Diversity, effectiveness and biochemical characterization of Bambara groundnut symbiotic Rhizobia strains in tropical Nigerian soils

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DIANDA, O.E. FAGADE, O. O. AJAYI This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4499028/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bambara groundnut (BG) has a high nutritious content, is under-utilized with the potential to eradicate malnutrition, yet has very low production rates. Rhizobia inoculant can enhance it’s production, but, inadequate information about the diversity and suitability of rhizobia strains is known. Diversities of Bambara-symbiotic-rhizobia in soils (54) collected across three states in Nigeria were characterized morphologically and biochemically. Strains were evenly distributed between; Niger (36%), Kaduna (35%), and Kano (29%), but significantly different between local governments. Rhizobia strains were Gram negative rods, 10% were highly effective, while 81% were infective. Strains couldn’t hydrolyse starch but showed varied utilization abilities for different carbon sources, 73% hydrolysed gelatin and 66% produced catalase enzyme. A wide diversity of Bambara-symbiotic-strains were present in the soils, but only 10% effectively fixed nitrogen. Although there is a rich diversity of Bamabara-symbiotic-strains in these soils, it is necessary to apply suitable effective rhizobia strains as inoculant. Biological sciences/Biotechnology Biological sciences/Microbiology Earth and environmental sciences/Environmental sciences Bambara groundnut rhizobia diversity efficiency nitrogen fixation indigenous Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1.0 INTRODUCTION Rhizobia species play significant roles for the amendment of the soil because they make associations which are symbiotic with several legumes and engage in nitrogen fixation through biological processes ( 1 ). These strains of rhizobium have been observed to allow for enhanced phytohormone productions, uptake of minerals and ameliorating the effects of toxic metal(s), and are therefore able to promote plant’s growth rates and developments ( 2 ) especially in soils which become highly polluted during agricultural practises. Current day agricultural practises has moved from using sustainable farming practises to less stressful and more environmentally friendly resources for farming eg. The use of biofertilizers such as rhizobia, phosphate solubilizing bacteria in the sub-saharan African tropical areas. This is mostly because they are cost saving, they improve soil health and most importantly the yields of crops ( 3 ). Bambara ground-nut (BG) a pulse crop plant that produces seed under the ground, is believed to have its originating point located in Africa ( 4 , 5 ), which is grossly under-utilised. It is mostly believed that it originated from West Africa (sahelian region) from among the Bambara tribes-people very near to Timbuktu in Mali ( 4 , 6 , 7 ). It then migrated to numerous parts of Oceanian, Asian and South American countries ( 8 , 9 ). It has been found to have many agronomical advantages in its nature this includes a increased nutrition values, high resistance and tolerance to drought, with abilities to survive in considerably harsh and intolerable soil types ( 10 , 11 , 12 , 13 ). It mostly is regarded as a crop that is able to survive famine ( 6 , 11 , 12 ), is probably due to the association it forms with mycorrhiza. Bambara groundnut by itself is an entire-completely fully-balanced food because it is fortified with Iron, contains 19% proteins such as lysines and methionines ( 14 ), 63% carbohydrates and 6% oil and fatty acid ( 9 , 15 , 16 ) (Fig. 1 ). Unfortunately, the lack of infrastructure, and adequate research to target the diverse and specific symbiotic rhizobia for leguminous beans such as Bambara groundnut, Mac has limited the use of biofertilizers for its production ( 17 ). The symbiotic relationships of rhizobia with legumes that are nitrogen fixers play key roles for the sustenance of most natural biological ecosystems especially within arid & dry areas of tropics and the sub-tropic zones (17, 18). Most research studies in relation to this were done to investigate the nitrogen-fixation status of other legumes and legume trees ( 19 , 20 , 21 ) in different ecological zones ( 22 , 23 , 24 ), in particular within Africa regions ( 25 , 26 , 27 , 28 , 29 , 30 ) but little has been done to identify suitable indeginous rhizobia strains for Bambara groundnut. The large numbers of morphologically and genotypically diversity of rhizobia found in northern Nigeria region may be harboring new strains of rhizobia that is worth being exploited to obtain strains with efficiency in biological nitrogen fixation for Bambara groundnut which is highly under utilized and has low production which doesn’t meet half of its demand to carry out the tests for their symbiotic ability and also ascertain their identities and functionalities in diverse environments. 2.0 METHODS 2.1 Sampling and Collection Site for Soils Soils were collected from farm sites where legumes are planted in 21 local governments from three states in Nigeria. Points in the middle of the field and not at the edges were selected for soil collection to ensure that active rhizobia strains were collected. The soils were collected at depths of 15cm- 20cm, using a soil auger, which was used to dig deep into the soil to collect soil at the required depths. The soil were then bagged in properly labelled transparent ziploc bags and stored in cold coolers till they were transferred to the fridge to be stored at 8ºC to allow for preservation of the bacteria diversity in the soil. They were then transferred to cold rooms and preserved until start of experiments. A total of 54 soils were collected from the different local governments. 2.2 Trapping of Rhizobia Low nutrient pot soils were prepared by mixing sea sand (the sea sand was washed thoroughly by using six changes of water or until pH increased to a values of 6.6–6-8, the most suitable pH range for the growth of rhizobium), small sized gravel stones and peat in ratios 6:1:6 this was thoroughly mixed with water till evenly distributed and sterilized at a temperature of 121 o C and pressure of 1.05 kg cm − 2 for 1 hour. The sterilized soils were then used to fill sterilized 250 ml pots (sterilization of pots was done using JIK bleaching agent which contains 3.5% w/v sodium hypochlorite, and thorough rinsing by submerging in six changes of sterile water and a final rinse again to ensure that all the residual traces the JIk were removed and did not remain to affect the bacteria during pot experiments).Viable seeds of Bambara groundnut were sterilised using sodium hypochlorite and ethanol (31, 32) and then two of each seeds (viable ones) were placed into each pots and were planted in enclosed and restricted screen house conditions till germination. The soil samples (54) which were collected from fields, and transferred to the lab to be stored under cool conditions were identified and brought to the lab for preparation of inoculum. Soil inoculant were prepared by weighing 10g of each soil in 90mls of sterile diluent water (K 2 HPO 4 (0.125g/L), MgSO 4 (0.05g/L)), the soil was then placed on a rotatory shaker and allowed to shake at low amplitudes for 1 hour to allow for revival and resuscitation of the soil bacteria ( 33 ) and 5 ml each was introduced into the Bambara groundnut plants at 2 weeks after planting (when germination has occurred) under screen house condition. Each soil was inoculated using 4 replicates. The rhizobia strains present in the soils were tapped in the nodules of the plants grown for 8 weeks when nodules had been formed in the plant and isolated from the undamaged nodules 2.3 Experimental Setup Sterile sand was prepared, and used to fill sterile pots (31, 32, 33). Sterile seeds were planted in pots and inoculated (1 ml) at 2 weeks after planting (WAP) using 4 replicates each along-side the un-inoculated and the N + control plant. Plants were grown for 8 week during which sterilized nutrient solution was applied to replenish nutrients lost during sterilization. Plant nutrients (solution) were made from micro and macro nutrient (Micronutrient–Stock Solution Constituents: H 3 BO 3 − 2.86g, ZnSO 4 .7 H 2 O − 0.22g, MnCl 2 .4 H 2 O − 1.81g, Na 2 MnO 4 . 2 H 2 O – 0.025g, CuSO 4 . 5 H 2 O – 0.08g, Distilled water − 1 L) (Macronutrient – stock solution constituents: CaHPO 4 -100g, MgSO 4 .7 H 2 O – 20g, K 2 HPO 4 – 20g, FeCl 3 -10g, NaCl − 20g, Distilled water − 1L) (100ml of the macro nutrient stock-solution, and 10ml micro nutrient stock-solution are added together and made up to 10 litres using sterile distilled water at 50 ml weekly per pot). An N + treatment (50 ml (48.5g of KNO 3 to 1.5L of distilled water) solutions was supplied to plant with nitrogen control (31, 32, 33). 2.4 Harvesting The plants were harvested with the use of a secateur cutting the plant stem very closely at the base of the plant. The shoot and stems were carefully collected and placed in labelled paper bags. The soil was poured out and the root was shaken gently to remove the sand the root were stored in labelled nylon. A sieve of an appropriate mesh size was placed under the root samples allowing the nodule to be caught catch when they become detached away from the surfaces and extensions of plant’s root ( 31 , 33 ). Sieves carrying the root were placed under gentle streams from running water from a tap and carefully washed in a bucket after washing it was immediately placed inside labelled bags and later transferred to the lab where the nodules were carefully detached before being placed in envelops (labelled) and some selected for nodule assay. The nodules were then dried naturally before being stored with silica gel (was placed in oven until colour changed to brown) (32, 33). 2.5 Morphology of Nodules The nodules were picked from the plant and used for assay, their sizes and colour was determined, their shapes and arrangement on the plant roots were observed and described. The sizes were classified into small, medium and large, the colours were classified into, green or white (which were consider non fixing), red, brown or pink (which were considered to be fixing). the shape was classified as round, irregular or clubbed shaped. The arrangements of the nodules on the roots of the experimental plants were classified as scattered or clustered (31, 32, 33). 2.6 Isolation of Bacteria Recovery of Rhizobia from nodules was done using the roots of the Bambara groundnut plants in the trap experiments on Congo red agar (32, 33, 34, 35, 36) using spread plate method. Unharmed nodules ( 3 ) were selected, rehydrated using sterilized water (distilled) for 15–20 mins and afterwards a thorough surface sterilization with JIK a household reagent containing 3.5% NaClO (sodium-hypochlorite) for a duration of 3 mins. The sterilized nodules went through thorough rinsing using sterilized water, afterwhich they were re-sterilized using 95% CH 3 CH 2 OH (ethanol) and then also re-rinsed again with six times of new different changes of sterilized water (distilled) ( 31 ). The sterilized nodules were crushed in sterilize Petri-dishes, and loops-full were streaked on congo-red agar ( 33 ) and inverted before being incubated using a favourable temperature of 28°C for a continued period of 5–14 days or till growth was seen (modified method of Somasegaram & Hoben, 2012). The selected isolates were then purified and identified appropriately as Rhizobia. All isolated rhizobia strains were subjected to gram reaction test ( 37 ), cell shape and growth on YMA-CR. Their growth characteristics and colour were used as a basis selection for further characterization. 