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ARS Home » Pacific West Area » Riverside, California » Agricultural Water Efficiency and Salinity Research Unit » Research » Research Project #434048

Research Project: Enhancing Specialty Crop Tolerance to Saline Irrigation Waters

Location: Agricultural Water Efficiency and Salinity Research Unit

2023 Annual Report

Objective 1: Generate new tools and techniques for studying and understanding plant responses to salt stress in high value specialty crop plants. Sub-objective 1A: Determine the importance of ion uptake and ion ratios during salinity stress, with emphasis on Na+ and Cl-. Sub-objective 1B: Evaluate the effect of endophytes on the salinity tolerance of horticultural crops. Sub-objective 1C: Determine the effect of priming using different biochemicals to increase salt tolerance in crop plants. Sub-objective 1D: Conduct expression analyses and characterize genes involved in salt tolerance in crop plants. Objective 2: Identify and develop plant material with improved salt tolerance, enabling use of low quality water/alternative waters for irrigation. Sub-objective 2A: Generate and screen alfalfa populations segregating for the component traits of the salt tolerance mechanism to select genotypes with high tolerance to salt. Sub-objective 2B: Identify markers (molecular or biochemical) for salt tolerance and use them in marker assisted selection (MAS) to improve alfalfa germplasm.

This project focuses on salinity responses and underlying mechanisms of high-value specialty crops that includes alfalfa, strawberry, almond, spinach, tomato, eggplant and pepper. In objective 1, we concentrate on understanding relative importance of sodium ions (Na+) and choride ions (Cl-) which will lead to improved prediction of plant response to salinity. Also, the relative importance of Na+ and Cl- may become instrumental in refining breeding or genetic improvement efforts of specific crops. Understanding the mechanism of how plants use Na+ to maintain growth and ion homeostasis may help in development of lines with higher Na+ tissue tolerance. We intend to explore new technologies such as effect of endophytes and priming on salinity tolerance of horticultural crops. The interactions of endophytes/priming with crops will increase knowledge of mechanisms used by plants against abiotic stresses. This knowledge has the potential to mitigate salinity effects on crops with rapid implementation. To understand the genetic changes happening in a genome in response to salinity, we plan to conduct expression and Ribonucleic acid sequencing (RNA-Seq) analyses followed by functional validation of selected genes using model plants. Studying expression of important genes characterized in model plants may help in identifying critical genes involved in salt tolerance of high-value crops. Global gene expression changes detected via RNA-Seq analysis may detect genes or mechanisms that are specific to a particular species. Furthermore, interactions among different pathways may provide a bigger picture of the whole process. Functional complementation of Arabidopsis mutants with candidate genes will confirm evolutionary conservation of the genes involved in the salt tolerance mechanism. This will facilitate development of molecular marker based assays for these genes to screen genotypes tolerant to salt. Additionally, these genes may be manipulated in alfalfa and strawberries for improved salt tolerance. In objective 2, we intend to generate and screen alfalfa populations segregating of the component traits of the salt tolerance mechanism to select genotypes with high tolerance to salt and develop markers for salt tolerance for marker assisted selection (MAS). Crossing genotypes differing for the component traits may lead to development of genotypes with combination of multiple component traits. Pyramiding genes for different component traits will provide enhanced salt tolerance in some of the alfalfa segregating lines. Screening these for salt tolerance will lead to identification of superior lines, which can then be molecularly tested for the presence of the component traits. In addition to selecting for salt tolerant lines we will be able to able to determine importance of different component traits of the salt tolerance mechanism. Once importance of the genes involved in the component traits of the salt tolerance mechanism has been established, molecular markers developed from these lines can be used in MAS. MAS will result in fast and efficient selection of genotypes for salt tolerance.

