Growth conditions and plant material.

The 2-year experiment, spanning from 2022 to 2023, was conducted at the John I. Haas Golding Farms, located near Toppenish, WA, USA (46.366°N, −120.388°W, 236 m of elevation), to investigate the impact of withholding irrigation water late in the season on physiological response, yield, and quality metrics of Humulus lupulus ‘Cascade’, a US public variety released in 1972.

This region is classified as cold semiarid climate (BSk), according to the Koppen Geiger climate classification system (Kottek et al. 2006), characterized by having dry, hot summers and freezing winters with low snowfall. Over the past decade, based on a weather station located 530 m north of the research site managed by Washington State University (weather.wsu.edu), the average (based on data from 2021 to 2023) annual air temperature was 11.4 °C, with summer (April–October) months averaging 18.8 °C [55% relative humidity (RH)] and winter (November–March) months 3.9 °C (78% RH). On average, the research site received 132 mm of precipitation annually, with 66% of the precipitation received during the winter months. The research site accumulated a cumulative reference evapotranspiration (ET_r) of 1336 mm during the hop-growing season, spanning from March to October. In addition, the Yakima Valley is ideal for hop cultivation because of its long days during the vegetation stage and significantly longer nights during the maturation phase.

The hop yard used for this experiment was planted in 2021. The soil in the top 30 cm at the research site is classified as loam, composed of 40% sand, 22% clay, and 38% silt. The soil’s water content at field capacity, readily available water point, and permanent wilting point is 0.29 m³·m⁻³, 0.22 m³·m⁻³, and 0.15 m³·m⁻³, respectively. These soil characteristic parameters were calculated using the SPAW software from the US Department of Agriculture (Rawls 1998; Saxton and Rawls 2006). The soil’s field capacity is considered the upper limit of soil water storage for plant use and is characterized as the amount of water content held in the soil after excess water has drained due to gravity to water potentials between −30 kPa and −10 kPa (Klute and Dirksen 1986; Monteith and Unsworth 2013). The permanent wilting point is considered the lower limit of soil water storage, defined as the point at which the soil water content is too low for plant roots to extract water, typically occurring when matric potential is about −1500 kPa (Monteith and Unsworth 2013).

Although water is theoretically available until the permanent wilting point, the crop water uptake efficiency decreases as it reaches this point. As the soil water content diminishes and approaches the permanent wilting point, soil water becomes more tightly bound to soil aggregates, making soil water uptake difficult for plants. Once the soil water content falls below a specific threshold, it can no longer be transported in the root system quickly enough to satisfy transpiration needs, causing the crop to undergo stress. However, for most crops, the permanent wilting point is not used as the soil water content lower limit or threshold (Datta et al. 2017). For hop irrigation management, the lower limit has been termed 50% readily available water. It can be calculated by taking the total available water (TAW) in m³·m⁻³, multiplying it by the crop’s depletion factor, and subtracting it from the soil’s water content at field capacity. The depletion factor for hops is 0.5, which means that the soil moisture in hop fields should not fall below 50% of the readily available water (Allen et al. 1998). The theoretically available soil water that the crop can access in the root zone has been termed TAW and is the difference between the soil water content between the soil’s field capacity and permanent wilting point (Allen et al. 1998).

Irrigation regimes.

The impact of a lapse in irrigation late in the season was investigated by withholding irrigation water for 15 d. This was accomplished by shutting off the irrigation water to the specified plots. In 2022 and 2023, the irrigation regimes included an untreated control (full irrigation; T1) and irrigation withheld 15 d before harvest (T2). In 2022, irrigation water was withheld starting on 17 Aug, with an average soil water content nearing soil saturation (0.42 ± 0.049 m³·m⁻³). In 2023, a third irrigation regime was added to evaluate the effects of a lapse in irrigation earlier in the season. In this third treatment, irrigation was withheld 30 d before harvest on 27 Jul, with an average soil water content of 0.25 ± 0.015 m³·m⁻³, corresponding to 71% available soil water. The T2 treatment in 2023 began on 11 Aug, with an average soil water content of 0.24 ± 0.009 m³·m⁻³, corresponding to 64% available soil water.