2.7 Preservation of Isolates The purified isolated strains were inoculated on YMA agar slants with 0.3% (W/V) CaCO 3 ( 34 ), the slants were kept in a fridge. Others that proved not to be rhizobia were stored using slants of nutrient agar (31, 32). 2.8 Authentication of the Strains Yeast Mannitol broth was weighed and then sterilized using heat-temperature of 121°C at 1 atm for a period of 15 mins and was left to cool. The yeast mannitol broth was then inoculated with pure Rhizobia strains and then incubated for a duration of 14 days at a heat-temperature of 28°C to allow for the adequate multiplication of the bacteria cells and the broth was standardized to meet required standard value (≥ 1x10 9 cfu/mL) ( 31 ). Bambara groundnut seeds were sterilized, planted in sterilised potted sand and allowed to grow for two weeks before being inoculated as a result of their slow germination. Plants under screen-house conditions were then left to grow for eight weeks to determine and evaluate the infectivity of rhizobial isolates by nodule formation. Plants were given nutrient solution 50 mL weekly to replenish nutrients that may have been depleted or unavailable due to the resulting absence of other plant-growth-promoting-bacteria (31, 32). 2.9 Cell Description: Gram Staining and Microscopic viewing This test was done for determining the nature of the peptidoglycan layer in the cell wall of a micro-organism i.e. either thick or thin. It was done to differentiate the isolates into gram positive or negative bacteria. Two loops full of water placed clean grease free slides were smeared with wire loop full of the bacteria on the slide. The sample was then passed through heat to fix the cell using a spirit lamp thrice. Crystal violet a primary stain was place on the slide covering the smear for 1 minute, rinsed off using clean water from a gently flowing tap to remove unbound stain. Gram’s iodine which is an active and effective mordant was then placed on the slide again covering the smear for 1 minute and then rinsed off. This was followed by the addition of ethanol which functioned as a decolourizer for 30 seconds. For cells with thin peptidogylcan layer, the primary stain is removed, the decolourizer was then washed off using running water and a counter stain, also called known as a secondary stain, safranin was placed on the slide entirely covering the smear for 30 seconds and rinsed off. The stained slides were then viewed under the microscope for gram negative long or short rod micro-organisms at x 100 objective lens ( 37 ). 2.10 Biochemical Tests Various tests were carried out to describe the organism based on its physiological activities, biochemical reactions, characteristic features and also possibly identify the organism. The biochemical identification tests carried out includes catalase test, citrate test, hydrolysis of gelatin, hydrogen sulphide test, starch hydrolysis test and sugar fermentation test. All procedures were carried out following standard methods ( 37 , 38 ). 2.10.1 Catalase test This test is used to detect whether the isolates produce the enzyme catalase or not. Catalase is found in most aerobic micro-organisms although some aerobic and microaerophilic micro-organisms do not produce catalase, but hydrogen peroxide is not produced in anaerobic metabolism. The reagent used was hydrogen peroxide (3%), 24 hour old culture of the micro-organism was used. a few drops of the reagent was placed on a slide and a little of the micro-organism was picked using a sterile wire loop under sterile condition. The micro-organism was then mixed with the reagent on the slide and observed for bubbles ( 37 , 38 ). 2.10.2 Citrate test Test is carried out to determine the abilities of the micro-organisms to produce the enzyme citrase thus enabling it to utilize citrate, converting it to oxaloacetate. The medium used was citrate medium which is composed of g of citrate medium per litre. This was homogenized and dispensed into vijou bottles and sterilized at a set pressure of 1.05 kg cm − 2 /atm for a duration of 15 minutes and slanted for 24 hours allowing it to set. The slants were inoculated with the micro-organisms followed by incubation for five days. Colour changes from greenish to bluish indicated the production of citrase enzyme ( 37 , 38 ). 2.10.3 Motility test Motility test was used to determine the abilities of the micro-organisms to migrate or move within the agar. The medium used for this test was nutrient agar of half strength. The agar was weighed homogenized and about 10mls was dispensed in test-tubes and corked with cotton wool and then sterilized at 1.05 kg cm − 2 for a quarter of a hour. The agar was allowed to cool and set and was inoculated with 24 hour old cultures of the micro-organisms by stabbing with an inoculating needle under sterile conditions. It was then incubated for 7 days after which the test-tubes were observed for movement from the original position both downwards and sideways ( 37 ). 2.10.4 Hydrolysis of gelatin Gelatin is a protein which can be metabolised only the organisms capable of producing proteolytic enzymes that can break it down. When broken down gelatin looses it gelling quality, thus this may be used as a method of identifying micro-organisms. The media is prepared by adding 15 g of gelatin to nutrient broth. The media is then sterilized using1.05 kg cm − 2 for 15 minutes, after dispensing into test-tubes. The medium was inoculated with 24 hour old cultures of the micro-organisms under sterile conditions using a sterile needle to stab the culture. The test-tube were then incubated for 7 days after which the test-tubes were placed in the refrigerator to solidify any un-denatured gelatin and the results were observed ( 37 ). 2.10.5 Starch hydrolysis test This is a test carried out to test the abilities of a micro-organisms to put to use starch as a sole carbon source, this can only be done if the micro-organisms produces the enzyme amylase which breaks down starch. The test reagent used was gram’s iodine. The media was prepared by addition of 1% starch to nutrient agar which was then sterilized at a standard pressure of 1.05 kg cm − 2 for a duration of 15 minutes and left to cool (about 45 o C), and dispensed in sterile disposable Petri-dishes allowing it to set properly. Twenty four hour old cultures of the micro-organism were then streaked across the Petri-dish and then incubator for 3–5 days after which the inoculated Petri-dish was flushed with gram’s iodine. The Unhydrolysed starch formed blue or black colours with iodine, while the hydrolysed starch forms clear zones around the bacteria resulting from the amylase activity. Partial hydrolysis is indicated by reddish brown zone. ( 37 , 38 ). 2.10.6 Sugar fermentation test The abilities of each organisms to ferment different sugars and thus their capability to metabolizing these sugars as carbon source was tested. Sugars such as galactose, mannitol, xylose, sucrose, glucose, arabinose, maltose, lactose and fructose were used. The products of the sugar metabolism, depends on the type of enzymes possessed or produced by the organism. The utilization of the sugar is termed fermentation. Sugar utilization is coupled with the production of acids which is indicated by the addition of an indicator which changes colour, the indicator used was phenol red ( 39 ). Presence of gas was detected by inserting Durham tubes in inverted positions into the reaction test-tubes the gas accumulated in the Durham tubes. Acid production were indicated as changes in the red colouration of phenol red indicator to yellowish. Attention was taken to the fact that gas could not be produced without acid production. The medium was prepared by adding 0.1% NaCl, 1% peptone, and 1% fermentable sugar to distilled water, then about 5 ml of the medium without the sugar was dispensed into test-tubes and corked with cotton wool, it was then sterilized at a standard pressure of 1.05 kg cm − 2 and a using heat-temperature of 121℃ for a time period of 15 minutes. The sugars were then sterilized separately for 10 minutes to prevent it from being denatured. The medium was allowed to cool and then about 0.5 ml of sugar was dispensed in test tubes aseptically. Under sterile conditions, a large amount of the micro-organism was scooped using a wire loop and mixed in sterile water until a milky colour was obtained.0.5 ml of this mixture was then dispensed into the labelled test-tube ( 37 ) 2.10.7 Hydrogen sulphide test In sterile Kligler iron agar (KIA), the test organism was inoculated using a sterile inoculating loop into and incubated overnight. Blackening on the medium indicates a positive result while negative is without blackening on the medium. 3.0. RESULTS 3.1 Distribution The percentage distribution of Rhizobia that was isolated from the three states were was not significantly different from each other with the highest population being found in Niger state and the least population in Kano state; 35% (Kaduna), 36% (Niger), 29% (Kano). The distribution between local governments on the other hand were significantly different from each other with Kajuru having 21% of the rhizobia isolated, 14% was from Paikoro, 12% from Shiroro and Doguwu, 10% from Igabi and Bosso, 7% from Bunkere, 6% from Tundun wada, 4% from Soba and Zongo kataf (Fig. 3 ). The highest significant distribution was obtained at Kajuru local govt. (21%) and the least significant distribution of 2% at Soba and Bichi Local govt. 3.2 Cell description The cells of the bacteria were rods as commonly described by previous research. 22% of the isolated rhizobia strains had long rod shape. The cells of most of the rhizobia population were mostly clustered while 18% were scattered in arrangement. 66% of the cells were clustered in chains while 17% were in pairs or single cells. The cells were mostly slow growing with 50% showing growth by the 5rd day and 30% showing growth by the 7th day while 20% of them were fast growing. The growth were mucoid with production of varied amounts of mucus some high and some in small amounts 60% of the isolates produced mucus. The growth were mostly dense and elastic but some isolates had diffuse and non-elastic growth (Fig. 4 ) 3.3 Efficiency and effectiveness of isolated strains The efficiency of the strains described their ability not to only form nodules but to also fix nitrogen sufficiently and adequately. Our findings showed that 81% of the strains were only infective forming nodules but not fixing nitrogen. 