Research Project: Enhancing Specialty Crop Tolerance to Saline Irrigation Waters

Location: Agricultural Water Efficiency and Salinity Research Unit

2019 Annual Report

Objectives
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.

Approach
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
To understand underlying genetic and biochemical mechanisms for the salt tolerance process, we evaluated 16 commercial almond rootstocks under five different treatments of irrigation water that included control, sulfate-dominant water with mixed cations, chloride dominant water with mixed cations, sodium-dominant water with mixed anions, and calcium- and magnesium- dominant water with mixed anions. An increased salinity from 1.4 deciSiemens per meter (dS/m) (control) to three dS/m (treatments) caused significant reductions in trunk diameter, chlorophyll Soil-Plant Analyses Development (SPAD), photosynthetic rate (Pn), stomatal conductance (gs), transpiration (Tr) and water use efficiency (WUE) for most rootstocks. The treatment in which sodium and chloride were the predominant ions presented maximum reduction in trunk diameter and survival rate (Sub-objective 1A). Expression analyses were carried out for a set of 10 genes selected for their involvement in salt stress. The expression analyses revealed that the treatment with chloride-dominant water and the treatment with sodium-dominant water both led to induction of majority of salt associated genes during salt stress, suggesting that both chloride and sodium accumulation can be toxic to almonds when using recycled waters high in salt (Sub-objective 1D). Important genes involved in salt tolerance mechanism were identified and the most tolerant rootstocks were selected. Correlations among gene expression, trunk diameter, biochemical markers and tissue ion concentrations allowed us to identify the most critical component of the salt tolerance mechanism in a particular almond genotype, which can then be genetically manipulated to improve its salt tolerance. In support of Sub-objective 1A research on spinach continued on the evaluation of the relationship between sodium and potassium and chloride and nitrate in the cultivars ‘Raccoon’ and ‘Gazelle’. Plants were irrigated with waters of varying salinity levels and two concentrations (low and sufficient) of potassium. Both cultivars substituted potassium for sodium in similar proportions in every salinity treatment, regardless of potassium dose. Plants absorbed significantly less sodium when potassium was sufficient, indicating that roots have a mechanism to favor potassium absorption over sodium. Interestingly, increasing salinity did not affect potassium accumulation in shoots, regardless of the potassium dose. Nevertheless, all plants accumulated enough potassium for growth even at low potassium concentration. The decreased biomass at the high salinity levels was attributed to high tissue chloride accumulation. Based on biomass similarities at the three lower salinity levels, regardless of potassium dose, we concluded that spinach needs much less potassium than previously thought and spinach benefited from sodium for growth when potassium was present in low concentrations, even at the highest salinity concentration. Our data suggest that in spinach, potassium is more efficient in reducing sodium absorption than the opposite. However, sodium seems to be required for plant growth, mainly when potassium is low in the growth medium, which was unexpected as sodium is not considered an essential nutrient for glycophytic plants. Thus, growing spinach with recycled waters of low to moderate salinity does not pose a risk to spinach production. Also, potassium recommendations for the successful growth of spinach, regardless of salinity, must be established to save fertilizer resources and decrease environmental pollution. Two experiments involving a diploid (Fragaria vesca) and a polyploid (Fragaria ananassa) commercial strawberry (‘Albion’) and the effect of two types of endophytes (plant symbiotic microbes), were conducted this year, as well, in support of Sub-objective 1A. The objective of the experiments was to test the effect of fungal and bacterial endophytes on the tolerance of strawberry to salinity provided by irrigation water. Both experiments have been concluded and the plants are being processed for shoot and root biomass while data for physiological parameters (e.g., photosynthetic efficiency) in response to salinity and endophytes are being analyzed. Although Albion has proven to be fairly tolerant to low-salinity irrigation, compared to 4 other commercial cultivars, we are searching for natural alternatives to enhance the salinity tolerance of commercial strawberries. In our experiment with diploid strawberries, both bacterial and fungal endophytes allowed plants grown with low-salinity water to accumulate more shoot biomass than plants without endophytes. However, when saline water was used for irrigation (ECiw = 7.5 dS/m), shoot biomass was reduced