It is important to note that we had no influence on the management of the research site before conducting the experiments. As a result, the initial soil water content at the start of the experiments reflected the growers’ management practices solely. Furthermore, although treatments T1 and T2 were repeated across both years, soil water conditions differed significantly. In 2022, soil moisture levels remained above field capacity across all irrigation regimes, whereas in 2023, soil moisture declined to drought conditions.

Cultural management.

Cascade plants were cultivated in an industry-standard hop yard, consisting of 4.3-m-wide rows, 1-m plant spacing, and a trellis height of 5.5 m. The hop yard was irrigated using single drip tubing, with emitters spaced 61 cm apart. Irrigation, nutrition management, and pest control during the season leading up to the irrigation treatments were exclusively managed by the farm staff, following industry guidelines that ensure maximum yield and quality. In the spring, the entire research site was strung with coconut coir rope. Subsequently, after reaching an average height of 1.83 m, the entire research site was treated with a post-emergent spray to remove unwanted suckers and vegetation at the base of the plants.

Plots were harvested when cone dry matter reached 23%, a typical indication for ripeness in Cascade hops in Washington State. The hop bines in each plot were cut and transported to a stationary experimental picking machine, where the cones were removed and separated from the bines. Cone wet biomass was recorded, and then a subsample of ∼10 kg of fresh cones was dried in a commercial kiln until the moisture content reached between 8% and 12%. After drying, cones were vacuum-sealed in nitrogen using mylar bags and stored at −14 °C until an analysis of brewing quality could be conducted.

Soil water content, plant performance, and weather.

During the 2022 and 2023 seasons, soil water content was assessed on the top 30 cm of the soil profile at the start and end of the experiment using a handheld soil moisture probe (HS2 HydroSense II; Campbell Scientific Inc., Logan, UT, USA). Soil water content was measured from each plot, from two different locations, between two healthy and representative plants to create an average soil water content per plot (n = 12).

Plant physiological performance was accessed only in 2023 on irrigation regimes T1 and T2. On 1 Sep 2023, 4 d before harvest, stomatal conductance, apparent transpiration (E), quantum yield of photosynthesis II (PhiPSII), and leaf surface temperature (°C) were measured using a steady-state porometer (LI-600 Porometer/Fluorometer; LI-COR, Lincoln, NE, USA). Stomatal conductance quantifies the extent of the stomatal opening and density on a leaf surface. This measurement reflects the plant’s physiological response to its environment (Faralli et al. 2019). Apparent transpiration refers to the rate at which water vapor is lost from the leaf surface to the atmosphere (Jensen et al. 1990). PhiPSII is expressed as a dimensionless ratio, a measure of the efficiency with which light absorbed by chlorophyll in photosynthesis II is used for photochemical reactions (Baker 2008). The evaluation was conducted on two leaves in the sunlight per plot. The leaves were selected at a height from the ground between 120 cm and 180 cm, ensuring the leaves were healthy and mature. The survey measurement was conducted on areas of the leaves excluding main veins and conducted between 1100 and 1300 HR to ensure consistent light and soil moisture conditions. A Washington State University AgWeatherNet weather station 530 m north of the research site recorded various weather parameters, including air temperature, relative humidity, wind speed and direction, solar radiation, precipitation, and reference evapotranspiration.

Hop cone dry matter.

