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.