in plant with and without endophytes compared to plants grown without salinity. Our data for ‘Albion’ biomass is currently being processed and plants have been sent to the companies that provided us with their bacterial and fungal endophytes to confirm that they were properly inoculated. Although there is no data available for the effect of endophytes on fruit yield, we believe that if endophytes provide a growth advantage for plants under salinity, it could be also an advantage for strawberry growers to maintain fruit yield when using recycled water for irrigation. Additionally, in support of Sub-objective 1A, a vegetable experiment was initiated in summer 2018 evaluating the salt tolerance of eight heirloom (non-hybrid) varieties of each of three species of the same family (tomato, eggplant and pepper). Varieties from regions with different climatic and potentially different salinity levels were selected. Yield was evaluated under control and salinity conditions, significant differences among varieties were identified, and the most and least tolerant of each species were selected for further experiments. Leaf ion analysis and expression of salt tolerance genes will be related to measured salt tolerance. These results are of interest to growers and extension specialists growing vegetables under saline conditions as well as plant breeders developing more salt tolerant varieties. This year collaborative work with the Federal University of Ceara (Brazil) concluded with a study evaluating carbon translocation from leaves to fruits when plants were irrigated with saline water at field level. Our data indicated that carbon translocation continues until the last week of harvest in plants irrigated with saline water. This indicates that irrigation should not be suspended for plants irrigated with saline water as their fruit development is somewhat delayed. Other research in support of Sub-objective 1A included, the completion of yield, soil salinity, and water budget data analysis on the four year grape experiment. 140 Ruggeri rootstock had the highest yield under all treatments. The salt tolerance, defined as yield under saline conditions/yield under control conditions, of Salt Creek grape rootstock was similar to Ruggeri rootstock but significantly greater than St. George rootstock. There were no significant differences in tolerance to water stress but all vines had yield loss when irrigated with water volumes below crop evapotranspiration. We determined that sensitivity to salinity increased over the course of the 4-year experiment, indicating that grape salt tolerance is lower than indicated in short-term experiments. The fruit yields of treatments with combined salt and water stress were not well predicted by the combined stress model due to treatment variability but yield was very close to the 1:1 line for two of the three rootstocks measured. A manuscript has been published in Agronomy journal. This research is of direct use by growers and extension personnel growing grapes under saline conditions and water scarcity. Research in support of Sub-objective 1D focused on further functional validation of almond genes involved in salt stress in which a high-affinity K+ transporter 1 (HKT1) gene from the almond rootstock ‘Nemaguard’ was inserted into Arabidopsis, which generated two transgenic lines. The HKT1 plays a role in removing sodium from the xylem and bringing it back to the root, hence protecting leaves from toxic concentrations of sodium. Both transgenic lines survived salt concentrations up to 120 millimolar sodium chloride (mM NaCl) as compared to the Arabidopsis line lacking HKT1. At 90 mM NaCl, the dry weight of Arabidopsis line lacking HKT1 decreased significantly compared to the transgenic lines. Both transgenic lines showed significantly longer lateral roots, lower electrolyte leakage and higher relative water content compared to the Arabidopsis line lacking HKT1. Our results indicate that transgenic plants coped well with increased salt concentration by maintaining the integrity of cell membranes. The expression analyses showed that HKT1 was induced in transgenic lines under salt treatment, which confirmed that the “Nemaguard” HKT1 can complement the salt tolerance function in Arabidopsis. In our previous work, the role of several genes associated with salt tolerance in alfalfa was established. In support of Sub-objective 2A, this data was used to identify elite alfalfa genotypes different in their genetic components of the salt tolerance mechanism. These parents were crossed to generate populations containing new genotypes with varying salt tolerance. One of the populations under high-salinity conditions is currently being tested (irrigation water salinity (ECiw) of 18 dS/m). Plant biomass is harvested every 35 days and data are recorded. As alfalfa is a perennial forage crop, the testing will continue in the second year of the experiment, which will allow us to identify novel genetic materials (lines) with enhanced salt tolerance.

