` EXPLORING AND SCREENING POTENTIAL RHIZOBIAL ISOLATES NODULATING FORAGE LEGUMES WHITE LUPIN (Lupinus albus) AND TREE LUCERNE (Chamaecytisus palmensis) GROWING IN ETHIOPIA M.Sc. THESIS BY HUSSIEN DAWUD ALEBACHEW NOVEMBER, 2023 ARBAMINCH, ETHIOPIA EXPLORING AND SCREENING POTENTIAL RHIZOBIAL ISOLATES NODULATING FORAGE LEGUMES WHITE LUPIN (Lupinus albus) AND TREE LUCERNE (Chamaecytisus palmensis) GROWING IN ETHIOPIA . BY HUSSEN DAWUD ALEBACHEW A THESIS SUBMITTED TO DEPARTMENT OF BIOLOGY, COLLEGE OF NATURAL AND COMUTATIONAL SCIENCES, SCHOOL OF GRADUATE STUDIES, ARBA MINCH UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR DEGREE OF MASTER OF SCIENCE IN BIOTECHNOLOGY PRINCIPAL ADVISOR: ASHENAFI HAILU (PhD) CO-ADVISORS: ENDALKACHEW WOLDEMESKEL (PhD) KINDU MOKONNEN (PhD) MELKAMU BEZABIH (PhD) NOVEMBER, 2023 ARBAMINCH, ETHIOPIA Declaration I hereby declare that this M.Sc. thesis is my original work and has not been presented for a degree in any other university, and all sources of material used for this have been duly acknowledged. Name: Hussien Dawud Signature: ___________ Date: _______________ i ARBA MINCH UNIVERSITY SCHOOL OF GRADUATE STUDIES ADVISORS’ THESIS SUBMISSION APPROVAL SHEET This is to certify that the thesis entitled “exploring and screening potential rhizobial isolates nodulating forage legumes white lupin (Lupinus albus) and tree lucerne (Chamaecytisus palmensis) growing in Ethiopia” submitted in partial fulfilment of the requirements for the degree of Master’s with specialization in biotechnology, and has been carried out by Hussen Dawud Id. No PRNS/028/14, under our supervision. Therefore we recommend that the student has fulfilled the requirements and hence hereby can submit the thesis to the department for defense. Ashenafi Hailu (PhD) _____________ ______________ Name of principal advisor Signature Date Endalkachew Woldemeskel (PhD) _____________ ________________ Co-advisor I Signature Date Kindu Mekonnen (PhD) _____________ ________________ Co-advisor II Signature Date Melkamu Bazabih (PhD) _____________ ________________ Co-advisor III Signature Date ii ARBA MINCH UNIVERSITY SCHOOL OF GRADUATE STUDIES EXAMINERS’ THESIS APPROVAL SHEET We, the undersigned, members of the board of examiners of the final open defense by Hussen Dawud have read and evaluated his thesis entitled ‟ exploring and screening potential rhizobial isolates nodulating forage legumes white lupin (Lupinus albus) and tree lucerne (Chamaecytisus palmensis) growing in Ethiopia’’ and examined the candidate’s oral presentation. Therefore, this is to certify that the thesis has been accepted in partial fulfilment of the requirements for the degree of Masters of Science in Biotechnology. ________________________ _______________ __________________ Chairperson Signature Date ________________________ ______________ _________________ External Examiner Signature Date ________________________ ________________ _______________ Internal Examiner Signature Date ________________________ _______________ ______________ Department head Signature Date iii Acknowledgements First and foremost, I would like to express my heartfelt gratitude and appreciation to my advisor Ashenafi Hailu (PhD), for his provision of essential reference materials, valuable comments, constructive advice, encouragement, and unreserved guidance starting from the beginning of the work to its end. My sincere gratitude also goes to my co-advisors Endalkachew Woldemeskel (PhD), Melkamu Derseh (PhD) and Kindu Mekonnen (PhD) from ILRI campus for arranging supports from Africa RISING, ILSSI projects, and Mixed Farming Initiative (MFS), guiding forage legume seed access, organizing soil sample and root nodule collections from various locations across Ethiopia and overseeing this collaborative research work. I extend my heartfelt appreciations to Africa RISING, ILSSI projects, and Mixed Farming Initiative (MFS) for their financial support through ILRI and Arba Minch University. I am very pleased to express my thanks and sincere appreciation to the laboratory technicians of Arbaminch University, department of biology: Awoke Mamo, Beyene Kushe, Dawit Albene and Fitsum Dejene and Yonas Siraj for their free laboratory service and unconditional laboratory support. I would also like to extend my appreciation to my friends Abenezer Tamirat, Bayu Fetene Yihunlamlak Wube, Amsalech Martsa, Meseretu Melese and Zikie Ataro who have technically guided and provided me with plenty of relevant literatures. Finally, my sincere gratitude and love goes to the beloved members of my family who have contributed a lot in my life. iv LIST OF ACRONYMS AND ABBREVIATIONS AMU Arba Minch University AMULR Arba Minch University Rhizobial Isolates of White lupin AMUTLR Arba Minch University Rhizobial Isolates of Tree lucerne ANOVA Analysis of Variance BNF Biological Nitrogen Fixation CR Congo Red GPS Global Positioning System μg /ml microgram per millilitre pH Hydrogen ion potential RSE Relative Symbiotic Effectiveness SDW Shoot Dry Weight UPGMA Unpaired Group Method Analysis YMA Yeast Extract Mannitol Agar YMB Yeast Extract Mannitol Broth v TABLE OF CONTENTS Contents Pages Declaration .................................................................................................................................. i Acknowledgements ................................................................................................................... iv LIST OF ACRONYMS AND ABBREVIATIONS .................................................................. v TABLE OF CONTENTS .......................................................................................................... vi LIST OF TABLES .................................................................................................................... ix LIST OF FIGURES ................................................................................................................... x Abstract ..................................................................................................................................... xi 1 INTRODUCTION .............................................................................................................. 1 Background ...................................................................................................................... 1 Statements of the problem ................................................................................................ 3 Objectives ......................................................................................................................... 3 1.3.1 General objective ....................................................................................................... 3 1.3.2 Specific objectives ..................................................................................................... 3 Significance of the study .................................................................................................. 4 Scope of the study ............................................................................................................ 4 2 REVIEW OF RELATED LITERATURE .......................................................................... 5 Legumes ........................................................................................................................... 5 Forage legumes ................................................................................................................ 5 2.2.1 White lupin ................................................................................................................ 6 2.2.2 Tree lucerne ............................................................................................................... 7 Biological nitrogen fixation ............................................................................................. 7 2.3.1 Rhizobia ..................................................................................................................... 8 2.3.2 Rhizobia-Legume symbiosis ..................................................................................... 9 2.3.3 Host specificity and mechanisms of nodule formation ............................................. 9 Ecological factors affecting legume–rhizobium symbiosis and BNF ............................ 10 2.4.1 Salt stress ................................................................................................................. 11 2.4.2 Drought stress .......................................................................................................... 11 2.4.3 Temperature stress ................................................................................................... 12 2.4.4 pH stress .................................................................................................................. 12 Rhizobia inoculants and its role in agriculture ............................................................... 12 vi Rhizobia inoculant selection and application ................................................................. 13 Rhizobia inoculant technology in Ethiopia .................................................................... 14 3 MATERIALS AND METHODS ....................................................................................... 1 Sampling sites .................................................................................................................. 1 Research design ................................................................................................................ 1 Soil and nodule sample collection .................................................................................... 2 Trapping rhizobia from the soil samples .......................................................................... 2 Isolation of rhizobia from nodules ................................................................................... 3 Purification and preservation of isolates .......................................................................... 3 Designation of the isolates ............................................................................................... 3 Authentication of the isolates ........................................................................................... 4 Preliminary symbiotic effectiveness of the rhizobial isolates .......................................... 4 Characterization of isolates ............................................................................................ 5 Morphological characteristics of isolates ....................................................................... 5 Eco-physiological tolerance test of isolates ................................................................... 5 3.12.1 Temperature tolerance test ....................................................................................... 5 3.12.2 Salt tolerance test ..................................................................................................... 5 3.12.3 pH tolerance test ...................................................................................................... 5 3.12.4 Intrinsic antibiotic resistance (IAR) test .................................................................. 6 3.12.5 Intrinsic heavy metals tolerance test ........................................................................ 6 Substrate utilization test of rhizobia isolates .................................................................. 6 3.13.1 Carbon source utilization ......................................................................................... 6 3.13.2 Nitrogen source utilization ...................................................................................... 7 Data analysis .................................................................................................................. 7 3.14.1 Numerical analysis ................................................ Error! Bookmark not defined. 3.14.2 Statistical analysis.................................................. Error! Bookmark not defined. 4 RESULTS AND DISCUSSIONS ...................................................................................... 1 Isolation of rhizobia from nodules ................................................................................... 1 Authentication of isolates ................................................................................................. 1 Morphological characteristics of isolates ......................................................................... 2 Eco-physiological characterization of isolates ................................................................. 3 4.4.1 Salt tolerance test ....................................................................................................... 3 vii 4.4.2 pH tolerance test ........................................................................................................ 4 4.4.3 Temperature tolerance test......................................................................................... 4 4.4.4 Intrinsic antibiotics resistance (IAR) test .................................................................. 5 4.4.5 Heavy metals resistance test ...................................................................................... 6 Substrate utilization test of isolates .................................................................................. 7 4.5.1 Carbon sources utilization test ................................................................................... 