is an acyltransferase 

responsible for the N-acylation of the amino-sugar backbone (Atkinson et al., 1994; Perret et al., 

2000; Lopez-Lara and Geiger, 2000). 

 

Figure 1.2. The nod factor core sythesis by nodA, nodB and nodC genes, nodE and nodF host-specific nodulation 

gene products. Image adapted from (Peters, 1997). 

 

 

 

 



6 
 

1.3.2 Nitrogen fixation genes 

The nitrogen fixation process is driven by a cluster of nif gene products (Thiel, 2019). The 

nitrogen fixing microbes are responsible for carrying out nitrogen fixation in the presence of 

nitrogenase which reduces nitrogen. Nitrogenase is comprised of two components, i.e. 

molybdenum iron (MoFe) and iron (Fe) protein and these componets are responsible for various 

functions. The molybdenum protein reduces nitrogen and the iron protein provides the electron 

during the nigrogen fixation process (Frank, 2014). The nitrogenase enzyme contains up to 20 

genes that encode amino proteins sequences in proteins, but the main structural genes are nifK 

nifD and nifH. The nifH gene is the Fe protein structural gene mostly essential in studies 

focusing on the ecology and evolution of nitrogen-fixing bacteria. The structural genes encoding 

α and β subunits of FeMo protein are nifD and nifK (Raymond et al., 2004; Dos Santos et al., 

2012). The nif gene regulation is controlled by nif and nifL whereby nif is the positive activator 

and nifL is a negative regulator. However, the overall expression of the nif gene is regulated by 

present levels oxygen (as indicated in Figure 1.3) and nitrogen (Monson et al., 1995). 

 

Figure 1.3. Oxygen level controls the expression of nifA and fixK expression in Sinorhizobium meliloti using fixL 
and fixJ regulators. Under low O2 conditions, fixJ regulator is phosphorylated and expresses nifA and fixK. In 
Bradyrhizobium japonicum, fixLJ controls the expression of fixK2 and its downstream targets. The nifA senses low 
O2 levels and activate nif-genes expression. Image adapted from (Stacey, 2007). 



7 
 

1.4 Problem identification 

Increasing profit and crop yield for farmers to meet food demand in Africa is challenging in the 

farming systems, specifically low-input (Tian and Yu, 2019). Moreover, the increasing cost and 

the environmental impact of inorganic nitrogen inputs have led to the reappraisal of legumes 

role. Legumes host rhizobia that fix atmospheric nitrogen, reducing the need for inorganic 

application (Harris and Ratnieks, 2022). Legumes establishes a symbiotic relationship with α and 

β-rhizobia where abundant atmospheric nitrogen (N2) is converted into N inside the nodules 

through the biological nitrogen fixation process (Udvari and Poole, 2013; Basu and Kumar, 

2020). Nitrogen fixation is essential in order to sustain agricultural production systems which 

benefit millions of smallholder farmers (Franke et al., 2018; Ulzen et al., 2018; Adjei-Nsiah et 

al., 2019). 

There is a need for agricultural technologies to focus on increasing production, system durability 

and improved profits so that household food security is enhanced for smallholder farmers 

(Helfenstein et al., 2020). The life cycles of rhizobia strains fixing nitrogen are complex whereby 

various abiotic stress factors may affect the biological nitrogen fixation process and the 

production of legumes (Poole et al., 2018; van Loon et al., 2018). Generally, rhizobia species are 

free-living in the rhizospheric soil, which provides unique nutritional and stress conditions where 

they must compete with other bacteria to form stable microbial population (Hinsinger et al., 

2009; Li et al., 2016). Although rhizobia have numerous traits that promote plant growth, 

arguably, the bacterial traits that facilitate legume nodulation especially under abiotic stress 

conditions are the possession of ACC deaminase enzyme and production of exopolysaccharides. 

These traits are key features in legume nodulation and in plant’s response to a vast range of 

abiotic stresses, thereby optimizing legumes and rhizobia interaction during nodulation. To help 



8 
 

overcome crop production limitations in stress prone areas, the screening and application of 

rhizobia strains that are stress tolerant could be a viable option. Rhizobia research on legume 

symbiosis in South Africa in the past focused mainly on the conventional applied aspects of 

inoculation with no records of the study of ACC deaminase and EPS impacts on nodulation and 

symbiotic nitrogen fixation. Therefore, the aim of the current study is to characterize selected 

rhizobia isolates from the SARCC collection for the occurrence of beneficial traits that promote 

nodulation and growth of legumes under abiotically stressed conditions. 

1.5 Aim and objectives 

1.5.1 Aim  

Characterization of rhizobia and their symbiotic performance on selected important legumes 

under abiotic stress conditions. 

1.5.1. Specific objectives 

 To evaluate rhizobia strains from the South African Rhizobium Culture Collection (SARCC) 

for their ability to produce ACC deaminase. 

 To screen the rhizobia isolates for in-vitro abiotic stress tolerance. 

 To detect the production of exopolysaccharide from the selected rhizobia strains. 

 To characterize the rhizobia isolates for genes involved in ACC deaminase (acdS) and 

exopolysaccharide (exoR) production as this are traits that aid in nodulation under abiotic 

stress conditions. 

 To conduct random mutagenesis of the acdS and exoR gene possessing rhizobia isolates.  

 To phylogenetically characterize the rhizobia strains possessing either ACC deaminase or 

EPS using symbiotic and housekeeping genes. 



9 
 

1.5 References 

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sources and rhizobium inoculation on growth and yield of soybean genotypes on Ferric Lixisols 

of Northern Guinea savanna zone of Ghana. Commun Soil Sci Plant Anal, 50, 853-868. 

Aminu, S.M., Shamsuddeen, U. & Dianda, M. (2015). Effects of inoculation on the growth of 

soybeans [Glycine max (L.) Merrill] planted in soils from different geographical location in 

North Western Nigeria. Int J. Adv Eng Technol, 3. 

Ardley, J., Parker, M., De Meyer, S., Trengove, R., O’Hara, G. et al. (2012). Strains of Gram-

negative, rod-shaped, non-spore-forming bacteria were isolated from nitrogen-fixing nodules of 

the native legumes Listia angolensis (from Zambia) and Lupinus texensis (from Texas, USA). Int 

J Syst Evol Microbiol, 62, 2579-2588. 

Atkinson, E.M., Palcic, M.M., Hindsgaul, O. & Long, S.R. (1994). “Proceedings of National 

Academy of Science” USA, 91, 8418-8422.  

Barreda, H. & Fikri-Benbrahim, K.  (2014). Taxonomy of rhizobia: current perspectives. Brit 

Microbiol Res J, 4, 616-639. 

Basu, S. & Kumar, G. (2020). “Nitrogen fixation in a legume-rhizobium symbiosis: the roots of a 

success story,” in Plant Microbe Symbiosis, eds A. Varma, S. Tripthi, and R. Prasad (Cham: 

Springer International Publishing), 35-53. 

Chen, W.F., Wang, E.T., Ji, Z.J. & Zhang, J.J. (2020). Recent development and new insight of 

diversification and symbiosis specificity of legume rhizobia: mechanism and application. J Appl 

Microbiol, 131, 553-563. 



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Chen, W.M., Laevens, S., Lee, T.M., Coenye, T., De Vos, P. et al. (2001). Ralstonia taiwanensis 

sp. nov., isolated from root nodules of Mimosa species and sputum of a cystic fibrosis patient. Int 

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Chen, W.M., Moulin, L., Bontemps, C., Vandamme, P., Bena, G. & Boivin-Masson, C. (2003). 

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Compant, S., Nowak, J., Coenye, T., Clement, C. & Ait Barka, E. (2008). Diversity and 

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Cummings, S.P., Gyaneshwar, P., Vinuesa, P., Farruggia, F.T., Andrews, M., et al. (2009). 

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De Meyer, S.E. & Willems, A. (2012). Multilocus sequence analysis of Bosea species and 

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11 
 

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species of the genera Burkholderia and Paraburkholderia. Int J Syst Evol Microbiol, 66, 2836-

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fixation and nitrogenase-like sequences amongst microbial genomes. BMC Genom, 13, 162. 