7% of the strains were non- efficient as they were unable to produce nodules under different soil types used under screen house conditions, 2% were efficient in fixing nitrogen in 3 different soil conditions but reduced while the most efficient were able to fix nitrogen under all soil types and conditions (Fig. 5 ). 3.4 Nodule arrangement on Roots Most of the isolated strains (70%) formed nodules which were scattered, with 47% having medium sized nodules, 28% having big nodules and 9% small nodules (Fig. 6 and Fig. 7 ). The arrangements of the nodules did not in any way affect the capacity and potential of the plant’s root nodules to fix nitrogen in the plants as both scattered and clustered nodules were shown to have pink or red coloured nitrogen fixing nodules. The colour of the plant’s root nodules were mostly pink with the larger number of the plants having pink nodules while fewer had white or green nodules and were not able to fix nitrogen at all being non-symbiotic with Bambara groundnut. The size of the nodules did not in any way influence the capacity and potential of the strain to biologically fix nitrogen in the plants. But plant which had white or green nodules were malnourished lacking nitrogen and therefore were yellowish in colour. 3.5 Enzyme production The ability of the isolated bacteria to produce four enzymes which include catalase, citrase, gelatinase and enzyme that breaks down Hydrogen sulphide were tested. 66%, 59% and 27% of these isolates were capable of producing the catalase, citrase and gelatinase enzymes respectively. 41% of the isolated bacteria were able to release H 2 S gas giving a blackish patch on the paper. These enzymes maybe responsible for the enhanced activities (Fig. 8) during biological nitrogen fixation but may not necessarily be a requirement for nitrogen fixation as some of the strains lacking these abilities were still able to fix nitrogen in Bambara groundnut plant. 3.6 Sugar utilization The ability of the rhizobia strains to use sugars including Arabinose, Sucrose, Glucose, Mannose and Galactose were classified as negative, positive and positive with the production of gas. Most of the rhizobia were able to utilize all the sugars some giving positive reactions (with a yellow coloration change in the media from red to yellow) while other were positive with the production of gas while the fewer percentage gave negative reactions. Arabinose (73%), Mannose (59%), Sucrose (70%), Galactose (37%), Glucose (59%) were positive without gas production. Arabinose (18%), Mannose (26%), Sucrose (24%), Galactose (50%), Glucose (24%) were positive with the production of gas. While Arabinose (9%), Mannose (15%), Sucrose (6%), Galactose (13%), Glucose (17%) had negative reactions (Fig. 9). Although we are unable to say cartigorically how, the capacity and potential of the rhizobium isolates to use these sugars which are commonly produced in the plants were shown to enhance the process of nitrogen fixation in Bambara groundnut. As the isolates which did not utilize any sugars were mostly unable to fix nitrogen in the plants. These may be due to the fact that the sugars could be metabolised and used for the provision of energy which is needed during the biological processes of nitrogen fixation particularly with the absence of oxygen within the plant nodules. This may also be an indication of the true symbiotic relationship between a legume plant and the rhizobia strains as they provide the plant with nutrients while the plant provides them with Nitrogen. 3.7 Hydrolysis of starch None of the isolated rhizobia strains were able to utilize starch as they all showed negative responses to breaking down of starch with implies that they were unable to produce the enzyme that can breakdown complex sugars such as starch which indirectly also shows why they need symbiotic relationships with legumes which are able to provide broken down carbohydrates for them (Fig. 10 ). The ability to breakdown starch which is a complex sugar did not in any way affect the capacity and potential of the isolates to biologically fix nitrogen in Bambara plants and was not an important ability required for the processes involved for biological nitrogen fixation in Bambara ground-nut plant. 3.8 Authentication of isolates in Bambara plants The authentication test carried out determined how effectively the strain could infect and fix nitrogen in the Bambara nut plant. The 34 strains were selected for authentication and efficiency test during which the fresh weight of plant’s shoot root, number of the plant’s nodules, dry weight of the shoot, root and the chlorophyll values were taken (Fig. 11 ). There was a positive effects on the plant’s shoot fresh weight and plant’s root fresh weight, total plant’s nodule weight and nodule number. The leaf chlorophyll was between 46.97 and 27.783 spad units, while the number of nodules was highest in isolate 30 and least in isolate 28. The strains which showed most efficiency were isolates 2, 7, 8, 10, 12, 13, 22, 30 and 35 as shown in Fig. 11 4.0 DISCUSSION This study gives report about the biochemical, morphological and fixing efficiency differences of bacteria that are symbiotic and form nodules with Bambara groundnut present in the 54 soil samples that were collected from Niger, Kano and Kaduna states in Nigeria regions which are characterized by sandy soils and low rainfall. One hundred and fifty Bambara symbiotic strains of rhizobia were trapped and recovered in the root nodules of Bambara plants. The isolated strains were designated as rhizobia based on their cell morphology, colony characteristics, and Congo red dye absorbance. All strains showed Gram-negative nature and having short or long rod-shaped cells and had cultures colonies that were whitish to pale pink on Congo red dye and didn’t absorb dye on incubation correlating with the reports made by (40, 41). Isolates lack of ability to take up Congo red dye is a distinct characteristic of rhizobia as shown in (41, 42, 43, 44) and also that of ( 44 ), where rhizobia strains isolated from cowpea showed similar characteristics. Following the growth rates, the bacteria family Rhizobiaceae are classified in two groups, the fast growers (FG) and slow growers (SG). Our study shows that the two rhizobia types were recovered in all the collected soils from the three state zones including Niger, Kano and Kaduna states in Nigeria which is a tropical region. Our finding concur with the studies by (17, 41, 45) that reports the presence of both types i.e. FG and SG rhizobia within the soils they selected and collected for their study in the sub-tropical and tropical regions. Our findings showed that Bambara groundnut can be symbiotic with both sub-groups of strains of rhizobia, but the SG were present in higher percentages. A total of 65% of strains of rhizobia isolated during this study had colonies with whitish or creamy growth on CR-YEMA agar after being incubated for 3 days at 28ºC and classifying them as fast growing rhizobia as described by ( 29 ). This differed from the work of ( 46 ) where the growth of all isolates obtained grew in 3–5 days and indicating they were FG and from the Rhizobium genus. Our findings show that there may be a relationship between rhizobial diversity and the invasion and colonization of Bambara groundnut and considering its promiscuity it is able to form symbiosis with several species of rhizobia hence the large collection of isolates that were obtained from the soils. This differed from the findings of ( 47 ) where in acacia there was no clearly stated relationship between the rhizobia diversity, it’s invasiveness and infection status, despite its known outstanding promiscuity, it mostly formed nodules with Bradyrhizobium spp. (above 90% of rhizobia strain isolates recovered from nodules). Our findings also correlate with the reports made by ( 48 ) where 9 types of different morphotype rhizobia were obtained showing the diversity in capacity and potential of strain isolates that colonized the plant’s root nodules of beans within eastern parts/ region of Kenya in Africa. The rhizobia strains recovered were effectively able to invade, infect and fix nitrogen biologically inside plant root nodules of Bambara plant and Bambara rhizobia symbionts were easily culturable on CR-YEMA media differing from the work of ( 47 ) where because of the poor infection by strains obtained the assumption was proposed that the infective and active bacteria have possibly lost inherent symbiotic informations, nod genes or nif genes in the symbiosis process or purification using artificial culture agar ( 49 ), or that active symbionts were not culturable or highly SG, and that only the non-nodulating bacteria endophytes that coexist with rhizobia within the nodules could be isolated or recovered (50, 51, 52), as was clearly shown in Agrobacterium spp. ( 27 ), the Burkholderia spp. (51) including many other numerous genera of bacteria ( 52 ). The isolates particularly the fast-growing ones formed larger colonies that were yellowish with creamish margins, convex, round and entire ( 2 , 53 ) with sizes between 2 to 5 mm. Rhizobia isolates that formed growth in 72 hours as was showen in the reports from the work of ( 42 ) in soybean, and by (3, 54, 55) in Cowpea, Bambara groundnut, and Soy-bean. The SG grew colonies mostly 5–10 days after incubation which had small or medium sized, white or light pinkish colonies similar to the reports by (41). In total, 150 isolates were recovered from the soil using trapping method differing from the total of 28 rhizobia isolates that were recovered using root nodules from cowpea plants in the evaluation study by (2, 41) where in total 201 bacterial strains were obtained from Mali, Ghana and South Africa from Bambara groundnut, this we assume may be due to the large number of soils that were sampled from the three states. The results of this study concurred with the conclusions in the reports given by ( 56 ) and (18) in Kenya and in Ecuador by ( 57 ) for the morphology and also biochemical characteristics for native strain rhizobium that nodulate common varieties of beans. Our findings differed to the findings of ( 58 ), were there was large numbers of FG isolates and was attributed to the prevalent presence of rapidly growing rhizobia in arid and semiarid lands, having the capacity to quickly multiply in the durations of shorter rains being more tolerative of stressed conditions than their counterpart slow-growing strains which can be considered to be a survival strategies. It also differed from the findings of ( 1 ) where there was a high frequency of fast-growing isolates obtained in cowpea plant which are mostly able to form nodules by associations with bacterial species Bradyrhizobium which are well known slow-growing strains and alkaline in nature indicating that leguminous cowpea plant doesn’t only form symbiosis with species type Bradyrhizobium but with several distinct species types of rhizobia. Our results also concur with the evaluation reports of ( 59 ) who obtained isolates of FG species rhizobia in the cowpea plants from their experiment. Our findings show that Bambara groundnut is highly promiscuous and potentially able to form