Spinach is a crop of economic importance in the Wintergarden region of Texas, where increasing soil salinity, irrigation water restrictions, and heat stress challenge its production. Improving abiotic stress tolerance and resource use efficiency are important goals for sustainable agricultural production. Although the effect of humic substances (HS) on abiotic stress tolerance in spinach has been evaluated under single stress, such as heat, drought, or salinity, their impact under combined abiotic stresses is unknown. We hypothesized that solid humic substances would be effective in alleviating multifactorial abiotic stress in spinach, which we examined in our first experiment, and further hypothesized that salinity stress in spinach would occur after a threshold intensity but that humic substance applications would ameliorate stress intensity past this threshold, which we examined in our second experiment. Three-week-old spinach seedlings (Spinacia oleracea L. cv. ‘Seaside’) were grown using an automatic gravimetric watering system in a climate-controlled high tunnel where daily maximum temperatures exceeded 32 °C. In a factorial experiment, we applied HS (control and 1 % v/v HS media amendment), drought (100 % and 50 % field capacity), and salinity (25 mL of RO water or 100 mM NaCl every other day) treatments. HS and HS×Salinity treatments more than doubled shoot and root FW and DW, leaf area, root length, and root surface area compared to other stresses, indicating HS improved growth and stress tolerance. Improved growth was matched with improved resource use efficiency, as HS and HS×Salinity treated plants exhibited the greatest WUE. Drought stress also increased WUE relative to the control but lowered transpiration rate responses to VPD in contrasts to HS and HS×Salinity treated plants, which increased their transpiration rates in response to increasing VPD. Salinity treatments increased sodium content (Na+) in both shoots and roots. No HS effect was observed in shoot Na+ accumulation, but HS reduced Na+ in roots under the Drought×Salinity treatment. Strong interactions between drought and salinity were observed for shoot and root Na+, Na+/K+, and shoot K+, showing that spinach accumulates Na+ and reduces K+ when drought and salinity stress are combined. HS had no apparent effect on leaf metabolites. Drought and salinity differentially impacted MDA, proline, and antioxidant content and activities, while HS showed no effect. Drought stress significantly increased MDA and proline while reducing TPC, TFC, ABTS, and FRAP. Salinity stress significantly decreased MDA, TPC, ABTS, FICA, and FRAP. In conclusion, HS improved overall spinach growth and WUE while decreasing root Na accumulation, and subsequently enhancing spinach stress tolerance.
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