Progress Report
This is the final report for project 2036-13210-012-000D (Enhancing Specialty Crop Tolerance to Saline Irrigation Waters), which began September 2018. The project achieved its goals and yielded positive results throughout. This project was replaced by new project 2036-13210-013-000D (Understanding and Improving Salinity Tolerance in Specialty Crops), which began March 15, 2023. In support of Sub-objective 1A, ARS researchers in Riverside, California, conducted several studies to explore salinity tolerance in different crops. One study focused on almond rootstocks, evaluating 16 commercial rootstocks under varying salinity levels, showed significant reductions in growth and physiological parameters, with sodium and chloride ions having the greatest impact. However, Rootpac 40, Empyrean 1, Cornerstone, and BB 106 were identified as the top-performing rootstocks under salinity stress, exhibiting low sodium and chloride accumulation. Another study investigated the interaction between almond rootstocks and scions. Further research in support of Sub-objective 1A focused on spinach. The study highlighted the preference of spinach plants for potassium over sodium, clearly showing that spinach plants reject sodium when potassium fertilization is sufficient. High-salinity water did not impact shoot nitrogen, phosphorus, and potassium levels, suggesting that spinach maintains its mineral homeostasis. The researchers also examined the effect of salinity and potassium availability on the phenolic profile and soluble sugars of spinach cultivars. While salinity reduced the leaf antioxidant capacity of 'Raccoon', it slightly increased total phenolics in 'Gazelle'. In another field trial focused on Sub-objective 1A, the salinity tolerance of eight Heirloom genotypes from eggplant, tomato, and pepper was evaluated. This study helped identify cultivars with high and low levels of salt tolerance. High salt tolerance was associated with low leaf sodium concentration, and salt-tolerant cultivars restricted sodium transport from roots to shoots. Additional research in support of Sub-objective 1A involved the evaluation of 24 diverse guar genotypes in a greenhouse lysimeter system. Salt-tolerant and salt-sensitive genotypes were identified based on the salt tolerance index and leaf sodium and chloride concentrations. The study revealed the effective regulation of sodium movement from roots to shoots in guar. Lastly, in support of Sub-objective 1A, research on yellow passion fruit under various salinity levels showed that salinity did not affect the mineral and antioxidant capacity of the leaves. Although leaf sodium and chloride concentrations increased with irrigation water salinity, macro and micronutrient levels remained stable. At higher salinity levels, a reduction in biomass and visual leaf symptoms were observed, probably caused by chloride accumulation. For Sub-objective 1B, ARS researchers conducted two experiments that investigated the impact of endophytes on strawberry's salinity tolerance. Diploid and polyploid strawberry plants were inoculated with fungal and bacterial endophytes and subjected to moderate salinity levels. Bacterial endophytes showed a significant advantage, promoting higher shoot biomass in both species under normal conditions. However, neither fungal nor bacterial endophytes provided benefits for salinity tolerance. Different endophyte strains should be explored in future research to determine their potential for enhancing strawberry's salinity tolerance. Experiments supported Sub-objective 1C by investigating the effect of priming agents on salt tolerance in almond rootstocks. Salinity and five priming agents were evaluated through trunk diameter change in grafted almond plants. However, the tested priming agents, including melatonin, H2S, salicylic acid, and zinc sulfate, did not significantly improve salt tolerance. Further research is needed to explore alternative strategies, or different priming agents, to enhance salt tolerance in almond rootstocks. Sub-objective 1D aimed to investigate gene expression patterns and characterization of genes related to salinity tolerance in various crops. In almond, gene expression analyses were conducted on 25 genes known to play important roles in salinity tolerance across 16 different rootstocks. The results revealed that both chloride-dominant and sodium-dominant waters induced most salt-associated genes during salt stress. The study also highlighted the significance of genes involved in sodium exclusion from roots for rootstock tolerance to salinity stress. To gain a deeper understanding of the genetic responses to salt stress, RNA sequencing (RNA-seq) analysis was performed on the most salt-tolerant and the most salt-sensitive almond genotypes. This analysis identified several genes involved in different pathways that are induced by salt treatment and exhibited differential expression between the sensitive and resistant rootstocks. Among