Nodule associated bacteria for plant growth promotion and nodulation enhancement in cowpea (Vigna unguiculata (L.) Walp

Abstract

The study entitled ‘Nodule associated bacteria for plant growth promotion and nodulation enhancement in cowpea (Vigna unguiculata (L.) Walp)’ was carried out at the Department of Agricultural Microbiology, College of Agriculture, Vellayani, Thiruvananthapuram, with the objective to isolate and characterize nodule associated bacteria from different varieties of cowpea and assess their nodulation efficiency and plant growth promotion in cowpea (Vigna unguiculata (L.) Walp). Root nodules of leguminous plants harbour diverse microbial communities that play crucial roles in nitrogen fixation and plant growth promotion. In this study, nodule- associated bacteria were isolated from healthy root nodules collected from different varieties of cowpea from various locations in the (AEU 8) southern laterites of Kerala. Surface-sterilized nodules were crushed aseptically, and the suspension was serially diluted and plated on different media including Yeast Extract Mannitol Agar (YEMA), Nutrient agar, N free malate bromothymol blue media, Tryptic soy agar, Burk’s medium, Jensen’s medium. The seventy-four isolates were purified and characterized based on colony morphology and Gram reaction. Of these, thirty-four morphologically distinct isolates were selected for further studies. The Indole Acetic Acid (IAA), Gibberellic acid (GA), Extracellular Ammonia (ECA) production, presence or absence of growth on N-free medium, phosphate solubilization potential and cellulolytic activity of the thirty-four bacterial isolates were assessed. The selected isolates produced IAA in the range of 2.64 μg mL-1 (NAB 57) to 63.98 μg mL-1 (NAB 1) of culture filtrate in the absence of tryptophan and 5.12 μg mL- 1 (NAB 63) to 98.18 μg mL-1 (NAB 1) in the presence of tryptophan. The isolate NAB 42 recorded significantly higher gibberellic acid production of 62.24 μg mL-1 and NAB 14 recorded highest extracellular ammonia production of 11.29 μmol mL−1. The isolate NAB 43 exhibited maximum cellulolytic activity of 5.45 and NAB 57 showed maximum phosphate solubilization potential of 4.6. Among these isolates NAB 1, NAB 2, NAB 42 and NAB 43 were showed growth on N-free medium. Plant growth promotion potential in cowpea was assessed by roll towel assay using standard protocol and NAB 2 exhibited the highest germination rate of 85.3% and longest root length (22.78 cm), while NAB 15 demonstrated the longest shoot length (19.81cm). The highest seedling vigour index (4121.5) was recorded for NAB 2, compared to 2223.75 in the control. Weighted average ranking was done and eleven isolates were selected for further experiments. Selected isolates were co-cultured with beneficial microbes, including rhizobial and non-rhizobial endophytes, to assess compatibility and mutual influence on growth and metabolic activity. The combinations exhibited compatibility with each other and with other beneficial microbes. Dual culture plate assay and agar well diffusion assay were employed to assess the antagonistic potential of the 34 isolates against Pythium sp., Fusarium oxysporum, Sclerotium rolfsii, and Rhizoctonia solani. Isolates NAB 51 and NAB 27 showed the maximum inhibitory activity against Fusarium oxysporum, while NAB 27 was most effective against Rhizoctonia solani. NAB 33 exhibited the maximum inhibition against Sclerotium rolfsii, and NAB 42 showed maximum antagonistic activity against Pythium sp. The maximum siderophore production was observed in NAB 2 as indicated by orange halo zone around the colony on CAS agar. All isolates produced volatile organic compounds, with NAB 2 exhibiting the maximum level and only one isolate NAB 43 tested positive for hydrogen cyanide (HCN) production. The growth performance and nodulation efficiency of the test cowpea plants were evaluated under controlled conditions using Leonard’s jar assemblies. Inoculated treatments with Rhizobium exhibited a marked improvement in growth and nodulation parameters compared to uninoculated controls. Among the isolates, strain NAB 2 recorded the significant increase in plant height (30.25 cm), root length (19.2 cm), shoot weight (4.35g), root weight (0.76g), nodule number (16), nodule weight (0.07g) compared to uninoculated controls, indicating effective symbiotic performance. Based on the results of the studies on plant growth promotion and nodulation enhancement, the isolates NAB 2 and NAB 43 were selected for pot culture experiment.

Morphological and biochemical characterization of the isolates revealed that the two isolates were Gram-negative, rods. The 16S rRNA gene sequencing showed maximum sequence similarity of the isolates with Stenotrophomonas hibscicola (NAB 2) and Enterobacter quasiroggenkampii (NAB 43). A pot culture experiment was carried out to assess the effectiveness of selected nodule-associated bacterial (NAB) isolates, both individually and in combination with Rhizobium, in promoting plant growth and improving nodulation in cowpea variety Anaswara by seed biopriming. The experimental design was Completely Randomized Design (CRD) with six treatments, each replicated four times. Among the NAB isolates, Stenotrophomonas hibiscicola NAB 2 in combination with Rhizobium sp. showed superior performance, significantly enhancing plant growth, yield, and nodulation parameters. This treatment resulted in the highest plant height, leaf and branch number, shoot and root biomass, root development, pod yield, and nodule number and weight, indicating its strong potential for improving crop productivity. The study concluded that the selected nodule-associated bacterial isolate NAB 2, particularly when used in combination with Rhizobium sp., significantly enhanced plant growth and nodulation in cowpea var. Anaswara. These findings suggest that co- inoculation of Stenotrophomonas hibscicola NAB 2 with Rhizobium sp. RZB1 can serve as an effective bioinoculant strategy to improve legume productivity and promote sustainable agriculture.