7 4.5.2 Nitrogen source utilization test .................................................................................. 7 Preliminary screening for relative symbiotic effectiveness of isolates ............................ 8 Correlation between symbiotic parameters .................................................................... 10 Phenotypic clustering analysis ....................................................................................... 11 5 CONCLUSIONS AND RECOMMENDATIONS ........................................................... 38 Conclusions .................................................................................................................... 38 Recommendations .......................................................................................................... 38 REFERENCES ........................................................................................................................ 39 APPENDICES ......................................................................................................................... 49 viii LIST OF TABLES Tables Pages Table 1: Enumeration and isolation sites of native bacterial isolates ........................................ 2 Table 2: Morphological characteristics of isolates ................................................................... 3 Table 3: Response of the test isolates to eco-physiological conditions ..................................... 5 Table 4: Response to intrinsic antibiotics and heavy metal test of isolates ............................... 6 Table 5: Carbon and nitrogen source utilization patterns .......................................................... 8 Table 6: Effects of eco-physiological and biochemical characteristics on phenotypic clusters of the strains ............................................................................................................................. 14 ix LIST OF FIGURES Figures Pages Figure 1: Model for biological nitrogen fixation in legumes ................................................... 10 Figure 2: Flow diagram for the development and use of rhizobial inoculants ........................ 14 Figure 3: Map of sampling sites................................................................................................. 1 Figure 4: Relative symbiotic effectiveness of isolates ............................................................. 10 Figure 5: Correlation coefficients among different variables .. Error! Bookmark not defined. B) Tree lucerne isolates ........................................................................................................... 37 Figure 6: Dendrogram clustering of isolates on the basis of phenotypic traits ........................ 37 x Abstract In Ethiopia, the production of livestock has been mainly constrained by low quality feed supply and seasonal fluctuations. The usage of good quality and drought-tolerant forage legumes like white lupin and tree lucerne are recommended to overcome feed constraints. Those legumes fix atmospheric nitrogen when being in symbiosis with rhizobia and thereby reduce the need for synthetic fertilizers. However, they show variations in terms of N2- fixation, calling for screening the best native symbionts. The aim of this study was therefore; to isolate, characterize, and screen potential rhizobia isolates nodulating white lupin and tree lucerne from Ethiopia. A total of 115 bacteria were isolated from both legumes, of which 48.7% were authenticated as rhizobia while the remaining were not. The authenticated rhizobia isolates showed a wide diversity in their response to different eco-physiological stresses and substrate utilization pattern. Majority of isolates grew at pH range of 5 - 8.5, temperatures of 15 - 35C, and NaCl (w/v) concentration of 0.5% - 2%. Regarding substrate utilization, almost all of the isolates metabolized all C- and N-sources tested. There was a significant variation (p < 0.05) among the strains in terms of inducing nodule number, nodule dry weight and shoot dry weight on sympatric (their own) hosts. Six strains nodulating white lupine induced higher shoot biomass than N-fertilized control plants while there was no significant difference between the highly effective strains of tree lucerne and the N-fertilized control plants. The numerical analysis of 56 phenotypic traits grouped white lupin isolates into four clusters at similarity coefficient of 77%, while tree lucerne isolates were grouped into three clusters at a similarity coefficient 69%, indicating phenotypic diversity of the strains. Isolates that had higher or equal symbiotic effectiveness with the N-fertilized control plants are recommended for field evaluation under different environmental conditions. We also recommend those isolates for molecular characterization and use as inoculants on their host legumes in soils where they perform best. Key words: forage legumes, rhizobia characterization and screening and symbiosis xi 1 INTRODUCTION Background Livestock production is a crucial component of agriculture specially in developing world (Mengistu et al., 2021). Ethiopia has the largest livestock population in Africa (Sintayehu, 2017). It is an integral part of the farming systems in the country. It plays a significant role in income generation, food and nutrition security, and source of manure for the majority of the populations in the country (Gashaw et al., 2014). Feed is the most important input in livestock production and its adequate supply throughout the year is an essential prerequisite for substantial expansion in livestock production (Riga et al., 2021). However, shortage of feed supply in terms of quantity and quality is the main factor limiting livestock productivity in Ethiopia (Tolera, 2008). Livestock feed resources in Ethiopia are mainly coming from natural grasslands, crop residues, crop aftermath, fodder trees, and shrubs followed by agro-industrial by-products, improved forage crops, and improved pastures (Tadele, et al., 2014). Crop residues account for 61-76% of cattle feed annually (Mulu, 2009), accompanied by having poor feeding value with low crude protein, metabolizable energy, minerals and vitamin contents (Riga et al., 2021), (Mekonnen et al., 2019a). As a result, livestock productivity is low in the country (Denkew, 2004). Therefore, looking for other alternative home-grown protein supplements is crucial to improve livestock production and productivity. Thus, growing and using forage legumes like white lupin and tree lucerne that high nutritive value is one option to solve this problem. Tree lucerne (Chamaecytisus palmensis), commonly known as tagasaste, is perennial legume indigenous to the Canary islands and well-adapted to many regions of the world, including the cool highlands of Ethiopia (Assefa et al., 2012). It is one of a good quality fodder legume with crude protein (18 - 24%), metabolizable energy (650 -700 g/kg DM) (Mengesha et al., 2017), making it a valuable feed option, thereby enhance the overall health and productivity of livestock (Mekonnen et al., 2017). It has also a great role to increase soil fertility when being in symbiosis with rhizobia. Another legume species called white lupin (Lupinus albus L.) is notably a multipurpose cool- season legume native to the Mediterranean region. It is cultivated and employed as a source of protein for animal and human nutrition in various parts of the world (Tounsi-Hammami et al., 1 2020). In Ethiopia, it is produced by smallholder farmers in the regional states of Amhara and Benishangul-gumuz (Nigussie, 2012). Lupin seeds are rich in protein (32.2%), fiber (16.2%), oil (5.95%), and sugar (5.85%) (Riga et al., 2021) (Lucas et al., 2015), making it an excellent source of feed. Like other legumes, white lupin can form a symbiosis with slow-growing (Velázquez et al., 2010) and fast-growing rhizobia (Valverde et al., 2005) and fixes atmospheric nitrogen. Studies show that, it can immobilize a total of 111 to 300 Kg N/ha (Yeheyis et al., 2023), making it an ideal to enrich the soil with this essential nutrient, thereby reducing the need for synthetic fertilizers. In Ethiopia, the production of white lupin and tree lucerne is hindered by various factors such as disease, low soil fertility and inappropriate agronomic practices. Thus, it is necessary to enhance their production capacity with modern agricultural practices. Nitrogen is one of the crucial nutrients required for plant growth and development, but its availability in Ethiopian soils is limited, resulting in very low forage yield. Inoculation of forage legumes with efficient rhizobia is believed to increase soil fertility which intern feed yield, thereby reducing the need for synthetic fertilizers. Numerous studies have shown that most of the symbiosis between legumes and rhizobia is very successful in restoring soil fertility through biological nitrogen fixation (Tesfaye, 2008). However, the success of this symbiotic association is dependent on the capability of legumes to form symbioses with rhizobia, the environment in which the legumes grow, agronomic practices (management) and the rhizobia strain itself (Gunnabo et al., 2021). Many agricultural soils, however, do not have an adequate amount of rhizobia in terms of number, quality, and effectiveness to form effective nitrogen fixation. Hence, it is required to adopt potential and sufficient inoculants to enhance yield. The development and application of rhizobial inoculants requires selection of efficient nitrogen-fixing strains that are effective in both laboratory and field setting. However, there is a lack of experience with screening and inoculating rhizobia for forage legumes in Ethiopia, as these legumes receive little attention. Thus, the aim of this research is to explore and screen symbiotically effective rhizobia isolates for the Ethiopian forage legumes like white lupin and tree lucerne. 2 Statements of the problem Forage legumes are an essential component of livestock production. White lupin and tree lucerne are among the main fodder legumes that are rich in protein and other nutrients, making them an excellent source of feed. In Ethiopia, they are frequently grown with little to no input supplies resulted in attaining low yield, contributing to low livestock production. Smallholder farmers tried to boost soil fertility and their productivity in an effort to lessen these issues. However, the use of chemical fertilizers has been restricted mainly because farmers cannot afford the ever-increasing price of mineral fertilizers and less accessibility in the area (Morris, 2007). This becomes a major challenge to sustainable production of feed in the region. Numerous studies from Ethiopia and other parts of Africa have demonstrated that BNF in various legume crops provides enough N for optimal and long-term crop productivity (Argaw, 2017). Maximizing the yield of legumes requires matching the plant with elite rhizobia that are both competitive for nodulation and capable of high rates of nitrogen fixation. But, there is no or little information on isolation and evaluation of symbiotic efficiency of rhizobia nodulating forage legumes, notably white lupin and tree lucerne in Ethiopia. As a result, isolating and characterising native rhizobia from the aforementioned forage legumes is an important first step in developing inoculants for the legumes. Objectives 1.3.1 General objective The general objective of this study was to isolate, characterize and screen potential rhizobial isolates nodulating forage legumes such as tree lucerne and white lupin growing Ethiopia. 1.3.2 Specific objectives ➢ To isolate bacterial isolates from root nodules of tree lucerne and white lupin. ➢ To authenticate the isolates by host plant re-infection method. ➢ To characterize rhizobial isolates eco-physiologically and biochemically. ➢ To screen potential rhizobial isolates based on their preliminary symbiotic effectiveness under greenhouse condition. 3 Significance of the study Low soil N content is a major factor limiting feed production in Ethiopia. In modern agriculture, synthetic nitrogen fertilization is widely used to improve agricultural yields. However, these days, access to mineral fertilizers is very challenging in developing countries especially in Ethiopia. Therefore, the use of microbial inoculants as a biofertilizer, is a great opportunity for smallholder farmers, for sustainable crop production and improved soil fertility. From an economic and environmental point of view, the use of rhizobial inoculants can reduce environmental pollution and production costs. Therefore, using locally isolated nitrogen-fixing rhizobia as bio fertilizers has a significant effect. Thus, the result of this study will serve as a foundation for the development and adoption of rhizobium inoculant technology to combat the urgent soil fertility problems and low feed production challenges. Additionally, it will improve the pool of rhizobia culture collection that can be used to screen superior strains. We think that isolates that have been preliminary symbiotically successful might benefit smallholder farmers in the study area. Scope of the study This study was concerned with the exploration, screening and evaluation of preliminary symbiotic efficiency of rhizobial isolates nodulating white lupin and tree lucerne growing in Ethiopia for biofertilizer production and recommendation in the region. 