The percent dry matter of hop cones was determined from a 50-g subsample collected during the picking process. To prevent desiccation before the hop cones were oven-dried, the hop cones were stored in polyethylene bags in an insulated cooler immediately after collection. The hop cone mass was measured before and after oven-drying at 105 °C for 24 h. The percent dry matter was calculated using the following formula:$$\text{\%}DM\hspace{0.17em}=\hspace{0.17em}100\text{*}\frac{\left(\mathit{Dry}\mathit{hop}\mathit{cone}\mathit{mass}\right)}{(\mathit{Fresh}\mathit{hop}\mathit{cone}\mathit{mass})}$$

Cone yield and chemical analyses.

Total cone yield was calculated from a subsample that contained 30% of wet hops collected from each plot. The wet hops were dried in an industrial hop kiln, and the mass was divided by 0.3 to account for the missing 70% that were not dried. To calculate cone yield in kilograms per hectare, the mass from the subsample was divided by the acreage that made up the plot.

Hop cone quality was evaluated using a third-party laboratory (Hollingbery and Son, Yakima, WA, USA), using the methods made available by the American Society of Brewing Chemists (ASBC Methods of Analysis 2011a, 2011b, 2011c). Total essential oil content was measured by steam distillation, α-acids and β-acids, humulone, and colupulone by high-performance liquid chromatography, and oil volatile compounds via gas chromatography with flame ionization detection.

Experiment design and statistical analysis.

A randomized complete block design with six replications was used to evaluate the effects of prolonged lapse in irrigation late in the season. Plots were composed of three rows containing seven plants. The outer rows were borders, and measures were only taken from the center row. The experimental design involved dividing the long three-row plots into six blocks, with each treatment randomly assigned to a block. Irrigation was discontinued in specific plots within each block by replacing the drip tubing in those sections with conventional tubing. A statistical analysis was conducted using JMP Pro v16 (SAS Institute Inc., Cary, NC, USA, 1989 to 2019). A fixed effect model one-way analysis of variance (ANOVA) was used to determine statistical significance at a level of 0.05. The ANOVA for the data replicated in both years included the following factors: irrigation regime, block, and year, which served as a repeated measure blocking factor. A detailed 2-year ANOVA can be found in Supplemental Table 1, with illustrated year*treatment interactions in Supplemental Fig. 1. Post hoc comparison analysis was conducted using the Student’s t test. Figures were developed using Sigmaplot v15 (Systat Software Inc., San Jose, CA, USA). The normality of the data were assessed using the Shapiro-Wilk test goodness of fit test, with all of the observations having normally distributed data. Levene’s test assessed the homogeneity of variances, which demonstrated no significant differences in variances across groups (P > 0.05), satisfying the assumption of homoscedasticity. The independence of errors was verified through residual analysis, revealing no discernible patterns or correlations among residuals.

Weather.

Weather conditions at the research site during the implementation of different irrigation regimes were consistent across both years of the study. Reference evapotranspiration, a standardized measure of the atmospheric demand for water, was significantly higher during the July and August months in 2023 than in 2022 (P = 0.0335) (Fig. 1A), although air temperatures during the July and August months between both years were not significantly different (P = 0.3409) (Fig. 1C). No precipitation was recorded during the application of the irrigation regimes in 2022, whereas 9.4 mm of precipitation was measured in 2023 (Fig. 1B). In 2022 and 2023, irrigation treatments were initiated shortly after the peak potential reference evapotranspiration (ET_r), and maximum air temperatures were recorded (Fig. 1A and C). This may have contributed to the performance in yield over the years.

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Soil water content.

