Unveiling the Impact of Organic Fertilizer on Rice (Oryza sativa L.) Salinity Tolerance: Insights from the Integration of NDVI and Metabolomics

Abstract

Soil salinization threatens global agriculture, reducing crop productivity and food security. Developing strategies to improve salt tolerance is crucial for sustainable agriculture. This study examines the role of organic fertilizer in mitigating salt stress in rice (Oryza sativa L.) by integrating NDVI and metabolomics. Using salt-sensitive (19X) and salt-tolerant (HHZ) cultivars, we aimed to (1) evaluate changes in NDVI and metabolite content under salt stress, (2) assess the regulatory effects of organic fertilizer, and (3) identify key metabolites involved in stress response and fertilizer-induced regulation. Under salt stress, survival rate of the 19X plants dropped to 6%, while HHZ maintained 38%, with organic fertilizer increasing survival rate to 25% in 19X and 66% in HHZ. NDVI values declined sharply in 19X (from 0.56 to <0.25) but remained stable in HHZ (~0.56), showing a strong correlation with survival rate (R² = 0.87, p < 0.01). NDVI provided a dynamic, non-destructive assessment of rice health, offering a faster and more precise evaluation of salt tolerance than survival rate analysis. Metabolomic analysis identified 12 key salt-tolerant metabolites, including citric acid, which is well recognized for regulating salt tolerance. HTPA, pipecolic acid, maleamic acid, and myristoleic acid have previously been reported but require further study. Additionally, seven novel salt-tolerant metabolites-tridecylic acid, propentofylline, octadeca penten-3-one, 14,16-dihydroxy-benzoxacyclotetradecine-dione, cyclopentadecanolide, HpODE, and (±)8,9-DiHETE-were discovered, warranting further investigation. Organic fertilizer alleviated salt stress through distinct metabolic mechanisms in each cultivar. In 19X, it enhanced antioxidant defenses and energy metabolism, mitigating oxidative damage and improving fatty acid metabolism. In contrast, HHZ primarily benefitted from improved membrane stability and ion homeostasis, reducing lipid peroxidation and oxidative stress. These findings primarily support the identification and screening of salt-tolerant rice cultivars while also highlighting the need for cultivar-specific fertilization strategies to optimize stress resilience and crop performance. Based on the correlation analysis, 26 out of 53 differential metabolites were significantly correlated with NDVI, confirming a strong association between NDVI shifts and key metabolic changes in response to salt stress and organic fertilizer application. By integrating NDVI and metabolomics, this study provides a refined method for evaluating salt stress responses, capturing early NDVI changes and key salinity stress biomarkers. This approach may prove valuable for application in salt-tolerant variety screening, precision agriculture, and sustainable farming, contributing to scientific strategies for future crop improvement and agricultural resilience.

Keywords: NDVI; metabolomics; organic fertilizer; rice stress tolerance; salinity stress.

Figure 1

Effects of salt stress and organic fertilizer on survival rate and NDVI dynamics of two rice (Oryza sativa L.) cultivars. (A): Pictures of 19X and HHZ seedlings on Day 5 under three treatments; (B): Survival rate of rice seedlings of 19X and HHZ under different conditions. Error bars are S.E. of the mean of 2 independent samples from two replications. Different letters indicated significant difference at 0.05 level. (B,C): NDVI dynamics of rice seedlings of 19X (C) and HHZ (D) after treatment under different conditions. Error bars are S.E. of the mean of 2 independent samples from two replications. ** indicates significant differences (p < 0.01), ns indicates no significant differences at 0.05 level. (E): Correlation between NDVI (Day 5) and survival rate of rice seedlings of two cultivars under three treatments (CK: standard Yoshida nutrient solution, CK-Salt: standard Yoshida nutrient solution + 0.6% (w/w) NaCl, Salt-OF: standard Yoshida nutrient solution + 0.6% (w/w) NaCl solution + 60-fold diluted fermented sheep manure solution).

Figure 2

Metabolomic profiling of 767 metabolites in rice seedlings of two rice (Oryza sativa L.) cultivars (19X and HHZ) under three treatments (CK: standard Yoshida nutrient solution, Salt: standard Yoshida nutrient solution + 0.6% (w/w) NaCl, OF: standard Yoshida nutrient solution + 0.6% (w/w) NaCl solution + 60-fold diluted fermented sheep manure solution); correlation matrix using Pearson correlation coefficient (A); principal component analysis of the relative metabolites identified from six treatments (B); clustering heatmap of the metabolites identified from six treatments, hierarchical cluster analysis was used for metabolite clustering (C); classification of metabolites (D).

Figure 3

Differential analysis of salt tolerance between 19X and HHZ. (A) DEMs found in 19X and HHZ under salt stress compared with CK. (B) Venn diagram of metabolites distribution in 19X and HHZ under salt stress compared with CK. (C) KEGG pathways related to the 93 DEMs.

Figure 4

Differential analysis of salt tolerance between salt treatment and CK groups. (A) DEMs between HHZ and 19X under salt treatment and CK. (B) Venn diagram of DEMs distribution in salt treatment and CK groups. (C) KEGG pathways related to the 62 DEMs.

Figure 5

Differential metabolites and KEGG pathway analysis in HHZ and 19X under OF and salt treatment. (A) DEMs found in OF and salt groups in HHZ. (B) DEMs found in OF and salt groups in 19X. (C) KEGG pathways related with the 10 annotated metabolites out of 28 DEMs found in HHZ between OF and salt groups.