4 2 REVIEW OF RELATED LITERATURE Legumes Legumes, belonging to the family Fabaceae, are the largest and most diversified group of flowering plants (Bahru, 2019). They are the second largest plant groups, comprising more than 650 genera and 20,000 species (Kudapa et al., 2013), distributed worldwide over a wide range of agro ecology, edaphic and climatic conditions. Legumes account for around 27% of crop production in agriculture worldwide (Graham & Vance, 2003). They play a central role in both human and animal consumption in their diet as source of proteins, fibers, carbohydrates, minerals and oils (Kirova & Kocheva, 2021). Legumes are an essential component of traditional crop-livestock agricultural systems in sub- Saharan African countries. They are regarded as low-cost substitutes for synthetic fertilizers for enhancing soil fertility and reclaiming land (Wolde-Meskel et al., 2005), due to their unique ability to associate with soil bacteria, collectively known as rhizobia, which invade and colonise roots (rarely stems), forming specialised organs known as nodules. Within the nodule, the bacteria reduce (“fix”) atmospheric nitrogen to its usable form that is passed over to the host plant for assimilation into organic compounds such as amino acids and nucleotides (Sprent et al., 2013). Forage legumes Forage legumes are used to feed livestock. In Ethiopia, forage legumes are chosen for their productivity, palatability, and grazing to boost livestock production (Mengistu, 2002). Forage legumes play a significant role in livestock production, serving as a valuable source of protein and other essential nutrients. They can also be a significant source of nitrogen (N) for the growth of succeeding cereal crops. Declining soil fertility and rising input costs have increased interest in the role of growing annual and short-term perennial forage legumes in rotations to increase soil N and subsequent cereal crop production (Strong et al., 2006). Forage legumes can supply considerable quantities of nitrogen comparable to 30-90 kg per hectare, which increases following crop grain output by 26-113% (Peoples et al., 2009). There are many forage legumes suited to Ethiopia, among which lupine and tree lucerne are common. 5 2.2.1 White lupin White lupin is one of the most cultivated and commercially important large seeded annual legumes. It originated from Mediterranean basin (Yeheyis et al., 2010) and has been cultivated in Greece, Italy, Egypt and Cyprus since ancient times (Nigussie, 2012). Today, it is a traditional pulse crop, grown around the Mediterranean and in Nile valley, extending to Sudan and Ethiopia. In Ethiopia, it is adapted to well-drained, light to medium textured soils with a pH range of 4.5-7.5 and altitudes between 1500 and 3000 m above sea level (Yeheyis et al., 2010), which makes it economically feasible and suitable for cultivation in wide climatic ranges. Its ability to grow in acidic soils is one of the most important features of the crop. Although white lupin is well known, widely cultivated and used by people around the world, its cultivation in Ethiopia lags far behind other legumes (Solomon, 2007). In Ethiopia, white lupin is produced by small holder subsistent farmers in Amhara and Benishangul-Gumuz regional states (Nigussie, 2012). In the main production season (June to December), a total of 17,241 tons of white lupin were produced in 2008 with an average productivity of 0.84 tons ha-1 (Oumer et al., 2015) which is far below the global potential of 3.92 tons/ ha-1 (Kalembasa et al., 2020a). In north-western Ethiopia, smallholder farmers use white lupin for its grain as snack and for preparation of local alcoholic beverage “areke” and to maintain soil fertility (personal communication with the local population). However, as a legume family, white lupin seeds are known for their relatively high protein value, ranging between 30 and 40%, and are rarely used as livestock feed in many other countries (Yeheyis et al., 2010). Even though white lupin is a protein rich forage, its contribution as livestock feed in Ethiopia has remained negligible. White lupin, as a legume crop can fix atmospheric nitrogen into its useable form for the companion or succeeding crop. Studies show that it can fix a total of 111 to 300 Kg N/ha. However, only one third to a quarter of this fixed N is left as residue to be used by the succeeding crop (Yeheyis et al., 2023) (Kalembasa et al., 2020b). 6 2.2.2 Tree lucerne Tree lucerne also known as “Tagasaste” is an evergreen, hardy browse legume that originates in the Canary Islands. It is native to Spain, but adapted to the cold and temperate regions of Australia, Ethiopia, New Zealand, Rwanda and South Africa. It was introduced to Ethiopia in the 1980’s and adapted at highlands with altitudes of above 2000 meter above sea level (Mekonnen et al., 2019b). Tree lucerne can withstand light frosts but affected by water logging. It grows best on fertile, well-drained soils. It can also be productive on acidic, infertile soils if they are well-drained. Once established, it can tolerate drought, but for best productivity, it needs more than 400 mm of rainfall annually (A. Mengistu, 2002). It is now growing in all agro-ecological and suited to sandy, well-drained soils of pH range 4 to7 (Rajan et al., 2019). Tree lucerne is one of the most recommended forage legumes in Ethiopia’s highland farming system and is grown in combination with garden crops and other food crops. As fodder legume, it supplies 23-27% of crude protein and 18-24% of crude indigestible fibre (Rajan et al., 2019). Farmers also use it for shelter, bee forage, sanitation purposes, farm implements, fences and firewood and improve soil fertility. It could play a significant role, where seasonal fodder shortages, poor soil fertility and firewood shortages are common. However, detailed studies on its management, performance and utilization to improve productivity and efficient utilization for various purposes are just beginning. According to studies, tree lucerne fixes 590 kg of N per hectare in a single year, making it the top N-fixer in the legume family (Habtemariam, 2021). However, there is no information on quantifying the amount of nitrogen fixed by this legume in Ethiopia. The demands for nitrogen are increasing worldwide with the increasing needs to feed the constantly growing human population. Thus, fixation of atmospheric nitrogen is a natural alternative to chemical fertilizers that enriches the soil with nitrogen more effectively and without polluting the environment. Biological nitrogen fixation Di-nitrogen (N2) is the most available gas, makes up 78% of the atmosphere (Verma et al., 2020). However, plants cannot directly assimilate it unless it is converted into its reactive 7 (oxidized and reduced) form either biologically or via industrial processes (Lindström & Mousavi, 2020a). Between 150 and 200 million tons of mineral N are required each year by plants in agricultural systems to produce the world’s food, animal feed and industrial products (Unkovich et al., 2008). To meet those requirements, close to 100 million tons of N are fixed annually via the industrial Haber Bosch process. However, the use of nitrogenous fertilizers has resulted in unacceptable levels of environmental pollution and the eutrophication of lakes and rivers (Al-Sherif, 1998). Therefore, finding another way to combat this issue becomes mandatory. Biological nitrogen fixation (BNF) is a process by which N2 in the atmosphere is reduced or oxidized into a biologically useful, combined form of N such as ammonium, nitrates and ammonia (Graham & Vance, 2000). BNF is considered to be more eco-friendly than industrial N fixation (Valentine et al., 2010), and therefore would be ideal for sustainable agriculture. It is an efficient source of nitrogen for resource poor farmers who are using little or no fertilizer, and plays a key role in sustainable grain legumes production. Given the high cost of fertilizer in developing countries and the limited market infrastructure for farm inputs, current research and extension efforts have been directed to integrated nutrient management, in which legumes play a crucial role (Chianu et al., 2009). More focus has been given to the symbiotic associations as they have the greatest quantitative impact on the nitrogen cycle. Assessing BNF is essential to manage N turnover in the soil and maximize crop yields while minimizing losses of reactive N to the environment (Lupwayi et al., 2011). Thus, quantifying BNF is a key factor for both economic viability and environmental performance of low-input farming systems. In spite of reviews examining BNF by crops and pasture legumes (Unkovich, 2012), there is still a strong need to estimate different rhizobia strains and host- legume interaction effect on N fixation in order to achieve full environmental potential and resource benefits of protein crop. 2.3.1 Rhizobia Rhizobia are Gram-negative soil bacteria. They can form nodules on root (rarely stems) of legumes and fix atmospheric N2 in symbiosis (Lindström & Mousavi, 2020b). Currently, over 200 known rhizobial species are reported within 19 genera of the α- and β-Proteobacteria subclasses, and the number increases every year (Ferraz Helene et al., 2022). Despite its designation as a centre of diversity for some of the forage legumes, little or no information in 8 Ethiopia on availability of rhizobia study and inoculant usage on such legumes. Exploration of such legumes for nodulation could help to uncover unknown rhizobia and support research efforts aimed at selecting effective combinations of rhizobium–legume (Wolde-meskel et al., 2005). 2.3.2 Rhizobia-Legume symbiosis The symbiotic interaction between rhizobia and legumes is crucial for agriculture, leading to the development of mutualistic associations (Ohyama, 2008). In this symbiotic relationship, rhizobia are hosted and supplied with carbon sources by legumes and in return legumes receive usable forms of nitrogen provided by rhizobia (Lindström & Mousavi, 2020a). The establishment of this relationship is supported by coordinated changes in gene expression in plant and rhizobia, which reprogram many biochemical and molecular processes in both symbionts (Kirova & Kocheva, 2021). Certain rhizobia have the ability to infect the plant roots and form nodules there, where atmospheric nitrogen is reduced to ammonia. This interaction is considered to be the most important source of biologically fixed nitrogen in agricultural systems (Graham & Vance, 2000). Between one-third and one-half of the total N added to agricultural land is attributable to the legume-rhizobia symbiosis (Herridge et al., 2008). Due to these symbiotic associations, legume plants can typically grow in nitrogen-deficient soils. However, each rhizobia species has a distinct host range allowing nodulation of a particular set of leguminous species. As a result, targeting for both partners would profit from the symbiosis (Kirova & Kocheva, 2021). 2.3.3 Host specificity and mechanisms of nodule formation Nitrogen-fixing bacteria have very high host specificity and a particular bacterial strain is able to form nodules with only a limited number of plant species. The interaction between macro and microsymbionts passes through distinct phases. Rhizobia are attracted by root exudates through chemotaxis and develop colonies in the rhizosphere of the host plant (Kirova & Kocheva, 2021). Signalling molecules such as flavonoids are found in root exudates and activate the transcription of rhizobial nod genes (Zhang et al., 2009). As a result, nod factors are synthesized (Liu et al., 2011), which mediate the attachment of rhizobia to initiate root trichomes, causing an inward curling of root hair tips thus enclosing the attached rhizobia. Through the secretion of nod factors, rhizobia induce root hair cell wall degradation at certain places which facilitates the entry of bacteria into cells. Afterward, 9 formation of the so-called infection thread is initiated within the curl where bacteria have been enclosed between root hair cell walls. At these spots, the formation of the infectious thread is initiated. Along the infection thread, rhizobia cross the entire root hair and reach the cork layer of the root. This intricate biochemical communication marks the first line of specificity in legume-rhizobium symbiosis. Figure 1: Model for biological nitrogen fixation in legumes Adapted from Lindström and Mousavi (2020a) Ecological factors affecting legume–rhizobium symbiosis and BNF Legume crops, unlike the majority of non-legumes, form a beneficial interaction with rhizobia where it converts plentiful N2 present in the atmosphere into its usable form at a specialised plant structures called "nodules". Through this process, legumes satisfy their N demand and that of other non-legume plants when grown in rotation. This process contributes to the agricultural sustainability of production systems for the benefit of millions of smallholder farmers (Franke et al., 2018). Despite having an important role in food security, the majority of legume crops demonstrate low productivity due to biotic and abiotic stresses (Kudapa et al., 2013). These factors can interpose survival of rhizobia in the soil, the infection process, nodule development and 10 nodule functioning and BNF. Thus, it is required to boost certain stress response components to increase crop output (Kudapa et al., 2013). 