In 2022, the average soil water content at the end of the season in plots subjected to a lapse in irrigation 15 d before harvest (T2; mean = 0.31 ± 0.062 m³·m⁻³) was 23% lower than in plots receiving full irrigation (T1; mean = 0.403 ± 0.072 m³·m⁻³). During the 15 d without irrigation, soil water content in T2 plots declined by 26% from an initial average of 0.428 ± 0.04 m³·m⁻³. A similar trend was observed in 2023, where the average soil water content at the end of the season in T2 plots (mean = 0.172 ± 0.018 m³·m⁻³) was 25% lower than in fully irrigated plots (T1; mean = 0.243 ± 0.024 m³·m⁻³). Across both T1 and T2 treatments, the average soil water content at the start of the experiment in 2023 was 0.25 ± 0.009 m³·m⁻³. In plots where irrigation was withheld for 30 d before harvest (T3), the soil water content at the end of the treatment on 11 Aug (mean = 0.182 ± 0.018 m³·m⁻³) was 24% lower than those irrigated with full irrigation (T1; mean = 0.239 ± 0.014 m³·m⁻³). Notably, plots that were subjected to T3 saw a decline in soil water content of 27% from the start of the treatment (mean = 0.248 ± 0.021 m³·m⁻³) on 27 Jul to the end of the treatment (mean = 0.182 ± 0.02 m³·m⁻³) on 11 Aug (Table 1).

Table 1. The average soil water content (SWC) measured among the plot treatments (TRT) at the start and end of each experiment across both years.

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It is important to emphasize that in 2022, the soil water content did not fall below the 50% readily available water threshold in plots subjected to a lapse in irrigation 15 d before harvest, indicating that these plants were not subjected to soil water stress conditions. In contrast, soil water content measured at the end of the experiments in 2023 fell below the 50% readily available water threshold in plots subjected to an irrigation lapse 15 and 30 d before harvest, indicating a high likelihood that plants in these conditions experienced soil water stress, as the soil water levels were insufficient to meet the plants’ physiological demands.

Physiological parameters.

At the end of the 2023 season, the mean stomatal conductance differed significantly between irrigation treatments (P < 0.0001). Plants subjected to a lapse in irrigation 15 d before harvest (T2; mean = 70 mmol H₂O m⁻²·s⁻¹) exhibited a stomatal conductance that was 260% lower than plants receiving full irrigation (T1; mean = 252 mmol H₂O m⁻²·s⁻¹) (Fig. 2A). The observed reduction in stomatal conductance under drought conditions aligns with prior findings by Eriksen et al. (2020) and Marceddu et al. (2022), reinforcing the sensitivity of stomatal regulation to water availability during critical phenological stages. It is noteworthy, however, that the stomatal conductance values for fully irrigated plants in our study were 90% lower than those reported by Eriksen et al. (2020) in a controlled growth chamber (mean = 480 mmol H₂O m⁻²·s⁻¹), potentially due to environmental conditions such as field temperatures and natural light intensity.

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A lapse in irrigation late in the season also significantly affected other physiological parameters such as apparent transpiration (E; P < 0.0001) and quantum efficiency of photosystem II (Φ_PSII; P = 0.0088). The transpiration rate in plants that were subjected to the lapse in irrigation (T2; mean = 3.18 mmol·m⁻²·s⁻¹) was 185% lower compared with that of fully irrigated plants (T1; mean = 9.09 mmol·m⁻²·s⁻¹) (Fig. 2B). This substantial decline in transpiration under such conditions corroborates similar trends observed by Eriksen et al. (2020) and reflects the critical role of stomatal closure in limiting water loss under water deficit conditions. The efficiency of photosystem II was reduced by 44% when plants were subjected to a lapse in irrigation late in the season (T2; mean = 0.266) compared with fully irrigated plants (T1; mean = 0.384) (Fig. 2C). This decrease in the efficiency of photosystem II under such conditions is consistent with the impairment of photosynthetic processes under water stress, as previously reported by Gräf et al. (2021), who observed similar patterns in response to various drought severities. The reduction in the efficiency of photosystem II indicates a decreased capacity for photochemical energy conversion (Wang et al. 2022), further illustrating the detrimental effects of water stress on plant physiology late in the season.

