Diversity of rhizobacteria associated with Vigna subterranea communally produced in Mpumalanga, KwaZulu-Natal, and Limpopo provinces, South Africa.

Abstract

Bambara groundnut (Vigna subterranea L. Verdc) is a pulse crop cultivated mostly by smallholder farmers in Africa and ranked the third most important legume crop after groundnut (Arachis hypogaea L.) and cowpea (Vigna unguiculata (L.) Walp.). Grain legumes such as Bambara groundnut form nitrogen-fixing association with bacteria in the roots collectively called rhizobia in a process that supplies sufficient nitrogen (N) for legumes and other crops under crop rotation and intercropping systems. Sustainable agriculture depends greatly on these mutualistic relations, especially a balanced interaction between a diversity of rhizosphere microorganisms, plants, and soil physical and chemical properties. Rhizosphere microorganisms perform a variety of functional processes that enhance the soil and promote plant growth, while the plant reciprocates this by providing the microbial communities with conducive endo and exo-microenvironment. The diversity and functional roles of rhizobia associated with Bambara groundnut rhizosphere have not been extensively studied. Hence, the aim of the current study was to (i) determine the incidence and diversity of the rhizobacterial associated with communally produced Bambara groundnut in Mpumalanga, KwaZulu Natal, and Limpopo provinces, South Africa, (ii) identify nutrient-cycling activities of rhizobacteria, and (iii) assess nutrient status, and enzyme activities of Bambara groundnut rhizosphere soil. Roots and soil samples from Bambara groundnut were collected from communal fields in Mpumalanga and Limpopo provinces. In KwaZulu-Natal province, soil samples were gathered from Bambara groundnut farmers' fields before planting the crop. Bambara groundnut root nodules were sterilized, crushed, and streaked on Nutrient Agar (NA) media to isolate bacteria, which were identified using morphological traits and 16S rRNA gene sequencing. The gene sequences were confirmed via BLASTn at NCBI. Nitrogen and phosphorus cycling activities of the bacterial isolates were tested, and the diversity of rhizobia in the samples was assessed using Shannon-Wiener, Simpson, and Pielou’s indices. Morphological characterization of the isolates resulted in the identification of 209 isolates, with 43 different isolates identified in all three provinces. About 89 % (186) of isolates from the root nodules tested positive for nitrogen cycling and 11 % (23) tested negative, while all isolates obtained from nodules in all three provinces tested negative for phosphate solubilization. The 16S rRNA gene sequences of the selected 153 isolates revealed a distinct evolutionary lineage mostly related to the genera Enterobacter, Leucobacter, Bacillus, Spingobacterium, Lysinibacillus, Stenotrophomonas, Cellulosimicrobium, Kaistella, Neorhizobium, Proteus, Micrococcus, and Mammalicoccus. with a significant E-value (≤ 0.000). In this study, 22 bacterial species (14.38%) were closely associated with Enterobacter absuriae, 19 (12.42%) with Leucobacter chromiiresistens, 18 (11.76%) with Bacillus pumilus, 14 (9.15%) with Sphingobacterium faecium, 13 (8.50%) each with Lysinibacillus sphaericus and Stenotrophomonas lactitubi, and 11 (7.19%) with Stenotrophomonas pavanii. Other species were associated with varying percentages, with the least common being Lysinibacillus pakistanensis, Mammalicoccus sciuri, and Sphingobacterium multivorum at 0.65% each.The N-cycling tests indicated that 186 (89 %) of isolates from the root nodules could fix nitrogen and 23 (11 %) could not, whereas all isolates from root nodules could not solubilize phosphate. Mpumalanga province had the highest number of bacterial isolates (107) from root nodules, followed by KwaZulu-Natal province (87) and last was Limpopo province (15). In Mpumalanga province, the University of Mpumalanga had the highest bacterial isolates at 40 and the highest species richness of 21, while Casteel and Mkhuhlu had 5 isolates each and lowest in species abundance. Hazyview had the least species richness (3). In KwaZulu-Natal province, the University of Zululand had higher number of isolates, 64, and 23 species richness when compared to Nhlangenyuke that had 23 isolates and 14 richness score. In Limpopo province, Gabaza village had 15 isolates and 10 richness score. In Mpumalanga province, the University of Mpumalanga had the highest genetic diversity index (H’) score of 2.81, followed by Hlamalani