nodules in their roots with several spp. of rhizobia as confirmed in the reported results of (41) and also similar to the results ( 60 ) which reinforced the wanton or promiscuous ability of P. vulgaris plant to form nodules with the different strains and types of Rhizobia. The distribution among the three states was not significantly different from 29% from Kano, 35% from Kaduna, and 36% from Niger which are in the same agroecological zones this differed from the work of ( 61 ) which reports a wider of indigenous diversity type of several bradyrhizobia spp. in soils from Kenya (western) compared with that of Kenya (eastern) which is an indication of the difference agroecology conditions. Both the isolates that were fast- and slow-growing produced mucus which in high to intermediate quantities and some of the isolates appeared as dense and elastic or as diffuse and non -elastic. The production of mucus is most likely a representation of the mechanisms used by rhizobia for adapting and enduring harsh climates and the edaphic soil conditions. It helps by preventing desiccation within the cells of the bacteria enabling or strengthening them to withstand variations in salinity, temperature, and acidity as shown in the work of ( 61 ). The evaluations by (62), also reports an increased production in the amount of mucus by Bradyrhizobium isolates as mechanisms for survival by adaptation. There was no consistency in the pH changes as both FG and SG isolates increased the pH in the media differing from the reports by ( 1 ) where all SG isolates increased the medium’s pH with the exception of one isolate (which was able to acidify the medium) and concurring with those of ( 63 ) where it is reported that SG rhizobia strains are able to produce acid and the work of ( 64 , 65 , 66 ) where 55 percent of the isolates which were fast growing produced acid. There was a wide diversity of Bambara symbiotic strains present in the soils collected from three of the legume producing northern states in Nigeria, and Bambara groundnut appears to be a highly compatible host because it was capable of allowing the formation of root nodules with wider ranges of diverse rhizobia spp. which included both FG species Ensifer, Rhizobium and Mesorhizobium, and the SG species the Bradyrhizobium. 5.0 CONCLUSION In soils obtained from northern Nigeria, there was a wide diversity of Rhizobia isolates that were recovered from the 54 soil samples and a total of 150 rhizobia isolates were recovered by trapping inside the surface root nodules in Bambara groundnut and were characterized both morpho-culturally and biochemically. The cells were mostly whitish, creamy, transparent or translucent colonies that were small in size and classified as Bradhyrhizobium but other several spp. were also recovered showing the promiscuous nature of Bambara groundnut. The soils had a rich reservoir of rhizobia spp., and the most prevalent rhizobia isolates were the slow-grower but also consisted of the fast growers, some of which were able to acidify the medium and producing substantial to minute amounts of mucus (exopolysaccharides production). These maybe considered as some of the attributes that assist the bacteria in survival and adaptation in the tropical regions. Our reports showed the large diverse FG and SG isolated rhizobia strains types that are able to nodulate the roots of Bambara groundnut and propose or imply morphological and biochemical variations within and amongst Bambara groundnut strains which are a criteria for the selection of potential isolates that can be used as biofertilizer. The effectiveness and efficiency of characterizing rhizobia with authentication method proves to be effective for discrimination of isolates. This study offers substantial informative data for carrying out future study about the large diversity and interactive relationship between Bambara symbiotic bacteria, the Bambara plant, and also the prevalent surrounding soil environment conditions. The isolated rhizobia strains which were characterized in this study need further screening to test their abilities for fixing nitrogen with different Bambara groundnut varieties or genotypes and some other major legumes that have high economic importance in northern Nigeria. Declarations CONFLICTS OF INTEREST All the participating authors hereby declare no resulting conflicts of interest whatsoever . COMPETING INTEREST There are no fundings to be reported for this research. The authors declare no competing interests. Author Contribution O.O. AJAYI and M. DIANDA wrote the main manuscript text , O.O. AJAYI prepared the figures and all authors reviewed the manuscript." 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strains\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4499028/v1/cc3391402fec85a053eb6feb.jpg"},{"id":61471060,"identity":"b60fde09-5156-4261-8eff-8c3d5cb0c707","added_by":"auto","created_at":"2024-07-31 06:47:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":34630,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEfficiency and effectiveness of isolated Bambara symbiotic strains.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlue bars- number of nodules\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4499028/v1/1abdcb09940041afad25cfc0.png"},{"id":61471772,"identity":"5de4f993-3bc8-4263-a20e-225a84e1080f","added_by":"auto","created_at":"2024-07-31 06:55:29","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":48547,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNodule description in experimental plants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGreen and red bars- percentage of nodules\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4499028/v1/da6d88a85db76205af605d20.jpg"},{"id":61470477,"identity":"3a008ddb-748c-4d47-ac2e-8c8d86f05e7f","added_by":"auto","created_at":"2024-07-31 06:39:29","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":118480,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNodule arrangement and colour in experimental plants.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlue bars- number of nodules for each colour\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4499028/v1/da6a816a9de91af48e82e76e.jpg"},{"id":61471770,"identity":"1aa276eb-2f6f-49e8-8dba-9326091d3c30","added_by":"auto","created_at":"2024-07-31 06:55:29","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":24279,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnzyme utilization among isolated Bambara symbiotic strains.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlue bars-positive, Brown bars- negative\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4499028/v1/d44353f16549e1ad2e3f4ab3.jpg"},{"id":61471064,"identity":"459cd453-026d-42ad-88d3-1d33d1959582","added_by":"auto","created_at":"2024-07-31 06:47:29","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":152786,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSugar utilization among isolated Bambara symbiotic\u003c/strong\u003e \u003cstrong\u003estrains\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"fig9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4499028/v1/db6f30a8eb6529704a83d0b6.jpg"},{"id":61470479,"identity":"8082eeb1-8c80-459e-96f3-f947ffa645f1","added_by":"auto","created_at":"2024-07-31 06:39:29","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":6991,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHydrolysis of starch in isolated Bambara symbiotic strains\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"fig.10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4499028/v1/c547f7ee2d5c12a3e8c41a7c.jpg"},{"id":61471773,"identity":"932a4771-2e76-42e7-8a96-d52bfd95e2c8","added_by":"auto","created_at":"2024-07-31 06:55:29","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":336400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlant biomass and leaf chlorophyll and nodule characterization of Bambara groundnut plants for authentication experiment.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig11.png","url":"https://assets-eu.researchsquare.com/files/rs-4499028/v1/9d9ea2b0e791bfd018d317a8.png"},{"id":87237814,"identity":"624ec4ec-2d45-4ea2-bc25-321a7fe7c7c2","added_by":"auto","created_at":"2025-07-21 23:16:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2993384,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4499028/v1/82a763e3-ee83-423f-8ac5-07b8e43d1566.pdf"},{"id":61472308,"identity":"fcfd9792-a750-4101-930a-56593ca845e7","added_by":"auto","created_at":"2024-07-31 07:03:29","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18229,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydatadiversity.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4499028/v1/6bd4b8f3fcf8083e83707547.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Diversity, effectiveness and biochemical characterization of Bambara groundnut symbiotic Rhizobia strains in tropical Nigerian soils","fulltext":[{"header":"1.0 INTRODUCTION","content":"\u003cp\u003eRhizobia species play significant roles for the amendment of the soil because they make associations which are symbiotic with several legumes and engage in nitrogen fixation through biological processes (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). These strains of rhizobium have been observed to allow for enhanced phytohormone productions, uptake of minerals and ameliorating the effects of toxic metal(s), and are therefore able to promote plant\u0026rsquo;s growth rates and developments (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) especially in soils which become highly polluted during agricultural practises. Current day agricultural practises has moved from using sustainable farming practises to less stressful and more environmentally friendly resources for farming eg. The use of biofertilizers such as rhizobia, phosphate solubilizing bacteria in the sub-saharan African tropical areas. This is mostly because they are cost saving, they improve soil health and most importantly the yields of crops (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBambara ground-nut (BG) a pulse crop plant that produces seed under the ground, is believed to have its originating point located in Africa (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), which is grossly under-utilised. It is mostly believed that it originated from West Africa (sahelian region) from among the Bambara tribes-people very near to Timbuktu in Mali (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). It then migrated to numerous parts of Oceanian, Asian and South American countries (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). It has been found to have many agronomical advantages in its nature this includes a increased nutrition values, high resistance and tolerance to drought, with abilities to survive in considerably harsh and intolerable soil types (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). It mostly is regarded as a crop that is able to survive famine (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), is probably due to the association it forms with mycorrhiza. Bambara groundnut by itself is an entire-completely fully-balanced food because it is fortified with Iron, contains 19% proteins such as lysines and methionines (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), 63% carbohydrates and 6% oil and fatty acid (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUnfortunately, the lack of infrastructure, and adequate research to target the diverse and specific symbiotic rhizobia for leguminous beans such as Bambara groundnut, Mac has limited the use of biofertilizers for its production (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). The symbiotic relationships of rhizobia with legumes that are nitrogen fixers play key roles for the sustenance of most natural biological ecosystems especially within arid \u0026amp; dry areas of tropics and the sub-tropic zones (17, 18).