EXPLORING THE BENEFITS OF BRACKISH WATER AND HALOPHYTES FOR HUMAN HEALTH

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1025361
• Grant No.: 2021-67014-33855
• Cumulative Award Amt.: $190,000.00
• Proposal No.: 2020-03393
• Project Start Date: Jan 15, 2021
• Project End Date: Jan 14, 2023
• Grant Year: 2021
• Program Code: [A1103]- Foundational Knowledge of Plant Products

Recipient Organization
NEW MEXICO STATE UNIVERSITY
1620 STANDLEY DR ACADEMIC RESH A RM 110
LAS CRUCES,NM 88003-1239

Performing Department
Plant Environmental Sciences

Non Technical Summary
Semiarid agriculture and nutritional security are at risk from long-term severe drought conditions. To ease the burden of irrigation withdrawals on the fresh water supply, significant brackish groundwater reserves are available. The stress of brackish water salinity activates plant antioxidant defense systems that include the production of antioxidants and other protective molecules. In most crops, production of these human health-promoting secondary metabolites is limited as salinity increases. By contrast, salinity is an undervalued property for salt tolerant halophytes in that their growth and metabolism are maintained with increasing salinity. The goal of this SEED project is to reveal brackish groundwater as a value-added product that, in turn, increases the value of native halophytes through increased production of human health-promoting secondary products for natural dietary supplements and functional food additives. Three native halophytes, Atriplex canescens (fourwing saltbush), A. lentiformis (big saltbush), and Lepidium alyssoides (mesa pepperwort), will be irrigated in a greenhouse for up to 12 weeks with brackish groundwater (5 dS/m) and desalination concentrate (8 dS/m). We will evaluate biomass production, ion uptake and distribution, accumulation of phenolics and glucosinolates, potential phytotoxins, and in vitro bioavailability of the extracted secondary metabolites. We will quantify total phenolics and total antioxidant activity, and identifyspecific phenolics, glucosinolates, and potential phytotoxins. Analytical results will be used to construct cost:return projections for greenhouse production, harvesting, processing, extraction, and analysis of phenolics and glucosinolates in these plant species. A webinar will be broadcast to share the results with local and regional stakeholders to include greenhouse and nursery growers, herbal dietary supplement manufacturers, herbalists, and the public. The potential outcomes of this study will include an increased interest in halophytes as an economically viable crop selection in greenhouse and nursery crop portfolios and in the phytomedicinal products industry, and a change in the perception of salinity from a threat to a value-added product for sustaining agriculture in semiarid regions. Experimental results will be integrated into a framework to launch our long-term goals of expanded study on more indigenous halophyte species and the isolation of new plant products with increased nutritional and medicinal value.

Animal Health Component

0%

Research Effort Categories

Basic

100%

Applied

0%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
102	2220	1020	50%
203	2299	1000	50%

Knowledge Area
102 - Soil, Plant, Water, Nutrient Relationships; 203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
2220 - Medicinal crops, non-narcotic; 2299 - Miscellaneous and new crops, general/other;

Field Of Science
1000 - Biochemistry and biophysics; 1020 - Physiology;

Keywords

brackish water

food quality

human health

pharmaceutical

plants

salinity

Goals / Objectives
The goal of this project is to reveal brackish water as a value-added product that increases the production of beneficial human health-promoting secondary metabolites by native halophytes.Objective 1: Evaluate biomass production and salt uptake of Atriplex canescens, A. lentiformis, and Lepidium alyssoides irrigated with brackish groundwater and desalination concentrate. Establish a brackish irrigation water:greenhouse production system and identify value and limitations of these impaired water sources.Objective 2: Assess yield and bioavailability of secondary metabolites (total phenolics, antioxidant activity, and glucosinolates) in A. canescens, A. lentiformis, and L. alyssoides using the greenhouse production system, spectrophotometry, and high performance liquid chromatography.Objective 3: Perform broadband characterization of specific phenolics, glucosinolates, and potential phytotoxins in A. canescens, A. lentiformis, and L. alyssoides from the greenhouse production system, whileusing ultra performance liquid chromatography and ultra high resolution mass specrometry.Objective 4: Construct simple variable cost:economic return projections for the production, harvesting, processing, extraction, and analysis of phenolics and glucosinolates in A. canescens, A. lentiformis, and L. alyssoides grown in the greenhouse production system.Objective 5: Hold a webinar for local and regional greenhouse and nursery producers, herbal dietary supplement manufacturers, and herbalists to report experimental findings and their implications for the phytomedicinal products industry, to collect feedback on information learned, and to strengthen the stakeholder and industry components for the next grant proposal submission.

