Ultraviolet-enhanced detoxification of chromate and protection of Brassica napus by Aspergillus sojae SH 20

Abstract

The pervasive issue of heavy metal contamination in agricultural lands poses significant concerns and has wide-ranging implications for ecosystems. However, an encouraging solution lies in exploiting the potential of fungal endophytes to alleviate these detrimental effects. This study emphasized on improving the growth-promoting and chromium-alleviating capabilities of fungal endophytes, particularly Aspergillus sojae strain SH20, through ultraviolet (UV) irradiation. Following UV treatment, SH20 exhibited significantly enhanced growth-promoting and chromium-alleviating capabilities in comparison to its non-irradiated counterpart. Distinctly, the UV-treated SH20 strain demonstrated an improved ability to accumulate and reduce toxic chromate in the soil, effectively addressing the growth constraints imposed by elevated chromium levels in Brassica napus L. The UV-irradiated SH20 variant boosted shoot length up to 3 times that of the control. Similarly, this fungal strain displayed a remarkable increase in the total fresh weight of the seedlings, recording nearly 17 times greater than the control. The isolate treated with UV light reduced the absorption of chromium by about 3 times in the roots, helping the young plants to grow well even when exposed to chromate stress. A drop in root colonization by the UV-treated strain further resulted in reduced chromate absorption by the roots. Also, the strain showed great skill in boosting the host's antioxidant defenses by reducing the buildup of harmful reactive oxygen species (ROS), increasing the removal of ROS, and improving the plant's antioxidant levels, including phenols and flavonoids. When the host plants were exposed to 25 ppm of Cr stress, the UV-irradiated variant SH 20 stimulated the production of flavonoids (246 μg/ml) and phenols (952 μg/ml) in comparison to the control (with 220 μg/ml of flavonoids and 919 μg/ml of phenols). In conclusion, this report highlights how exposing the A. sojae strain SH20 to UV light has the potential to enhance its abilities to promote growth and bioremediate. This suggests a promising solution for addressing heavy metal contamination in agricultural lands.

Keywords: Aspergillus sojae; Bioremediation; Brassica napus; Endophytes; UV-radiations.

Fig. 1

Characterization of the chromate-resistant endophytic fungus Aspergillus sojae SH20 included the following aspects: (A) Examination of colony morphology on PDA, (B) Light microscopy analysis of hyphae and conidiophore stained with lactophenol cotton blue, and (C) Phylogenetic relationship illustrated through a neighbor-joining tree, showcasing the ITS rDNA sequence similarity between the isolate and closely related sequences obtained from the NCBI GenBank database.

Fig. 2

Production of A) IAA, B) Salicylic acid, C) Flavonoids and D) Phenols by fungal strain SH 20 and its mutant (SH 20 45f) under varying concentration of chromate stress. Data shown are mean ± SE of 3 replicates and labels on bars denotes significance among treatments (Duncan test P < 0.05).

Fig. 3

Impact of Aspergillus sojae (SH 20) and its mutant SH 20 (45f) on A) Bio-reduction of Cr⁺⁶ to Cr⁺³ in Fungal cultural filtrate (FCF), B) Biosorption and C) Bio-concentration factor. Ctrl CB = Czapek broth spiked with 100 ppm of Cr without endophytes; T1 = Czapek broth spiked with 100 ppm of Cr and inoculated with SH 20; T2 = Czapek broth spiked with 100 ppm of Cr and inoculated with SH 20 (45f); T3 = Czapek broth spiked with 300 ppm of Cr and inoculated with SH 20; T4 = Czapek broth spiked with 300 ppm of Cr and inoculated with SH 20 (45f); T5 = Czapek broth spiked with 600 ppm of Cr and inoculated with SH 20; T6 = Czapek broth spiked with 600 ppm of Cr and inoculated with SH 20 (45f); T7 = Czapek broth spiked with 900 ppm of Cr and inoculated with SH 20; T8 = Czapek broth spiked with 900 ppm of Cr and inoculated with SH 20 (45f).

