A biostimulant prepared from red seaweed Kappaphycus alvarezii induces flowering and improves the growth of Pisum sativum grown under optimum and nitrogen-limited conditions

Abstract

Nitrogen (N) is one of the critical elements required by plants and is therefore one of the important limiting factors for growth and yield. To increase agricultural productivity, farmers are using excessive N fertilizers to the soil, which poses a threat to the ecosystem, as most of the applied nitrogen fertilizer is not taken up by crops, and runoff to aquatic bodies and the environment causes eutrophication, pollution, and greenhouse gas emissions. In this study, we used LBS6, a Kappaphycus alvarezii-based biostimulant as a sustainable alternative to improve the growth of plants under different NO₃ ^- fertigation. A root drench treatment of 1 ml/L LBS6 significantly improved the growth of Pisum sativum plants grown under optimum and deficient N conditions. No significant difference was observed in the growth of LBS6-treated plants grown with excessive N. The application of LBS6 induced flowering under optimum and deficient N conditions. The total nitrogen, nitrate and ammonia contents of tissues were found to be higher in treated plants grown under N deficient conditions. The LBS6 treatments had significantly higher chlorophyll content in those plants grown under N-deficient conditions. The root drench application of LBS6 also regulated photosynthetic efficiency by modulating electron and proton transport-related processes of leaves in the light-adapted state. The rate of linear electron flux, proton conductivity and steady-state proton flux across the thylakoid membrane were found to be higher in LBS6-treated plants. Additionally, LBS6 also reduced nitrogen starvation-induced, reactive oxygen species accumulation by reduction in lipid peroxidation in treated plants. Gene expression analysis showed differential regulation of expression of those genes involved in N uptake, transport, assimilation, and remobilization in LBS6-treated plants. Taken together, LBS6 improved growth of those treated plants under optimum and nitrogen-limited condition by positively modulating their biochemical, molecular, and physiological processes.

Keywords: Kappaphycus alvarezii; Pisum sativum; biostimulants; gene expression; nitrogen metabolism; sustainable agriculture.

Figure 1

LBS6 improved the growth of Pisum sativum grown under different levels of nitrate supplementation. Pea plants were grown under (A) optimum-N, (B) N-deficient, and (C) excessive-N for 45 days. The plants were drenched near root-zone with water, LBS6 1mL/L and 0.5mL/L on 14th and 21st day after sowing. Effect of LBS6 on (D) Plant height, (E) fresh and (F) dry weight of shoots grown under N-limited conditions. Treatment details: plants are grown under optimum (C1, C2 and C3), N-deficient (T1, T2 and T3) and excessive-N (T4, T5 and T6) conditions. The plants sprayed with water (control) (C1, T1, T4), 1ml/L of LBS6 (C2, T2 and T5), and 0.5mL of LBS6 (C3, T3 and T6). The values were presented as mean ± SE and significantly different mean values at p ≤ 0.05 were represented by different letters. Each experiment was carried out in triplicate, and each experimental unit had ten plants (n=30).

Figure 2

LBS6 root drenching positively influenced the rate of emergence of leaves of pea plants under under different nitrate supplementation. (A) The plastochron arrangement in 45 days old pea plants treated with water, LBS6 1mL/L and 0.5mL/L under different N contents. Effect of LBS6 on (B) Average number of leaves and (C) Rate of emergence of leaves. Plants are grown under optimum (C1, C2 and C3), N-deficient (T1, T2 and T3) and excessive-N (T4, T5 and T6) conditions. The plants sprayed with water (control) (C1, T1, T4), 1ml/L of LBS6 (C2, T2 and T5), and 0.5mL of LBS6 (C3, T3 and T6). The values were presented as mean ± SE and significantly different mean values at p ≤ 0.05 were represented by different letters. Each experiment was carried out in triplicate, and each experimental unit had ten plants (n=30).

Figure 3

LBS6 application showed higher number of flowers in pea plants grown under different nitrate supplementation. Total number of flowers at 45 days after second treatment. Plants are grown under optimum (C1, C2 and C3), N-deficient (T1, T2 and T3) and excessive-N (T4, T5 and T6) conditions. The plants sprayed with water (control) (C1, T1, T4), 1ml/L of LBS6 (C2, T2 and T5), and 0.5mL of LBS6 (C3, T3 and T6). The values were presented as mean ± SE and significantly different mean values at p ≤ 0.05 were represented by different letters. Each experiment was carried out in triplicate, and each experimental unit had ten plants (n=30).

Figure 4

LBS6 improved the pigment content of pea plants grown under different nitrate supplementation. (A) Chlorophyll a and (B) Chlorophyll b content of pea leaves. Plants are grown under optimum (C1, C2 and C3), N-deficient (T1, T2 and T3) and excessive-N (T4, T5 and T6) conditions. The plants sprayed with water (control) (C1, T1, T4), 1ml/L of LBS6 (C2, T2 and T5), and 0.5mL of LBS6 (C3, T3 and T6). The values are presented as mean ± SE of three independent experiments which was repeated thrice (n=9), and significantly different mean values are represented by different letters.

Figure 5

LBS6 regulates different biochemical parameters to improve plant growth under different nitrate supplementation. Effect of root drench with 0.5mL and 1mL/L of LBS6 on (A) Nitrogen starvation index, (B) Nitrate (C) Ammonia (D) Total amino acids and (E) Malondialdehyde content of leaves and (F) Electrolyte leakage. Plants are grown under optimum (C1, C2 and C3), N-deficient (T1, T2 and T3) and excessive-N (T4, T5 and T6) conditions. The plants sprayed with water (control) (C1, T1, T4), 1ml/L of LBS6 (C2, T2 and T5), and 0.5mL of LBS6 (C3, T3 and T6). The values are presented as mean ± SE of three independent experiments which was repeated thrice (n=9), and significantly different mean values are represented by different letters.

Figure 6

LBS6 regulate the expression of (A) Nitrate Transporter 2.1 (NTR 2.1), (B) Nitrate Transporter 2.3 (NTR 2.3), (C) Nitrite Reductase (NiR), (D) Glutamate Synthase 1 (GOGAT), (F) Glutamine Synthetase 1 (GS1), (E) Glutamine Synthetase 2 (GS2), (G) Calcineurin B-like Interacting Protein kinase 1 (CIPK1), (H) SYM29, (I) Root Determined Nodulation 1 (RDN1) and (J) Nodulation Inception-like protein (NIN) involved in N uptake, transport, assimilation, and remobilization under different nitrate supplementation. C1, T1 and T4 are control and C2, T2 and T5 are treated with 1ml/L of LBS6 under optimum, N-deficient, and excessive-N conditions respectively. The values are presented as mean ± SE of three independent experiments which was repeated thrice (n=9), and significantly different mean values are represented by different letters.

Figure 7

A schematic diagram of regulation of N-uptake and assimilation by the root drench application of LBS6 under N limited conditions. The green arrow shows the upregulation of the gene expression and enzyme activity, whereas the red arrow shows the downregulation.