\u003c/p\u003e \u003cp\u003eMost research studies in relation to this were done to investigate the nitrogen-fixation status of other legumes and legume trees (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e21\u003c/span\u003e) in different ecological zones (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e24\u003c/span\u003e), in particular within Africa regions (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e30\u003c/span\u003e) but little has been done to identify suitable indeginous rhizobia strains for Bambara groundnut.\u003c/p\u003e \u003cp\u003eThe large numbers of morphologically and genotypically diversity of rhizobia found in northern Nigeria region may be harboring new strains of rhizobia that is worth being exploited to obtain strains with efficiency in biological nitrogen fixation for Bambara groundnut which is highly under utilized and has low production which doesn\u0026rsquo;t meet half of its demand to carry out the tests for their symbiotic ability and also ascertain their identities and functionalities in diverse environments.\u003c/p\u003e"},{"header":"2.0 METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Sampling and Collection Site for Soils\u003c/h2\u003e \u003cp\u003eSoils were collected from farm sites where legumes are planted in 21 local governments from three states in Nigeria. Points in the middle of the field and not at the edges were selected for soil collection to ensure that active rhizobia strains were collected. The soils were collected at depths of 15cm- 20cm, using a soil auger, which was used to dig deep into the soil to collect soil at the required depths. The soil were then bagged in properly labelled transparent ziploc bags and stored in cold coolers till they were transferred to the fridge to be stored at 8\u0026ordm;C to allow for preservation of the bacteria diversity in the soil. They were then transferred to cold rooms and preserved until start of experiments. A total of 54 soils were collected from the different local governments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Trapping of Rhizobia\u003c/h2\u003e \u003cp\u003eLow nutrient pot soils were prepared by mixing sea sand (the sea sand was washed thoroughly by using six changes of water or until pH increased to a values of 6.6\u0026ndash;6-8, the most suitable pH range for the growth of rhizobium), small sized gravel stones and peat in ratios 6:1:6 this was thoroughly mixed with water till evenly distributed and sterilized at a temperature of 121\u003csup\u003eo\u003c/sup\u003e C and pressure of 1.05 kg cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e for 1 hour. The sterilized soils were then used to fill sterilized 250 ml pots (sterilization of pots was done using JIK bleaching agent which contains 3.5% w/v sodium hypochlorite, and thorough rinsing by submerging in six changes of sterile water and a final rinse again to ensure that all the residual traces the JIk were removed and did not remain to affect the bacteria during pot experiments).Viable seeds of Bambara groundnut were sterilised using sodium hypochlorite and ethanol (31, 32) and then two of each seeds (viable ones) were placed into each pots and were planted in enclosed and restricted screen house conditions till germination.\u003c/p\u003e \u003cp\u003eThe soil samples (54) which were collected from fields, and transferred to the lab to be stored under cool conditions were identified and brought to the lab for preparation of inoculum. Soil inoculant were prepared by weighing 10g of each soil in 90mls of sterile diluent water (K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e(0.125g/L), MgSO\u003csub\u003e4\u003c/sub\u003e (0.05g/L)), the soil was then placed on a rotatory shaker and allowed to shake at low amplitudes for 1 hour to allow for revival and resuscitation of the soil bacteria (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e33\u003c/span\u003e) and 5 ml each was introduced into the Bambara groundnut plants at 2 weeks after planting (when germination has occurred) under screen house condition. Each soil was inoculated using 4 replicates. The rhizobia strains present in the soils were tapped in the nodules of the plants grown for 8 weeks when nodules had been formed in the plant and isolated from the undamaged nodules\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Experimental Setup\u003c/h2\u003e \u003cp\u003eSterile sand was prepared, and used to fill sterile pots (31, 32, 33). Sterile seeds were planted in pots and inoculated (1 ml) at 2 weeks after planting (WAP) using 4 replicates each along-side the un-inoculated and the N\u0026thinsp;+\u0026thinsp;control plant. Plants were grown for 8 week during which sterilized nutrient solution was applied to replenish nutrients lost during sterilization. Plant nutrients (solution) were made from micro and macro nutrient (Micronutrient\u0026ndash;Stock Solution Constituents: H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e \u0026minus;\u0026thinsp;2.86g, ZnSO\u003csub\u003e4\u003c/sub\u003e.7 H\u003csub\u003e2\u003c/sub\u003eO \u0026minus;\u0026thinsp;0.22g, MnCl\u003csub\u003e2\u003c/sub\u003e.4 H\u003csub\u003e2\u003c/sub\u003eO \u0026minus;\u0026thinsp;1.81g, Na\u003csub\u003e2\u003c/sub\u003eMnO\u003csub\u003e4\u003c/sub\u003e. 2 H\u003csub\u003e2\u003c/sub\u003eO \u0026ndash; 0.025g, CuSO\u003csub\u003e4\u003c/sub\u003e. 5 H\u003csub\u003e2\u003c/sub\u003eO \u0026ndash; 0.08g, Distilled water \u0026minus;\u0026thinsp;1 L) (Macronutrient \u0026ndash; stock solution constituents: CaHPO\u003csub\u003e4\u003c/sub\u003e -100g, MgSO\u003csub\u003e4\u003c/sub\u003e .7 H\u003csub\u003e2\u003c/sub\u003eO \u0026ndash; 20g, K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e \u0026ndash; 20g, FeCl\u003csub\u003e3\u003c/sub\u003e -10g, NaCl \u0026minus;\u0026thinsp;20g, Distilled water \u0026minus;\u0026thinsp;1L) (100ml of the macro nutrient stock-solution, and 10ml micro nutrient stock-solution are added together and made up to 10 litres using sterile distilled water at 50 ml weekly per pot). An N\u003csup\u003e+\u003c/sup\u003e treatment (50 ml (48.5g of KNO\u003csub\u003e3\u003c/sub\u003e to 1.5L of distilled water) solutions was supplied to plant with nitrogen control (31, 32, 33).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Harvesting\u003c/h2\u003e \u003cp\u003eThe plants were harvested with the use of a secateur cutting the plant stem very closely at the base of the plant. The shoot and stems were carefully collected and placed in labelled paper bags. The soil was poured out and the root was shaken gently to remove the sand the root were stored in labelled nylon. A sieve of an appropriate mesh size was placed under the root samples allowing the nodule to be caught catch when they become detached away from the surfaces and extensions of plant\u0026rsquo;s root (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Sieves carrying the root were placed under gentle streams from running water from a tap and carefully washed in a bucket after washing it was immediately placed inside labelled bags and later transferred to the lab where the nodules were carefully detached before being placed in envelops (labelled) and some selected for nodule assay. The nodules were then dried naturally before being stored with silica gel (was placed in oven until colour changed to brown) (32, 33).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Morphology of Nodules\u003c/h2\u003e \u003cp\u003eThe nodules were picked from the plant and used for assay, their sizes and colour was determined, their shapes and arrangement on the plant roots were observed and described. The sizes were classified into small, medium and large, the colours were classified into, green or white (which were consider non fixing), red, brown or pink (which were considered to be fixing). the shape was classified as round, irregular or clubbed shaped. The arrangements of the nodules on the roots of the experimental plants were classified as scattered or clustered (31, 32, 33).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Isolation of Bacteria\u003c/h2\u003e \u003cp\u003eRecovery of Rhizobia from nodules was done using the roots of the Bambara groundnut plants in the trap experiments on Congo red agar (32, 33, 34, 35, 36) using spread plate method. Unharmed nodules (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) were selected, rehydrated using sterilized water (distilled) for 15\u0026ndash;20 mins and afterwards a thorough surface sterilization with JIK a household reagent containing 3.5% NaClO (sodium-hypochlorite) for a duration of 3 mins. The sterilized nodules went through thorough rinsing using sterilized water, afterwhich they were re-sterilized using 95% CH\u003csub\u003e3\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eOH (ethanol) and then also re-rinsed again with six times of new different changes of sterilized water (distilled) (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). The sterilized nodules were crushed in sterilize Petri-dishes, and loops-full were streaked on congo-red agar (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e33\u003c/span\u003e) and inverted before being incubated using a favourable temperature of 28\u0026deg;C for a continued period of 5\u0026ndash;14 days or till growth was seen (modified method of Somasegaram \u0026amp; Hoben, 2012). The selected isolates were then purified and identified appropriately as Rhizobia. All isolated rhizobia strains were subjected to gram reaction test (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e37\u003c/span\u003e), cell shape and growth on YMA-CR. Their growth characteristics and colour were used as a basis selection for further characterization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Preservation of Isolates\u003c/h2\u003e \u003cp\u003eThe purified isolated strains were inoculated on YMA agar slants with 0.3% (W/V) CaCO\u003csub\u003e3\u003c/sub\u003e (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e34\u003c/span\u003e), the slants were kept in a fridge. Others that proved not to be rhizobia were stored using slants of nutrient agar (31, 32).