The leaf temperature also showed a significant effect when plants were subjected to the irrigation regimes (P = 0.0490). Plants subjected to a lapse in irrigation late in the season (T2; mean = 37 °C) experienced a leaf temperature that was 1.3 °C higher than plants under full irrigation (T1; mean = 35.8 °C) (Fig. 2D). This modest but significant increase in leaf temperature caused by a lapse in irrigation is consistent with the thermal stress responses documented in previous studies, where Gräf et al. (2021) reported temperature increases of 6 to 8 °C in plants experiencing varying severities of drought. Although the temperature increase in our study was smaller, it still reflects the well-documented trend of elevated leaf temperatures in water-stressed plants due to reduced transpiration and evaporative cooling (Eriksen et al. 2022).

Hop cone yield.

The 2-year analysis of hop cone yield revealed a statistically significant difference between the irrigation regimes (P = 0.0026) and years (P = 0.0007) (Supplemental Table 1). Plants subjected to a lapse in irrigation during the final 15 d before harvest (T2; mean = 1961 kg·ha⁻¹) had a 9.5% lower yield compared with fully irrigated plants (T1; mean = 2157 kg·ha⁻¹) (Fig. 3A). The hop cone yield was nearly 11% higher in 2023 (mean = 2174 kg·ha⁻¹) than in 2022 (mean = 1944 kg·ha⁻¹); even though the plants were subjected to water stress conditions (Table 1). However, it has been demonstrated that hop plants are not fully established until the third year (Chochran 2016). The data collected in 2023 revealed that plants subjected to a lapse in irrigation 30 d before harvest (T3; mean = 1654 kg·ha⁻¹) resulted in a hop cone yield reduction of 28.8% compared with fully irrigated plants (T1; mean = 2324 kg·ha⁻¹) (Fig. 3B). The substantial yield loss observed in plants deprived of water for 15 d, at both 15 and 30 d before harvest, underscores that stress from extended irrigation lapses during cone development can severely reduce productivity. Although plants subjected to a lapse in irrigation 15 d before harvest did not experience water stress conditions in 2022, the yield reduction (13%) tells us the plants were significantly affected by the sudden loss of irrigation over the 15-d period. However, the effect was much lower (5%) than in plants that experienced a lapse in irrigation 30 d before harvest, which did experience water stress conditions in 2023. These findings highlight that both timing and duration of drought stress are key factors, among many, that determine the extent of yield loss. These results align with findings by Mozny et al. (2023), who reported that water stress near harvest decreases yield proportionately to drought duration. Similarly, our results echo those of Nakawuka et al. (2017), who observed yield declines with deficit irrigation throughout the growing season. Collectively, these findings underscore the vital importance of water availability in the late stages of plant development, particularly as cone maturation approaches, when water stress can markedly impact yield outcomes.

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Hop cone dry matter and total oil.

The 2-year analysis of hop cone dry matter content revealed no statistically significant differences between the irrigation regimes (P = 0.2834), with a stable mean dry matter content of 23.04% across the study (Fig. 4A). This stability across the irrigation regimes was evident in plants that were subjected to a lapse in irrigation 30 d before harvest (P = 0.5224; Fig. 4B) in 2023, suggesting that hop cone dry matter is relatively unaffected by short-term irrigation lapses late in the growing season. The consistent dry matter accumulation under drought conditions supports the hypothesis that hop plants can effectively regulate water use and maintain structural biomass stability despite limited water availability. This observation aligns with findings from Gloser et al. (2013), who demonstrated that hop plants maintain structural biomass stability despite limited water availability. The resilience of cone dry matter to drought stress is likely attributable to the plant's adaptive mechanisms—such as increased water use efficiency and conservative resource allocation—which preserve essential biomass components despite reductions in transpiration and photosynthesis during water-limited conditions (Yan et al. 2023).