Project Methods
Stakeholder involvement. Local stakeholders support our project plan and implementation, although the need for our study throughout the U.S. greenhouse and nursery industry is apparent. Our proposal also addresses needs identified by members of the USDA W-3170 and by the dairy industry.Sequential list of project activities. Experiments 1, 2 completion; plant processing, analysis; data analysis; stakeholder and AFRI-PD meetings; NIFA final report; NM-WRRI, ASHS meetings; peer-reviewed journal article submissions; prepare for next proposal. Greenhouse cultivation and harvest. Our 90-d greenhouse protocol for plant establishment, irrigation, fertilization, and harvest has been perfected and used repeatedly (Flores et al., 2016, 2017; Ozturk et al., 2018; Hooks et al., 2018a, 2018b; Picchioni et al., 2020). The plants will be arranged in a randomized complete block design with four replications, each comprised of three, 14-cm-tall grow cells supporting single seedlings. Three sub-irrigation treatments will include a tap water control (0.6 dS/m), brackish groundwater from BGNDRF (4 to 5 dS/m), and brackish groundwater RO concentrate from BGNDRF (8 dS/m). Two duplicate experiments will be run, each with a total treatment duration of 12 wk. Plant populations will be established to provide destructive harvests at0, 2, 4, 8, and 12 wk.Ion analysis and sample preparation. Monthly analyses of the irrigation treatment solutions by the PES plant, soil, and water testing lab will include alkalinity, EC, pH, cations, Cl, NO3, P, S, and 20 heavy metals. At all harvests shoots will be frozen in liquid N2, lyophilized, weighed, and stored at -80C to await analysis. Dried tissues will be ground to pass a 40-mesh screen and 0.25-mg subsamples will be extracted and analyzed for Na, Ca, Mg, Cl, and S (Hooks et al., 2018a). At only the final harvest, roots will be washed and processed as described for shoots.Total phenolics and antioxidant activity. In methanol extracts of ground subsamples, total phenolics will be determined by the Folin-Ciocalteu procedure (Guzman et al., 2015). Antioxidant activity will be measured by the DPPH method (Sharma and Bhat, 2009).Glucosinolate analysis. For L. alyssoides only, glucosinolates will be extracted using the hot methanol method described in Peterson et al. (2002). Following extraction, HPLC methods for quantifying glucosinolates will follow Doheny-Adams (2017). Solvents will be 0.02 M tetrabutylammonium bromide (A) and a 70:30 mixture of tetrabutylammonium bromide and acetonitrile (B). The initial concentration will start at 0% B and increase to 100% B over 30 min, holding for 5 min and then returning to the initial concentration. External glucosinolate standards at 1.25, 2.5, 5, 10, and 20 mM of sinigrin, glucoiberin, glucocherolin, glucotropeolin, glucoraphanin, glucobrassicin and gluoconapin (the primary gluocosinolates in Lepidium spp.; Kaur et al., 2013) will be prepared in triplicate and analyzed with the extracted samples.Simulated human digestion model. To exert their medicinal properties, glucosinolates and phenolics must be absorbed by the human gut. Thus, we will conduct bioavailability assays using an accepted and well-established in vitro digestion model for gut absorption of phytocompounds with medicinal bioactivity in humans (Nielson and Ferruzzi, 2013). The model is currently being used in I. Guzman's lab through a funded project looking at simulated digestions of medicinal compounds. The first step is the oral phase where the raw material is mixed with a synthetic saliva and human oral enzymes. The second phase is the gastric phase where the plant-containing oral phase undergoes acidification (pH 2) in the presence of pepsin. The sample enters an intestinal simulation where the pH is increased to 6.5, and pancreatin, lipase and bile are added. The samples are then centrifuged for two hours to mimic peristalsis, or intestinal contraction. A digesta and an aqueous sample are collected. The digesta will represent all material entering the small intestine, while the aqueous fraction will represent only the bioavailable metabolites. Total phenolics and glucosinolates will be measured as described previously, and the percent amounts that are available for absorption (bioavailability) will be determined as [(aqueous fraction ÷ digesta) X 100].Preliminary identification of phenolic compounds and toxins. During the grant performance period, I. Guzman will develop an HPLC method for phenolic compound identification and quantification for future proposals; this effort will be expedited by preliminary identifications in T. Schaub's lab. Using an Orbitrap Fusion Tribrid mass spectrometer, ultrahigh resolution mass spectrometry (MS) will be performed in T. Schaub's lab onrepresentativemethanolic extracts produced in I. Guzman's lab, to include all treatments and species. We have budgeted for50 such samples in T. Schaub's analyses. Direct infusion MS will initially identify the distribution of potential phenolic compounds. Extracts will then be analyzed by ultraperformance liquid chromatography/ultrahigh resolution MS for identification of toxins and for confirmation of phenolic compound molecular identifications combined with library matching of fragment ion mass spectra. Available spectral libraries include WEIZMASS, METLIN, mzCloud and NIST 2017. For more information, see budget and documents of collaboration (T. Schaub).Expected results, data analysis, and use of data. Total phenolic and glucosinolate concentrations will be multiplied by total root or shoot biomass to obtain metabolite yields (amounts). The plant response variables will include dried biomass, ion concentrations and amounts, and total phenolic and glucosinolate concentrations and amounts. We will assess the possibility that quality of the raw material is positively affected by salt accumulation in specific organs by intensifying the stress response. A three-way (factorial) analysis of variance will be performed (two experiments X three species X three saline treatments) using the general linear model of SAS software version 9.3 (SAS Institute, Cary, NC). Significant treatment main effects will be evaluated by mean separation using Duncan's multiple range test at an alpha of 0.05. Preliminary identification of phenolic compounds and of toxins will be included in our reporting. A supplemental objective will include the construction of simple variable cost:return analysis using our laboratory data followed by scaling to greenhouse and medicinal product industry standards. The latter results will be shared in a cooperative extension circular for reporting the economic potential for native halophytes to provide new high valueplant-based industrial and food products. A broader intent is to use our data as a case for further testing, the identification of additional halophytes suitable for high-value secondary products production with brackish groundwaters.Communication plan. The project director (PD) will present progress at the AFRI PD meeting and submit annual andfinal project reports to NIFA. We will host a stakeholder webinarto report findings to regional greenhouse and nursery producers, herbal and dietary supplement manufacturers, and practicing herbalists. The webinar will be archived for public viewing. Following pre-approval from our IRB, stakeholder and public feedback will inform the research team for strengthening the project, assessing the outcomes of the project (see outcomes section), constructing the cost:return analysis, and refininglong-term research goals.The graduate student will present the findings at the annual conferences of the New Mexico Water Resources Research Institute (NM-WRRI) and the American Society for Horticultural Science (ASHS). The PD will mentor the graduate student in preparing and submitting two peer-reviewed journal article manuscripts.