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Authentication of the Strains\u003c/h2\u003e \u003cp\u003eYeast Mannitol broth was weighed and then sterilized using heat-temperature of 121\u0026deg;C at 1 atm for a period of 15 mins and was left to cool. The yeast mannitol broth was then inoculated with pure Rhizobia strains and then incubated for a duration of 14 days at a heat-temperature of 28\u0026deg;C to allow for the adequate multiplication of the bacteria cells and the broth was standardized to meet required standard value (\u0026ge;\u0026thinsp;1x10\u003csup\u003e9\u003c/sup\u003e cfu/mL) (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Bambara groundnut seeds were sterilized, planted in sterilised potted sand and allowed to grow for two weeks before being inoculated as a result of their slow germination. Plants under screen-house conditions were then left to grow for eight weeks to determine and evaluate the infectivity of rhizobial isolates by nodule formation. Plants were given nutrient solution 50 mL weekly to replenish nutrients that may have been depleted or unavailable due to the resulting absence of other plant-growth-promoting-bacteria (31, 32).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Cell Description: Gram Staining and Microscopic viewing\u003c/h2\u003e \u003cp\u003eThis test was done for determining the nature of the peptidoglycan layer in the cell wall of a micro-organism i.e. either thick or thin. It was done to differentiate the isolates into gram positive or negative bacteria. Two loops full of water placed clean grease free slides were smeared with wire loop full of the bacteria on the slide. The sample was then passed through heat to fix the cell using a spirit lamp thrice. Crystal violet a primary stain was place on the slide covering the smear for 1 minute, rinsed off using clean water from a gently flowing tap to remove unbound stain. Gram\u0026rsquo;s iodine which is an active and effective mordant was then placed on the slide again covering the smear for 1 minute and then rinsed off. This was followed by the addition of ethanol which functioned as a decolourizer for 30 seconds. For cells with thin peptidogylcan layer, the primary stain is removed, the decolourizer was then washed off using running water and a counter stain, also called known as a secondary stain, safranin was placed on the slide entirely covering the smear for 30 seconds and rinsed off. The stained slides were then viewed under the microscope for gram negative long or short rod micro-organisms at x 100 objective lens (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Biochemical Tests\u003c/h2\u003e \u003cp\u003eVarious tests were carried out to describe the organism based on its physiological activities, biochemical reactions, characteristic features and also possibly identify the organism. The biochemical identification tests carried out includes catalase test, citrate test, hydrolysis of gelatin, hydrogen sulphide test, starch hydrolysis test and sugar fermentation test. All procedures were carried out following standard methods (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.10.1 Catalase test\u003c/h2\u003e \u003cp\u003eThis test is used to detect whether the isolates produce the enzyme catalase or not. Catalase is found in most aerobic micro-organisms although some aerobic and microaerophilic micro-organisms do not produce catalase, but hydrogen peroxide is not produced in anaerobic metabolism. The reagent used was hydrogen peroxide (3%), 24 hour old culture of the micro-organism was used. a few drops of the reagent was placed on a slide and a little of the micro-organism was picked using a sterile wire loop under sterile condition. The micro-organism was then mixed with the reagent on the slide and observed for bubbles (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.10.2 Citrate test\u003c/h2\u003e \u003cp\u003eTest is carried out to determine the abilities of the micro-organisms to produce the enzyme citrase thus enabling it to utilize citrate, converting it to oxaloacetate. The medium used was citrate medium which is composed of g of citrate medium per litre. This was homogenized and dispensed into vijou bottles and sterilized at a set pressure of 1.05 kg cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e/atm for a duration of 15 minutes and slanted for 24 hours allowing it to set. The slants were inoculated with the micro-organisms followed by incubation for five days. Colour changes from greenish to bluish indicated the production of citrase enzyme (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.10.3 Motility test\u003c/h2\u003e \u003cp\u003eMotility test was used to determine the abilities of the micro-organisms to migrate or move within the agar. The medium used for this test was nutrient agar of half strength. The agar was weighed homogenized and about 10mls was dispensed in test-tubes and corked with cotton wool and then sterilized at 1.05 kg cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e for a quarter of a hour. The agar was allowed to cool and set and was inoculated with 24 hour old cultures of the micro-organisms by stabbing with an inoculating needle under sterile conditions. It was then incubated for 7 days after which the test-tubes were observed for movement from the original position both downwards and sideways (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.10.4 Hydrolysis of gelatin\u003c/h2\u003e \u003cp\u003eGelatin is a protein which can be metabolised only the organisms capable of producing proteolytic enzymes that can break it down. When broken down gelatin looses it gelling quality, thus this may be used as a method of identifying micro-organisms. The media is prepared by adding 15 g of gelatin to nutrient broth. The media is then sterilized using1.05 kg cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e for 15 minutes, after dispensing into test-tubes. The medium was inoculated with 24 hour old cultures of the micro-organisms under sterile conditions using a sterile needle to stab the culture. The test-tube were then incubated for 7 days after which the test-tubes were placed in the refrigerator to solidify any un-denatured gelatin and the results were observed (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.10.5 Starch hydrolysis test\u003c/h2\u003e \u003cp\u003eThis is a test carried out to test the abilities of a micro-organisms to put to use starch as a sole carbon source, this can only be done if the micro-organisms produces the enzyme amylase which breaks down starch. The test reagent used was gram\u0026rsquo;s iodine. The media was prepared by addition of 1% starch to nutrient agar which was then sterilized at a standard pressure of 1.05 kg cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e for a duration of 15 minutes and left to cool (about 45\u003csup\u003eo\u003c/sup\u003eC), and dispensed in sterile disposable Petri-dishes allowing it to set properly. Twenty four hour old cultures of the micro-organism were then streaked across the Petri-dish and then incubator for 3\u0026ndash;5 days after which the inoculated Petri-dish was flushed with gram\u0026rsquo;s iodine. The Unhydrolysed starch formed blue or black colours with iodine, while the hydrolysed starch forms clear zones around the bacteria resulting from the amylase activity. Partial hydrolysis is indicated by reddish brown zone. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e2.10.6 Sugar fermentation test\u003c/h2\u003e \u003cp\u003eThe abilities of each organisms to ferment different sugars and thus their capability to metabolizing these sugars as carbon source was tested. Sugars such as galactose, mannitol, xylose, sucrose, glucose, arabinose, maltose, lactose and fructose were used.\u003c/p\u003e \u003cp\u003eThe products of the sugar metabolism, depends on the type of enzymes possessed or produced by the organism. The utilization of the sugar is termed fermentation. Sugar utilization is coupled with the production of acids which is indicated by the addition of an indicator which changes colour, the indicator used was phenol red (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Presence of gas was detected by inserting Durham tubes in inverted positions into the reaction test-tubes the gas accumulated in the Durham tubes. Acid production were indicated as changes in the red colouration of phenol red indicator to yellowish. Attention was taken to the fact that gas could not be produced without acid production.\u003c/p\u003e \u003cp\u003eThe medium was prepared by adding 0.1% NaCl, 1% peptone, and 1% fermentable sugar to distilled water, then about 5 ml of the medium without the sugar was dispensed into test-tubes and corked with cotton wool, it was then sterilized at a standard pressure of 1.05 kg cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e and a using heat-temperature of 121℃ for a time period of 15 minutes. The sugars were then sterilized separately for 10 minutes to prevent it from being denatured. The medium was allowed to cool and then about 0.5 ml of sugar was dispensed in test tubes aseptically. Under sterile conditions, a large amount of the micro-organism was scooped using a wire loop and mixed in sterile water until a milky colour was obtained.0.5 ml of this mixture was then dispensed into the labelled test-tube (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e37\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e2.10.7 Hydrogen sulphide test\u003c/h2\u003e \u003cp\u003eIn sterile Kligler iron agar (KIA), the test organism was inoculated using a sterile inoculating loop into and incubated overnight. Blackening on the medium indicates a positive result while negative is without blackening on the medium.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3.0. RESULTS","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Distribution\u003c/h2\u003e \u003cp\u003eThe percentage distribution of Rhizobia that was isolated from the three states were was not significantly different from each other with the highest population being found in Niger state and the least population in Kano state; 35% (Kaduna), 36% (Niger), 29% (Kano). The distribution between local governments on the other hand were significantly different from each other with Kajuru having 21% of the rhizobia isolated, 14% was from Paikoro, 12% from Shiroro and Doguwu, 10% from Igabi and Bosso, 7% from Bunkere, 6% from Tundun wada, 4% from Soba and Zongo kataf (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The highest significant distribution was obtained at Kajuru local govt. (21%) and the least significant distribution of 2% at Soba and Bichi Local govt.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Cell description\u003c/h2\u003e \u003cp\u003eThe cells of the bacteria were rods as commonly described by previous research. 22% of the isolated rhizobia strains had long rod shape. The cells of most of the rhizobia population were mostly clustered while 18% were scattered in arrangement. 