at 1.79, Nkomazi at 1.77, Boschfontein and Bushbuckridge at 1.63, and Hazyview had the lowest score of 1.01. The trend was different for Simpson index (D) scores, with Hazyview having the highest score index of 0.27, while the lowest diversity score of 0.05 was recorded at the University of Mpumalanga. When rhizobia evenness (J) was studied, Casteel had a score closest to even level at 0.83, while Hazyview and Boschfontein both had scores of 0.56. In KwaZulu-Natal province genetic diversity (H’) of the microbial population was higher at the University of Zululand at 2.85 and Nhlangenyuke had lower H’ index of 0.5. Simpson index also indicated higher diversity (D) score of 0.94 at the University of Zululand and lower at Nhlangenyuke with a diversity (D) score of 0.04. The physicochemical properties of soil samples collected from Bambara groundnut fields in three provinces were assessed at the KwaZulu-Natal Department of Agriculture and Rural Development’s Analytical Services Unit, Cedara, South Africa. Phosphorus (P), total nitrogen (N), potassium (K+), calcium (Ca2+), magnesium (Mg2+), zinc (Zn), manganese (Mn), copper (Cu), pH, organic carbon (OC), organic matter (OM), clay, exchangeable acidity, and acid saturation in the soils were determined using previously described methods. Phosphorus and nitrogen enzyme cycling activities (acid phosphatase, β-glucosidase, β-glycosaminidase, and alkaline phosphatase) were determined according to the method adapted from Jackson, Tyler, and Millar (2013) and conveyed in nmolh-1 g-1 using 5 g of each soil sample, while nitrate reductase activities method was adapted from Bruckner, Wright, Kampichler, Bauer and Kandeler (1995). Collected data were subjected to analysis of variance (ANOVA) using Statistix 10 software. Mean separation was achieved using Fisher’s Least Significant Differences (LSD) at 5 % probability. All physico-chemical properties such as pH, total nitrogen, organic clay content, magnesium, manganese, soil density, exchangeable bases, effective cation exchange capacity (ECEC), zinc, and copper were statistically (P ≤ 0.05) different among localities, except for phosphorus, exchangeable acidity, acid saturation and the soil enzyme activities (acid phosphatase, β-glucosaminidase, and alkaline phosphatase). The pH of the soil in all sample sites was acidic, ranging from 4.5 to 5.8 with Hazyview, Bushbuckridge, and Nkomazi being slightly acidic, and University of Zululand and Mpumalanga pH were rated as strongly acidic. Nhlangenyuke had the highest percentage of soil organic carbon and organic matter of 1.86 % and 3.20 %, respectively. Nhlangenyuke soil had a high K+ value of 0.45 cmolc kg-1 when compared to the other sample sites. Hazyview had the highest clay content, Ca2+, and effective cation exchangeable capacity (ECEC) of 38.00 %, 4.98 cmolc kg-1, and 6901.9 cmolc kg-1, respectively. Copper (Cu), magnesium (Mg2+), and manganese (Mn) of 9.39 mg kg-1, 2.11 mg kg-1, and 49.55 mg kg-1, respectively, were high in soils collected from Gabaza village whereas Boschfontein had the lowest Cu, Mg2+, Ca2+, K+, ECEC, and clay content of 0.30 mg kg-1, 0.23 mg kg-1, 0.99 cmolc kg-1, 0.10 cmolc kg-1, 1344 cmolc kg-1, and 7.50 %, respectively. The University of Zululand had the lowest Mn at 6.01 mg kg-1. Soil collected from Casteel had a high Zn level of 28.47 mg kg-1 and the lowest Zn of 0.22 mg kg-1 was found in Hlamalani. Nhlangenyuke soil had the highest N of 1.10 mg kg-1 whereas the lowest N of 0.43 mg kg-1 was in Hlamalani. Soil enzyme activities; N-cycling and P-cycling, which involves acid phosphatase, β-glucosidase, β-glycosaminidase, and alkaline phosphatase were not different among localities. Hlamalani soil had the highest nitrate reductase of 19710 nmolh-1 g-1 and Bushbuckridge was the lowest nitrate reductase of 6243.33 nmolh-1 g-1. In conclusion, rhizobia isolates identified in root nodules varied amongst the locations with Mpumalanga province having the highest number of isolates followed by KwaZulu-Natal province and the least was Limpopo Province. In all three provinces nodules had both nitrogen cycling bacteria and non-cycling bacteria with no isolates able to solubilize phosphate. The physico-chemical soil properties varied with the location. Hlamalani had the highest nitrate reductase and Bushbuckridge the lowest. The current study indicated that there is a huge diversity of rhizobacterial organisms associated with Bambara groundnuts with potential for commercialisation after testing their efficiency in nutrient cycling.