Progress 01/15/21 to 01/14/23

Outputs
Target Audience:Our academic audience includes research scientists at New Mexico Water Resources Research Institute, American Society for Horticultural Science, and American Society of Agricultural and Biological Engineers-New Mexico Section. Our federal, county, industry, and individual stakeholders are from US-Bureau of Reclamation's Brackish Groundwater National Desalination Facility, practicing herbalists, specialty crop producers, regional greenhouse and nursery managers, cooperative extension service, and members of the public. Changes/Problems:Methods changes. Per the project initiation document, there was one methods deletion and one methods addition. Due to lack of saline solution treatment effects on secondary metabolite production in the halophyte experiments, we did not conduct the planned study on our simulated human digestion model to determine bioavailability of the metabolites produced. In lieu of the digestion model, we designed and completed duplicate greenhouse experiments on two domesticated leafy green vegetable crops, leaf lettuce (glycophyte) and Swiss chard (halophyte). In these experiments, we compared salt stress effects with salt shock effects on total phenolics and total antioxidants. The results are discussed in the accomplishments section. Problems, personnel time, and practicability of brackish groundwater treatment. • Greenhouse ventilation problems diverted personnel time away from the project on two occasions. • Appearance of insect pests in the greenhouse, including whiteflies, loopers, fungus gnats, and thrips, necessitated unplanned time for the development of an aggressive non-chemical control plan aided with recommendations by the head grower at Masson Farms of New Mexico Inc. • RO concentrate was not available at BGNDRF for greenhouse halophyte experiment #2 and for both vegetable crop experiments. Thus, unexpected efforts were necessary to develop a laboratory salt formula to successfully simulate the RO concentrate solution treatment. • The pandemic imposed unintended effort to deal with new greenhouse, laboratory, and travel protocols for this research project. The pandemic forced campus instruction to 100% online delivery. These issues diverted all the PD's time in spring 2021 that slowed progress on the project. • The RO concentrate solution was at the limit of gypsum solubility. We noted a gradual, 5% decline in the electrical conductivity of the RO concentrate solution over a 6-wk period in greenhouse halophyte experiment #1, due to precipitation and formation of calcium-bearing minerals like dolomite, calcite, aragonite, and gypsum. A realistic level of RO treatment of gypsiferous well waters that are typical for BGNDRF must be carefully determined to ensure ion solubility and prevent scaling-induced perturbations in conveyance, storage, treatment, and use of the brine concentrate. This underappreciated side of Ca SO4-dominant groundwaters has important implications for as much as 34% of the BGW in the U.S. which has been classified as gypsiferous by U.S. Geological Survey. Especially noteworthy for brackish well #3 water used in the experiments is the low recovery of permeate water (i.e., about 50%) to avoid calcium precipitation, i.e., "scaling". This limitation supports our project. That is, the low recovery results in a high volume of RO concentrate to be managed which is costly by conventional means. This demonstrates the importance of finding cost-effective and value-added ways of dealing with this water rather than through costly disposal. • In the RO concentrate supply, the concentration of cadmium exceeded the USEPA primary drinking water maximum contaminant level of five parts per billion. This represented another negative side effect of RO processing of brackish groundwater. What opportunities for training and professional development has the project provided?One postdoctoral researcher increased their training in LC-MS for plant analysis, particularly in providing preliminary identification of phenolic compounds. One undergraduate student and one graduate student received training in the following areas to execute the experiments safely and successfully: Fundamentals of Laboratory Safety and HazCom. Laboratory Safety Refresher Course. Annual Strategic and Essential Training. Setting up greenhouse sand culture system. Preparing complete Hoagland nutrient solution concentrated stock and irrigation solutions. Preparing laboratory saline solutions for irrigation use. Gravimetric method for determining plant irrigation frequency and amount. Calculating and graphing plant evapotranspiration. Harvesting experiments and preserving samples by freezing and lyophilization. Weighing, grinding and extraction of plant tissues for secondary metabolite and ion analysis. Conducting total phenolic and total antioxidant activity assays using spectrophotometry. Conducting analyses on HPLC, ICP, and autoanalyzer. Using LC-MS data and Van