66% of the cells were clustered in chains while 17% were in pairs or single cells. The cells were mostly slow growing with 50% showing growth by the 5rd day and 30% showing growth by the 7th day while 20% of them were fast growing. The growth were mucoid with production of varied amounts of mucus some high and some in small amounts 60% of the isolates produced mucus. The growth were mostly dense and elastic but some isolates had diffuse and non-elastic growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Efficiency and effectiveness of isolated strains\u003c/h2\u003e \u003cp\u003eThe efficiency of the strains described their ability not to only form nodules but to also fix nitrogen sufficiently and adequately. Our findings showed that 81% of the strains were only infective forming nodules but not fixing nitrogen. 7% of the strains were non- efficient as they were unable to produce nodules under different soil types used under screen house conditions, 2% were efficient in fixing nitrogen in 3 different soil conditions but reduced while the most efficient were able to fix nitrogen under all soil types and conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Nodule arrangement on Roots\u003c/h2\u003e \u003cp\u003eMost of the isolated strains (70%) formed nodules which were scattered, with 47% having medium sized nodules, 28% having big nodules and 9% small nodules (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The arrangements of the nodules did not in any way affect the capacity and potential of the plant\u0026rsquo;s root nodules to fix nitrogen in the plants as both scattered and clustered nodules were shown to have pink or red coloured nitrogen fixing nodules. The colour of the plant\u0026rsquo;s root nodules were mostly pink with the larger number of the plants having pink nodules while fewer had white or green nodules and were not able to fix nitrogen at all being non-symbiotic with Bambara groundnut. The size of the nodules did not in any way influence the capacity and potential of the strain to biologically fix nitrogen in the plants. But plant which had white or green nodules were malnourished lacking nitrogen and therefore were yellowish in colour.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Enzyme production\u003c/h2\u003e \u003cp\u003eThe ability of the isolated bacteria to produce four enzymes which include catalase, citrase, gelatinase and enzyme that breaks down Hydrogen sulphide were tested. 66%, 59% and 27% of these isolates were capable of producing the catalase, citrase and gelatinase enzymes respectively. 41% of the isolated bacteria were able to release H\u003csub\u003e2\u003c/sub\u003eS gas giving a blackish patch on the paper. These enzymes maybe responsible for the enhanced activities (Fig.\u0026nbsp;8) during biological nitrogen fixation but may not necessarily be a requirement for nitrogen fixation as some of the strains lacking these abilities were still able to fix nitrogen in Bambara groundnut plant. \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Sugar utilization\u003c/h2\u003e \u003cp\u003eThe ability of the rhizobia strains to use sugars including Arabinose, Sucrose, Glucose, Mannose and Galactose were classified as negative, positive and positive with the production of gas. Most of the rhizobia were able to utilize all the sugars some giving positive reactions (with a yellow coloration change in the media from red to yellow) while other were positive with the production of gas while the fewer percentage gave negative reactions. Arabinose (73%), Mannose (59%), Sucrose (70%), Galactose (37%), Glucose (59%) were positive without gas production. Arabinose (18%), Mannose (26%), Sucrose (24%), Galactose (50%), Glucose (24%) were positive with the production of gas. While Arabinose (9%), Mannose (15%), Sucrose (6%), Galactose (13%), Glucose (17%) had negative reactions (Fig.\u0026nbsp;9). Although we are unable to say cartigorically how, the capacity and potential of the rhizobium isolates to use these sugars which are commonly produced in the plants were shown to enhance the process of nitrogen fixation in Bambara groundnut. As the isolates which did not utilize any sugars were mostly unable to fix nitrogen in the plants. These may be due to the fact that the sugars could be metabolised and used for the provision of energy which is needed during the biological processes of nitrogen fixation particularly with the absence of oxygen within the plant nodules. This may also be an indication of the true symbiotic relationship between a legume plant and the rhizobia strains as they provide the plant with nutrients while the plant provides them with Nitrogen. \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Hydrolysis of starch\u003c/h2\u003e \u003cp\u003eNone of the isolated rhizobia strains were able to utilize starch as they all showed negative responses to breaking down of starch with implies that they were unable to produce the enzyme that can breakdown complex sugars such as starch which indirectly also shows why they need symbiotic relationships with legumes which are able to provide broken down carbohydrates for them (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003e). The ability to breakdown starch which is a complex sugar did not in any way affect the capacity and potential of the isolates to biologically fix nitrogen in Bambara plants and was not an important ability required for the processes involved for biological nitrogen fixation in Bambara ground-nut plant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Authentication of isolates in Bambara plants\u003c/h2\u003e \u003cp\u003eThe authentication test carried out determined how effectively the strain could infect and fix nitrogen in the Bambara nut plant. The 34 strains were selected for authentication and efficiency test during which the fresh weight of plant\u0026rsquo;s shoot root, number of the plant\u0026rsquo;s nodules, dry weight of the shoot, root and the chlorophyll values were taken (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e11\u003c/span\u003e). There was a positive effects on the plant\u0026rsquo;s shoot fresh weight and plant\u0026rsquo;s root fresh weight, total plant\u0026rsquo;s nodule weight and nodule number. The leaf chlorophyll was between 46.97 and 27.783 spad units, while the number of nodules was highest in isolate 30 and least in isolate 28. The strains which showed most efficiency were isolates 2, 7, 8, 10, 12, 13, 22, 30 and 35 as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e11\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4.0 DISCUSSION","content":"\u003cp\u003eThis study gives report about the biochemical, morphological and fixing efficiency differences of bacteria that are symbiotic and form nodules with Bambara groundnut present in the 54 soil samples that were collected from Niger, Kano and Kaduna states in Nigeria regions which are characterized by sandy soils and low rainfall. One hundred and fifty Bambara symbiotic strains of rhizobia were trapped and recovered in the root nodules of Bambara plants. The isolated strains were designated as rhizobia based on their cell morphology, colony characteristics, and Congo red dye absorbance. All strains showed Gram-negative nature and having short or long rod-shaped cells and had cultures colonies that were whitish to pale pink on Congo red dye and didn\u0026rsquo;t absorb dye on incubation correlating with the reports made by (40, 41). Isolates lack of ability to take up Congo red dye is a distinct characteristic of rhizobia as shown in (41, 42, 43, 44) and also that of (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e44\u003c/span\u003e), where rhizobia strains isolated from cowpea showed similar characteristics. Following the growth rates, the bacteria family \u003cem\u003eRhizobiaceae\u003c/em\u003e are classified in two groups, the fast growers (FG) and slow growers (SG). Our study shows that the two rhizobia types were recovered in all the collected soils from the three state zones including Niger, Kano and Kaduna states in Nigeria which is a tropical region. Our finding concur with the studies by (17, 41, 45) that reports the presence of both types i.e. FG and SG rhizobia within the soils they selected and collected for their study in the sub-tropical and tropical regions. Our findings showed that Bambara groundnut can be symbiotic with both sub-groups of strains of rhizobia, but the SG were present in higher percentages. A total of 65% of strains of rhizobia isolated during this study had colonies with whitish or creamy growth on CR-YEMA agar after being incubated for 3 days at 28\u0026ordm;C and classifying them as fast growing rhizobia as described by (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e29\u003c/span\u003e). This differed from the work of (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e46\u003c/span\u003e) where the growth of all isolates obtained grew in 3\u0026ndash;5 days and indicating they were FG and from the Rhizobium genus.\u003c/p\u003e\u003cp\u003eOur findings show that there may be a relationship between rhizobial diversity and the invasion and colonization of Bambara groundnut and considering its promiscuity it is able to form symbiosis with several species of rhizobia hence the large collection of isolates that were obtained from the soils. This differed from the findings of (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e) where in acacia there was no clearly stated relationship between the rhizobia diversity, it\u0026rsquo;s invasiveness and infection status, despite its known outstanding promiscuity, it mostly formed nodules with Bradyrhizobium spp. (above 90% of rhizobia strain isolates recovered from nodules). Our findings also correlate with the reports made by (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e) where 9 types of different morphotype rhizobia were obtained showing the diversity in capacity and potential of strain isolates that colonized the plant\u0026rsquo;s root nodules of beans within eastern parts/ region of Kenya in Africa. The rhizobia strains recovered were effectively able to invade, infect and fix nitrogen biologically inside plant root nodules of Bambara plant and Bambara rhizobia symbionts were easily culturable on CR-YEMA media differing from the work of (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e) where because of the poor infection by strains obtained the assumption was proposed that the infective and active bacteria have possibly lost inherent symbiotic informations, nod genes or nif genes in the symbiosis process or purification using artificial culture agar (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e), or that active symbionts were not culturable or highly SG, and that only the non-nodulating bacteria endophytes that coexist with rhizobia within the nodules could be isolated or recovered (50, 51, 52), as was clearly shown in Agrobacterium spp. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e27\u003c/span\u003e), the Burkholderia spp. (51) including many other numerous genera of bacteria (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e52\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe isolates particularly the fast-growing ones formed larger colonies that were yellowish with creamish margins, convex, round and entire (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e53\u003c/span\u003e) with sizes between 2 to 5 mm. Rhizobia isolates that formed growth in 72 hours as was showen in the reports from the work of (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e42\u003c/span\u003e) in soybean, and by (3, 54, 55) in Cowpea, Bambara groundnut, and Soy-bean. The SG grew colonies mostly 5\u0026ndash;10 days after incubation which had small or medium sized, white or light pinkish colonies similar to the reports by (41). In total, 150 isolates were recovered from the soil using trapping method differing from the total of 28 rhizobia isolates that were recovered using root nodules from cowpea plants in the evaluation study by (2, 41) where in total 201 bacterial strains were obtained from Mali, Ghana and South Africa from Bambara groundnut, this we assume may be due to the large number of soils that were sampled from the three states. The results of this study concurred with the conclusions in the reports given by (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e56\u003c/span\u003e) and (18) in Kenya and in Ecuador by (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e57\u003c/span\u003e) for the morphology and also biochemical characteristics for native strain rhizobium that nodulate common varieties of beans.\u003c/p\u003e\u003cp\u003eOur findings differed to the findings of (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e58\u003c/span\u003e), were there was large numbers of FG isolates and was attributed to the prevalent presence of rapidly growing rhizobia in arid and semiarid lands, having the capacity to quickly multiply in the durations of shorter rains being more tolerative of stressed conditions than their counterpart slow-growing strains which can be considered to be a survival strategies. It also differed from the findings of (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) where there was a high frequency of fast-growing isolates obtained in cowpea plant which are mostly able to form nodules by associations with bacterial species \u003cem\u003eBradyrhizobium\u003c/em\u003e which are well known slow-growing strains and alkaline in nature indicating that leguminous cowpea plant doesn\u0026rsquo;t only form symbiosis with species type \u003cem\u003eBradyrhizobium\u003c/em\u003e but with several distinct species types of rhizobia. Our results also concur with the evaluation reports of (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e59\u003c/span\u003e) who obtained isolates of FG species rhizobia in the cowpea plants from their experiment.\u003c/p\u003e \u003cp\u003eOur findings show that Bambara groundnut is highly promiscuous and potentially able to form nodules in their roots with several spp. of rhizobia as confirmed in the reported results of (41) and also similar to the results (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e60\u003c/span\u003e) which reinforced the wanton or promiscuous ability of P. vulgaris plant to form nodules with the different strains and types of Rhizobia. The distribution among the three states was not significantly different from 29% from Kano, 35% from Kaduna, and 36% from Niger which are in the same agroecological zones this differed from the work of (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e61\u003c/span\u003e) which reports a wider of indigenous diversity type of several bradyrhizobia spp. in soils from Kenya (western) compared with that of Kenya (eastern) which is an indication of the difference agroecology conditions. Both the isolates that were fast- and slow-growing produced mucus which in high to intermediate quantities and some of the isolates appeared as dense and elastic or as diffuse and non -elastic. The production of mucus is most likely a representation of the mechanisms used by rhizobia for adapting and enduring harsh climates and the edaphic soil conditions. It helps by preventing desiccation within the cells of the bacteria enabling or strengthening them to withstand variations in salinity, temperature, and acidity as shown in the work of (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e61\u003c/span\u003e). The evaluations by (62), also reports an increased production in the amount of mucus by \u003cem\u003eBradyrhizobium\u003c/em\u003e isolates as mechanisms for survival by adaptation.\u003c/p\u003e \u003cp\u003eThere was no consistency in the pH changes as both FG and SG isolates increased the pH in the media differing from the reports by (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) where all SG isolates increased the medium\u0026rsquo;s pH with the exception of one isolate (which was able to acidify the medium) and concurring with those of (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e63\u003c/span\u003e) where it is reported that SG rhizobia strains are able to produce acid and the work of (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e66\u003c/span\u003e) where 55 percent of the isolates which were fast growing produced acid. There was a wide diversity of Bambara symbiotic strains present in the soils collected from three of the legume producing northern states in Nigeria, and Bambara groundnut appears to be a highly compatible host because it was capable of allowing the formation of root nodules with wider ranges of diverse rhizobia spp. which included both FG species Ensifer, Rhizobium and Mesorhizobium, and the SG species the \u003cem\u003eBradyrhizobium.\u003c/em\u003e\u003c/p\u003e"},{"header":"5.0 CONCLUSION","content":"\u003cp\u003eIn soils obtained from northern Nigeria, there was a wide diversity of Rhizobia isolates that were recovered from the 54 soil samples and a total of 150 rhizobia isolates were recovered by trapping inside the surface root nodules in Bambara groundnut and were characterized both morpho-culturally and biochemically.\u003c/p\u003e \u003cp\u003eThe cells were mostly whitish, creamy, transparent or translucent colonies that were small in size and classified as \u003cem\u003eBradhyrhizobium\u003c/em\u003e but other several spp. were also recovered showing the promiscuous nature of Bambara groundnut. The soils had a rich reservoir of rhizobia spp., and the most prevalent rhizobia isolates were the slow-grower but also consisted of the fast growers, some of which were able to acidify the medium and producing substantial to minute amounts of mucus (exopolysaccharides production). These maybe considered as some of the attributes that assist the bacteria in survival and adaptation in the tropical regions. Our reports showed the large diverse FG and SG isolated rhizobia strains types that are able to nodulate the roots of Bambara groundnut and propose or imply morphological and biochemical variations within and amongst Bambara groundnut strains which are a criteria for the selection of potential isolates that can be used as biofertilizer. The effectiveness and efficiency of characterizing rhizobia with authentication method proves to be effective for discrimination of isolates.\u003c/p\u003e \u003cp\u003eThis study offers substantial informative data for carrying out future study about the large diversity and interactive relationship between Bambara symbiotic bacteria, the Bambara plant, and also the prevalent surrounding soil environment conditions. The isolated rhizobia strains which were characterized in this study need further screening to test their abilities for fixing nitrogen with different Bambara groundnut varieties or genotypes and some other major legumes that have high economic importance in northern Nigeria.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCONFLICTS OF INTEREST\u003c/h2\u003e \u003cp\u003eAll the participating authors hereby declare no resulting conflicts of interest whatsoever .\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCOMPETING INTEREST\u003c/h2\u003e \u003cp\u003eThere are no fundings to be reported for this research. The authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eO.O. AJAYI and M. DIANDA wrote the main manuscript text , O.O. AJAYI prepared the figures and all authors reviewed the manuscript.\"\u003c/p\u003e\u003ch2\u003eACKNOWLEDGMENTS\u003c/h2\u003e \u003cp\u003eThe participating authors wish to thank the Soil microbiology unit of the international institute for tropical agriculture (IITA) Ibadan Nigeria, for providing research facilities for use in this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eOrrell, P., and Bennett, A. E. How can we exploit above-belowground interactions to assist in addressing the challenges of food security? Front. PlantSci. 2013. 4:432. doi: 10.3389/fpls.2013.00432\u003c/li\u003e\n\u003cli\u003eKarthik, C., Oves, M., Sathya, K., Sri Ramkumar, V., and Arulselvi, P. I. Isolation and characterization of multi-potential Rhizobium strain ND2 and its plant growth-promoting activities under Cr (VI) stress. Arch. Agron. 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Microbiol. 2018. 9:968. doi: 10.3389/fmicb.2018.00968\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bambara groundnut, rhizobia diversity, efficiency, nitrogen fixation, indigenous","lastPublishedDoi":"10.21203/rs.3.rs-4499028/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4499028/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBambara groundnut (BG) has a high nutritious content, is under-utilized with the potential to eradicate malnutrition, yet has very low production rates. Rhizobia inoculant can enhance it’s production, but, inadequate information about the diversity and suitability of rhizobia strains is known. Diversities of Bambara-symbiotic-rhizobia in soils (54) collected across three states in Nigeria were characterized morphologically and biochemically. Strains were evenly distributed between; Niger (36%), Kaduna (35%), and Kano (29%), but significantly different between local governments. Rhizobia strains were Gram negative rods, 10% were highly effective, while 81% were infective. Strains couldn’t hydrolyse starch but showed varied utilization abilities for different carbon sources, 73% hydrolysed gelatin and 66% produced catalase enzyme. A wide diversity of Bambara-symbiotic-strains were present in the soils, but only 10% effectively fixed nitrogen. Although there is a rich diversity of Bamabara-symbiotic-strains in these soils, it is necessary to apply suitable effective rhizobia strains as inoculant.\u003c/p\u003e","manuscriptTitle":"Diversity, effectiveness and biochemical characterization of Bambara groundnut symbiotic Rhizobia strains in tropical Nigerian soils","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-31 06:39:24","doi":"10.21203/rs.3.rs-4499028/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e4ae5c72-c8c2-4764-a029-43e95ee2c19a","owner":[],"postedDate":"July 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":34839994,"name":"Biological sciences/Biotechnology"},{"id":34839995,"name":"Biological sciences/Microbiology"},{"id":34839996,"name":"Earth and environmental sciences/Environmental sciences"}],"tags":[],"updatedAt":"2025-07-21T23:08:17+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-31 06:39:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4499028","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4499028","identity":"rs-4499028","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}