2.4.1 Salt stress Salt stress is one of the most important abiotic factors limiting plant growth and productivity especially in arid and semi-arid regions (Bertrand et al., 2015). It is estimated that 6% of total world area and 30% of arable land could be qualified as deteriorated and not suitable for conventional agriculture owing to high salinity (Kirova & Kocheva, 2021). In saline soils yield of leguminous crops is decreased due to the lack of successful symbiosis, negatively affecting survival, growth, nodulation and nitrogen fixation in the legume- rhizobium symbiosis (Rao et al., 2002). Salinity also affects the infection process by inhibiting root hair growth, decreasing the number of nodules per plant and the amount of nitrogen fixed per unit weight of nodules (Predeepa & Ravindran, 2010). Rhizobial strains significantly differ in their salt stress sensitivity and some strains can survive in saline soils. However, most rhizobia are not able to tolerate the harmful effects of high saline concentrations. Usually, fast-growing rhizobia are more salt-tolerant in comparison with the ones having slower growth rate (Kirova & Kocheva, 2021). In saline environments, the efficiency of the symbiotic relationship is significantly lowered due to the decreased rhizobial cell number which restricts root infection, inhibits nodule development and leads to reduced activity. Finding salt-tolerant rhizobia may alleviate the toxic effect of salinity and favour the growth of host plants in saline environments (Bertrand et al., 2015). 2.4.2 Drought stress The legume-rhizobium symbioses are impacted by soil moisture deficiency, which is a major barrier to plant growth, productivity, and microorganism proliferation. Despite the fact that rhizobial populations thrive in extremely dry conditions and have efficient nodulation, it has been noted that during drought stress, population densities fall (Basu et al., 2017) The microbial population's sensitivity to drought varies with the rhizobial strains. Therefore, drought-tolerant rhizobial strains must be chosen within their legume host range for successful legume-rhizobium interaction and improved N2 fixation 11 2.4.3 Temperature stress Temperature is one of the limiting factors for biological nitrogen fixation. It adversely affects effectiveness of rhizobia and reduces host legume growth and development. High soil temperatures decrease rhizobia populations and BNF in legumes, as result of a delay in nodulation (Hungria & Kaschuk, 2014). It has also a profound influence in nitrogen metabolism. Saha et al.(2017) reported that, little activity is observed at low temperature and warming promotes the microbial nitrogen fixation and uptake of fixed gas. According to findings reported by Hungria and Kaschuk (2014), the plant nitrogenase activity reduces dramatically as a result of formation of ineffective nodules at high temperature above 40°C. In addition, the relative activity of the rhizobia is altered by temperature, so that rhizobium that is highly effective at a specified range of temperature is less active at another range of temperatures. For these reasons, greater nitrogen gains probably can be achieved by improvements in the heat resistance of the symbiosis. 2.4.4 pH stress A soil with low pH contains high amounts of aluminium and iron oxides, but deficient in phosphorus. Extreme or low soil pH can inhibit rhizobial activity in the rhizosphere around legume roots. Low levels of phosphorus, calcium, and molybdenum, as well as the toxicity of aluminium and manganese, are hallmarks of extremely acidic soils and have an impact on both plants and rhizobia (Ferreira et al., 2016). Nodulation and N fixation are therefore more adversely impacted by low pH of the soil. Rhizobia inoculants and its role in agriculture Inoculation is the process of introducing rhizobia into the soil to enable proper nodulation and nitrogen fixation if certain and effective rhizobia are absent from the soil or are present in insufficient amounts. Soil contains millions of rhizobia but they may not be specific to the particular legume grown and in an active stage to induce infection and initiate N2 fixation. This brings the necessity of inoculation of particular rhizobium inoculants into their specific legume partner (Adal, 2009). Rhizobium inoculants are carefully chosen strains of beneficial soil microorganisms that are cultured in a laboratory and packed in with or without a carrier. They are host-specific, inexpensive, and an environmentally friendly source of nitrogen that improves the growth 12 and yield of legume crops, and provides organic nitrogen for subsequent crops (Bahru, 2019) Rhizobia inoculants used in cropping systems can aid to increase the soil fertility levels for following crops planted in the same field, boosting production and productivity, improving soil health, increasing the hormones and enzymes that encourage plant growth (Bahru, 2019) Rhizobial inoculants are environmentally friendly and economically advantageous because they fix nitrogen, solubilize phosphorus, mobilise phosphorus, solubilize potassium, solubilize micronutrients, enhance organic matter, and lessen the negative effects of inorganic fertilisers. Rhizobia inoculant selection and application The maximum global of atmospheric N fixation by legumes requires the selection and development of elite inoculum (Sessitsch et al., 2002). The ability of the inoculum to induce nodules on the host plant, its efficiency in increasing yield under a wide range of soil and climatic conditions, and its resistance to biotic and abiotic stresses could be considered as criteria for selecting a premium indigenous inoculant (Temesgen, 2017). In addition, the inoculum should also be genetically stable and able to grow well in media and survive during manufacturing processes and in soil. 13 Figure 2: Flow diagram for the development and use of rhizobial inoculants Rhizobia inoculant technology in Ethiopia Legume inoculation is an old established agricultural practice used since the end of the last century. It has contributed to increased N2 fixation and yield in legume crops in situations where the natural N2 fixation was not optimum. The technology is relatively recent to Ethiopia as compared to a six decades old mineral fertilizer use. However, quite efficient indigenous rhizobial inoculants have been obtained and developed for major cool and warm season food legumes by research institutions and universities. These elite rhizobial isolates are now used as commercial inoculants. Currently, the country has two inoculant multiplying institutions: National Soils Testing Center (NSTC) and Menagesha Biotechnology Industry PLC (MBI). These institutions produce nearly 400,000 sachets of rhizobial inoculant per year, which is too low to meet the national demand. To ensure a sustainable inoculant supply system at the national level, bioprospecting of efficient rhizobial strains, reliable inoculant production, distribution, and quality control schemes are needed to be in place and integrated (Mnalku et al., 2020). The afro mentioned institutions have been focusing on food legume but have never worked with rhizobia nodulating forage legumes. Thus, this study was intended to explore forage legume rhizobia for screening elite strains to develop inoculants for the forage legumes 14 3 MATERIALS AND METHODS Sampling sites Soil and nodule samples were collected from diverse agro-ecological locations of Ethiopia particularly from Bahir Dar Zuria, Chencha, Debrebirhan, Gummer, Lemo and Selale (appendix I and Fig 3), where forage legumes like tree lucerne and white lupin are commonly grown with no history of previous rhizobia inoculation. These sampling sites were selected based on their known potential for growing the selected forage legumes. Figure 3: Map of sampling sites Research design Soil and nodule samples were collected from different agro-ecological sites in Ethiopia, and nitrogen-fixing rhizobia were isolated from the collected nodules and soils (using the plant trap method). The isolates were examined for their ability to reinfect their respective host plants. The experimental design consists of randomly assigned treatments in a completely randomized design with three replications. The treatment groups were uninoculated and unfertilized controls, N-fertilized controls, and rhizobial isolates. At the flowering stage, the 1 host plant was uprooted and data on plant growth parameters such as nodule number, nodule dry weight and shoot dry weight per plant were recorded. The data generated from experiments were subjected to statistical analysis to determine the mean variations between the treatments using the R package agricolae. The isolates were also subjected to phenotypic, eco-physiological and biochemical characterization. A multivariate computer clustering analysis of phenotypic variables was carried out to group the isolates using similarity coefficients by the Unweighted Pair Group Mean with the Average (UPGMA) clustering method with the NTSYSpc21 program. Soil and nodule sample collection Large and healthy nodules from field standing forage legumes (tree lucerne and white lupin) were carefully collected, maintained in vials containing silica gel and cotton (Somasegaran and Hoben, 1994) and transported to laboratory. Simultaneously, about 3 kg of soil samples were also excavated randomly from five M pattern points in the field at a depth of 0–20 cm, pooled, mixed and resampled following procedures described previously (Wolde-meskel et al., 2004), sealed in dry clean plastic polythene bags, labelled and transported to greenhouse at Abaya Campus for trapping rhizobia using plant trap method. During the time of sample collection, passport information of each sampling site was recorded (Appendix I). Trapping rhizobia from the soil samples Rhizobia were trapped from soil by inducing nodules on the aforementioned forage legumes using the ‘‘plant-trap’’ method, following procedures adopted by Somasegaran and Hoben (1994). Briefly, each soil sample was filled into surface sterilized (using 70% ethanol) 3kg capacity plastic pots. Undamaged and evenly sized seeds of tree lucerne (scarified with hot water for 30 min) and white lupine (acquired from of International Livestock Research Institute) were surface sterilized with 95% ethanol for 10 seconds followed by 3% (v/v) sodium hypochlorite (NaHClO3) for 4 minutes. The seeds were then repeatedly washed with six changes of sterile distilled water to avoid possible effects of the sterilizing chemicals. The seeds were then transferred to 1% water agar and kept in the dark at room temperature for germination. The germinated seedlings were aseptically thinned down into the pots containing the soil samples. The pots were kept in the greenhouse and watered with water as needed. At the early flowering stage, the plants were carefully uprooted, and the roots were 2 gently washed under running tap water to remove the adhering soils. Finally, healthy and large nodules were sorted and maintained as described above. Isolation of rhizobia from nodules Rhizobia were isolated from the harvested nodules following Somasegaran and Hoben (1994). Dehydrated or desiccated root nodules were immersed in sterile distilled water overnight. The imbibed nodules were surface-sterilized with 95% ethanol for 10 seconds, followed by 3% (v/v) solution of sodium hypochlorite (NaHClO3) for 4 minutes and rinsed in six changes of sterile distilled water to completely remove the sterilizing chemicals. The sterile nodules were aseptically crushed using flamed sterilized blunt-tipped forceps in a drop of sterile distilled water inside a laminar flow hood. Finally, one loop-full of nodule suspension was streaked on a yeast extract mannitol agar (YMA) medium containing 0.25% (w/v) Congo red (CR) and incubated at 28 ± 2oC for 3-12 days. Purification and preservation of isolates The purification of isolates was done by picking a single well-grown rhizobial colony with a disinfected inoculating loop and streaking on freshly prepared sterile YMA medium and incubated at 28 ± 2°C for 3–12 days. The purity and uniformity of colony was attained through repeated re-streaking. A loopful of purified colonies were then transferred to a test tube containing 10ml of sterile yeast-extract mannitol broth (YMB), incubated on rotary shaker adjusted for 120 revolutions per minute (rpm) at room temperature for 3-12 days. The growth of the bacteria on the broth media was confirmed by the formation of cloudy appearance. At log bacterial growth phase (~1 OD), 700µl of the broth culture was taken and pipetted into sterile screw capped Eppendorf tubes containing 300µl of glycerol (50% v/v), and maintained at -20°C for further characterization. Designation of the isolates The purified rhizobia isolates were designated as AMULR and AMUTLR, with the first, second and third letters indicating the name of the university by which the rhizobia are isolated, followed by the remaining letter for legume plants used as trap host (LR – White lupin rhizobia, TL- tree lucerne rhizobia) with different serial numbers for each strain. 