Krevelen diagrams to identify a concise list of plant phenolic compounds. Data management with lab notebooks, spreadsheets, and SAS. Creating posters and powerpoint presentations. Interacting with US-BOR BGNDRF technical support staff. Stakeholder meeting: organizing, conducting, recording minutes, Q&A, follow-up correspondence. Protocol for simulating ROC with lab salts to produce experimental irrigation solutions. Insect biocontrol and use of biopesticides. Statistical analysis. Data processing. Biochemistry. Technical writing. Presenting findings at national technical conferences. How have the results been disseminated to communities of interest? Virtual stakeholder's conference from Las Cruces, NM on December 20, 2022. The conference included clientele from local greenhouse and nursery production industries, U.S. Bureau of Reclamation (Brackish Groundwater National Desalination Research Facility), specialty crop groups, growers, cooperative extension, and faculty from the NMSU Department of Plant and Environmental Sciences. Graduate student poster presentation on brackish groundwater effects on native halophytes. American Society for Agricultural and Biological Engineers-New Mexico Section. Las Cruces, NM. October 21, 2022. Graduate student poster presentation at the American Society for Horticultural Sciences Annual Conference in Chicago, IL on July 31, 2022. Lucker A, Consford J, Guzman I, Schutte B, Shukla M, Trainor P, Picchioni G. 2022. Comparing secondary metabolite production in Swiss chard and leaf lettuce irrigated with brackish groundwater. HortScience 57(9):S199. Graduate student poster presentation at the American Society for Horticultural Sciences Annual Conference in Chicago, IL on July 31, 2022. Lucker A, Consford J, Guzman I, Schutte B, Shukla M, Trainor P, Picchioni G. 2022. Utilization of brackish groundwater in halophytes for human health. HortScience 57(9):S21. Graduate student poster presentation on brackish groundwater effects on native halophytes. College of ACES Open House. Las Cruces, NM. April 9, 2022. Graduate student poster presentation on brackish groundwater effects on native halophytes. Fabian Garcia Agricultural Science Center Field Day. Las Cruces, NM. September 22, 2021. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1: Evaluate biomass production and salt uptake of Atriplex canescens (Pursh) Nutt., A. lentiformis (Torr.) S. Watson, and Lepidium alyssoides A. Gray var. alyssoides irrigated with brackish groundwater and desalination concentrate. Identify value and limitations of the impaired water sources. Significant effects of saline treatment, plant species, and the treatment X species interaction were observed (P < 0.05). Key Outcomes and Impact. Brackish groundwater stimulated growth of A. lentiformis and all species utilized Ca and SO4-S (the major inorganic constituents) as beneficial resources. Lepidium alyssoides and A. lentiformis recovered 10% of the Ca and SO4-S applied in the RO concentrate treatment. The plants could use these electrolytes in growth, osmotic adjustment, nutrient storage, and secondary metabolism. Thus, rather than a waste stream, RO concentrate should be considered as a value-added asset. Sodium chloride effects dominate the crop salinity database. Our data showed that the mixed saline solutions of BGW and RO concentrate produce comparable results as their counterpart NaCl irrigation solutions. This indicates that the diverse BGW chemistries of the U.S. may provide similar effects on growth and physiology of native halophytes, which is a timely addition to the database. Objective 2: Assess concentration, yield, and bioavailability of secondary metabolites (total phenolics, antioxidant activity, and glucosinolates) in A. canescens, A. lentiformis, and L. alyssoides using the greenhouse production system, spectrophotometry, and high-performance liquid chromatography. We evaluated the concentrations and yields (amounts) of total phenolics (TP), total antioxidants (TA), and total glucosinolates of A. canescens, A. lentiformis, and L. alyssoides subjected to the experimental conditions described in Objective 1. Our results showed no effect of saline solution on TP, TA, or glucosinolates in any of the species at an alpha of 0.05. A question arose as to whether shorter salt acclimation times than 48 h could elicit a greater response in TP and TA. This possibility was investigated in expanded study on short cycle domesticated leafy green vegetable crops, glycophyte leaf lettuce (Lactuca sativa L. 