the differentially expressed genes (DEGs), transporter proteins such as SOS2, NHX2, and SOS3 were found. Additionally, genes coding for transmembrane receptors, ion transporters, organic solutes, and hormones showed differential expression. To validate the functionality of some candidate genes, the Prunus genes PpHKT1 and PpSOS2 were transformed into Arabidopsis mutants, and both genes fully complemented the lost salt-tolerance function in these mutants. Investigation in support of Sub-objective 1D, focusing on the conservation of the Salt Overly Sensitive (SOS) pathway, crucial for extruding sodium ions from plant cells under high-salinity conditions, was carried out in spinach. Although the SOS pathway was conserved in spinach, it exhibited some differences in protein-protein interactions compared to the model plant Arabidopsis. This suggests variations in the mode of action of the SOS pathway between spinach and Arabidopsis. Additional research supported Sub-objective 1D with guar RNA expression profiles analyzed in roots and leaves of two genotypes with contrasting salt tolerance. The study revealed the significance of various component traits involved in salinity tolerance mechanisms, including the exclusion of sodium/chloride from roots, sequestration of chloride in root vacuoles, retrieval of sodium/chloride from the xylem, translocation of sodium/chloride from roots to shoots, and the maintenance of potassium-sodium homeostasis. Research on passion fruit, in support of Sub-objective 1D, focused on the expression analyses of 12 transporter genes related to sodium and chloride transport. These genes exhibited higher expressions in roots than leaves, indicating the crucial role of roots in ion transport. The exclusion of sodium from roots into the soil was found to be vital for regulating sodium concentration in passion fruit tissues, while the xylem loading of chloride and the compartmentalization of chloride into vacuoles played important roles in regulating tissue chloride concentration under salinity stress. Still in support Sub-objective 1D, eight genotypes of heirloom eggplant, tomato, and pepper (all Solanaceae) with different salinity tolerance, centered on gene expression analyses of 12 genes involved in sodium and chloride transport. Gene induction in response to salinity outweighed genotype-specific expression. Results showed that sodium exclusion was crucial for eggplant and pepper, while the sequestration of sodium into vacuoles played a critical role in tomato plants. For Sub-objective 2A, alfalfa research focused on evaluating a population generated from crossing two salt-tolerant lines with different traits. Among the F2 genotypes (2nd generation), several showed improved salt tolerance under high-salinity, and 24 salt-tolerant F2 segregants were further assessed for survival at even higher salinity. The most salt-tolerant F2 genotypes were used to produce F3 progenies, which were evaluated at a seawater-salinity level of 50 deciSiemens per metre (dS/m). Only a few genotypes survived without ion toxicity, demonstrating the unexplored potential for breeding salt-tolerant alfalfa varieties for use in salt-affected regions of the United States. Additionally, in support of Sub-objective 2A, almond rootstocks from breeding populations were screened for salinity tolerance. These rootstocks had undergone comprehensive evaluations for various traits. A total of 45 and 36 elite hybrids developed by the University of California (UC) were assessed for salinity tolerance in the 2020-21 and 2021-22 seasons, respectively, based on their survival rate and relative change in trunk diameter. Selected genotypes are now used by our UC collaborators in their breeding programs. For Sub-objective 2B, ARS researchers conducted genome-wide association studies to identify genetic markers linked to maize's salinity tolerance. Using a panel of 399 maize lines, we investigated associations between salt tolerance and early vigor traits. This analysis successfully identified 57 markers significantly associated with early vigor under salinity stress, providing insights into candidate genes for salt tolerance. These findings have implications for breeders, aiding the development of salt-tolerant maize cultivars for marginal lands impacted by high salinity. Additional support of Sub-objective 2B included research that revealed a significant correlation between leaf-K concentrations under salinity and salt tolerance in various crop plants, including tomato, eggplant, pepper, guar, and almond rootstocks. The leaf-K-salinity/leaf-K-control ratio emerged as a more effective marker for selecting salt-tolerant genotypes than the widely used leaf-Na/leaf-K ratio. Additionally, the ratio of leaf-proline salinity/leaf-proline control in almond rootstocks exhibited an inverse correlation with salt tolerance, suggesting that leaf proline is a useful biochemical marker for screening almond genotypes for salt tolerance.