3 Authentication of the isolates Each of the purified isolates was subjected to host plant re-infection test to confirm the isolates as rhizobial strains. Each isolate was inoculated into its respective host plant in modified Leonard jars (MLJ) containing sterilized and nitrogen-free river sand as indicated in Somasegaran and Hoben (1994). MLJ were prepared from two plastic cups. The upper cups were filled with pre-treated, nitrogen-free river sand, which was soaked in 1N sulphuric acid (H2SO4) solution for overnight and washed with tap water till the pH becomes nearly 7.0. The second cups were connected to the upper cups by cotton wick. The two cups were covered with aluminum foil and autoclaved as usual. The seeds of white lupin and tree lucerne were surface sterilized and pre-germinated as described before and the germinated seedlings were aseptically transplanted into the MLJ. One ml of each inoculum at its logarithmic growth phase was inoculated to the base of the seedlings growing in the jars. The experiment was laid out using a randomized complete block design (RCBD) in the greenhouse under natural light and day/night temperatures with three replicates including an unfertilized and an uninoculated check as a negative and uninoculated but nitrogen fertilized checks as a positive controls. All the treatments were supplemented with 300-500ml quarter strength of Jenson’s N-free nutrient solution and sterile deionized water once a week and every two days, respectively. The positive control was further supplied with 1% KNO3 (w/v) solution once every week as nitrogen source (Howieson & Dilworth, 2016). At the early flowering stage, the plants were uprooted and carefully rinsed with tap water for nodule assessment. Preliminary symbiotic effectiveness of the rhizobial isolates Plants were harvested at the early flowering stage and the number nodules; shoot and nodule dry biomass of each treatment group was measured to evaluate the preliminary relative symbiotic effectiveness of the isolates. The dry weight of the nodules and shoots was measured after they were dried in the oven at 70 °C for 48 h (Prévost & Antoun, 2007). The relative symbiotic efficiency (RSE) of each isolate was calculated following a previously applied method (Gunnabo et al., 2019) as follows SDWi−SDWN− 𝑅𝑆𝐸 = X100 SDWN+−SDWN− Where: RSE represents relative symbiotic effectiveness, SDWi = the shoot dry weight (in grams) of inoculated plants; SDWN- = the shoot dry weight of negative-control plants and SDWN+ = shoot dry weight of N-fertilized positive controls. 4 The isolates were categorized into four efficiency groups using the method suggested by (Purcino et al., 2000) as: ineffective (< 35%), lowly-effective (35 to 50%), effective (50 to 80%) and highly effective ( ≥80%) Characterization of isolates All isolates that were confirmed as nitrogen-fixing bacteria, were characterized as follow as below. Morphological characteristics of isolates The colony characteristics of authenticated isolates such as color, size in diameter, shape, and texture were determined according to Somasegaran and Hoben (1994). Eco-physiological tolerance test of isolates All purified isolates were evaluated in vitro for the following eco-physiological stresses in the laboratory. The development of the colony was inspected and the result was recorded qualitatively either as “+” for the presence of growth or “–” for the absence of growth after 3–12 days of incubation. 3.12.1 Temperature tolerance test The growth of each isolate at different incubation temperatures was evaluated by inoculating them solid YMA medium and incubating at 5oC, 10oC, 15oC, 20oC, 35oC 40oC, and 45oC (Lupwayi & Haque, 1994). The growths of colonies at each temperature gradient were recorded as positive while absence of colonies was considered as negative. 3.12.2 Salt tolerance test The capability of the isolates to tolerate different salt concentrations was determined by inoculating each isolate on the YMA media containing 0.5 %, 1%, 2%, 3%, 4%, 5%,and 6%, of NaCl (w/v) as indicated in Lupwayi and Haque (1994). Tolerance was evaluated qualitatively as (+) for growth and (-) for absence of growth. 3.12.3 pH tolerance test The capacity of each rhizobial isolate to grow at a variant pH values was evaluated by inoculating them on YMA medium adjusted at a pH of 4.0, 4.5, 5.0, 5.5, 8.0, 8.5, and 9.0 using sterile 0.1 N (normal) HCl and 1 N NaOH as described by Bernal (2001), Kebede et al.(2020). 5 3.12.4 Intrinsic antibiotic resistance (IAR) test The intrinsic antibiotic resistance of isolates to different antibiotics at different concentration was evaluated by streaking each isolate on YMA medium containing freshly prepared filter sterilized antibiotics using 0.22 µm sized membrane filters following the methods described by (Somasegaran and Hoben, 1994). The antibiotics were amoxicillin (50µg /ml), ampicillin (50µg /ml), bacitracin (50µg /ml), cephalexin (25 and 100 µg /ml), kanamycin (10 and 100 µg /ml), penicillin (10µg/ml), streptomycin (25 and 100 µg/ml). Each antibiotic was added to YMA medium at 50oC via filter paper with the Luer-Lock system using hypodermal syringes in a laminar flow hood. Strains were considered resistant when growth occurred and sensitive when no growth was detected. 3.12.5 Intrinsic heavy metals tolerance test According to Maatallah et al (2002) the resistance of isolates to different heavy metals were also tested on YMA plates supplemented with filter sterilized heavy metals at different concentrations. The test heavy metals were ZnSO4 (100µg/ml), CuSO4 (100µg/ml), CsCl2 (50µg/ml), Pb(CHOOCOO)2 (100µg/ml), CoCl2 (250µg/ml), Al2(SO4 (250µg/ml), ZnCl2 (50µg/ml) and MnSO4 (500µg/ml). Substrate utilization test of rhizobia isolates 3.13.1 Carbon source utilization The rhizobia isolates were tested for their ability to utilize different carbohydrates as the sole carbon source. The test was carried out in a standard basal YMA (without D-mannitol and reducing yeast extract from 0.5g/l to 0.05g/l) medium as previously indicated by Somasegaran and Hoben (1994). The tested carbon sources were citric acid, dextrose, D- fructose, D-glucose, lactose, sorbitol, and sucrose. The test carbon sources were prepared as 10% (w/v) solutions in distilled water. Heat labile carbon sources were filter sterilized using disposable membrane filter of 0.22µm size and plastic hypodermic syringes with the Luer- Lok system and added to the autoclaved carbohydrate-free basal medium before pouring plates, but 10ml of 10% (w/v) heat stable carbohydrates were autoclaved together with the medium. The medium modified with the test carbohydrate was poured into sterile petri dishes and solidified in the laminar flow hood. Broth culture of the test strains at their logarithmic growth phase was inoculated onto cells drawn on the medium by sterile micropipettes and 6 incubated at at 28 ± 2°C for 3-12 days. Appearances of colonies were recorded as positive (+) for utilization while absence of colonies was noted as negative (-) for no utilization. 3.13.2 Nitrogen source utilization The pattern of our isolates to assimilate different nitrogen sources was tested using seven amino acids (L-leucine, L-alanine, glycine, L- asparagine, L-arginine L-proline, L- tryptophan). They are also tested for three vitamins (ascorbic acid, thiamine, and nicotinic acid). The test nitrogen sources were sterilized by 0.22µm sized membrane filters. Each amino acid and vitamins was added to pre-autoclaved and cooled basal medium at a final concentration of 0.5 g/l as reported in Amarger et al (1997). Finally, broth culture of each isolate was inoculated into those basal media and incubated at 28±2oC for 3-12 days. Then growth of the colonies on plates were observed and taken as (+) if growth was there and (-) if colonies did not grew. Data analysis The data from eco-physiological and biochemical characterization of isolates were used for numerical taxonomic studies. The presence (+) and absence (-) of growth were converted to binary data, with 1 representing bacterial growth and 0 representing no growth. A computer cluster analysis of 56 phenotypic variables for 56 isolates was carried out using similarity coefficient by the Unweight Pair Group Mean with the Average (UPGMA) clustering method with NTSYSpc21 version 2.1 The data generated from the experiment were subjected to statistical analysis using R package agricolae software program. The mean variations between treatments were assessed by one-way ANOVA and least significant difference (LSD) at p < 0.05 values were calculated to test the significant differences between treatment means. 7 4 RESULTS AND DISCUSSIONS Isolation of rhizobia from nodules In this study, a total of 115 bacterial isolates were recovered from root nodules of the two forage legumes white lupine (65 isolates) and tree lucerne (50 isolates) grown at different locations in Ethiopia. In some cases, more than one kind of isolate was obtained from a single nodules. This is in agreement with Girmaye et al. (2018) who found multiple occupants of bacterial isolates from root nodule of cowpea. Most of isolates were originated from Bahir Dar zuria using white lupin and least number of isolates recovered from Selale using tree lucerne as a trap host. The variation in the number of isolates at specific sampling locations could be due to the morphological difference between the primary isolates on the YMA plate and further purification practices. Authentication of isolates The ability of each isolate to re-infect its host plant was tested under greenhouse conditions. Consequently, 60% of white lupin isolates and 34% of tree lucerne isolates were capable of inducing nodules on the roots of their respective hosts and were subsequently authenticated as rhizobia (Somasegaran and Hoben, 1994), while the rest of the isolates were rejected as they failed to nodulate. This was somewhat consistent with the findings of Pudelko (2010a), who found that 42.86% of strains isolated from white lupin induced nodules on the corresponding hosts. Sintie (2018) also reported that 39% of isolates recovered from root nodules of white lupin from north-western Ethiopia formed nodules upon verification under greenhouse. On the otherhand, Gault et al. (1994) reported that strains isolated from tree lucerne formed nodules when eximined under greenhouse. The isolates that were not induced nodules on the roots of their hosts mentioned above might be contaminants that are capable of co-inhabiting with rhizobia. Several studies demonastrated the presence of non rhizobial bacteria in the root nodules of legumes. In agreement with this, Mhamdi et al. (2005) showed that agrobacteria were able to colonize root nodules of common bean. The positive and negative control plants did not form any nodules, confirming aseptic experimental conditions. 1 Table 1: Enumeration and isolation sites of native bacterial isolates Legumes sampling sites Bacterial isolates Confirmed Rhizobia % of proportion Bahir Dar Zuria 29 20 30.7 Chencha 27 15 23.1 Lemo 9 4 6.2 Total 65 39 60 Debrebirhan 17 8 16 Gummer 20 7 14 Lemo 9 0 0 Selale 4 2 4 Total 50 17 34 Sum total 115 56 48.7 Morphological characteristics of isolates Our rhizobial isolates showed different morphological characteristics (Table 2), suggesting that there is a diversity among the isolates. Accordingly, 53.8% of the strains isolated from white lupin were found to be large mucoid (LM), buttery in texture, with a colony size ranging from 1.5 to 3 mm, while the remaining 46.2% of the isolates were small mucoid (SM), elastic in texture, with a colony size of less than 1 mm diameter. The colony color of the isolates was also found to vary from white-opaque to milky-translucent with a dome or flat bump on YMA medium. These features confirmed previous findings by Sintie (2018) and Tounsi-Hammami et al.(2020). Furthermore, nearly half (53.8%) of the white lupin isolates appeared on YMA after 3-7 days, while the remaining (46.2%) of the isolates required 7-12 days to form visible colonies. This was in line with the finding of Pudelko (2010b) and Msaddak et al. (2023), each of them reported that, isolates of white lupin plant exhibited growth on 3-12 days. Regarding isolates recovered from tree lucerne, all of them (100%) were small mucoid, had elastic texture and flat elevation with a colony size of less than 1 mm. The colony color varied from milky-transparent to creamy mucoid. Our results were consistent with previous reports on tagasate rhizobia by Messaoud et al.(2015). Furthermore, all of the isolates from tree lucerne formed visible colonies after 7-12 days of incubation on YMA. All isolates of the two legumes were rod in shape. 2 Tree lucerne White lupin Table 2: Morphological characteristics of isolates Characteristics White lupin Tree lucerne No. of isolates % No. of isolates % Colony color White opaque 21 53.8% 12 70.6% Milky 18 46.2% 5 29.4% translucent Colony < 1mm 18 46.2% 17 100% diameter 1-2mm 7 18% 0 0% >2mm 14 35.8% 0 0% Colony shape Round dome 21 53.8% 6 35.3% Round flat 18 46.2% 11 64.7% Colony shape LM 21 53.8% 0 0% SM 18 46.2% 17 100% Texture Buttery 21 58.3% 0 0% Elastic 18 46.2% 17 100% Date of colony 3-5 days 8 20.5% 0 0% Appearance 5-7 days 13 33.3% 0 0% 7-12 days 18 46.2% 17 100% Eco-physiological characterization of isolates 4.4.1 Salt tolerance test Our isolates showed tolerance and susceptibility to the tested salt with different concentrations ranging from 0.5 to 6% (w/v) (Table 3; Appendix III). Accordingly, 30.8% of isolates from white lupine and 41.2% from tree lucerne were able to grow at 0.5% NaCl, but showed a steady decline in growth when inoculated into the medium with 1% and 2% salt concentration. However; it was found that, all isolates of the two forage legumes were intolerant of a salt concentration of more than 2%, reflecting the serious effects of high salt concentration on the growth of our isolates. These results are slightly consistent with the findings of Alshaharani and Shetta (2015) who found that all isolates of woody legumes from Saudi Arabia grew at lower salt concentrations. Baljinder et al. (2008) also found that rhizobial isolates from fenugreek plants were unable to grow salinities above 1% NaCl concentrations. 