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L., Khan, N., Maluk, M. et al. (2018). Whole genome analyses suggests that Burkholderia sensu 

lato contains two additional novel genera (Mycetohabitans gen. nov., and Trinickia gen. nov.): 

implications for the evolution of diazotrophy and nodulation in the Burkholderiaceae. Genes 

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17 
 

CHAPTER 2 
 

Nodulation efficacy test of Rhizobia spp. on selected important legumes in vivo under 

normal and abiotic stress conditions 

2.1. Legumes 

Legumes belong to the Leguminosae family comprising of over 20,000 species of herbs, 

climbers, trees and shrubs and only a few species are used for human consumption (Yorgancilar 

and Bilgicli, 2014; Stagnari et al., 2017). They are mainly a source of reduced nitrogen in the last 

decade and are currently cultivated and consumed worldwide (Goncalves et al., 2016; Rajan and 

Ankur, 2017). In human diet, legumes are good sources of proteins as they provide dietary fibre, 

unsaturated fats, amino acids, essential minerals, carbohydrates, vitamins, and cheaper when 

compared to animal proteins (Bouchenak and Lamri-Senhadji, 2013; Annor et al., 2014; Rebello 

et al., 2014). Moreover, legumes are called ‘poor man’s meat’ and this is based on their 

consumption in different regions while taking into account the inverse relationship beween their 

consumption and income observation (Messina et al., 2016). Legumes are a staple food 

worldwide for numerous cultures and are amongst other first crops cultivated by mankind as 

inexpensive meat alternatives (Kouris-Blazos and Belski, 2016). 

Worldwide, health organizations have recommended legume consumption as a healthy diet. This 

is because of the role they play in controlling and preventing chronic non-communicable 

diseases (NCDs), e.g diabetes, cancer and cardiovascular diseases (Iqbal et al., 2006; Clifton, 



18 
 

2010; United Nations, 2014). They have a number of beneficial health traits such as 

hypocholesterolaemia, antiatherogenic, anticarcinogenic and hypoglycaemic properties (Ndidi et 

al., 2014; Messina, 2016). Legumes can also aid in controlling body weight because they give a 

greater satiety, prevents fat accumulation in the abdomen and lastly, they regulate sugar levels in 

the blood (Williams et al., 2008; Clifton, 2010). 

Cultivation of legumes by subsistence farmers at household level could be a source of income. 

However, irrigation systems and fertilizers are expensive so legumes are excellent crops for local 

farmers who cannot afford. Therefore, planting legumes reduce soil erosion and because of the 

legume-rhizobia symbiosis, they are considered an excellent rotation crops (Kalidass and 

Mahapatra, 2014; Nedumaran et al., 2015). Commonly cultivated legumes and subsequently 

used for human consumption are lentils peas (Pisum sativum), (Lens culinaris), broad beans 

(Vicia faba), soybeans (Glycine max), lupins (Lupinus), lotus (Nelumbo nucifera), mung bean 

(Vigna radiata), green beans (Phaseolus vulgaris) and peanuts (Arachis hypogaea) (Anonymous, 

2013; Yorgancilar and Bilgicli, 2014). However, just like any other crops, the production of 

legumes is adversely affected by a variety of abiotic stresses. 

 

Figure 2.1. Variety of legume seeds, Lens culinaris, Pisum sativum, Vicia faba, Cicer arietinum, Glycine max and 

Vigna unguiculata commonly cultivated for human consumption. Image adapted from (FAO, 2016). 



19 
 

2.2 Climate change and abiotic stress  

Agriculrural practices and crops have evolved over the years due to climate change which is 

constantly altering the environment. This directly affects the agricultural sector as it is mostly 

vulnerable. Agricultural production such as complex legume crop management, especially 

nitrogen nutrition is affected by climate change as it causes significant fluctuations in rainfall and 

temperature (Banerjee and Adenaeuer, 2014; Vadez et al., 2012; Hu et al., 2017; Peng et al., 

2020). In order to maintain soil health as agriculture faces climate change, sustainable land 

management practices have been known for centuries. These practices are potential mitigation 

strategies for climate change and have garnered new attention (Tilman et al., 2002; Norris and 

Congreves, 2018). Crop rotation restores nutrients in the soil and this is an ancient land 

management strategy. Legumes (e.g soybean, alfafa and peanut) are essential in crop rotations to 

increase the nitrogen content in the soil, thus reducing the application of synthesized nitrogen 

fertilizers (Foyer et al., 2019). However, this is not possible using legumes alone; they rely on 

rhizobia (Gibson et al., 2008; Oldroyd et al., 2011). 

Moreover, human society depends on agriculture for basic means of living, however, 

agriculture’s sustainable development is directly related to the development and survival of the 

human society (Banerjee and Adenaeuer, 2014; Peng et al., 2020). The production and 

distribution of plants is influenced greatly by various abiotic stresses (salinity, drought, low or 

high temperature) which are the negative impacts of non-living factors on living organisms in a 

specific environment, thus reducing crop yield and poor plant growth worlwide (Acquaah, 2012). 

These abiotic stressors have the ability to restrict the global use of arable lands and negatively 

impact production in agriculture (He et al., 2018). The advesre impacts in relation to abiotic 

stresses are more severe in Africa and South Asia and already these countries are experiencing 



20 
 

inadequate amount of food (Hasegawa et al., 2018). However, rhizobia plays a vital role under 

abiotic stress to aid in plant growth whereby the root system is modified, the mobilization and 

essential elements uptake is enhanced and physiological parameters are modulated 

(Egamberdieva et al., 2018). The development of rhizobia inoculants with an increased abiotic 

stress tolerance is of paramount importance for mitigating challenges related to climate change 

(Wongdee et al., 2021). Therefore, in order to adjust to climate change depicted by rainfall 

variability, feasible strategies are within reach. These include rhizobia inoculation, planting 

varieties that are resilient to water stress and timely planting to reduce drought impact 

(Tumuhairwe et al., 2018). 

2.3. Nitrogen fixation 

Nitrogen is an essential element required for good crop yield in agriculture (Andrews et al., 

2013; Dall’Agnol et al., 2014). All eukaryotic organisms use inorganic nitrogen in an ammonium 

(NH4) or nitrate (NO3) form (Giller, 2001; Loganathan et al., 2014), the abundant amount of 

nitrogen in the atmosphere is in a N2 form which cannot be readily utilized by plants. Rhizobia 

and archaea bacteria reduces nitrogen into ammonia through the nitrogen fixation process 

(Giller, 2001; Lindstrom and Mousavi, 2010). In agriculture, the biological nitrogen fixation 

technology is currently used to overcome challenges associated with the depleted of soil fertility 

and to reduce the application of inorganic fertilizers (Tairo and Ndakidemi, 2013; Nyoki and 

Ndakidemi, 2014). 

Nitrogen fixation is an affordable and inexpensive way to enhance crop yield when compared to 

the nitrogen chemical fertilizers (Jonah et al., 2012; Nyoki and Patrick, 2013; Mfilinge et al., 

2015). Inoculating legumes with rhizobia in agriculture is an essential nitrogen fixing technology 



21 
 

to improve productivity and yield of different legume crops while enhancing the fertility of the 

soil (Deshwal and Chaubey, 2014). Most legumes can take in and absorb NO3
- and NH4

+, 

however they can also obtain their required N amount from symbiotic microorganisms capable 

of fixing nitrogen (France et al., 2009; Sprent, 2009; Andrews et al., 2011; Andrews et al., 

2013). These microorganisms are termed “rhizobia” and are able to form nodules which are 

specialized structures on their host’s roots where rhizobia fixes atmospheric N2 into NH3 in the 

presence of nitrogenase (Sprent and Sprent, 1990; Andrews et al., 2009). 

2.4. Nodule formation  

Nodules are specialized organs located on legume’s species roots and stems and are associated 

with symbiotic nitrogen fixing bacteria, rhizobia. Rhizobia occupy an exclusive ecological niche 

for biological nitrogen fixation which reduces atmospheric dinitrogen to ammonia. Legume-

rhizobium symbiosis enables rhizobia to convert nitrogen into a readily available state to be used 

by the host legume for their growth which in turn provides a built in supply of nitrogen fertilizer 

for almost all legumes worldwide (Martinez-Romero, 2003; Brewin, 2010; Oldroyd et al., 2011). 

Each and every nodule can contain up to 109 rhizobia in a niche, this is perfect for reducing N2 

by providing the bacteria with malate (carbon supply) and a low O2 environment (Downie, 

2014). The legume and rhizobia association is mostly studied as one of the beneficial plant 

microbe interactions and it basically begins with a molecular interaction between the two 

partners. This interaction is induced by the initial signal exchange whereby when nitrogen is 

scarce in the soil, legume roots exude flavonoids and isoflavonoids (phenolic compounds) into 

the rhizospheric soil (Peters and Verma, 1990; Liu and Murray, 2016). Rhizobia sense these 

molecules and activate the nodulation protein D (nodD) which is a transcriptional regulator; this 



22 
 

causes genes responsible for Nod factors synthesis to be transcribed (D’Haeze and Holsters, 

2002). 

