Salinity, pH tolerance, and efficiency of indigenous nitrogen-fixing bacteria on nodulation and growth of Vigna subterranea (L. Verdc.).

Abstract

Bambara groundnut (Vigna subterranea L. Verdc.) is an underutilized and highly nutritious crop with a variety of seed coat colours, influenced by secondary metabolites known to possess antimicrobial properties. Despite its potential, production remains low due to ecological stresses, such as extreme temperatures, drought, and poor rainfall, as well as plant genotype selection and seed coat properties, all of which contribute to soil salinity and pH fluctuations that limit plant growth and beneficial microorganisms. The study hence had three objectives (i) to assess the survival of indigenous nitrogen-fixing bacteria on Bambara groundnut seed coat extracts, (ii) their tolerance to low pH leve and high soil salinity levels, and (iii) the efficiency of these bacteria in promoting nodulation and growth of Bambara groundnut. Objective 1, a 3×10×3 factorial experiment in a CRD with 3 replications was used to test three Bambara groundnut (BR-W, B-W, and B-B) variety seed coats extracted using water, ethanol, and acetone, on 10 Bambara groundnut extracted bacterial strains (Proteus columbae, Mammaliicoccus sciuri, Bacillus pumilus P12, Stenotrophomonas pavanii , Bacillus pumilus P4, Kaistella daneshvariae, Stenotrophomonas maltophilia, Cellulosimicrobium cullulans, Sphingobacterium faecium, and Enterobacter asburiae) under laboratory conditions. The inhibitory effects of the three Bambara groundnut seed coat extracts varied with bacteria. Proteus columbae, B. pumilus P12, B. pumilus P4, and S. pavanii were inhibited by all three extracts, whereas the growth of M. sciuri, S. maltophila, E. asburiae, and C. cullulans was inhibited by water and acetone extracts only, with ethanol extracts having no negative effects on the four. The growth of S. faecium was inhibited by water and ethanol extracts only, while acetone extracts had no negative effect, and K. daneshvariae growth was inhibited by water extracts only. A laboratory experiment was established to assess the tolerance of the same bacterial isolates to pH levels (3.5, 4.5, 5.5) with a media pH of 6.9 as the control, and salinity levels (2–128 mM/m³ of NaCl + CaCl₂). Cellulosimicrobium cellulans, S. pavanii, and P. columbae had the highest absorbance, ranging from 0.519 to 0.69, while S. faecium, M. sciuri, and B. pumilus P4 had the lowest absorbance values, ranging from 0.286 to 0.398 under various pH levels. The salinity levels of 0 mM exhibited the highest radial growth, followed by 4 mM and 8 mM, ranging from 11.833 mm to 12.222 mm, whereas the salinity levels of 128 mM, 64 mM, and 32 mM showed the lowest radial growth, ranging from 7.6 to 11.133 mm for all bacterial isolates. Three Bambara varieties inoculated with the ten aforementioned bacterial strains, compared to uninoculated and KNO₃-fertilized controls under greenhouse conditions. Mammaliicoccus sciuri, K. daneshvariae, and S. maltophila had the highest stem diameters, while S. maltophila, B. pumilus P4, and P. columbae produced the highest nodule colour and abundance score. All bacterial isolates had high relative nitrogen fixing effectiveness (>80), with K. daneshvariae, M. sciuri, and S. faecium treated plants demonstrating the highest. In conclusion, the Bambara groundnut seed coats exhibited notable antimicrobial activity against certain indigenous nitrogen-fixing bacterial strains, highlighting the potential influence of seedcoat compounds on beneficial soil microorganisms. The isolates demonstrated tolerance to acidic conditions (pH ≥ 4.12) but were unable to withstand salinity levels above 4.24 mM/m³. Despite these stress limitations, all isolates showed high relative nitrogen-fixing effectiveness, indicating strong potential for biofertilizer development in acidic and mildly saline soils. Notably, Mammaliicoccus sciuri, Kaistella daneshvariae, Stenotrophomonas maltophilia, Bacillus pumilus P4, and Proteus columbae enhanced Bambara groundnut growth and are promising candidates for an inoculant.