3 4.4.2 pH tolerance test The tested isolates revealed differences in their growth response to a pH ranging from 4 to 9 (Table 3; Appendix III). In light of this, almost all isolates from each of the two legumes thrived between pH values of 5 and 8.5, which was consistent with previous findings reported by Kebede et al. (2020) who noted that isolates from cowpea grew well at pH values ranging between 6.0 and 8.5. Raza et al. (2001) also reported that, all rhizobial isolates of lupin grew in pH levels ranging from 4 - 10. On the other hand, 33.3% of isolates from white lupin were grown at pH 4, indicating they were more tolerant of acidic conditions. But none from tree lucerne withstood at these pH value. These was in agreement with previous reports by Alshaharani and Shetta (2015) who noted that most isolates of woody legumes from Saudi Arabia grew at pH 5.5- 8.5, but no strains grew at pH 4.0. One isolate (AMULR47) from white lupin were found to be intolerant to all tested pH values. In general, the isolates, which were found to have grown in a wider pH ranges, may have practical application with respect to selection of a wide-range pH tolerant strains that can perform well under acidic, neutral and alkaline soils. 4.4.3 Temperature tolerance test The current study showed that, our isolates had different pattern of growth at different incubation temperatures ranging from 5 - 45oC (Table 3; Appendix III). Consequently, almost all isolates from the two legumes grew well at an incubation temperature of 10 - 35oC but none was able to grow at 5 and 45oC, suggesting that our isolates are capable of adapting in a wide range of soil temperature. This result collaborates with Hilali et al.(2016) who founded that, the optimum temperature of growth for all the strains isolated from white lupin ranged between 10°C and 35°C. It also appeared that, 94.9% of our isolates from white lupin were found able to grow at 40oC, indicating that most of the isolates can overcome high soil temperature. The growth of those isolates at 40 ºC is not surprising as previous studies already identified thermo-tolerant isolates from chickpea and alfalfa that can grow at 40 °C (Shimekite, 2006). Practically, the existence of the isolates that could tolerate high temperature could potentially be helpful to develop inoculant that can perform better at high temperature. 4 Table 3: Response of the test isolates to eco-physiological conditions White lupin isolates Tree Lucerne isolates Test parameters No of isolate % No of isolates % 0.5 12 30.8 7 41.2 1.0 9 23.1 1 5.9 2.0 2 5.1 0 0 3.0 0 0 0 0 4.0 0 0 0 0 5.0 0 0 0 0 60 0 0 0 0 4.0 13 33.3 0 0 4.5 20 51.2 5 29.4 5.0 28 71.7 15 88.2 5.5 33 84.6 17 100 8.0 38 97.4 14 82.3 8.5 36 92.3 13 76.4 9.0 35 89.7 8 47.05 5oC 0 0 0 0 10oC 39 100 13 76.4 15oC 39 100 13 76.4 20 39 100 17 100 35 37 94.8 5 29.4 40oC 37 94.8 0 0 45oC 0 0 0 0 4.4.4 Intrinsic antibiotics resistance (IAR) test Our rhizobial isolates were treated to several antibiotics at varying concentrations, showed wide variations in tolerance or sensitivity (Table 4). It was noted that, about 94.6%, 66.6% and 61.1% of isolates from white lupin and 100%, 17.6% and 35.3% from tree lucerne were found to be resistant to ampicillin (50μg/ml), bacteriocin (50μg/ml) and amoxicillin (50μg/ml) respectively. These could enable them to coexist with antibiotic-producing microorganisms in the soil. In addition to this, 41% of isolates from white lupin tolerated 1mg/ml of penicillin. However; no isolate from all legumes showed growth on streptomycin, 5 NaCl concentration ranges Temperature range pH ranges (%) kanamycin, and cephalexin at different concentrations, showing the series effects of these antibiotics on the growth of our isolates. This is consistent with the findings of Hilali et al. (2016) who found that tetracycline, streptomycin, and kanamycin significantly inhibited the growth of rhizobia isolated from white lupin. Similarly, Sintie (2018) reported that streptomycin and kanamycin were found to be the most growth inhibitor antibiotics for the tested rhizobial isolates of white lupin. 4.4.5 Heavy metals resistance test Our isolates varied in tolerance to different concentrations of heavy metals. Accordingly, all isolates exhibited resistance to ZnSO4 and Al2(SO4)3, making them good candidates for inoculant development in Al and Zn toxic soils. However; all isolates were found to be sensitive to CuSO4, CoCl and pb(CH3COO)2. On the other hand, majority (76.2%) of the isolates from white lupin and all from tree lucerne were found to be resistant to ZnCl2. In addition to this, 97.4% and 94.9% of isolates from white lupin were tolerated MnSO4 and CsCl respectively, but none from tree lucerne resisted them. This was in agreement with previous report by Sintie (2018) who claimed that all isolates of white lupin grew on the growth medium containing the given concentrations of manganese and zinc, but sensitive to cobalt and copper. The intrinsic heavy metal resistance of the test isolates observed in this study implies that the resistant isolates could be regarded as potential candidates to develop inoculant for environments polluted with heavy metals. Table 4: Response to intrinsic antibiotics and heavy metal test of isolates Concentrations of the antibiotics and White lupin Tree Lucerne heavy metals (μg/ml) No of isolate % No of isolate % Cephalexin (25) 0 0 0 0 Cephalexin (100) 0 0 0 0 Kanamycin (10) 0 0 0 0 Kanamycin (100) 0 0 0 0 Streptomycin (25) 0 0 0 0 Streptomycin (100) 0 0 0 0 Ampicillin (50) 37 94.8 17 100 Penicillin (50) 16 41.2 0 0 Amoxicillin (10) 25 64.1 7 41.1 Bacteriocin (50 26 66.6 3 17.6 6 Antibiotics MnSO4 (500µg/ml) 38 97.4 0 0 CsCl (50µg/ml) 37 94.8 0 0 ZnCl2 (50µg/ml) 30 76.9 17 100 CuSO4 (100µg/ml) 0 0 0 0 CoCl (250µg/ml) 0 0 0 0 Pb(CH3COO)2 (500µg/ml) 0 0 0 0 ZnSO4 (100µg/ml) 39 100 17 100 Al2SO4 (250µg/ml) 39 100 17 100 Substrate utilization test of isolates 4.5.1 Carbon sources utilization test The current investigation revealed that isolates from white lupin and tree lucerne utilized almost all carbon sources tested, suggesting that they are versatile in using substrates and can survive under soil conditions with limited nutrients. In agreement with this, Haile et al. (2016) claimed that rhizobial isolates utilized a wide variety of carbon sources that may be crucial in improving their survival in different soil conditions. Degefu et al.(2018) further delineated that, rhizobia isolates that use a broader range of carbon sources are of paramount importance for the production of inoculum that can be used in soils with different carbon sources. Furthermore, all isolates utilized D-glucose, D-lactose, sucrose and dextrose. Relatively, sorbitol and fructose were the most recalcitrant substrates, utilized by only 85% and 88% of isolates from white lupine and tree lucerne, respectively. This indicates that, our isolates were versatile in utilizing a broader carbon sources. This coincides with previous reports by Kebede et al. (2020) who tested the ability of rhizobial isolates of cowpea to utilize different carbon sources and revealed that all isolates were able to utilize and grow well on most carbon sources. 4.5.2 Nitrogen source utilization test This study revealed that all tested isolates from both legumes were able to degrade L-alanine and L-leucine, thiamine and nicotinic acid, making them predominantly significant for the production of inoculum for soils with different nitrogen sources. However, no isolate used L- tryptophan. The ability of our isolates to utilize diverse nitrogen sources would give them an ecological advantage, allowing them to compete with other microbes and thrive in nutrient- poor soil conditions. The versatility of our isolates in using different substrates also ensures 7 Heavy metals their heterotrophic competence, which allows them to survive in the soil. Assefa, and Jida, (2011) indicated that the capability of isolates to use a broader range of nitrogen sources gave them survival and competitive advantage in the soil. Table 5: Carbon and nitrogen source utilization patterns White lupin Tree lucerne C- and N-Sources No of isolate % No of isolate % Glucose 39 100 17 100 Sorbitol 33 84.6 16 94.1 Lactose 39 100 17 100 Dextrose 39 100 17 100 Fructose 39 100 15 88.2 Sucrose 39 100 17 100 Citric Acid 33 92.30 17 100 L-Asparagine 27 69.2 15 88.2 L-Proline 38 97.4 5 29.4 Thiamine 39 100 17 100 Glycine 39 100 15 88.2 L-Alanine 39 100 17 100 L-Arginine 39 100 5 29.4 L-Leucine 39 100 17 100 Nicotinic Acid 39 100 17 100 L-Tryptophan 0 0 0 0 Ascorbic acid 26 66.6 1 5.8 Preliminary screening for relative symbiotic effectiveness of isolates Our isolates induced pink coloured nodules upon re-inoculation on their respective host plants on sand culture under greenhouse conditions, which is indications for effective nitrogen fixation (Somasegaran and Hoben, 1994). There was a significant variations in the number of nodule, nodule dry weight and shoot dry weight plant-1 at p < 0.05 level of significance. Regarding nodule number, white lupin plants inoculated with isolate AMULR17 formed higher nodule number (58 nodules plant-1) while those inoculated with AMULR60 attained the minimum nodule number (1 plant-1), demonstrating the different potential of the 8 rhizobial isolates to induce nodules on the host plant. This was slightly consistent with a related study by Abd-Alla (1999), who reported that white lupin plants inoculated with rhizobia could induced 3-85 nodules per plant. The maximum mean NN plant-1 recorded in this study were less than similar findings reported by Abd-Alla (1999) but greater than Sintie (2018). Furthermore, white lupin plant inoculated with AMULR52 showed a maximum NDW (0.05 g/p-1) while a minimum NDW (0.01 g/p-1) was recorded from isolate AMULR60. The highest (1.09 g/p-1) and lowest (0.45 g/p-1) shoot dry weight was obtained in plants inoculated with isolate AMULR52 and AMULR51, respectively. All white lupin isolates induced higher shoot dry weight than the negative control but 15.4% isolates induced higher shoot dry weight than the positive controls. This was consistent with the reports of Tounsi-hammami et al. (2020), who found that inoculated white lupine plants had higher shoot dry weight than non- inoculated plants, showing that inoculated plants benefited more from symbiosis with the native rhizobial strains. Similarly, from tree lucerne plants, variations were observed between strains in terms of nodulation, nodule dry weight, shoot biomass and symbiotic effectiveness. Accordingly, highest number of nodules (62 plant-1) was recorded from plants treated with isolate AMUTLR70 whereas the lowest (5 plant-1) was obtained from AMUTLR95. The plant inoculated with AMUTLR78 showed a maximum NDW (0.036 g/p-1); while a minimum NDW (0.004 g/p-1) was recorded from isolate AMUTLR95. The maximum shoot dry weight of 0.17 g/plant was recorded in the plant inoculated with isolate AMUTLR70, while the lowest shoot dry weight of 0.10 g/plant was obtained in plants inoculated with isolate AMULR74 Based on the relative dry matter enrichment of the shoots of inoculated plants with nitrogen- fertilized control plants (Purcino et al., 2000), the relative symbiotic efficiency of white lupin isolates was between 17.45 and 133.3% (Fig 4). Interestingly, 23.1% of the isolates were found to be highly effective and the remaining 46.2%, 15.4% and 15.4% were effective, moderately effective and ineffective, respectively. Regarding relative symbiotic effectiveness of isolates from tree lucerne, 35.3%, 52.9% and 11.8% were found to be highly effective, effective and moderately effective, respectively. 9 A. White lupin isolates B. Tree lucerne isolates Figure 4: Relative symbiotic effectiveness of isolates Where E= effective, HE = highly effective, ME = moderately effective, IE = ineffective, SymRel= relative symbiotic effectiveness Correlation between symbiotic parameters The correlation between different symbiotic parameters revealed that, there was a significant or highly significant positive correlation among all parameters. Accordingly, relative symbiotic effectiveness of the isolates was positively correlated with nodule number, nodule and shoot dry biomass of the two aforementioned legumes. Shoot dry weight was also positively correlated with nodule number and nodule dry weight, indicating isolates with higher nodulation capacity provide a better benefit to their host plants. This was in consistent 10 with Argaw (2012) who reported that shoot dry weight of cowpea was positively correlated with nodule number and nodule dry weight, and symbiotic effectiveness was positively correlated with nodule number, nodule dry weight, and shoot dry weight. The presence of positive relationship between the test parameters confirmed reliability SDW and symbiotic effectiveness as an indicator for efficiency in N fixation. Tree lucerne Lupin F i g u r e 1 : Correlation coefficients among different v ariables. Where *=significant correlation, **and***=highly significant correlation, NN=nodule number, NDW=nodule dry weight, SDW=shoot dry weight, SymRel= Relative symbiotic effectiveness. Phenotypic clustering analysis Based on the similarity coefficients of the Unweighted Pair Group Mean with the Average (UPGMA) clustering method, a computer multivariate cluster analysis of 56 phenotypic variables on 39 rhizobial isolates obtained from white lupin was performed using the NTSYSpc21 software. At a cut point of 77% similarity, it generated four heterogeneous clusters with two independent lineages formed by the strains AMULR12 and AMULR17. This suggests that phenotypic diversity exists among the white lupin nodulating rhizobia groups. 