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In contrast to the stable dry matter content, the total oil content was affected by withholding irrigation in plants late in the season (P = 0.0011; Fig. 4C). Plants subjected to a lapse in irrigation 15 d before harvest (T2; mean = 0.88%) showed a 19% reduction in total oil content compared with fully irrigated plants (T1; mean = 1.09%). Among the 2 years, the total oil content was 32% higher in 2022, which had more than enough water after withholding irrigation, compared with 2023, which experienced drought conditions (P < 0.0001) (Table 1). The analysis conducted from the 2023 data, which assessed the implications of an earlier lapse in irrigation revealed that withholding irrigation 30 d before harvest (T3; mean = 0.71%) resulted in a 13.2% reduction in total oil content compared with those fully irrigated (T1; mean = 0.82%) (P < 0.0001; Fig. 4D). This decline is consistent with observations in other crops, where drought stress has been shown to impair the production of secondary metabolites, including volatile oils and essential compounds. As reported by Kapoor et al. (2020), water stress often leads to lower oil yields as plants prioritize survival mechanisms over the synthesis of secondary metabolites. In hops, the reduction in total oil content under drought conditions likely reflects a downregulation of oil biosynthesis pathways, which are highly sensitive to water availability (Eriksen et al. 2022). This decrease in metabolic activity may limit the production of essential oils, which are crucial for hop quality, particularly in brewing applications where aromatic compounds are highly valued.

Bittering compounds.

The 2-year analysis on α-acid (P = 0.3592; Fig. 5A) and β-acid (P = 0.1056; Fig. 5B) content did not reveal a significant impact following a lapse in irrigation late in the season. The mean α-acid and β-acid content across the irrigation regimes was 4.3% and 6.4%, respectively. In 2023, when assessing the impact of a lapse in irrigation 30 d before harvest, there was also no significant effect on either α-acids (P = 0.3976; Fig. 5E) or β-acids content (P = 0.1076; Fig. 5F).

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However, a significant difference was observed in cohumulone content, a subclass of α-acids (P = 0.0022). Plants subjected to a lapse in irrigation in the final 15 d before harvest (T2; mean = 30.42%) experienced a 1.4% reduction in cohumulone content compared with hops irrigated with full irrigation (T1; mean = 30.01%) (Fig. 5C). A similar observation was made in 2023 when assessing a lapse in irrigation 30 d before harvest (P < 0.0001). A lapse in irrigation 30 d before harvest (T3; mean = 30.3%) resulted in a 3.2% reduction in cohumulone compared with plants receiving full irrigation (T1; mean = 31.3%) (Fig. 5F). Colupulone content was unaffected by a lapse in irrigation late in the season in either the 2-year analysis (P = 0.0581; Fig. 5D) or the analysis conducted in 2023 (P = 0.0719; Fig. 5H).

The 2-year analysis also revealed a significant year effect when evaluating α-acids (P < 0.0001), β-acids (P < 0.0001), and cohumulone (P < 0.00011) (Supplemental Table 1). For α- and β-acids, higher percentages were produced in 2022 (did not experience drought conditions), with α-acids at 5.5% and β-acids at 7.33%, compared with 2023 (experienced drought conditions), when α-acids dropped to 3.05% and β-acids to 5.55%. Conversely, cohumulone content was 4% higher in 2023 than in 2022.

These findings indicate that a loss of irrigation 15 or 30 d before harvest for 15 d does not affect hop cone α-acid and β-acid contents. These findings align with those of Nakawuka et al. (2017), who reported that hop cultivars are relatively stable in their α-acid and β-acid content across varying soil moisture conditions, including water deficit stress. Similarly, Fandiño et al. (2015) found no significant change in α-acid and β-acid levels, supporting the conclusion that these compounds are less responsive to irrigation variability. However, a closer examination of the effects of soil moisture on alpha and beta acids reveals that drought conditions experienced by plants in 2023 led to lower concentrations of these compounds compared with plants subjected to excess soil moisture in 2022. This aligns with the findings of Keukeleire et al. (2007), who reported higher α-acids levels during growing seasons that received significantly more precipitation.

Monoterpene compounds.