Nod factors are lipo-chitooligosaccharides (LCOs) that are host determinant and interact with 

flavonoids (Downie, 1998) and recognised by receptors in the root cells plasma membrane. In 

plants, Nod factors recognition induces molecular and physiological responses, e.g. the build up 

of genes induced during the symbiotic nitrogen fixation, curling of the root hair and the infection 

thread formation (Gage, 2004). The role of the infection thread is to facilitate root hair 

penetration (Szczyglowski et al., 1998). The cortical and pericycle cell division occurs 

simultaneously, resulting in nodule formation (Figure 2.2). Rhizobia bacteria then travels 

through the infection facilitated by the division of bacterial cell and ultimately freed into the 

induced nodule primordium cells (Calvert et al., 1984; Mathews et al., 1989). Nitrogen (N2) is 

fixed into ammonia (NH3) in the bacteroids, then utilised by the plants for amino acids and 

proteins synthesis (Poole et al., 2018). 

 

Figure 2.2. Schematic overview of the nodule development processes. The signals released from the host legume 

triggers a chain of events which allows rhizobia to invade root cells of the plant. Successful signaling leads to 

cortical cell divisions and nodule development. Image adapted from (Deakin and Broughton, 2009). 



23 
 

Legumes have two types of nodules i.e. determinate and inderterminate. The meristem activity is 

the main difference between the nodules (Kereszt et al., 2011). The determinate nodules 

meristem is short-lived with a spherical shape (Figure 2.3b), for example they are generally 

produced by tropical legumes e.g soybean (Glycine max) and bird food (Lotus japonicas). The 

indeterminate nodules meristem is persistent and tip-localised; resulting in a continuous nodule 

growth and a cylindrical shape with branches (Figure 2.3a). They are mainly produced by 

temperate legumes, e.g. pea (Pisum sativa), white clover (Trifolium repens) and barrel clover 

(Medicago truncatula) (Sprent, 2009). 

 

Figure 2. 3. Types of nodules, indeterminate nodules of Trifolium repens (a) and derterminate nodules of Vigna 

unguiculata (b). Image adapted from the current research. 

When soils are devoid of native rhizobia that fix atmospheric nitrogen, the BNF technology 

becomes successful provided the isolation, screening and characterization of indigenous soil 

rhizobia strains is properly done. This may result in the discovery of novel and effective strains 

a b 



24 
 

with a potential to be used in the production of inoculants (Lindstrom et al., 2010). The selection 

and isolation of stress tolerant rhizobia strains may enhance growth in legume plants through 

nodulation and the ability of plants to fix nitrogen under abiotic stresses (Zou et al., 1995). 

Moreover, the selection of rhizobia strains that are effective is a crucial aspect to ensure that 

biological nitrogen fixation benefits are obtained (Biswas et al., 2008). 

The selection of host plants in this study was based on the availability of isolates from the 

legumes of choice in the SARCC, legumes commonly cultivated in South Africa and their 

importance. The following host plants were selected, i.e. Vigna unguiculata, Phaseolus vulgaris, 

Medicago sativa, Trifolium repens and Lupinus albus. Vigna unguiculata is a staple crop in most 

African countries and serves as food for human consumption and fodder for livestock (Singh et 

al., 2003; Ndema, 2010). Phaseolus vulgaris is a protein power house and an affordable grain 

legume highly consumed globally and their production is central, ensuring food security in 

Africa and Latin America’s poor households (Siddiq and Uebersax, 2012; Wheeler and Von 

Braun, 2013; Ron et al., 2015; Bitocchi et al., 2017). Medicago sativa is a perennial legume crop 

grown worldwide with high effects on soil fertility, quality forage, nutritional quality and protein 

content. It is the most important forage crop, provides protein to dairy and beef, cattle, sheep, 

horses, birds and other livestock (Huyghe, 2003; Radovic et al., 2009; Li and Brummer, 2012). 

Trifolium repens is a forage crop consisting of about 240 species, high protein and minerals 

when compared to other grasses and species of Trifolium. It has the ability to fix atmospheric 

nitrogen and improves the quality of the feed (Shrestha, 2002; Ellison et al., 2006). Lupinus 

albus is a temperate legume with agronomic potential due to high protein content in the seeds 

and positive effect on soil fertility. It has become popular as protein source in the last decade, 



25 
 

used for animal and human feed and manure in agriculture (Rosolem et al., 2002; Jensen et al., 

2004). 

The South African Rhizobium Culture Collection (SARCC) based in Pretoria, South Africa hosts 

a large collection of rhizobia strains previously collected from various root nodules of legumes. 

A study done on legumes of South Africa using one of the strains from the collection has 

resulted in the development of rhizobia inoculants that were commercialized and currently 

available in the local markets (Strijdom, 1998). Abiotic stresses reduce plant biodiversity and 

this result in nutrients imbalance. Poor soil fertility limit crop production and is a challenge 

worldwide regarding food security (Vanlauwe et al., 2015; Chasek et al., 2019; Zhu et al., 2020). 

However, legume hosts are able to survive and grow under abiotic stresses; this improves when 

stress-tolerant rhizobia strains inoculate legumes (Wei et al., 2008; Kajic et al., 2019). Therefore, 

the present research work aims to perform a nodulation efficacy test on selected rhizobia strains 

from the collection under normal and abiotic stress conditions. 

2.5. Materials and methods 

2.5.1 Sampling  

Rhizobia isolates (25) previously collected from the root nodules of Medicago sativa, Trifolium 

repens Lupinus albus, Phaseolus vulgaris and Vigna unguiculata and deposited at the South 

African Rhizobium Culture Collection (SARCC) were used in this study. Pure cultures of 

rhizobia isolates were originally deposited by various reseachers and stored at -80 ℃ (ultra-low 

freezer) in 20% glycerol stocks. The collection is at the Agricultural Research Council-Plant 

Health and Protection (ARC-PHP), Biological Nitrogen Fixation (BNF) unit, Pretoria, South 

Africa. 



26 
 

2.5.2 Rhizobia isolates and growth media 

A total number of 25 rhizobia strains isolated from the root nodules of Medicago sativa, 

Trifolium repens, Lupinus albus, Vigna unguiculata and Phaseolus vulgaris as the target hosts 

were used in this study. The isolates were cultured onto yeast mannitol agar plates supplemented 

with 10% Congo red (YM-CR). The YM-CR media composition (g/L) consisted of: 10g 

mannitol, 0.5g K2HPO4, 0.1g NaCl, 0.2g MgSO4.7H2O, 0.4g yeast extracts, 15g agar, 10 mL 

congo red, pH 6.8-7.2 and autoclave at 121 ℃ for 20 min. The isolates were grown on YM-CR 

agar plates and incubated for 2-3 days at 26 ± 2 ℃ until colony formation. 

 

2.6. Glasshouse nodulation efficacy test 

2.6.1. Seeds germination 

The seeds of Medicago sativa, Trifolium repen, Lupinus albus, Vigna unguiculata and Phaseolus 

vulgaris host plants were surface sterilized as described by (Somasegaran and Hoben, 1994). 

Briefly, the seeds were firstly immersed in 95% ethanol for 30 seconds and 1% sodium 

hypochlorite solution for 1 minute. The excess bleach on the seeds was drained off and the seeds 

were rinsed repeatedly (5x) using sterile distilled water. To imbibe the seeds, they were soaked 

for 4 hours in sterile water and rinsed twice. The seeds were then placed in a petri dish (10-15 

seeds per plate, depending on the size of the seed) containing sterile 0.75% (w/v) water agar (10 

mL). Subsequently, the plates were incubated for 2-3 days at 28 ºC to germinate or until the 

radicle emerge (Figure 2.4). 



27 
 

 

Figure 2.4. Germination and development of Medicago sativa (a), Trifolium repens (b) and Vigna anguiculata (c) 

seedlings on water agar to be transplanted in Leonard jars. Image adapted from the current research. 

2.6.2 Bacterial inoculum preparation 

Rhizobial isolates were cultured onto YMCR agar plates and incubated at 28 ± 2 ℃ for 2-3 days. 

A single colony of the freshly grown culture for each isolate was further inoculated into 50 mL 

of yeast mannitol broth (YMB). The inoculated flasks were incubated at 28 ± °C for 2-3 days at 

120 rpm on a rotary shaker until visible growth; this was indicated by the broth turning milky. 