Fig. 4

Effect of heavy metal (Cr⁺⁶) and A.sojae inoculation on A) IAA, B) Salicylic acid C) Flavonoids and D) Phenol contents in Brassica napus seedlings. Ctrl = endophyte free B. napus seedlings without undergoing Cr stress; T1 = endophyte free B. napus seedlings exposed to 25 ppm Cr⁺⁶; T2 = endophyte free B. napus seedlings exposed to 50 ppm Cr⁺⁶; T3 = A. sojae SH 20 associated B. napus seedlings without undergoing Cr stress; T4 = A. sojae SH 20 associated B. napus seedlings exposed to 25 ppm Cr⁺⁶; T5 = A. sojae SH 20 associated B. napus seedlings exposed to 50 ppm Cr⁺⁶; T6 = A. sojae SH 20 (45f) associated B. napus seedlings without undergoing Cr stress; T7 = A. sojae SH 20 (45f) associated B. napus seedlings exposed to 25 ppm Cr⁺⁶; T8 = A. sojae SH 20 (45f) associated B. napus seedlings exposed to 50 ppm Cr⁺⁶. Data shown are mean ± SE of 3 replicates and labels on bars denotes significance among treatments (Duncan test P < 0.05).

Fig. 5

Effect of Aspergillus sojae on A) Chromium in soil, B) Chromium translocated to roots, C) Chromium translocated to leaves, D) Bio-concentration factor and E) Bio-reduction of Cr. Ctrl = endophyte free B. napus seedlings without undergoing Cr stress; T1 = endophyte free B. napus seedlings exposed to 25 ppm Cr⁺⁶; T2 = endophyte free B. napus seedlings exposed to 50 ppm Cr⁺⁶; T3 = A. sojae SH 20 associated B. napus seedlings without undergoing Cr stress; T4 = A. sojae SH 20 associated B. napus seedlings exposed to 25 ppm Cr⁺⁶; T5 = A. sojae SH 20 associated B. napus seedlings exposed to 50 ppm Cr⁺⁶; T6 = A. sojae SH 20 (45f) associated B. napus seedlings without undergoing Cr stress; T7 = A. sojae SH 20 (45f) associated B. napus seedlings exposed to 25 ppm Cr⁺⁶; T8 = A. sojae SH 20 (45f) associated B. napus seedlings exposed to 50 ppm Cr⁺⁶.

Fig. 6

Effect of Cr⁺⁶ stress and SH 20 inoculation on A) Catalase, B) Ascorbate peroxidase, C) Peroxidase and D) DPPH in host plants. Ctrl = endophyte free B. napus seedlings without undergoing Cr stress; T1 = endophyte free B. napus seedlings exposed to 25 ppm Cr⁺⁶; T2 = endophyte free B. napus seedlings exposed to 50 ppm Cr⁺⁶; T3 = A. sojae SH 20 associated B. napus seedlings without undergoing Cr stress; T4 = A. sojae SH 20 associated B. napus seedlings exposed to 25 ppm Cr⁺⁶; T5 = A. sojae SH 20 associated B. napus seedlings exposed to 50 ppm Cr⁺⁶; T6 = A. sojae SH 20 (45f) associated B. napus seedlings without undergoing Cr stress; T7 = A. sojae SH 20 (45f) associated B. napus seedlings exposed to 25 ppm Cr⁺⁶; T8 = A. sojae SH 20 (45f) associated B. napus seedlings exposed to 50 ppm Cr⁺⁶. Data shown are mean ± SE of 3 replicates and labels on bars denotes significance among treatments (Duncan test P < 0.05).

Fig. 7

ROS accumulation in SH 20 and UV irradiated SH 20 (45f) inoculated Brassica napus leaves in comparison to control under varying levels of Cr⁺⁶ stress. A) Control, B) 25 ppm chromate, C) 50 ppm chromate, D) SH 20, E) SH 20 + 25 ppm chromate, F) SH 20 + 50 ppm chromate, G) SH 20 (45 F), H) SH 20 (45 F) + 25 ppm chromate and I) SH 20 (45 F) + 50 ppm chromate. Fresh leaves of 16 days old seedlings were detached from the plants and were stained with DAB in order to visualize ROS.

Fig. 8

Colonization of endophytic fungus Aspergillus sojae SH20 and UV irradiated SH 20 (45F) under varying Cr⁺⁶ stresses. A) SH 20, B) SH 20 + 25 ppm chromate, C) SH 20 + 50 ppm chromate, D) SH 20 (45 F), E) SH 20 (45 F) + 25 ppm chromate and F) SH 20 (45 F) + 50 ppm chromate in the cortical constituency of Brassica napus root stained with lactophenol cotton blue dye after 21 days of successful colonization.