This study analyzed seven monoterpene compounds, α-pinene, β-pinene, citral, geraniol, myrcene, linalool, and ocimene, in hops subjected to different irrigation regimes across 2 years. Of these, three compounds, geraniol (P = 0.0004), linalool (P = 0.0346), and ocimene (P = 0.0048), exhibited significant differences when plants were subjected to different irrigation regimes. Geraniol content was reduced by 20.8% when water was withheld 15 d before harvest (T2; mean = 0.10%) compared with those that received full irrigation (T1; mean = 0.12%). Similarly, linalool content was reduced by 11.4% when plants were subjected to a lapse in irrigation 15 d before harvest (T2; mean = 0.31%) compared with those subjected to full irrigation (T1; mean = 0.35%) (Fig. 6F). A lapse in irrigation 15 d before harvest (T2; mean = 0.33%) also led to a 13% reduction in ocimene content compared with those that were fully irrigated (T1; mean = 0.38%) (Fig. 6G).

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A lapse in irrigation earlier in the season, just 30 d before harvest, affected five monoterpenes, including β-pinene (P = 0.0250), citral (P = 0.0065), geraniol (P = 0.0017), myrcene (P = 0.0020), and ocimene (P = 0.0002). β-pinene content was reduced by 19% when plants experienced a lapse in irrigation 30 d before harvest (T3; mean = 0.68%) compared with those fully irrigated (T1; mean = 0.84%) (Fig. 6I). Myrcene content was reduced by 20.2% in plants that experienced a lapse in irrigation 30 d before harvest (T3; mean = 38.3%) compared with those fully irrigated (T1; mean = 48%) (Fig. 6M). A lapse in irrigation 30 d before harvest (T3; mean = 0.35%) also led to a 28.6% reduction in ocimene content compared with plants that received full irrigation (T1; mean = 0.49%) (Fig. 6L).

However, in the case of citral, a lapse in irrigation 30 d before harvest (T3; mean = 0.60%) resulted in an increase of 10% in citral content compared with those irrigated with full irrigation (T1; mean = 0.55%) (Fig. 6K). Geraniol content saw an increase of 23.3% when irrigation water was withheld 30 d before harvest (T3; mean = 0.21%) compared with plants fully irrigated (T1; mean = 0.15%). Of the seven monoterpenes studied, four showed a significant year effect: citral (P < 0.0001), geraniol (P < 0.0001), myrcene (P = 0.0015), and ocimene (P < 0.0001) (Supplemental Table 1). Except for myrcene, the concentrations of these compounds increased significantly in 2023, when plants were exposed to drought conditions, compared with 2022, when soil moisture levels remained above full capacity.

These findings suggest that the timing of water stress can lead to varying outcomes in monoterpene biosynthesis. The increase in certain monoterpenes under drought conditions aligns with findings in conifers, where drought was shown to enhance monoterpene concentrations (Kainulainen et al. 1992). The observed reduction in several monoterpenes, such as β-pinene, linalool, and ocimene, under drought conditions is consistent with previous research, which suggests that water stress can inhibit monoterpene biosynthesis due to disruptions in photosynthesis and altered carbon allocation to secondary metabolic pathways (Loreto and Schnitzler 2010; Peñuelas and Staudt 2010). In contrast, the increase in citral and geraniol, particularly when drought stress occurred earlier, points to a more complex regulatory mechanism that may vary depending on the plant’s developmental stage (Chen et al. 2023) or interaction with other environmental factors.

The increase in monoterpene concentrations earlier in the season under specific drought conditions also may be influenced by elevated soil temperatures, which were not directly measured in this study but are known to fluctuate more sharply in drought scenarios due to reduced soil moisture (Al-Kayssi et al. 1990). Previous studies, such as those by Snow et al. (2003), have demonstrated that elevated temperatures can enhance monoterpene production in plant species like Douglas fir. Overall, these findings underscore the nuanced response of monoterpenes to environmental stressors, particularly water availability, and highlight the importance of considering both the timing and duration of drought when assessing its effects on secondary metabolite production.