The bacterial inoculum was prepared as previously described by Hassen and Labuschagne (2010) 

with modifications, the final cell concentration of each rhizobia isolate was adjusted with 0.85 % 

of sterile saline solution to ± 1 x 108 cfu/mL using the T60 spectrophotometer (OD600nm of ≥ 0.8-

1). The adjusted cell concentration of each isolate was used as an inoculum. 

2.6.3 Glasshouse nodulation efficacy test 

The Leonard jar assembly containing sterile sand and nitrogen free Hoagland solution (Hoagland 

and Arnon, 1938) was used. Three seedlings per legume were transplanted per jar (Leonard, 

1943). The seedlings (Medicago sativa, Trifolium repen, Lupinus albus, Vigna unguiculata and 

Phaseolus vulgaris) in each jar were inoculated with 2 mL suspension of the rhizobia culture 

a b c 



28 
 

with a concentration level of ±1 x 108 cfu/mL and the seedlings were covered with sand 

immediately. This was done for the nodulation efficacy test under normal conditions. To induce 

the salinity, acidity/alkalinity abiotic stress conditions, three seedlings per legume were 

transplanted per jar (Leonard, 1984), containing Hoagland’s solution with added different NaCl 

concentrations (50mM, 100mM and 150mM) to induce salinity stress condition. The 

acidity/alkalinity condition was achieved by adjusting the Hoagland solution to pH5, pH7 and 

pH8. The jars were covered with half a petri dish to protect against contamination and the covers 

were removed when the seedlings reached a ± 2 cm height. Each treatment contained a legume 

seedlings (Medicago sativa, Trifolium repen, Lupinus albus, Vigna unguiculata and Phaseolus 

vulgaris) and rhizobia inoculum with three replications. Un-inoculated Leornard jars were the 

negative controls and only contained the nitrogen free solution. The inoculated (experiment) and 

non-inoculated (negative control) were both supplied with Hoagland solution (Broughton and 

Diworth, 1971). All treatments were replicated 3 times and the experiment was arranged in a 

completely randomized design (CRD) in a controlled glasshouse with day and night temperature 

of 27 oC and 18 oC respectively (Figure 2.5). The Leonard jars assemblies were maintained for 

five to eight weeks in the glasshouse during which N-free nutrient solution in the reservoir was 

monitored. The N-free nutrient solution may be depleted during the glasshouse trial and new 

solution may be added into reservoir, this depends on the size and growth conditions of the 

plants. After eight weeks, plants were carefully removed from the Leonard jars and the roots 

were rinsed thoroughly with water to remove the sand. Nitrogen deficiency in non-inoculated 

plants and treatments that failed to form nodules upon inoculation was evident by the lack of 

vibrant green colour in the leaves. The leaves were pale yellow and the overall growth of the 



29 
 

plants was stunted. Furthermore, the following data was evaluated: shoot fresh biomass, shoot 

dry biomass and number of nodules. 

 

Figure 2.5. Nodulation efficacy test under glasshouse conditions (a &b). Image adapted from the current research. 

2.6.4 Statistical analysis 

The analysis was conducted with the help of a statistician at ARC. The glasshouse gnotobiotic 

study data (nodule number, fresh biomass and dry biomass) gathered were subjected to analysis 

of variance (ANOVA) using the general linear model procedure (PROC GLM) in SAS-9.4 

statistical software (SAS, 2016). The mean values were compared by using the least significant 

difference (LSD) test and Duncan’s Multiple Range test at 5 % (p ≤ 0.05) level of significance. 

2.7 Results 

The 25 rhizobia isolates tested for authentication trial in this study were confirmed as the same 

root nodule microsymbionts of legumes from which they were initially isolated as they resulted 

in nodulation upon inoculation. The rhizobia isolates nodulated their original host legumes i.e. 

Medicago sativa (strain RAK1, RF12, RF14, RF15, RF35 and RF36) , Trifolium repens (strain 

SAC1, SAC2, SAC3 and SR4), Lupin (strain VK10) , Vigna unguiculata (strain XBD2, XBQ5, 

a b 



30 
 

XCR6, XCV14, XFH1, XFH12, XFH13, XFH14, XFH3 and XS21) and Phaseolus vulgaris 

(strain UC10, UC11, UC12 and UD5). Nodulation test on Medicago sativa, Trifolium repens, 

Vigna unguiculata and Phaseolus vulgaris resulted in the formation of pink nodules. Legumes 

without inoculation (negative control) showed nitrogen deficiency symptoms whereby the plant 

leaves turned pale yellow (chlorosis) with stunted growth and did not form any nodules.  

It was evident that the most effective treatments resulted in the formation of leaves dark green in 

colour and the negative controls formed leaves that were pale green to yellow in colour. 

However, the symbiotic efficiency of the isolates in their original host plants was reduced in 

some of the legumes when grown under abiotic stress conditions in vivo, especially under 150 

mM NaCl and pH9 stresses. The results revealed that most isolates exhibited tolerance to 50 mM 

NaCl, 100 mM NaCl, pH 5 and pH 7 stresses while few isolates could not tolerate most of the 

abiotic stresses tested. In addition to the difference in the leaves and nodules color, the 

inoculated plants also showed a clear difference in terms of plant growth where the un-inoculated 

plants resulted in stunted growth (Figure 2.7b-j). 

Although nodules were formed on most of the inoculated host legumes, the performance of 

nodution varied based on the response of each rhizobia isolates. The nodules were mainly 

located on the tap roots, forming clusters of nodules and only a few nodules were located on 

lateral roots. The nodule size, colour and shape varied amongst the legumes. Medicago sativa 

and Trifolium repens resulted in indeterminate nodules whereas Vigna unguiculata, Phaseolus 

vulgaris and Lupinus albus resulted in determinate nodules. The number of nodules recorded 

ranged from 3.38 to 59.11 per legume plant under normal conditions. The highest number of 

nodules was formed by rhizobia isolates RF36 (32.28) and isolate RF12 (29.11) inoculated on 



31 
 

Medicago sativa, Isolate SAC3 (59.11) and isolate SAC1 (38.89) inoculated on Trifolium repens 

and isolate XS21 (35.55) and XBQ5 (31.11) inoculated on Vigna unguiculata (Table 2.1). 

The number of nodules formed and fresh biomass decreased with an increased salt concentration 

in the legume hosts. Isolate XCV14 inoculating Vigna unguiculata, UC10 inoculating Phaseolus 

vulgaris and VK10 inoculating Lupinus albus displayed no nodulation in all salt concentrations 

tested. Higher salt concentrations (100 and 150 mM) also impaired nodulation in isolate RF12 

inoculating Medicago sativa and UC10 inoculating Phaseolus vulgaris. The isolates (XS21, 

XFH13, XFH14, XBD2 and XBQ5) inoculating Vigna unguiculata and isolates (RF15, RF35 

and RF36) inoculating Medicago sativa were tolerant to salinity and resulted in nodulation at 

various NaCl concentrations tested (Table2.1-2.3 and 2.5). 

The negative effect of salinity on fresh biomass was mostly significant in Trifolium repens, 

Lupinus albus and Phaseolus vulgaris at 100 and 150 mM NaCl concentrations. Vigna 

unguiculata and Medicago sativa showed the most tolerance and the fresh biomass was 

consistent throughout the NaCl concentrations. Rhizobia inoculation also significantly increased 

the fresh biomass of the inoculated plants under acidity and alkalinity stress treatment when 

compared to the non-inoculated. Isolates inoculating Vigna unguiculata exhibited more tolerance 

to acidity or alkalinity conditions as there was a noticeable difference on the fresh biomass under 

increased or decreased pH stress. The fresh biomass of Lupin albus, Trifolium repens and 

Phaseoulus vulgaris was decreased at pH 9 and pH 5 stresses, plants exhibited lower fresh 

biomass (Table 2.6-2.10). 

Significant (p ≤ 0.05) differences amongst the isolates in terms of nodulation, shoot fresh and dry  

biomass were observed, this was in comparison to the non-inoculated controls under normal and 



32 
 

abiotic stress conditions (Table 2.1-2.10). Under normal conditions, a significant difference 

between the inoculated and non-inoculated controls in terms of fresh and dry biomass was 

observed. Isolate XFH12 and XBD2 exhibited the highest fresh biomass (8.730g and 8.729g 

respectively) on Vigna unguiculata, followed by UD5 and UC11 (8.154g and 7.957g 

respectively) on Phaseolus vulgaris. Isolate SAC3 and SAC2 (1.823g and 1.703g respectively) 

also exhibited the highest fresh biomass on Trifolium repens, isolate RF35 and RF15 also 

exhibited 1.579g and 1.575g respectively inoculating Medicago sativa. 