'Red Salad Bowl') and halophyte Swiss chard (Beta vulgaris L. var. cicla 'Bright Lights'). Salt stress (gradual, stepwise exposure to salinity) with RO concentrate (CaSO4-dominant) and NaCl at 8 dS/m increased TP concentrations in lettuce by 20% (as expected from the literature) with no increase in Swiss chard. Salt shock (sudden exposure to salinity) increased TP concentrations of Swiss chard by 15% to 20%. Identical results were obtained with the CaSO4-dominant RO concentrate and NaCl-only solutions. Key Outcomes and Impact. Salt shock results from this study should be of interest to the scientific body. It is an unconventional research practice and an equally uncommon natural phenomenon but may be a practical approach for improving plant secondary metabolite concentrations. Our findings may stimulate more research to refine salt shock treatments for phytonutrient production. Saline irrigation solution composition effects in phytochemical research are a grossly underappreciated subject as NaCl is the dominant salt used in research trials. Our findings show that NaCl and CaSO4 produced similar effects on secondary metabolic traits of four halophyte and one glycophyte species. Considering the project outcomes, the body of knowledge in irrigated agriculture and plant secondary products could aim higher by accounting for the diversity in water quality of our aquifers and waste streams. Most studies report concentrations of secondary metabolites rather than amounts or yields. The lack of appreciation for secondary metabolite yields is holding back the assessment of economic return of bioactive products. Our study showed species differences in metabolite yields (i.e., amounts or quantities) per experimental unit, and the differential impact of salt stress and salt shock on total phenolic yields. Thus, our findings may encourage future research on plant secondary products to adopt a reporting protocol that adds metabolite yields to allow practical application of the results. Our findings will highlight the potential for BGW and brine concentrate to be used as irrigation sources without any treatment while simultaneously increasing the value of crops as an enticing proposition. It could provide a revenue stream for farmers and desalination operations, and help turn impaired water sources into financial assets rather than liabilities. Objective 3: Perform broadband characterization of specific phenolics, glucosinolates, and potential phytotoxins in A. canescens, A. lentiformis, and L. alyssoides from the greenhouse production system, whileusing ultra performance liquid chromatography and ultra-high resolution mass spectrometry. Key Outcomes and Impact. Preliminary identifications were made of phenolic compounds known to be beneficial to human health, including piceatannol 3-o-glucoside, ursolic acid, protocatechuic acid, catarrhine, and chlorogenic acid. Objective 4: Construct simple variable cost:economic return projections for the production, harvesting, processing, extraction, and analysis of phenolics and glucosinolates in A. canescens, A. lentiformis, and L. alyssoides grown in the greenhouse production system. Simple cost and return analysis for phenolics and glucosinolates in the native halophytes was not conducted due to the lack of saline irrigation solution treatment effects noted previously. However, we made an approximation for estimated potential revenue that could arise from the leaf lettuce experiments in Objective 2. Key Outcomes and Impact. Considering our salt shock data on leafy greens--dried shoot biomass and TP concentrations--along with a range of retail prices for polyphenols in dietary supplements, number of plants per square foot, and number of crops per year in greenhouse conditions, an additional $1 to $2 per square foot in revenue could be realized. This approximate dollar value does not include the basal crop value and other dietary components like total protein, fiber, vitamins, and minerals. It considers only the incremental value attributable to the increases in TP concentration by RO concentrate or NaCl solution irrigation (8 dS/m) over a 15-wk cycle. These findings are a positive step to increase awareness on value-added health benefits attributable to impaired water sources. Objective 5: Hold a webinar for local and regional greenhouse and nursery producers, herbal dietary supplement manufacturers, specialty crop growers, federal partners, and herbalists. We held a virtual stakeholder's conference on December 20, 2022, that included clientele from local greenhouse and nursery production industries, U.S. Bureau of Reclamation (Brackish Groundwater National Desalination Research Facility), specialty crop groups, growers, cooperative extension, and our Department of Plant and Environmental Sciences. Key Outcomes and Impact. We reported on our research results, after which a Q & A period followed. We recorded comments and suggestions to aid in dissemination of our findings and in future research. Several attendees commented that the meeting was "fun" and enjoyed being briefed on our research. Specific interests by stakeholders in attendance were expressed as follows: Protocol and infrastructure for salt shock treatments. Market potential for secondary metabolite enriched specialty crops. Research on diversity of RO concentrates with different compositions. Consumer "buy-in" for halophytes as dietary supplements. Shock salinity as an "elicitor".

