Enhancement of Drought-Stress Tolerance of Brassica oleracea var. italica L. by Newly Isolated Variovorax sp. YNA59

Abstract

Drought is a major abiotic factor and has drastically reduced crop yield globally, thus damaging the agricultural industry. Drought stress decreases crop productivity by negatively affecting crop morphological, physiological, and biochemical factors. The use of drought tolerant bacteria improves agricultural productivity by counteracting the negative effects of drought stress on crops. In this study, we isolated bacteria from the rhizosphere of broccoli field located in Daehaw-myeon, Republic of Korea. Sixty bacterial isolates were screened for their growth-promoting capacity, in vitro abscisic acid (ABA), and sugar production activities. Among these, bacterial isolates YNA59 was selected based on their plant growth-promoting bacteria traits, ABA, and sugar production activities. Isolate YNA59 highly tolerated oxidative stress, including hydrogen peroxide (H₂O₂) and produces superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) activities in the culture broth. YNA59 treatment on broccoli significantly enhanced plant growth attributes, chlorophyll content, and moisture content under drought stress conditions. Under drought stress, the endogenous levels of ABA, jasmonic acid (JA), and salicylic acid (SA) increased; however, inoculation of YNA59 markedly reduced ABA (877 ± 22 ng/g) and JA (169.36 ± 20.74 ng/g) content, while it enhanced SA levels (176.55 ± 9.58 ng/g). Antioxidant analysis showed that the bacterial isolate YNA59 inoculated into broccoli plants contained significantly higher levels of SOD, CAT, and APX, with a decrease in GPX levels. The bacterial isolate YNA59 was therefore identified as Variovorax sp. YNA59. Our current findings suggest that newly isolated drought tolerant rhizospheric Variovorax sp. YNA59 is a useful stress-evading rhizobacterium that improved droughtstress tolerance of broccoli and could be used as a bio-fertilizer under drought conditions.

Keywords: Variovorax sp. YNA59; antioxidant activity; broccoli; drought stress; phytohormones.

Fig. 1. Catalase and exopolysaccharide (EPS) activity as displayed on orange media and Congo red medium.

(A) Shows the capability of catalase production, (B) EPS production, and (C) growth of isolate YNA59 at different concentrations of PEG6000.

Fig. 2. Characteristics of bacterial isolates.

(A) Quantification of abscisic acid (ABA) observed in culture broth (LB broth) of YNA59 and (B) Quantification of sucrose content in LB of YNA59. Each data point is the mean of three replicates. Error bars represent standard errors. The bars represented by different letters are significantly different from each other as evaluated by Duncan’s multiple range tests (DMRTs).

Fig. 3. Isolate YNA59 tolerance to oxidative stress.

(A) showed resistance to H₂O₂, (B) produce significant amount of superoxide dismutase SOD activity, (C) Catalase activities (CAT) and (D) Ascorbic peroxidase (APX) in the YNA59 broth (culture filtrate) and YNA59 cell (pellets were mix in distilled water). Each data point is the mean of at least three replicates. Error bars represent standard errors. The bars presented with different letters are significantly different from each other as evaluated by DMRT.

Fig. 4

Effects of selected bacterial isolate YNA59 on the growth of broccoli plants under normal and drought stress.

Fig. 5. Endogenous abscisic acid (ABA), jasmonic acid (JA), and salicylic acid (SA) quantification in broccoli plants inoculated with YUNA59.

(A) Demonstrates ABA, (B) JA, and (C) shows the amount of SA under normal and drought stress. Each data point is the mean of at least three replicates. Error bars represent standard errors. The bars presented with different letters are significantly different from each other as evaluated by DMRT.

Fig. 6

Detection of hydrogen peroxide in broccoli leaves under normal and drought stress using 3,3’-diaminobenzidine (DAB) staining method.

Fig. 7. The effect of bacterial isolates YNA59 on different antioxidants.

(A) Superoxide dismutase (SOD); (B) Catalase (CAT); (C) Ascorbic peroxidase (APX), and (D) Guaiacol peroxidase (GPX) contents in broccoli plants under normal and drought stress. Each data point is the mean of three replicates. Error bars represent standard errors. The bars presented with different letters are significantly different from each other as evaluated by DMRT.

Fig. 8. Phylogenetic tree of YNA59 was constructed using 16S rRNA sequences by neighbor joining (NJ) and maximum likelihood methods.

(A) Phylogenetic tree of YNA59 was constructed using 16S rRNA sequences by neighbor joining (NJ) and maximum likelihood methods; (B) Microscopic observation of YNA59 using a scanning electron microscope.