Sesquiterpene compounds.

This study evaluated the concentration of four sesquiterpenes in hop cones to varying irrigation regimes. Among the compounds, three sesquiterpenes, caryophyllene (P = 0.0348), caryophyllene oxide (P = 0.0024), and humulene (P = 0.0088), were found to be significantly affected by the irrigation regimes. Specifically, withholding irrigation during the final 15 d before harvest (T2, mean = 9.1%) resulted in a 12.4% increase in caryophyllene content compared with fully irrigated plants (T1; mean = 8.1%) (Fig. 7A). Similarly, humulene content increased by 9.1% under the same conditions (T2; mean = 22.7%) compared with those fully irrigated plants (T1; mean = 20.8%) (Fig. 7D). In contrast, caryophyllene oxide showed a different response. A lapse in irrigation during the final 15 d before harvest (T2; mean = 0.31%) resulted in a 16.1% reduction in caryophyllene oxide content compared with fully irrigated plants (T1; mean = 0.26%) (Fig. 7B). Farnesene content remained unaffected by the irrigation lapse late in the season (Fig. 7C).

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The 2023 data, which evaluated sesquiterpenes after withholding irrigation 30 d before harvest, revealed more pronounced changes in sesquiterpene. Under these conditions, all four sesquiterpenes, caryophyllene (P = 0.0075), caryophyllene oxide (P = 0.0110), farnesene (P = 0.0008), and humulene (P = 0.0088), were significantly affected. Withholding irrigation 30 d before harvest (T3; mean = 0.33%) resulted in a 37.7% decrease in caryophyllene oxide content compared with fully irrigated plants (T1; mean = 0.53%) (Fig. 7F). However, humulene content exhibited the opposite trend, increasing by 17.2% in plants subjected to drought stress 30 d before harvest (T3; mean = 25.2%) compared with fully irrigated plants (T1; mean = 21.5%) (Fig. 7H). When comparing caryophyllene oxide levels in plants subjected to irrigation lapses 15 d before harvest (T2; mean = 0.43%) vs. 30 d before harvest (T3; mean = 0.33%), a 23% reduction in caryophyllene oxide was observed in the latter treatment (Fig. 7F). Post hoc comparisons also highlighted that although humulene content increased significantly in T3 compared with T1, the difference between T3 (mean = 25.2%) and T2 (mean = 23.3%) was 7.5%, reflecting a more moderate increase (Fig. 7H).

Although there was a significant effect in the caryophyllene content from plants that experienced a lapse in irrigation 30 d before harvest (T3; mean = 9.6%) compared with those irrigated with full irrigation (T1; mean = 8.1%), the post hoc analysis revealed no significant difference in caryophyllene content from plants subjected to a lapse in irrigation 15 d before harvest (T2; mean = 9.6%) (Fig. 7E). Similarly, farnesene content was unaffected when irrigation was withheld from plants 30 d before harvest (T3; mean = 7%) compared with those that experienced a lapse in irrigation 15 d before harvest (T2; mean = 6.9%) (Fig. 7G).

The increases in sesquiterpene concentrations in humulene, farnesene, and caryophyllene, during times of irrigation loss, underscore the significant influence of plant-perceived stress on sesquiterpene biosynthesis. This is especially for the results observed in 2022 when the plants were subjected to a lapse in irrigation, but soil moisture levels remained above optimal. In contrast to monoterpenes, which often decrease under drought stress, sesquiterpenes appear to be upregulated for the most part, linking drought stress to enhanced terpenoid biosynthesis. This phenomenon may be attributed to the activation of enzymes such as terpene synthases (Tholl 2006). Furthermore, the more pronounced sesquiterpene increases observed during earlier drought stress (30 d before harvest) compared with later stress (15 d before harvest) suggest that the timing of water loss is critical in regulating sesquiterpene production, among many others.