Stable water isotopes have been used extensively to study the water use strategy of plants in various ecosystems. In deep vadose zone (DVZ) regions, the rooting depth of trees can reach several meters to tens of meters. However, the existence of roots in deep soils does not necessarily mean the occurrence of root water uptake, which usually occurs at a particular time during the growing season. Therefore, quantifying the contribution of deep-layer soil water (DLSW) in DVZ regions using the natural abundance of stable water isotopes may not be accurate because this method assumes that trees always extract shallow- and deep-layer soil water. We propose a multi-step method for addressing this issue. First, isotopic labeling in deep layers identifies whether trees absorb DLSW and determines the soil layer depths from which trees derive their water source. Next, calculate water sources based on the natural abundance of stable isotopes to quantify the water use strategy of trees. We also compared the results with the natural abundance of stable water isotopes method. The 11- and 17-year-old apple trees were taken as examples for analyses on China’s Loess Plateau. Isotopic labeling showed that the water uptake depth of 11-year-old apple trees reached 300 cm in the blossom and young fruit (BYF) stage and only 100 cm in the fruit swelling (FSW) stage, whereas 17-year-old trees always consumed water from the 0–320 cm soil layer. Overall, apple trees absorbed the most water from deep soils (> 140 cm) during the BYF stage, and 17-year-old trees consumed more water in these layers than 11-year-old trees throughout the growing season. In addition, the natural abundance of stable water isotopes method overestimated the contribution of DLSW, especially in the 320–500 cm soil layer. Our findings highlight that determining the occurrence of root water uptake in deep soils helps quantify the water use strategy of trees in DVZ regions.
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