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Kankarla V, Shukla MK, Picchioni GA. 2021. Root growth, architecture, and ion uptake of alfalfa and triticale irrigated with brackish groundwater and reverse osmosis concentrate. Agrosystems, Geosciences, Environment. https://doi.org/10.1002/agg2.20180.
• Type: Other Status: Published Year Published: 2021 Citation: Shukla MK, Ben Ali A, Cerra S, Schutte B, Picchioni GA, Gard C. 2021. Irrigation with brackish groundwater and desalination concentrate: Effect on microbial properties, plant uptake, and ion deposition in soil. New Mexico Water Resources Research Institute Technical Completion Report TR 397 (47 p). NM-WRRI, Las Cruces. Available online at https://cduaws.nmwrri.nmsu.edu/wp-content/uploads/PDF/tr397.pdf.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Lucker A, Consford J, Guzman I, Schutte B, Shukla M, Trainor P, Picchioni G. 2022. Comparing secondary metabolite production in Swiss chard and leaf lettuce irrigated with brackish groundwater. HortScience 57(9):S199.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Lucker A, Consford J, Guzman I, Schutte B, Shukla M, Trainor P, Picchioni G. 2022. Utilization of brackish groundwater in halophytes for human health. HortScience 57(9):S21.
• Type: Theses/Dissertations Status: Under Review Year Published: 2023 Citation: Lucker, A. 2023. Brackish groundwater effects on biomass, water use, mineral uptake, and secondary metabolite production in native halophytes. M.S. Thesis, New Mexico State University, Las Cruces, NM.