33 
 

  

     

Figure 2.6. Effect of inoculation of Vigna inguiculata and Trifolium repens with rhizobia strains. Vigna unguiculata inoculation with isolate XFH13 and 
Trifolium repens inoculation with isolate SAC2 resulted in healthy looking shoots and roots with many pink nodules (left) as compared to uninoculated plants 
(right) with no nodules and stunted growth with yellow leaves in each case, a&g) Normal conditions, b&h) 50 mM NaCl, c) 100 mM NaCl, i) 150 mM NaCl, 
d&j) pH 5, e&k) pH 7 and f&l) pH 9. 

 

 

 

a b c d e 

i 

f 

g k j l h 



34 
 

Table 2.1. Evaluation of the authentication test of rhizobia strains inoculated on Vigna inguiculata for nodulation and plant biomass  

  
Rhizobia 
strains   

 
0 stress (normal conditions 

    
Salinity stress (50mM NaCl) 

 
Salinity stress (100mM NaCl) 

 
Salinity stress (150mM NaCl) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass 

(g) 

  Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass 

(g) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass 

 (g) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass  

(g) 
XBD2 27.28cd 8.729a 1.087a 22.11e-j  6.643e-i 0.752c-h  20.00i-k  6.642e-i  0.565h-o  20.00d-f  6.643d  0.583h-n 

XBQ5 31.11b 8.392ab 0.998ab 21.44g-j  7.215d-f 0.669d-k  18.67j-l  6.087g-j  0.515j-q  20.67h-k  6.198g-j  0.527j-q 

XCR6 16.11l 6.233g-j 0.759c-g 11.89mn  5.955ij  0.473l-r  9.22n  4.980kl  0.517j-q  12.00mn  4.978kl  0.428m-r 

XCV14 10.22n 4.987k 0.506k-q 0.00o  4.358l-o  0.401n-r  0.00o  3.989no  0.363p-r  0.00o  3.756no  0.352qr 

XFH1 21.17h-j 5.753j 0.706d-i 12.78mn  5.782j  0.519j-q  10.44n  4.731k-m  0.480k-r  11.78mn  4.728k-m  0.421m-r 

XFH12 25.33c-f 8.733a 0.995ab 15.22lm  7.331c-f  0.698d-j  23.11e-i  6.626f-i  0.515j-q  18.78j-l  6.695e-h  0.553i-p 

XFH13 27.89c 7.371d 0.785c-f 24.44d-g  7.993bc  0.727d-i  21.78e-j  6.008h-j  0.629e-l  21.78e-j  6.008j-j  0.433m-r 

XFH14 27.89c 5.768j 0.799cd 20.44h-k  6.738e-g  0.513j-q  20.67h-k  5.938ij  0.430m-r  17.66kl  5.940ij  0.503k-r 

XFH3 23.28e-h 8.241ab 0.887bc 0.00o  4.801k-m  0.432m-r  11.33n  4.839k-m  0.378o-r  9.67n  4.454k-n  0.478l-r 

XS21 35.55a  8.324ab 1.059a 19.67i-k  8.358ab  0.789c-e  25.33c-e  7.347c-e  0.599e-m  25.33c-e  7.191d-f  0.638d-l 

CONTROL 0.00o 3.894no 0.393p-r 0.00m  4.268m-o  0.371p-r  0.00o  3.974no  0.354qr  0.00m  3.662o  0.315r 

LSD0.05 3.7460 0.7062 0.0190 
 

3.7460 0.7062 0.0190 3.7460 0.7062 0.0190 3.7460 0.7062 0.0190 

Pr>F <0.001 <0.001 <0.001 
 

 <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001 

 

 

 



35 
 

Table 2.2. Evaluation of the authentication test of rhizobia strains inoculated on of Medicago sativa for nodulation and plant biomass  

  
Rhizobia   
 strains   

0 stress (normal conditions) 
 

Salinity stress (50mM NaCl) 
 

Salinity stress (100mM NaCl) 
 

Salinity stress (150mM NaCl) 
Nodule 
number 

Fresh 
biomass(g) 

Dry 
biomass 

(g) 

Nodule 
number 

Fresh 
biomass(g) 

Dry 
biomass 

(g) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass 

(g) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass 

(g) 
RAK1 24.22f-h 0.4095k 0.1367cd  20.33k  0.894fg  0.072hi  21.33g-k  0.922e-g  0.094f-h  13.00l  0.558ij  0.053ij 

RF12 29.11bc 0.6903hi 0.0767g-i  11.00l  0.798gh  0.071hi  0.00m  0.210[  0.011l  0.00m  0.210l  0.011j 

RF14 19.78k 1.4273c 0.1473c  20.11k  1.609b  0.132c-e  25.67d-f  1.426c  0.157bc  0.00m  0.426jk  0.041jk 

RF15 23.94f-j 1.6700b 0.1682ab  21.33g-k  1.668ab  0.157b  31.33ab  1.657ab  0.179ab  12.33l  1.212d  0.114d-f 

RF35 27.72cd 1.7940a 0.1820a  24.33e-g  1.564bc  0.136cd  27.33c-e  1.537bc  0.156bc  21.00g-k  1.203d  0.118d-f 

RF36 32.28a 0.9468ef 0.1112ef  11.33l  1.068de  0.100fg  24.00e-i  1.416c  0.141cd  20.67jk  1.395c  0.136cd 

CONTROL 0.00m 0.2035l 0.0193kl  0.00m  0.186l  0.013kl  0.00m  0.153l  0.012l  0.00m  0.120l  0.008l 

LSD0.05 3.561 0.1703 0.0276 3.561 0.1703 0.0276 3.561 0.1703 0.0276 3.561 0.1703 0.0276 

Pr>F <0.001 <0.001 <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001 

 

 

 

 

 

 

 



36 
 

Table 2.3. Evaluation of the authentication test of rhizobia strains inoculated on Phaseolus vulgaris for nodulation and plant biomass  

 
Rhizobia   
 strains  

0 Stress (normal conditions) 
 
Salinity stress (50mM NaCl) 

 
Salinity stress (100mM NaCl) 

 
Salinity stress (150mM NaCl) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass 

(g) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass 

(g) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass  

(g) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass 

(g) 
UC10 12.389b 8.855b 0.8927cd  13.00b  6.969ef  0.679fg  0.00f  6.727f  0.673g  0.00f  5.634gh  0.511hi 

UC11 20.666a 9.595a 0.9672bc  5.78de  7.773cd  0.744e-g  12.44b  7.530c-e  0.784ef  10.00bc  5.295hi  0.495hi 

UC12 22.000a 9.877a 1.000b  0.00f  7.060e-f  0.687b  0.00f  7.197d-f  0.738b  0.00f  4.974i  0.468hi 

UD5 12.611b 9.588a 1.0812a  4.56e  7.466c-e  0.680fg  8.111cd  8.083c  0.827a  8.67cd  6.045g  0.556h 

CONTROL 0.00f 7.320de 0.7392e-g  0.00f  5.073hi  0.437i  0.00f  5.110hi  0.493hi  0.00f  4.770i  0.458hi 

LSD0.05 3.033 0.640 0.1066 3.033 0.640 0.1066 3.033 0.640 0.1066 3.033 0.640 0.1066 

Pr>F <0.001 <0.001 <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001 

 

 

 

 

 

 

 

 



37 
 

Table 2.4. Evaluation of the authentication test of rhizobia strains inoculated on Trifolium repens for nodulation and plant biomass  

 
Rhizobia 
 strains  

0 Stress (normal conditions) 
 

Salinity stress (50mM NaCl) 
 

Salinity stress (100mM NaCl) 
 

Salinity stress (150mM NaCl) 
Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass 

(g) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass 

(g) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass 

(g) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass 

(g) 
SAC1 38.89b 1.2878e 0.1207d  15.00ef  1.409d  0.138b  12.22fg  0.375i  0.031g-i  12.89e-g  0.418hi  0.036g 

SAC2 32.89c 1.7033b 0.1643ab  10.00gh  0.686g  0.061f  4.78i  0.210k  0.011ij  12.22fg  0.503h  0.058f 

SAC3 59.11a 1.8227a 0.1778a  21.00d  1.550c  0.153bc  5.89i  0.346ij  0.031g-i  16.00e  0.368i  0.033gh 

SR4 32.56c 1.0657f 0.0972e  7.33hi  0.419hi  0.045fg  0.00j  0.225jk  0.014h-j  16.00e  0.502h  0.046fg 