11 Cluster I contained two isolates AMULR1 and AMULR2 originating from the same location. The first isolate (AMULR1) was able to survive on all pH levels tested, utilize all carbon sources and tolerated NaCl concentrations up to 2%. Regarding nitrogen source assimilation, both strains assimilated majority of the amino acids. In addition to this, they grew at a wide range of temperatures (10 - 35oC), resistant to half of the tested heavy metals. However; they were found to be intolerant to all antibiotics. Sixteen isolates, two from Bahir Dar Zuria and fourteen from Chencha, were grouped under cluster II, showing that the location of the isolates did not alter their phenotypic clustering. More than half (62.5%) of the isolates in this cluster were characterized by utilizing all carbon sources. They utilized 70–90% of the nitrogen sources and grew in a wide temperature range (10–15 °C), but none of them were tolerant to lower (5 °C) and upper (45 °C) temperatures, They could not grew on NaCl concentration above 2% as well as various antibiotics such as streptomycin (25 and 100 µg/ml), kanamycin (10 and 100 µg/ml), cephalexin (25 and 100 µg/ml). Regarding heavy metals, all isolates of this cluster were resistant to MnSO4 (500 g/ml), but none of them proved resistant to CuSO4 (100 µg/ml), CoCl2 250 (µg/ml) and Pb(CHOOCOO) 2 (250g/ml). Cluster III consisted of 16 isolates that were characterized by surviving at a pH ranging from 4.5 to 9.0, whereas none survived at temperatures of 5 °C and 45 °C. Regarding the carbon source utilization pattern, all but one of the isolates (AMULR50) metabolized all tested carbohydrates as their sole carbon sources. The isolates also used the majority of the tested amino acids as a nitrogen source, however; none of them assimilated L-tryptophan. It is tricky to find that they were hardly growing on L-tryptophan, while it is one of essential aminoacids. All isolates were unable to resist most antibiotics tested, but only a few isolates were resistant to penicillin (1 mg/ml) and amoxicillin (5 mg/ml). In addition, all isolates were found to be resistant to ampicillin at a concentration of 5 mg/ml. The fourth cluster was formed by three isolates AMULR47, AMULR48 and AMULR49, which were characterized by their ability to metabolize almost all carbohydrates tested. In addition, they used vast majority (70%) of the nitrogen sources evaluated. However, they did not use L-asparagine, L-tryptophan, or ascorbic acid. All isolates in this cluster were found to be sensitive to each of the salt concentrations tested. Regarding heavy metal resistance, they proved to be resistant to 40% of the heavy metals tested, but sensitive to CuSO4, CoCl and pb(CH3COO)2. All isolates in this cluster grew at 10–35°C, but none were grown at lower 12 (5°C) or higher (45°C) temperatures. They were sensitive to most (80%) of the antibiotics tested, but resistant to amoxicillin and ampicillin at a concentration of 5 mg/ml. The dendrogram obtained from a multivariate computer cluster analysis of 56 phenotypic variables on 17 rhizobial strains isolated from tree lucerne also revealed three distinctive clusters and two ungrouped isolates with a similarity level of 69% Cluster one consisted of five isolates, most of which were able to survive in a wide pH range (4.5–9.0) and grew at a temperature of 10–35°C. Regarding their salt tolerance, the majority (60%) were able to grow with 0.5% NaCl, but none were able to grow above this salt concentration. All isolates in this cluster were tolerant only to ampicillin (5 mg/ml), but sensitive to other antibiotics tested. Regarding to substrate utilization, the strains in the cluster showed interesting uptake patterns for carbon and nitrogen sources. Thus, all nitrogen sources except L-tryptophan were used, but the use of the carbon sources was quite different than the use of the nitrogen sources. Cluster two is the largest cluster, comprising 9 isolates. No isolate in this cluster grew at a lower pH (4.0), but all were able to grow between 5.5 and 8.5. Almost all isolates in this cluster (88.8%) were able to grow at a temperature of 10 °C, while 22.2% of the isolates withstood temperatures of 35 °C and all were sensitive to 5 °C, 40 °C, and 45 °C. Almost half of the isolates in this cluster (44.4%) can tolerate 0.5% NaCl, but none are able to grow beyond this salt concentration. Approximately 22.2% and 44.4% of the isolates were resistant to bacitracin (5 mg/ml) and amoxicillin (5 mg/ml), respectively. All isolates were resistant to ampicillin (5 mg/ml) but intolerant to the other antibiotics tested. Regarding carbon source utilization, all isolates assimilated all tested carbon sources except fructose. Cluster III was the lowest cluster, consisting of only two isolates, specifically AMUTLR97 and AMUTLR99. Isolates in this cluster manifested themselves by metabolizing all tested carbon sources and half of the nitrogen sources, growing at moderately acidic to neutral pH, but highly sensitive to alkaline solutions. They resist only a few heavy metals, including ZnCl, ZnSO4 and Al2 (SO4)2. They were sensitive to most antibiotics but were still resistant to amoxicillin and ampicillin. 13 Table 6: Effects of eco-physiological and biochemical characteristics on phenotypic clusters of the strains Characteristics White lupin Tree lucerne isolates isolates Clusters Uncluste Clustures Unclust red ered 4 1 10 - - + - - - - - - 4.5 + + 1 - + - 3 2 - - + 5 + + 7 - + + + 8 + - + 5.5 + + 11 2 + + + + + + + 8 + + + 2 + + 4 + - + - 8.5 + + + - + + 3 + - + - 9 + + + - + - 2 5 - - - 0.5% + 7 - 1 + + 3 4 1 - - 1% + 5 - - + + - - - - - 2% 1 1 - - - - - - - - - 3-6% - - - - - - - - - - - 5oC - - - - - - - - - - - 10oC + + + + + + 1 8 1 - - 15oC + + + + + + 3 + + + + 20oC + + + + + + + + + + + 35oC + + + + - - 2 2 1 1 - 40oC + 15 + + - - - - - - - 45oC - - - - - - - - - - - Bacitracin (5) - + 10 - - + 1 2 - - + Penicillin (1) - 12 3 - - + - - - - - Streptomycin (2.5) - - - - - - - - - - - Streptomycin (10) - - - - - - - - - - - Kanamycin (1) - - - - - - - - - - - 14 Intrinsic antibiotics Temprature (oC) NaCl (%) pH resistance pH I ( N=2) II (N =16) III (N =16) IV (N =3) AMULR12 AMULR17 I ( N=2) II (N =16) III (N =16) AMUTLR78 AMUTLR74 Kanamycin (10) - - - - - - - - - - - Cephalexin (2.5) - - - - - - - - - - - Cephalexin(10) - - - - - - - - - - Amoxicillin (5) 1 15 5 + + + - 4 + - - Ampicillin (5) - 16 16 + + + + + + + + MnSO4 (500) + + + + - + - - - - + CsCl2 (50 ) + 15 + + - + - - - - - ZnCl2 (50 ) - 14 14 1 - + + + + + - CuSO4 (100) - - - - - - - - - - - CoCl2 (250) - - - - - - - - - - - Pb (CHOOCOO)2(250) - - - - - - - - - - - ZnSO4 (100) + + + + + + + + + + + Al2SO4 (250) + + + + + + + + + + + Glucose + + + + + + + + + + + Sorbitol 1 12 15 2 + + 4 + + + + Lactose + + + + + + + + + + + Dextrose + + + + + + + + + + + Fructose + + + + + + + 8 + - + Sucrose + + + + + + + + + + + Citric Acid 1 14 + + + + + + + + + L-Asparagine + 14 11 - - + + 7 + + + L-Proline + + + + - + + - - - + Thiamin + + + + + + + + + + + Glycine + + + + + + + 8 1 - + L-Alanine + + + + + + + + + + + L-Arginine + + + + + + + - - - + L-Leucine + + + + + + + + + + + Nicotinic Acid + + + + + + + + + + + L-Tryptophan - - - - - - - - - - - Ascorbic acid + 12 11 - - + - - - - - ‘+’ the growth of the isolates on the provided test pattern; “-” the inability of the isolates to grow on the provided test parameter 15 Nitrogen sources utilization Carbon sources utilization Intrinsic heavy metals(µg/ml) AMULR1 AMULR2 I AMULR5 AMULR7 AMULR16 AMULR19 AMULR22 AMULR29 AMULR31 II AMULR26 AMULR30 AMULR34 AMULR27 AMULR23 AMULR28 AMULR38 AMULR25 AMULR33 AMULR36 AMULR12MW AMULR64 AMULR58 AMULR59 AMULR63 AMULR65 III AMULR60 AMULR50 AMULR53 AMULR55 AMULR54 AMULR56 AMULR51 AMULR52 AMULR57 AMULR61 AMULR47 AMULR48 IV AMULR49 AMULR17 AMULR12 0.53 0.65 0.77 0.88 1.00 Coefficient A) White lupin isolates AMUTLR67 . AMUTLR72 I AMUTLR70 AMUTLR76 AMUTLR74 AMUTLR78 AMUTLR80 AMUTLR83 AMUTLR67MW AMUTLR92 AMUTLR88 AMUTLR90 II AMUTLR89 AMUTLR94 AMUTLR87 AMUTLR95 AMUTLR97 III AMUTLR99 0.38 0.54 0.69 0.84 1.00 Coefficient B) Tree lucerne isolates Figure 6: Dendrogram clustering of isolates on the basis of phenotypic traits . 37 5 CONCLUSIONS AND RECOMMENDATIONS Conclusions The rhizobial isolates examined in this study were found to be diverse in their host re- infection ability, symbiotic effectiveness, morphology, substrate utilization and response to various eco-physiological stresses. Preliminary symbiotic screening revealed that 23.1% of white lupin isolates and 35.3% of tree lucerne isolates were found to be highly potent and showed a significant increase in nodulation, shoot and nodule dry weight. Six isolates (AMULR28, AMULR58, AMULR55, AMULR49, AMULR57 and AMULR52) from white lupin and one isolate (AMUTLR70) from tree lucerne were even better than plants fertilized with nitrogen. The tested isolate have better and broader utilization of carbon and nitrogen sources, providing them an ecological advantage and enhances their chance for survival, but fastidious to their resistance to the test antibiotics and heavy metals. Recommendations On the basis of the above conclusion, the following recommendations are suggested: The variation of our isolates to respond for different ecological conditions and utilization of various nutrients might be an indication of the possible diversity. However; it does not allow in identifying rhizobia at the species level. Therefore; we recommended to identify them through genetic analysis using molecular techniques. Symbiotically highly effective isolates on sand under greenhouse conditions may not give the same results under field conditions. Therefore, this needs to be supported by in field conditions to realize the development of inoculants for large-scale production. 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Journal of the Plant, 57(1), 171–183. 48 APPENDICES Appendix I: Description of the sampling sites Designations for rhizobial isolates Sampling locations Altitude Latitude Longtude AMULR1 Bahir Dar Zuria 1850 11o41ʹ 08.3ʺ 37o 27ʹ 03.7ʺ AMULR2 Bahir Dar Zuria 1840 11o 41ʹ 16.4ʺ 37o 26ʹ 56.2ʺ AMULR5 Bahir Dar Zuria 1830 11o 41ʹ 34.7ʺ 37o 26ʹ 50.1ʺ AMULR7 Chencha 2676 6° 15' 20.1" 37° 34' 45.1" AMULR12 Bahir Dar Zuria 1825 11o 41ʹ 33.4ʺ 37o 26ʹ 53.0ʺ AMULR16 Chencha 2672 6° 15' 20.6" 37° 34' 38.6" AMULR17 Chencha 2672 6° 15' 20.6" 37° 34' 38.6" AMULR19 Chencha 2685 6° 15' 21.5" 37° 34' 42.7" AMULR22 Chencha 2672 6° 15' 20.6" 37° 34' 38.6" AMULR23 Chencha 2685 6° 15' 21.5" 37° 34' 42.1" AMULR25 Chencha 2676 6° 15' 20.1" 37° 34' 45.1" AMULR26 Chencha 2672 6° 15' 20.6" 37° 34' 38.6" AMULR27 Chencha 2685 6° 15' 21.5" 37° 34' 42.1" AMULR28 Chencha 2672 6° 15' 20.6" 37° 34' 38.6" AMULR29 Chencha 2672 6° 15' 20.6" 37° 34' 38.6" AMULR30 Chencha 2685 6° 15' 21.5" 37° 34' 42.1" AMULR31 Chencha 2672 6° 15' 20.6" 37° 34' 38.6" AMULR33 Chencha 2672 6° 15' 20.6" 37° 34' 38.6" AMULR34 Chencha 2685 6° 15' 21.5" 37° 34' 42.1" AMULR36 Bahir Dar Zuria 1850 11o41ʹ 08.3ʺ 37o 27ʹ 03.7ʺ AMULR38 Bahir Dar Zuria 1840 11o 41ʹ 16.4ʺ 37o 26ʹ 56.2ʺ AMULR47 Lemo 2176 7o 30ʹ 25.9ʺ 37o 48ʹ 21.9ʺ AMULR48 Lemo 2176 7o 30ʹ 25.9ʺ 37o 48ʹ 21.9ʺ AMULR49 Lemo 2176 7o 30ʹ 25.9ʺ 37o 48ʹ 21.9ʺ AMULR50 Bahir Dar Zuria 1840 11o 41ʹ 16.4ʺ 37o 26ʹ 56.2ʺ AMULR51 Bahir Dar Zuria 1840 11o 41ʹ 16.4ʺ 37o 26ʹ 56.2ʺ AMULR52 Bahir Dar Zuria 1830 11o 41ʹ 34.7ʺ 37o 26ʹ 50.1ʺ AMULR53 Bahir Dar Zuria 1840 11o 41ʹ 16.4ʺ 37o 26ʹ 56.2ʺ AMULR54 Bahir Dar Zuria 1840 11o 41ʹ 16.4ʺ 37o 26ʹ 56.2ʺ 49 AMULR55 Bahir Dar Zuria 1830 11o 41ʹ 34.7ʺ 37o 26ʹ 50.1ʺ AMULR56 Bahir Dar Zuria 1840 11o 41ʹ 16.4ʺ 37o 26ʹ 56.2ʺ AMULR57 Bahir Dar Zuria 1830 11o 41ʹ 34.7ʺ 37o 26ʹ 50.1ʺ AMULR58 Bahir Dar Zuria 1840 11o 41ʹ 16.4ʺ 37o 26ʹ 56.2ʺ AMULR59 Lemo 2176 7o 30ʹ 25.9ʺ 37o 48ʹ 21.9ʺ AMULR60 Bahir Dar Zuria 1840 11o 41ʹ 16.4ʺ 37o 26ʹ 56.2ʺ AMULR61 Bahir Dar Zuria 1840 11o 41ʹ 16.4ʺ 37o 26ʹ 56.2ʺ AMULR63 Bahir Dar Zuria 1840 11o 41ʹ 16.4ʺ 37o 26ʹ 56.2ʺ AMULR64 Bahir Dar Zuria 1840 11o 41ʹ 16.4ʺ 37o 26ʹ 56.2ʺ AMULR65 Bahir Dar Zuria 1830 11o 41ʹ 34.7ʺ 37o 26ʹ 50.1ʺ AMUTLR67 Debrebirhan 2924 9o47ʹ 22.5ʺ 39o 39ʹ 35.7ʺ AMUTLR70 Gummer 2919 7o54ʹ 52.8ʺ 38o 4ʹ 01ʺ AMUTLR72 Gummer 2919 7o54ʹ 52.8ʺ 38o 4ʹ 01ʺ AMUTLR74 Gummer 2915 7o54ʹ 52.3ʺ 38o 03ʹ 57.5ʺ AMUTLR76 Debrebirhan 2919 9o47ʹ 22.5ʺ 39o 39ʹ 35.8ʺ AMUTLR78 Debrebirhan 2919 9o47ʹ 22.5ʺ 39o 39ʹ 35.8ʺ AMUTLR80 Gummer 2919 7o54ʹ 52.8ʺ 38o 4ʹ 01ʺ AMUTLR83 Debrebirhan 2919 9o47ʹ 22.5ʺ 39o 39ʹ 35.8ʺ AMUTLR87 Debrebirhan 2959 9o47ʹ 12.6ʺ 39o 39ʹ 54.6ʺ AMUTLR88 Selale 3015 9o49ʹ 00.6ʺ 38o 35ʹ 26.2ʺ AMUTLR89 Gummer 2915 7o54ʹ 50.3ʺ 38o 03ʹ 56.8ʺ AMUTLR90 Gummer 2913 7o54ʹ 51.3ʺ 38o 03ʹ 50.1ʺ AMUTLR92 Debrebirhan 2924 9o47ʹ 22.5ʺ 39o 39ʹ 35.7ʺ AMUTLR94 Gummer 2913 7o54ʹ 51.3ʺ 38o 03ʹ 50.1ʺ AMUTLR95 Selale 3082 9o48ʹ 44.7ʺ 38o 36ʹ 25.1ʺ AMUTLR97 Debrebirhan 2919 9o47ʹ 22.5ʺ 39o 39ʹ 35.8ʺ AMUTLR99 Debrebirhan 2961 9o47ʹ 12.6ʺ 39o 39ʹ 54.3ʺ 50 Appendix II Growth media A. Components of YEMA-CR medium growth media Components Amount Mannitol 10g K2HPO4 0.5g MgSO4.7H2O 0.2g NaCl 0.1g Yeast extracts 0.5g Agar 15g Congo red 10ml Distilled water 1000ml B. Ingredients of modified Jensen’s N-free medium Constituents amount (g/l) CaHP4 1 K2HP4 0.2 MgSO4.7H2O 0.2 NaCl 0.2 FeCl 0.1 Trace elements stock solution 1ml C. Constituents of trace elements stock solution. Constituents Amount (g/l) H3BO3 2.86 MnSO4. 4H2O 2.03 ZnSO4. 7H2O 0.22 CuSO4. 5H2O 0.08 Na2MoO. 2H2O 0.14 Adapted from Somasegaran and Hoben (1994). 