Progress 01/15/21 to 01/14/22

Outputs
Target Audience:Four commercial greenhouse and nursery managers visited our greenhouse experiments in progress,representing Sunland Nursery Inc., Masson Farms of New Mexico Inc., and Plant Propagation Technologies Inc.Technical and supervisory collaborating personnel offederal partner,U.S. Bureau of Reclamation's Brackish Groundwater National Desalination Research Facility (BGNDRF),were briefed on our project andprovided samples of brackish groundwater (BGW)and BGW reverse osmosis(RO) brine concentrate.Members of the border region publicattendeda local field day that, in part, highlighted his project. Changes/Problems:There have been no substantive changes to the project. The following is a list of problems that have been circumvented with necessary but slight adjustments to our greenhouse plant production and laboratory protocols. Greenhouse fan and ventilator malfunction resulted in short-term periods of supraoptimal air temperatures in the greenhouse. Appearance of insect pests in the greenhouse, including whiteflies, loopers, fungus gnats, and thrips, necessitated the development of an aggressive non-chemical control plan aided with recommendations by the head grower at Masson Farms of New Mexico Inc. More information on the control plan is available from the PD. RO concentrate was not available at BGNDRF for greenhouse experiment #2.Thus, unplanned time was necessary to successfully simulate the brine concentrate solution irrigation treatment with laboratory salts during experiment #2. The RO concentrate solution irrigation treatment was at the limit of gypsum solubility.We noted a gradual, 5% decline in the electrical conductivity in the RO concentrate solution over a 6-wk period in greenhouse experiment #1, due to precipitation and formation of calcium-bearing minerals like dolomite, calcite, aragonite, and gypsum. A realistic level of RO treatment of gypsiferous well waters (characteristic of BGNDRF) must be carefully determined to ensure ion solubility and prevent scaling-induced perturbations in conveyance, storage, treatment, and use of the brine concentrate.This underappreciated side of calcium sulfate dominated groundwaters has important implications for as much as 34% of the BGW in the U.S. which has been classified as gypsiferous by U.S. Geological Survey. Especially noteworthy for brackish well #3 water used in the experiments is the relatively low recovery of permeate water (i.e., about 50%) to avoidcalcium precipitation. This limitation supports our project. That is, the low recovery results in a relatively high volume of RO concentrate to be managed, which further justifies cost-effective and value-added ways of dealing with this water rather than through costly disposal. The pandemic imposed unexpected time constraints to deal with new greenhouse, laboratory and travel protocols for this research project. The pandemic also forced campus instruction to 100% online delivery, which required all of the PD's time in spring 2021. What opportunities for training and professional development has the project provided?One postdoctoral Research Associate increased their training inacidified methanolicplant extracts onLC-MS analysis from our plant samples to provide preliminary identification of phenolic compounds.One undergraduate student and one graduate student received training in the following areas in order to execute the experiments: Fundamentals of Laboratory Safety and HazCom by NMSU Department of Environmental Health, Safety, and Risk Management. Annual Strategic and Essential Training Program (law, policy, regulation, and compliance) by NMSU Center for Learning and Professional Development. Setting up greenhouse sand culture system. Preparing complete Hoagland nutrient solution concentrated stock and irrigation solutions. Preparing laboratory saline solutions for irrigation use. Gravimetric method for determining plant irrigation frequency and amount. Calculating and graphing plant evapotranspiration. Harvesting experiments and preserving samples by freezing and lyophilization. Weighing, grinding and extraction of plant tissues for secondary metabolite and ion analysis. Conducting total phenolic and total antioxidant activity assays using spectrophotometry. Conducting analyses on HPLC, ICP, and autoanalyzer. Data management with lab notebooks, spreadsheets, and SAS. Using linear regression analysis. Creating posters and powerpoint presentations. Interacting with US-BOR BGNDRF technical support staff. How have the results been disseminated to communities of interest?One poster was presentedand one poster abstract submitted by the graduate student (see products and other products sections). Two publications resulted from our team's work onbrackish groundwater (seeproducts). What do you plan to do during the next reporting period to accomplish the goals? Complete greenhouse and lab analyses for both experiments #1 and #2. Complete all SAS analyses. Conduct stakeholder meeting. Complete and submit peer reviewed manuscript on biomass, water use and ion uptake of experiments #1 and #2. Run in vitro human digestion and bioavailability assay on selected samples. Complete manuscript draft for secondary metabolite production in experiments #1 and #2. Discuss secondary metabolite results with CES personnel in reference to a possible CES publication, pending outcomes of the experiments. Conduct simple cost:return analysis of phenolics and glucosinolates from the greenhouse production system. Present results at NM-WRRI and ASHS. Begin work on next grant proposal. Submit final report.