Accomplishments

Review Publications
Zrig, A., Ferreira, J.F., Hamouda, F., Tounekti, T., Selim, S., Jaouni, S.A., Khemira, H., Abdelgawad, H. 2019. The impact of foliar fertilizers on growth, and biochemical responses of Thymus vulgaris to salinity stress. Arid Land Research and Management. 33(3):297-320. https://doi.org/10.1080/15324982.2018.1551817.
Ferreira, J.F., Sandhu, D., Liu, X., Halvorson, J.J. 2018. Spinach (Spinacea oleracea, L.) response to salinity: nutritional value, physiological parameters, antioxidant capacity, and gene expression. Agriculture. 8(10):163. https://doi.org/10.3390/agriculture8100163.
Kaundal, A., Sandhu, D., Duenas, M., Ferreira, J.F. 2019. Expression of the high-affinity K+ transporter 1 (PpHKT1) gene from almond rootstock 'Nemaguard' improved salt tolerance of transgenic Arabidopsis. PLoS One. 14(3):e0214473. https://doi.org/10.1371/journal.pone.0214473.
Sandhu, D., Pudussery, M.V., Ferreira, J.F., Liu, X., Pallete, A., Grover, K.K., Hummer, K.E. 2019. Variable salinity responses and comparative gene expression in woodland strawberry genotypes. Scientia Horticulturae. 254:61-69. https://doi.org/10.1016/j.scienta.2019.04.071.
Katiki, L.M., Araujo, R.C., Ziegelmeyer, L., Gomes, A.C., Gutmanis, G., Rodrigues, L., Bueno, M.S., Verissimo, C.J., Louvndini, H., Ferreira, J.F., Amarante, A.F. 2019. Evaluation of encapsulated anethole and carvone in lambs artificially- and naturally-infected with Haemonchus contortus. Experimental Parasitology. 197:36-42. https://doi.org/10.1016/j.exppara.2019.01.002.
Wetzstein, H.Y., Porter, J.A., Janick, J., Ferreira, J.F., Mutui, T.M. 2018. Selection and clonal propagation of high artemisinin genotypes of Artemisia annua. Frontiers in Plant Science. 9:358. https://doi.org/10.3389/fpls.2018.00358.
Sandhu, D., Coleman, Z., Atkinson, T., Rai, K.M., Mendu, V. 2018. Genetics and physiology of the nuclearly inherited yellow foliar mutants in soybean. Frontiers in Plant Science. 9:471. https://doi.org/10.3389/fpls.2018.00471.
Grant, N., Mohan, A., Sandhu, D., Gill, K.S. 2018. Inheritance and genetic mapping of the reduced height (Rht18) gene in wheat. Plants. 7(3):58. https://doi.org/10.3390/plants7030058.
Pacheco, P.A., Rodrigues, L.N., Ferreira, J.F., Gomes, A.C., Verissimo, C.J., Louvandini, H., Coasta, R.L., Katiki, L.M. 2018. Inclusion complex and nanoclusters of cyclodextrin to increase the solubility and efficacy of albendazole. Parasitology Research. 117(3):705-712. https://doi.org/10.1007/s00436-017-5740-3.
Ferreira, J.F., Liu, X., Suarez, D.L. 2019. Fruit yield and survival of five commercial strawberry cultivars under field cultivation and salinity stress. Scientia Horticulturae. 243:401-410. https://doi.org/10.1016/j.scienta.2018.07.016.
Liu, S., Ferreira, J.F., Tian, D., Tang, Y., Liu, L., Yang, W., Liu, Z., Tian, N. 2017. Preparative separation of high-purity dihydroartemisinic acid from artemisinin production waste by combined chromatography. Chemical and Pharmaceutical Bulletin. 66(3):319-326. https://doi.org/10.1248/cpb.c17-00927.
Suarez, D.L., Celis, N., Anderson, R.G., Sandhu, D. 2019. Grape rootstock response to salinity, water and combined salinity and water stress. Agronomy. 9(6):321. https://doi.org/10.3390/agronomy9060321.