CONTROL 0.00j 0.1408kl 0.0128j  0.00j  0.078l  0.006j  0.00j  0.059l  0.004j  0.00j  0.043l  0.008j 

LSD0.05 3.140 0.1276 0.0208 3.140 0.1276 0.0208 3.140 0.1276 0.0208 3.140 0.1276 0.0208 

Pr>F <0.001 <0.001 <0.001  <0.001  <0.001  <0.001  <0.001  <0.001   <0.001  <0.001   <0.001  <0.001 

 

 

 

  

 

 

 

 

 

 



38 
 

Table 2.5. Evaluation of the authentication test of rhizobia strains inoculated on Lupinus albus for nodulation and plant biomass  

  
Rhizobia 
strains 
  

0 Stress (normal conditions) 
 

Salinity stress (50mM NaCl) 
 

Salinity stress (100mM NaCl) 
 

Salinity stress (150mM NaCl) 
Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass 

(g) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass 

(g) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass 

(g) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass 

(g) 
VK10 3.3872a 6.487b 0.7132a  0.00b  7.313a  0.489b  0.00a  4.232c  0.360cd  0.00b  4.212c  0.387c 

CONTROL 0.00b 3.984c 0.3700c  0.00b  6.156b  0.696a  0.00a  3.254d  0.298cd  0.00b  3.154d  0.286d 

LSD0.05 0.889 0.502 0.090 0.889 0.502 0.090 0.889 0.502 0.090 0.889 0.502 0.090 

Pr>F <0.001 <0.001 <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001 

 

 

 

 

 

 

 

 

 

 

 

 

 



39 
 

Table2.6. Effect of Acidity/alkalinity on nodulation and plant biomass of Vigna unguiculata with or without rhizobia inoculation 

 
Rhizobia 
Strains 

 
Acidity/alkalinity (pH 5) 

 
Acidity/alkalinity (pH 7) 

 
Acidity/alkalinity (pH 9) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass  

(g) 

Nodule 
number 

Fresh 
biomass  

(g) 

Dry 
Biomass 

(g) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass 

(g) 
XBD2  20.89h-j  7.943hi  0.822e-g  26.55b-e  9.392b-d  0.897b-f  24.67d-g  8.323f-h  0.904b-f 

XBQ5  25.78c-f  10.179a  1.053a  26.78b-d  9.313b-e  0.891b-f  22.00g-i  7.520i-k  0.810fg 

XCR6  4.56m  5.183o-q  0.523l-n  6.55m  5.183o-q  0.528l-n  16.56kl  5.420n-p  0.506mn 

XCV14  0.00n  5.025pq  0.496mn  0.00n  5.320o-q  0.516l-n  0.00n  5.982mn  0.613j-l 

XFH1  23.89e-g  7.610ij  0.731g-i  18.33jk  5.610no  0.540k-m  15.44l  5.527n-p  0.538k-m 

XFH12  23.55f-h  9.835a-c  0.969a-c  21.11hi  9.894ab  0.986ab  19.78ij  7.027j-l  0.634i-k 

XFH13  25.33c-f  8.794e-g  0.868c-f  26.67b-d  8.239gh  0.868c-f  32.22a  7.955hi  0.808f-h 

XFH14  16.33kl  7.131jk  0.705h-j  28.00bc  6.492kl  0.678ij  32.56a  8.259f-h  0.946b-d 

XFH3  16.89kl  6.973kl  0.656ij  20.33ij  6.974kl  0.697ij  26.78b-d  8.023hi  0.849d-f 

XS21  27.67bc  8.840d-f  0.887b-f  28.78b  9.801a-c  0.922b-e  29.00b  9.269c-e  0.988ab 

CONTROL  0.00n  4.186r  0.431n  0.00n  5.034o-q  0.501mn  0.00n  4.766qr  0.463mn 

LSD0.05  2.702 0.206  0.103  2.702 0.206  0.103  2.702  0.206  0.103 

Pr>F  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001 

 

 

 

 



40 
 

Table 2.7. Effect of Acidity/alkalinity on nodulation and plant biomass of Medicago sativa with or without rhizobia inoculation 

 
Rhizobia 
Strains 

 
Acidity/alkalinity (pH 5) 

 
Acidity/alkalinity (pH 7) 

 
Acidity/alkalinity (pH 9) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass  

(g) 

Nodule 
number 

Fresh 
biomass  

(g) 

Dry 
biomass  

(g) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass  

(g) 
RAK1  14.67d  0.218kl  0.028gh  24.67ab  0.490e  0.050d-f  11.56ef  0.339h-j  0.038e-g 

RF12  13.67de  0.383f-i  0.040d-g  22.33a-c  0.497e  0.057d  11.89ef  0.265jk  0.039e-g 

RF14  21.56c  0.430e-g  0.049d-f  22.44a-c  1.072b  0.119a  10.00f  0.372g-i  0.054de 

RF15  12.78de  0.335ij  0.033fg  24.89a  1.003b  0.112a  12.11d-f  0.452ef  0.074c 

RF35  21.33c  0.356g-i  0.035fg  22.44a-c  1.195a  0.120a  24.89a  0.411f-i  0.039e-g 

RF36  22.00bc  0.412f-h  0.043d-g  24.89a  0.827c  0.083bc  24.78a  0.605d  0.092b 

CONTROL  0.00g  0.133m  0.009i  0.00g  0.121m  0.007i  0.00g  0.172lm  0.014hi 

LSD0.05  2.742  0.076  0.017  2.742 0.076  0.017  2.742  0.076  0.017 

Pr>F  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001 

 

 

 

 

 

 

 



41 
 

Table 2.8. Effect of Acidity/alkalinity on nodulation and plant biomass of Phaseolus vulgaris with or without rhizobia inoculation 

 
Rhizobia 
Strains 

 
Acidity/alkalinity (pH 5) 

 
Acidity/alkalinity (pH 7) 

 
Acidity/alkalinity (pH 9) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass 

(g) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass  

(g) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass 

(g) 
UC10  21.11b  7.088b-d  0.728cd  6.33f  7.590bc 0.838bc  3.45fg  5.124h  0.472gh 

UC11  28.33a  6.782c-e  0.667de  0.00g  7.861b 0.720cd  0.00g  5.149gh  0.473gh 

UC12  0.00g  9.421a  1.001a  17.00cd  6.726c-e 0.748b-d  0.00g  5.351f-h  0.556e-g 

UD5  14.00de  6.206d-f  0.649d-f  17.67bc  7.207bc 0.886ab  11.00e  6.030e-g  0.557e-g 

CONTROL  0.00g  4.975hi  0.471gh  0.00g  6.129ef 0.500f-h  0.00g  4.169i  0.369h 

LSD0.05  3.581  0.884  0.157 3.581  0.884  0.157  3.581  0.884  0.157 

Pr>F  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001 

 

 

 

 

 

 

 

 

 

 



42 
 

Table 2.9. Effect of Acidity/alkalinity on nodulation and plant biomass of Trifolium repens with or without rhizobia inoculation 

 
Rhizobia 
Strains 

 
Acidity/alkalinity (pH 5) 

 
Acidity/alkalinity (pH 7) 

 
Acidity/alkalinity (pH 9) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass 

(g) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
Biomass 

(g) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass 

(g) 
SAC1  29.56ab  0.478hi  0.043ef  30.89ab  0.777d  0.079c  9.33e  0.593fg  0.053de 

SAC2  25.22cd  0.439i  0.041e-g  33.33a  1.650a  0.138a  11.11e  0.591fg  0.049e 

SAC3  28.11b-d  0.660ef  0.070cd  29.11bc  1.560b  0.144a  8.33e  0.477hi  0.079c 

SR4  33.56a  0.540gh  0.054de  25.00d  1.124c  0.116b  9.33e  0.679e  0.070cd 

CONTROL  0.00f  0.0342j  0.0267fg  0.00f  0.3367j  0.0380e-g  0.00f  0.3180j  0.0233g 

LSD0.05  4.023  0.076  0.018  4.023  0.076  0.018  4.023  0.076  0.018 

Pr>F  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001  <0.001 

 

 

 

 

 

 

 

 



43 
 

Table 2.10. Effect of Acidity/alkalinity on nodulation and plant biomass of Lupinus albus with or without rhizobia inoculation 

 
Rhizobia 
Strains 

 
Acidity/alkalinity (pH 5) 

 
Acidity/alkalinity (pH 7) 

 
Acidity/alkalinity (pH 9) 

Nodule 
number 

Fresh 
Biomass 

(g) 