51 Appendix III: Substrate utilization pattern of isolates Isolates AMULR1 + + + + + + + + + + + + + + + - + AMULR2 + - + + + + - + + + + + + + + - + AMULR5 + - + + + + + + + + + + + + + - + AMULR7 + - + + + + + + + + + + + + + - + AMULR12 + + + + + + + - - + + + + + + - - AMULR16 + - + + + + + + + + + + + + + - + AMULR17 + + + + + + + + + + + + + + + - + AMULR19 + + + + + + + + + + + + + + + - + AMULR22 + + + + + + + + + + + + + + + - + AMULR23 + + + + + + + + + + + + + + + - + AMULR25 + + + + + + - - + + + + + + + - - AMULR26 + + + + + + + + + + + + + + + - + AMULR27 + - + + + + + + + + + + + + + - + AMULR28 + + + + + + + + + + + + + + + - + AMULR29 + + + + + + + + + + + + + + + - + AMULR30 + + + + + + + - + + + + + + + - - AMULR31 + + + + + + + + + + + + + + + - + AMULR33 + + + + + + - + + + + + + + + - + AMULR34 + + + + + + + - + + + + + + + - - 52 Glucose Sorbitol Lactose Dextrose Fructose Sucrose Citric Acid L-Asparagine L-Proline Thiamine Glycine L-Alanine L-Arginine L-Leucine Nicotinic Acid L-Tryptophan Ascorbic acid AMULR36 + + + + + + + + + + + + + + + - + AMULR38 + + + + + + + + + + + + + + + - + AMULR47 + + + + + + + - + + + + + + + - - AMULR48 + - + + + + + - + + + + + + + - - AMULR49 + + + + + + + - + + + + + + + - - AMULR50 + - + + + + + - + + + + + + + - - AMULR51 + + + + + + + + + + + + + + + - + AMULR52 + + + + + + + + + + + + + + + - + AMULR53 + + + + + + + - + + + + + + + - - AMULR54 + + + + + + + - + + + + + + + - - AMULR55 + + + + + + + - + + + + + + + - - AMULR56 + + + + + + + - + + + + + + + - - AMULR57 + + + + + + + + + + + + + + + - + AMULR58 + + + + + + + + + + + + + + + - + AMULR59 + + + + + + + + + + + + + + + - + AMULR60 + + + + + + + + + + + + + + + - + AMULR61 + + + + + + + + + + + + + + + - + AMULR63 + + + + + + + + + + + + + + + - + AMULR64 + + + + + + + + + + + + + + + - + AMULR65 + + + + + + + + + + + + + + + - + AMUTLR67 + - + + + + + + + + + + + + + - - AMUTLR70 + + + + + + + + + + + + + + + - - AMUTLR72 + + + + + + + + + + + + + + + - - AMUTLR74 + + + + + + + + + + + + + + + - - AMUTLR76 + + + + + + + + + + + + + + + - - AMUTLR78 + + + + - + + + - + - + - + + - - AMUTLR80 + + + + + + + + - + + + - + + - - 53 AMUTLR83 + + + + + + + + - + - + - + + - - AMUTLR87 + + + + + + + - - + + + - + + - - AMUTLR88 + + + + + + + + - + + + - + + - - AMUTLR89 + + + + + + + + - + + + - + + - - AMUTLR90 + + + + + + + + - + + + - + + - - AMUTLR92 + + + + + + + + - + + + - + + - - AMUTLR94 + + + + - + + + - + + + - + + - - AMUTLR95 + + + + + + + - - + + + - + + - - AMUTLR97 + + + + + + + + - + + + - + + - - AMUTLR99 + + + + + + + + - + - + - + + - - Appendix IV: Salt, pH and temperature tolerance test of isolates Isolates AMULR1 + + + - - - - + + + + + + + - + + + + - AMULR2 + + - - - - - - + + + + + + - + + + + - AMULR5 + + - - - - - - + + + + + + - + + + - - AMULR7 + + - - - - - - + + + + + + - + + + + - AMULR12 + + - - - - - + + + + + + + - + + + - - AMULR16 + + - - - - - - + + + + + + - + + + + - AMULR17 + + - - - - - - - + + + + - - + + + - - AMULR19 - - - - - - - + + + + + + + - + + + + - AMULR22 - - - - - - - + + + + + + + - + + + + - AMULR23 - - - - - - - + + + + + + + - + + + + - AMULR25 + - - - - - - + + + + + + + - + + + + - 54 0.5% NaCl 1% NaCl 2% NaCl 3% NaCl 4% NaCl 5% NaCl 6% NaCl pH 4.0 pH4.5 pH5.0 pH5.5 pH8.0 pH8.5 pH9.0 To 5oC To 10oC To 15oC To 20oC To 35oC To 40oC To 455oC AMULR26 + - - - - - - + + + + + + + - + + + + - AMULR27 - - - - - - - - + + + + + + - + + + + - AMULR28 + + - - - - - + + + + + + + - + + + + - AMULR29 - - - - - - - + + + + + + + - + + + + - AMULR30 - - - - - - - + + + + + + + - + + + + - AMULR31 - - - - - - - + + + + + + + - + + + + - AMULR33 - - - - - - - - + + + + + + - + + + + - AMULR34 - - - - - - - - + + + + + + - + + + + - AMULR36 - - - - - - - - + + + + + + - + + + + - AMULR38 + + + - - - - + + + + + + + - + + + + - AMULR47 + - - - - - - - - - - - - - - + + + + - AMULR48 - - - - - - - - - - + + - - - + + + + - AMULR49 - - - - - - - - - - + + - - - + + + + - AMULR50 - - - - - - - - - - - + + + - + + + + - AMULR51 - - - - - - - - - - - + + + - + + + + - AMULR52 - - - - - - - - - - + + + + - + + + + - AMULR53 - - - - - - - - - - - + + + - + + + + - AMULR54 - - - - - - - - - - - + + + - + + + + - AMULR55 - - - - - - - - - - + + + + - + + + + - AMULR56 - - - - - - - - - - + + + + - + + + + - AMULR57 - - - - - - - - - - + + + + - + + + + - AMULR58 - - - - - - - - - + + + + + - + + + + - AMULR59 - - - - - - - - - + + + + + - + + + + - AMULR60 - - - - - - - - - + + + + + - + + + + - AMULR61 - - - - - - - + - - - + + + - + + + + - AMULR63 - - - - - - - - - + + + + + - + + + + - AMULR64 - - - - - - - - - + + + + + - + + + + - AMULR65 - - - - - - - - - + + + + + - + + + + - AMUTLR67 + - - - - - - - + + + + + - - - + + - - AMUTLR70 + - - - - - - - - + + + - - - - - + - - AMUTLR72 + - - - - - - - + + + + + + - + + + - - 55 AMUTLR74 - - - - - - - - + + + - - - - - + + - - AMUTLR76 - - - - - - - - - + + + + + - - - + - - AMUTLR78 - - - - - - - - - - + + + - - - - + - - AMUTLR80 - - - - - - - - + + + + + + - + + + - - AMUTLR83 - - - - - - - - - + + + + + - + + + - - AMUTLR87 + - - - - - - - - - + + + + - - + + - - AMUTLR88 + - - - - - - - - + + + + - - + - + - - AMUTLR89 + - - - - - - - - + + + + + - + + + - - AMUTLR90 + - - - - - - - - + + + + - - + + + - - AMUTLR92 - - - - - - - - + + + + + - - + + + - - AMUTLR94 - - - - - - - - - + + + + - - + + + - - AMUTLR95 - - - - - - - - - + + + + + - + + + - - AMUTLR97 - - - - - - - - - + + - - - - - + + - - AMUTLR99 + - - - - - - - - + + - - - - + + + - - Appendix V: Heavy metals and antibiotics resistance test of isolates Isolates AMULR1 + + - - - - + + - - - - - - - - + - AMULR2 + + - - - - + + - - - - - - - - - - AMULR5 + + + - - - + + + + - - - - - - + + AMULR7 + - + - - - + + + - - - - - - - + + AMULR12 - - - - - - + + - - - - - - - - + + AMULR16 + + - - - - + + + + - - - - - - - + 56 MnSO4 CsCl, Zncl CuSO4 CoCl pb(CH3COO)2 ZnSO4 Al2(SO4)2 50μg/ml BAC 50μg/ml PEN 25μg/ml STR 100μg/ml STR 10μg/ml KAN 100μg/mlKAN 25μg/ml CEP 100μg/ml CEP 10μg/ml AMO 50μg/ml AMP AMULR17 + + + - - - + + + + - - - - - - + + AMULR19 + + + - - - + + + + - - - - - - + + AMULR22 + + + - - - + + + + - - - - - - + + AMULR23 + + + - - - + + - - - - - - - + + AMULR25 + + + - - - + + + - - - - - - - + + AMULR26 + + + - - - + + + + - - - - - - + + AMULR27 + + + - - - + + + + - - - - - - + + AMULR28 + + + - - - + + + + - - - - - - + + AMULR29 + + + - - - + + + + - - - - - - + + AMULR30 + + + - - - + + + + - - - - - - + + AMULR31 + + + - - - + + + + - - - - - - + + AMULR33 + + - - - - + + + - - - - - - - + + AMULR34 + + + - - - + + + + - - - - - - + + AMULR36 + + + - - - + + - + - - - - - - + + AMULR38 + + + - - - + + - + - - - - - - + + AMULR47 + + + - - - + + - - - - - - - - + + AMULR48 + + - - - - + + - - - - - - - - + + AMULR49 + + - - - - + + - - - - - - - - - + AMULR50 + + + - - - + + + - - - - - - - - + AMULR51 + + + - - - + + + + - - - - - - + + AMULR52 + + + - - - + + + - - - - - - - + + AMULR53 + + + - - - + + + - - - - - - - - + AMULR54 + + - - - - + + + - - - - - - - - + AMULR55 + + + - - - + + + - - - - - - - - + AMULR56 + + - - - - + + + - - - - - - - - + AMULR57 + + + - - - + + + - - - - - - - + + AMULR58 + + + - - - + + - - - - - - - - - + AMULR59 + + + - - - + + - - - - - - - - - + AMULR60 + + + - - - + + + - - - - - - - - + AMULR61 + + + - - - + + + - - - - - - - - + AMULR63 + + + - - - + + - - - - - - - - - + 57 AMULR64 + + + - - - + + - + - - - - - - + + AMULR65 + + + - - - + + - - - - - - - - - - AMUTLR67 - - + - - - + + - - - - - - - - - + AMUTLR70 - - + - - - + + - - - - - - - - - + AMUTLR72 - - + - - - + + - - - - - - - - - + AMUTLR74 - - + - - - + + + - - - - - - - - + AMUTLR76 - - + - - - + + - - - - - - - - - + AMUTLR78 - - + - - - + + - - - - - - - - - + AMUTLR80 - - + - - - + + - - - - - - - - - + AMUTLR83 - - + - - - + + - - - - - - - - - + AMUTLR87 - - + - - - + + - - - - - - - - + + AMUTLR88 - - + - - - + + - - - - - - - - + + AMUTLR89 - - + - - - + + + - - - - - - - - + AMUTLR90 - - + - - - + + - - - - - - - - - + AMUTLR92 - - + - - - + + - - - - - - - - - + AMUTLR94 - - + - - - + + + - - - - - - - + + AMUTLR95 - - + - - - + + - - - - - - - - + + AMUTLR97 - - + - - - + + - - - - - - - - + + AMUTLR99 - - + - - - + + - - - - - - - - + + 58 Appendix V: Symbiotic parameters of white lupin rhizobia Strains NN NDW SDW RSE Rate AMULR52 35.00a-g 0.15a 1.09 a 133.03a HE AMULR49 13.00c-g 0.04abc 1.05ab 122.13ab HE AMULR57 44.67a-e 0.09abc 1.04abc 122.02ab HE AMULR55 32.33a-g 0.11abc 1.00a-d 114.35abc HE AMULR58 48.33abc 0.15ab 0.99 a-e 112.94abc HE AMULR28 45.33a-d 0.07abc 0.94 a-f 106.45a-d HE N+ 0.00g 0.00c 0.92a-g 100.00a-e HE AMULR17 57.67a 0.08abc 0.85a-h 90.89a-f HE AMULR23 25.67a-g 0.05abc 0.80a-h 84.04a-f HE AMULR1 22.00a-g 0.03abc 0.79a-h 80.22a-f HE AMULR65 30.00a-g 0.03abc 0.79a-h 78.01a-g E AMULR63 4.67fg 0.05abc 0.78a-h 76.05a-g E AMULR31 22.67a-g 0.05abc 0.77a-h 74.65a-g E AMULR50 53.00ab 0.11abc 0.76a-h 72.58a-g E AMULR61 6.67fg 0.03abc 0.75a-i 71.22a-g E AMULR22 38.00a-f 0.07abc 0.75a-i 70.85a-g E AMULR25 57.33a 0.07abc 0.75a-i 70.76 a-g E AMULR7 35.33a-g 0.06abc 0.74a-i 70.55a-g E AMULR12 19.67b-g 0.04abc 0.73a-i 68.95a-g E AMULR36 10.67 d-g 0.02abc 0.73a-i 67.44a-g E AMULR64 16.67b-g 0.02abc 0.72a-i 66.98 a-g E AMULR16 4.00fg 0.02abc 0.68a-i 59.46a-g E AMULR38 8.00eg 0.02abc 0.68a-i 58.03a-g E AMULR59 4.33fg 0.02abc 0.65b-i 54.29a-g E AMULR54 21.67a-g 0.05abc 0.63b-i 52.88b-g E AMULR2 28.00a-g 0.06abc 0.63b-i 50.52b-g E AMULR30 5.33fg 0.02abc 0.62c-i 50.39b-g E AMULR34 23.00a-g 0.06abc 0.62c-i 50.28 b-g E AMULR5 39.67 a-f 0.09abc 0.59d-i 45.99b-g ME AMULR29 33.00a-g 0.03abc 0.59d-i 45.51b-g ME AMULR26 17.67b-g 0.03abc 0.59d-i 44.42b-g ME 59 AMULR27 28.67a-g 0.11abc 0.59d-i 42.43c-g ME AMULR47 13.67c-g 0.03abc 0.58e-i 42.41c-g ME AMULR60 1.00g 0.01abc 0.55f-i 38.38c-g ME AMULR19 4.67fg 0.02abc 0.52ghi 33.62d-g IE AMULR33 15.33c-g 0.02abc 0.51ghi 30.77d-g IE AMULR48 40.00 a-f 0.05abc 0.50ghi 26.27efg IE AMULR53 17.00b-g 0.06abc 0.48hi 24.62efg IE AMULR56 19.67b-g 0.08abc 0.46hi 20.50fg IE AMULR51 32.33a-g 0.07abc 0.45hi 17.45fg IE N- 0.00g 0.00c 0.34i 0.00g IE CV% 1.99 1.99 1.99 1.99 _ LSD0.05 17.64 0.07 0.20 37.65 _ Where, NN= Nodule Number, NDW = Nodule dry weight, SDW = Shoot dry weight, SymRes = Relative symbiotic effectiveness, CV = Critical value, LSD = Least significant difference. Means within a column followed by the same letter(s) are not significantly different at P< 0.05. NN N SDW SE 60 Appendix VI: Symbiotic parameters of tree lucerne rhizobia Strains NN NDW SDW SymRes Rate AMUTLR70 61.67a 0.022abc 0.17a 103.21a HE N+ 0.00e 0.000c 0.17a 99.98a HE AMUTLR99 29.33ae 0.021abc 0.16a 93.90a HE AMUTLR78 52.33ab 0.036a 0.16a 92.94a HE AMUTLR87 48.00abc 0.016abc 0.15a 90.35a HE AMUTLR67 19.33b-e 0.013abc 0.15a 87.54a HE AMUTLR72 16.33b-e 0.011bc 0.14a 80.56a HE AMUTLR83 22.67b-e 0.008bc 0.14a 79.35a E AMUTLR90 39.67a-d 0.014abc 0.14a 75.09a E AMUTLR94 26.67a-e 0.012bc 0.13a 69.82a E AMUTLR92 13.00cde 0.010bc 0.13a 69.63a E AMUTLR80 22.00b-e 0.012bc 0.12ab 69.23a E AMUTLR88 40.67a-d 0.026ab 0.12ab 67.09ab E AMUTLR95 5.33de 0.004bc 0.12ab 66.76ab E AMUTLR97 19.00b-e 0.010bc 0.12ab 62.74ab E AMUTLR76 12.00cde 0.012bc 0.12ab 60.97ab E AMUTLR89 32.33a-e 0.012bc 0.10ab 49.41ab ME AMUTLR74 11.33de 0.007bc 0.10 ab 45.22ab ME N- 0.00e 0.000c 0.04 b 0b IE CV% 2.02 2.02 2.02 2.02 _ LSD0.05 19.76 0.01 0.05 35.01 _ Where, NN= Nodule Number, NDW = Nodule dry weight, SDW = Shoot dry weight, SymRes = Relative symbiotic effectiveness, CV = Critical value, LSD = Least significant difference. Means within a column followed by the same letter(s) are not significantly different at P< 0.05. NN SDW SE 61 Table 12: ANOVA table for symbiotic parameters for white lupin isolates Response Explanatory Df Sum Sq Mean Sq F value Pr(>F) Rep 2 46 22.93 0.1906 0.8269 NN Strain 40 31785 794.63 6.6021 4.826e-13 *** Residuals 80 9629 120.36 Rep 2 0.004786 0.0023931 1.3847 0.2563359 NDW Strain 40 0.1664941 0.0041235 1.3860 0.0004815*** Residuals 80 0.138259 0.0017282 Rep 2 0.0042 0.002114 0.1374 0.8718 SDW Strain 40 3.7831 0.094577 6.1475 3.099e-12*** Residuals 80 1.2308 0.015384 SymRel Rep 2 6560 3280 69964 0.001584*** Strain 40 114911 2872.8 61278 3.366e-12*** Residuals 80 37505 468.8 Table 13: ANOVA table of symbiotic parameters for tree lucerne isolates Response Explanatory Df Sum Sq Mean Sq F value Pr(>F) Rep 2 212.9 106.44 0.7347 0.4867 NN Strain 18 16350.2 908.35 6.2703 0.00000157*** Residuals 36 5215.1 144.86 Rep 2 0.0001218 0.000060912 1.1960 0.3141 NDW Strain 18 0.00039949 0.000221940 4.3577 0.00008513*** Residuals 36 0.0018335 0.000050931 Rep 2 0.000949 0.0004746 0.5962 0.5562736 SDW Strain 18 0.054262 0.0030146 3.7867 0.0003305*** Residuals 36 0.028659 0.0007961 SymRel Rep 2 956.5 478.23 1.070 0.3536681*** Strain 18 30233.6 1679.64 3.758 0.0003546*** Residuals 36 16090.2 446.95 62 Appendix VII: Pictures taken during the experiment White lupin plants growing at the farm land Trapping rhizobia from the soil Preserved nodules Isolation of rhizobia 63 White lupin isolates grown on YMA-CR medium Tree lucerne isolates grown on YMA-CR medium Broth culture of isolates 64 Authentication of white lupin isolates under greenhouse conditions Authentication of tree lucerne isolates under greenhouse conditions 65 Inoculated Nitrogen Negative fertilized Control Leaf colour variation between treatments groups Root nodules of white lupin plants Inoculated Nitrogen Negative fertilized control Leaf colour variation between treatments groups Root nodules of tree lucerne plants 66