Impacts
What was accomplished under these goals? Objectives 1, 2, and 3.Picchioni and colleagues received funding from USDA-NIFA-AFRI to investigate the effects of brackish groundwater (BGW) and reverse osmosis (RO) concentrate on biomass, water use, ion uptake, and secondary metabolites in the halophytes, L. alyssoides, Atriplex canescens, and A. lentiformis. The greenhouse culture system was established with special precautions made for gypsum solubility limit in the RO concentrate solution treatment. Two duplicate greenhouse experiments were conducted and included a tap water control (0.8 dS/m), BGW (4 dS/m), NaCl (4 dS/m), BGW RO concentrate (8 dS/m), and NaCl (8 dS/m) as solution irrigation treatments. After 0, 3, and 6 wk, plants were harvested and processed for analysis.Data analysis in experiment #1 and lab analysis for experiment #2 are in progress.To disseminate the preliminary data of this research, the graduate student presented a poster at the annual Fabian Garcia Agricultural Science Center Field Day, and submitted a poster abstract to the New Mexico Water Resources and Research Institute (NM-WRRI) Annual Conference. Objectives 4 and 5. Nothing to report.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Kankarla, V., M.K. Shukla, and G.A. Picchioni. 2021. Root growth, architecture, and ion uptake of alfalfa and triticale irrigated with brackish groundwater and reverse osmosis concentrate. Agrosystems, Geosciences, Environment. https://doi.org/10.1002/agg2.20180.
• Type: Other Status: Published Year Published: 2021 Citation: Shukla, M.K., A. Ben Ali, S. Cerra, B. Schutte, G.A. Picchioni, and C. Gard. 2021. Irrigation with Brackish Groundwater and Desalination Concentrate: Effect on Soil Microbial Properties, Plant Uptake, and Ion Deposition in Soil. New Mexico Water Resources Research Institute Tech. Compl. Rept. TR 397 (47 pp.). NM-WRRI, Las Cruces. Available online at https://cduaws.nmwrri.nmsu.edu/wp-content/uploads/PDF/tr397.pdf.
• Type: Conference Papers and Presentations Status: Submitted Year Published: 2021 Citation: Lucker, A., Consford, J., and Picchioni, G. Exploring the Benefits of Brackish Groundwater on Native Halophytes for Human Health. Poster abstract submitted to 66th Annual New Mexico Water Conference, New Mexico Water Resources and Research Institute (October 26, 2021).