Dry 
biomass 

(g) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
Biomass 

 (g) 

Nodule 
number 

Fresh 
biomass 

(g) 

Dry 
biomass 

(g) 
VK10 4.00a 6.649a 0.635a 0.00b 6.740a 0.650a 0.00b 4.022c 0.494b 

CONTROL 0.00b 5.652b 0.5513b 0.00b 5.102b 0.492b 0.00b 3.412c 0.366c 

LSD0.05 1.453 0.678 0.082 1.453 0.678 0.082 1.453 0.678 0.082 

Pr>F <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 

 



44 
 

2.8 Discussion 

This study evaluated the nodulation efficacy of 25 rhizobia isolates from SARCC under normal 

and abiotic stress conditions. Although a total number of 40 isolates were selected from the 

SARCC, only 25 isolates were evaluated for the nodulation efficacy based on the availability of 

legume seeds of their original hosts and their previous performance under normal conditions in a 

glasshouse nodulation efficacy test. The authentication of rhizobia is required to screen effective 

native rhizobia isolates to determine their symbiotic efficiency. The glasshouse nodulation 

efficacy test in this study revealed that inoculating Medicago sativa, Trifolium repens, Vigna 

unguiculata and Phaseolus vulgaris with rhizobia isolates previously isolated from these 

legumes induced nodulation on their original host. The current results concur with previous 

studies reporting that inoculation with efficient rhizobia strains can significantly increase 

nodulation (Hungria and Mendes, 2015; Tounsi-Hammami et al., 2016). Osei et al. (2018) 

reported that rhizobia can induce nodule formation subsequently capable to fix atmospheric 

nitrogen is commonly used to evaluate the inherent relation between rhizobia and their legume 

hosts. Control legumes or legumes without inoculation did not form any nodules revealing that 

there was no external contamination during the glasshouse experiment. The absence of 

contamination is crucial for the bacteria nodulating legume authentication experiment (Ogutcu et 

al., 2008; Hassen et al., 2014). 

Among the SARCC isolates tested, most isolates inoculating Vigna inguiculata were highly 

effective and resistant to the abiotic stresses tested, followed by isolates inoculating Medicago 

sativa and Trifolium repens. Isolates inoculating Lupinus albus and Phaseolus vulgaris were the 

least effective. Effective isolates in most of the abiotic stresses tests provided the highest plant 

biomass and number of nodules when tested under acidity or alkalinity and salinity conditions. 



45 
 

This finding concurs with results by Beshah and Assefa (2019), they reported that stress tolerant 

isolates have the ability to thrive under various environmental stresses as they can occupy root 

nodules, fix atmospheric nitrogen to improve crop production.  

Nodulation varied based on the legume species. Amongst the legumes, Vigna unguiculata, 

Trifoloium repens, Phaseolus vulgaris and Medicago sativa plants were the most nodulated 

when compared to Lupinus albus which was the least nodulated. This could be as a result of 

genetic differences among the species, it has been demonstrated that legume species and 

microsymbionts interactions are highly specific (Qiang et al., 2013). Moreover, although 

nodulation was observed in most of the inoculated legumes and the nodules were pink, however, 

some nodules were not pink in color and the un-inoculated treatments did not form any nodules. 

A number of authors have demonstrated that for a bacterial isolate to be classified as a legume 

nodulating bacteria, its identity must first be confirmed through plant nodulation efficacy test on 

relevant legume host. Therefore, nodulation is used as a confirmatory test to determine whether 

the bacteria have the ability to nodulate legumes or not (Lafay and Burdon, 2007; Arora et al., 

2017). 

In the current study, aunthetication test revealed that most of the rhizobia isolates inoculating 

Medicago sativa, Trifolium repens, Vigna unguiculata and Phaseolus vulgaris resulted in pink 

nodules formation. Nodules that are actively fixing nitrogen contain leghaemoglobin and its 

presence is indicated by the red color on the interior of the nodules, which shows whether the 

bacteria is alive and active (Virtanen et al., 1947). Leghaemoglobin is a red haemeprotein 

specifically accumulated in nodule; it plays a vital role in nitrogen fixation and this is achieved 

by protecting nitrogenase from inactivation by molecular oxygen (Appleby, 1984). Moreover, 

pink nodules contain nifH genes and expreses them actively, these genes codes for the synthesis 



46 
 

of nitrogenase enzymes used to reduce N to NH3 (Rondon et al., 2007). A previous study by 

Farid and Navabi (2015) also revealed that most of the root nodules were pink in colour; 

indicating the presence of a protein containing iron (leghaemoglobin) which plays an important 

role during effective nitrogen fixation process. The current results also revealed that the color of 

Medicago sativa, Trifolium repens and Vigna unguiculata leaves inoculated with rhizobia were 

dark green. Morever, Phaseolus vulgaris showed nodulation but the leaves were not dark green; 

they were pale green to yellow. A study by Degefu et al. (2018) revealed similar results to the 

current findings; their study was based on the authentication and efficacy test of Vigna 

unguiculata. The study’s findings reported that, isolates effective in fixing atmospheric nitrogen 

results in deep green leaves and deep red or pink nodules. Dungu (2017) also reported that cow 

pea formed dark green leaves upon inoculation with Bradyrhizobium strains when compared to 

the non-inoculated plants which formed yellowish leaves. 

The current findings demonstrated that inoculating Medicago sativa, Trifolium repens, Vigna 

unguiculata, Lupinus albus and Phaseolus vulgaris with rhizobial isolates enhanced nodulation 

and nitrogen fixation. This in turn improves nitrogen concentration on the plant’s shoot and 

biomass. The results of the glasshouse nodulation efficacy test demonstrated significant increases 

in nodulation, fresh and dry biomass. Previous reports indicated that efficient rhizobia strains 

stimulate the formation of more nodules which provide legumes with more fixed nitrogen 

resulting in greater biomass yield, increased nodulation, nitrogen fixation and plant growth 

(Thalikarathna et al., 2019). 

Nodulation and plant growth were highly affected by different concentrations of NaCl in this 

current study. Plants growth was retarded with increased NaCl concentration compared to plant 

growth and nodulation under normal conditions. The relationship between legumes and rhizobia 



47 
 

under saline conditions to be successful requires isolates that can show tolerance to higher NaCl 

concentration. The impact of salt depends on plant species, salinity levels and ionic composition 

(Yadav et al., 2010). Vigna unguiculata exhibit tolerance to high temperature, drought, high 

salinity, ability to reduce soil erosion and fix atmospheric nitrogen, therefore it is used in 

sustainable farming (Timko et al., 2007; Agbicodo et al., 2009; Merwad et al., 2018). Munns and 

Tester (2008) also revealed that compared to other crops, alfalfa is relatively tolerant to salt 

stress. These current results also revealed that tolerance to higher NaCl concentrations was 

observed in isolates XFH13, XFH14, XS21, XBD2 and XBQ5 inoculating Vigna unguiculata, 

isolates RF15, RF35 and RF36 inoculating Medicago sativa. 

In comparison with the glasshouse authentication test results under normal conditions, plants 

showed a decrease in a number of nodules, fresh and dry biomass plant biomass under abiotic 

stress conditions. However, inoculation of the legume plants with rhizobial isolates increased 

plant tolerance to saline conditions when compared to the negative controls (non-inoculated) 

with the same Hoaglands solution. The current results obtained from this study revealed that 

isolate XFH13 (Vigna unguiculata), RF35 (Medicago sativa), UC11 (Phaseolus vulgaris), and 

SAC1 (Trifolium repens) resulted in maximum nodulation under 50, 100 and 150 mM NaCl 

concentrations. Swaraj and Bishnoi (1999) reported that rhizobia thrive under saline conditions 

because of the ionic adjustment they make. Furthermore, rhizobia cope more efficiently with 

salinity in comparison with their host legumes. However, their rate of growth and survival of 

rhizobia varies under saline conditions as this is based on the type of strain used. Khaitov et al. 

(2020) have also recently reported that legume inoculation with rhizobia decreases ethylene 

levels under saline and water deficit conditions. However, this increases plants tolerance to 

stress, increases nodulation and photosynthesis. In this sudy, tolerance to salinity also varied per 



48 
 

strain used to inoculate their original host under saline conditions and inoculation enhanced plant 

tolerance to salinity and nodulation when compared to the non-inoculated. 