1. Genetic regulation of salinity tolerance in guar. Guar is a drought-tolerant crop adapted to semiarid conditions and its gum is widely used in both oil and natural gas industries. However, literature on guar's tolerance to salinity is lacking. ARS researchers in Riverside, California, evaluated the salinity tolerance in 28 guar genotypes. The study found a strong correlation between the ratio of leaf-potassium (K) concentrations under salinity to control (K-salinity/K-control) and the salt tolerance index for stem and root length, highlighting its importance as a marker for salt tolerance. Salinity treatment had a greater impact on gene expression differences than genotype. The complex network of component traits involved Na and Cl exclusion, as well as tissue tolerance. RNA sequencing of tolerant (Matador) and sensitive (PI 340261) genotypes supported the significance of salinity treatment on gene expression patterns. These findings provide insights into the biological pathways underlying salinity stress and identify candidate genes for salt tolerance in guar. This understanding may become instrumental in developing new guar genotypes tolerant to saline conditions without compromising crop productivity. Our results will help breeders and geneticists develop new salt-tolerant guar varieties that can be irrigated with low-quality saline waters that are not suitable for staple crops and will allow farmers to increase crop yields in marginal lands.

Review Publications
Giglioti, R., Ferreira, J.F., Luciani, G., Louvandini, H., Okino, C., Niciura, S., Oliveira, M., Amarante, A., Katiki, L. 2022. Potential of Haemonchus contortus first-stage larvae to characterize anthelmintic resistance through P-glycoprotein gene expression. Small Ruminant Research. 217. Article 106864.
Sandhu, D., Pallete, A., William, M., Ferreira, J.F., Kaundal, A., Grover, K.K. 2023. Salinity responses in 24 guar genotypes are linked to multigenic regulation explaining the complexity of tolerance mechanisms in planta. Crop Science. 63(2):585-597.
Araújo, A.F., Cavalcante, E.S., Lacerda, C.F., Albuquerque, F.A., Sales, J.R., Lopes, F.B., Ferreira, J.F., Costa, R.N., Lima, S.C., Bezerra, M.A., Gheyi, H.R. 2022. Fiber quality, yield, and profitability of cotton in response to supplemental irrigation with treated wastewater and NPK fertilization. Agronomy. 12(10). Article 2527.
Ferreira, J.F., Liu, X., Suddarth, S., Nguyen, C., Sandhu, D. 2022. NaCl accumulation, shoot biomass, antioxidant capacity, and gene expression of Passiflora edulis f. flavicarpa Deg. in response to irrigation waters of moderate to high salinity. Agriculture. 12(11). Article 1856.
Silva Filho, J., Fontes, P., Ferreira, J.F., Cecon, P., Crutchfield, E. 2022. Optimal nutrient solution and dose for the yield of nuclear seed potatoes under aeroponics. Agronomy. 12(11). Article 2820.
Freitas, E.D., Lacerda, C.F., Amorim, A.V., Ferreira, J.F.S., Costa, C.A.G., Silva, A.O., Gheyi, H.R. 2022. Leaching fraction impacts water use efficiency and nutrient losses in maize crop under salt stress. Revista Brasileira de Engenharia Agricola e Ambiental. 26(11):797-806.
Ashworth, D.J., Ibekwe, A.M., Men, Y., Ferreira, J.F. 2022. Dissemination of antibiotics through the wastewater–soil–plant–earthworm continuum. Science of the Total Environment. 858. Article 159841.