Ascorbic acid formulation for survivability and diazotrophic efficacy of Azotobacter chroococcum Avi2 (MCC 3432) under hydrogen peroxide stress and its role in plant-growth promotion in rice (Oryza sativa L.)

Abstract

Oxidative stress generates reactive oxygen species which causes cell damage of living organisms and are normally detoxified by antioxidants. Indirect reports signify the damages caused by reactive oxygen species and neutralized by antioxidant, but the direct evidence to confirm this hypothesis is still unclear. To validate our hypothesis, an attempt was made in a diazotrophic bacterium (Azotobacter chroococcum Avi2) as a biological system, and hydrogen peroxide (H₂O₂) and ascorbic acid were used as oxidative stress and antioxidant supplement, respectively. Additionally, rice plant-growth attributes by Avi2 was also assessed under H₂O₂ and ascorbic acid. Results indicated that higher concentration of H₂O₂ (2.5 mM-4.5 mM) showed the complete mortality of Avi2, whereas one ppm ascorbic acid neutralized the effect of H₂O₂. Turbidity, colony forming unit, DNA quantity, nifH gene abundance, indole acetic acid and ammonia productions were significantly (p < 0.5) increased by 11.93%, 17.29%, 19.80%, 74.77%, 71.89%, and 42.53%, respectively in Avi2-treated with 1.5 mM H₂O₂ plus ascorbic acid compared to 1.5 mM H₂O₂ alone. Superoxide dismutase was significantly (p < 0.5) increased by 60.85%, whereas catalase and ascorbate peroxidase activities were significantly (p < 0.05) decreased by 64.28% and 68.88% in Avi2-treated with 1.5 mM H₂O₂ plus ascorbic acid compared to 1.5 mM H₂O₂ alone. Germination percentage of three rice cultivars (FR13a, Naveen and Sahbhagi dhan) were significantly (p < 0.5) increased by 20%, 13.33%, and 4%, respectively in Avi2-treated with 0.6 mM H₂O₂ plus ascorbic acid compared with uninoculated control. Overall, this study indicated that ascorbic acid formulation neutralizes the H₂O₂-oxidative stress and enhances the survivability and plant growth-promoting efficacy of A. chroococcum Avi2 and therefore, it may be used as an effective formulation of bio-inoculants in rice under oxidative stress.

Keywords: Antioxidant; Ascorbic acid; Diazotrophs; Hydrogen peroxide; Oxidative stress; Rice.