Enhanced nodulation resulting from rhizobia inoculation was positively associated with fresh 

biomass. Some rhizobia isolates were more efficient based on the fresh and dry biomass and the 

number of nodules formed. The maximum growth-promoting activity based on the plant’s fresh 

biomass was recorded in isolate XFH12 (8.730g), XBD2 (8.729g) inoculating Vigna 

unguiculata, SAC3 (1.823g), SAC2 (1.703g) inoculating Trifolium repens, RF35 (1.579g), RF15 

(1.575g) inoculating Medicago sativa and UC10 (8.154g), UC11 (7.957g) inoculating Vigna 

unguiculata. These rhizobia isolates improved significantly both nodulation and fresh biomass. 

The glasshouse test results further indicated that efficient rhizobia strains stimulate more nodules 

formation and consequently improved plant growth. There were no nodules formed on the roots 

of legumes that were not inoculated (negative control) with rhizobia isolates revealing that there 

was no contamination. 

Previous reports also revealed that due to the absence of rhizobia in the non-inoculated 

treatments (inorganic nitarate/ammonium limiting conditions), plants resulted in no nodule 

formation which decreased the biomass of the plants (Fatima et al., 2007; van Noorden et al., 

2016). Increased salinity in the substrate has been previously reported to decrease the number of 

nodules and size in Vigna inguiculata (Al-Saedi et al., 2016). This decreases in the presence of 

rhizobia in the rhizosphere because the synthesis exopolysccharides, glucans and 

lipopolysaccharides (LPS) is inhibited and these are essential superficial molecules produced by 

the bacteria required for their interaction with plants (Tewari and Sharma, 2020). Therefore, the 

nodule number and mass, leghaemoglobin synthesis and nitrogenase activity decreases as the 

synthesis of exopolysaccharides, glucans and lipopolysaccharides is inhibited (Sunita et al., 



49 
 

2019). Although Vigna inguiculata was more tolerant to salinity, isolate XCR6, XFH3 and 

XFH1 resulted in a decreased number of nodules and isolate XCR6 did not nodulate at all. Our 

current result also revealed similar results for some isolates inoculating Medicago sativa, 

Phaseolus vulgaris, Trifolium repens and Lupin albus. The decrease in the number of nodules 

with the increase in NaCl concentration (salinity) was not only observed in Vigna inguiculata. 

Some of the nodules were not pink, indicating a decrease in leghaemoglobin and nitrogenase 

activity. 

Salinity affects plant growth, fresh and dry plant biomass (Raptan et al., 2001; Yupsanis et al., 

2001; Goulam et al., 2002; Lobato et al., 2009). However, this can be overcomed by the 

treatment of plants with rhizobacteria, this will increase the germination, root and shoot growth, 

overall  plant biomass, seed weight, and the yield under saline conditions (Joseph et al., 2007; 

Yasmin et al., 2007). Ertan et al. (2008) reported that radish plants treated with rhizobacteria 

increased growth parameters significantly under saline and normal conditions. Current results in 

this study are similar to these finding, most of the isolates were able to significantly increase 

plant growth and nodulation under salinity and normal conditions, results similar to this were 

reported in beans (Yildrim and Taylor, 2005). Moreover, Keneni et al. (2010) reported that 

isolates tolerant to higher NaCl concentrations are extremely competitive when they colonize the 

rhizosphere and nodulate their host plants in adverse environmental conditions, e.g. soils affected 

by salt. Therefore, the tolerance to NaCl concentration by these isolates mentioned above in this 

current study can potentially enhance the growth and yield of legumes under saline prone 

conditions. 

Previously, it has been reported that soil acidity affects the persistence of rhizobia in the soil, 

plant’s rhizosphere and nodulation efficiency in tropical areas (Graham et al., 1994). However, 



50 
 

in the current study, most of the isolates were tolerant to acidic pH as they were still able to form 

nodulation at pH 5. This could be due to the fact that adaptive strategies designed to minimize 

damage induced by acid have been evolved by rhizobia because acid tolerance mechanisms that 

are inducible are important to symbionts growing in acidic soils (Foster, 2000). In most 

instances, the transcriptional activation of genes is due to the bacterial response and products that 

helps cope with physicochemical stress. At least 15-20 genes have been reported to contribute to 

rhizobia acid tolerance (Foster, 2000; Vinuesa et al., 2003). To be specific, actA, exoR, actP, 

actR, lpiA, and phrR genes are important for the growth of rhizobia at low pH (de Lucena et al., 

2010). In this study, the exoR gene was detected in isolate UC11, SAC1, SAC2 and SR4 

(Chapter 3) and these isolates had the ability to form nodulation at a low pH of 5. 

Soil reaction, pH in particular influences numerous soil properties and processes that affect plant 

growth and this can be considered as a variable key. The activity of microorganisms, nutrients 

solubility and availability are vital processes that are pH dependent. For an example, in acidic 

soils, most micro-nutrients are more available than in neutral-alkaline soil conditions and this 

generally favors plant growth (Loncaric et al., 2008). In this study, isolates were more sensitive 

to alkaline pH when compared to the acidic pH. However, some isolates showed tolerance under 

acid and alkaline stresses as they resulted in plant roots nodulation and an increased plant growth 

when compared to non-inoculated plants. This was evident in isolates XS21, XFH12, XFH13, 

XS21, XBQ5, XFH3 and XFH12 inoculated on  Vigna unguiculata and isolates RF12, RF14, 

RF15, RF36 and RAK1 inoculated on Medicago sativa (Table 6 and 7). Farissi et al. (2014) 

revealed that environmental conditions which are highly acidic and alkaline limit rhizobia 

growth and the successful nitrogen fixation with the legume host. Higher soil pH negatively 

affects the growth and survival of rhizobia because the essential mineral nutrients required by 



51 
 

rhizobia are no longer available. Moreover, some of the micro-nutrients and elements that are not 

essential can be toxic when available at a higher concentration. In contrast, although the 

availability of macro-nutrients is increased, the availability of phosphorus and micro-nutrients is 

reduced, resulting in lower levels which can in turn negatively affect plant growth (Jiang et al., 

2017). Therefore, in agricultural production, it is best to select rhizobia isolates capable of 

tolerating acid and alkaline stresses. 

Legume inoculation with rhizobia isolates varied per legume at different pH stresses. Isolate 

XCV14 did not show any response to inoculation. In a previous study, Giller (2001) reported the 

absence of some legumes responses to rhizobia inoculation under various environments. This can 

be due to the host plant’s inherent characteristics and the type of rhizobia species used, including 

the nitogen fixation sensitivity to the acidity, dryness, salinity, low fertility and high 

temperatures of the soil (Graham et al., 1994; Wolde-meskel et al., 2018). In this study, the 

isolates showed more sensitivity to alkaline conditions as they resulted in reduced nodulation or 

no nodulation upon inoculating their original legume hosts. The survival or multiplication of 

rhizobia is greatly influenced by alkaline or acidic agricultural soil. Agricultural fields generally 

have an average pH of 7.0-8.5. Alkaline stress can hinder rhizobia growth and subsequently the 

establishment of a symbiotic relationship with legumes. Therefore, it makes good agricultural 

practice to select rhizobia isolates that are tolerant to alkaline conditions and capable of 

nodulating legumes (Farissi et al., 2014). Isolates XS21, XFH12, XFH13, XBQ5, XFH3 and 

XFH12 all inoculated on Vigna unguiculata and isolates RF36, RF12, RF14, RF15, RF36 and 

RAK1 on Medicago sativa in the current study were the most tolerant to acid and alkaline 

conditions. 

 



52 
 

2.9 Conclusion  

The current study confirmed that the selected rhizobia strains preserved at the SARCC formerly 

isolated from the root nodules of Trifolium repens, Lupinus albus, Vigna unguiculata, Medicago 

sativa and Phaseolus vulgaris have the ability to nodulate their original host under normal and 

abiotic stress conditions. The results of the glasshouse nodulation efficacy test conducted 

following the Koch’s postulate experiment demonstrated significant (p≤0.05) increases in nodule 

number, fresh and dry biomass under normal and abiotic stress conditions in comparison with the 

non-inoculated control. The nodulation efficacy and the biomass of the plants declined under 

abiotic stress conditions. However, some of the isolates (RF36, RF12, RF14, RF15 and RAK1) 

inoculating Medicago sativa, isolates (XS21, XFH12, XFH13, XBQ5 and XFH3) inoculating 

Vigna inguiculata and isolates (SAC1, SAC2 and SAC3) inoculating Trifolium repens showed 

more tolerance. Therefore, these results provide baseline information in the use of rhizobia as 

inoculant and that these isolates might have a practical advantage when selecting rhizobia 

isolates to promote sustainable agricultural production systems under abiotic stress conditions. 

 

 

 

 

 

 



53 
 

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