Evaluation of European-developed hemp cultivars (Cannabis sativa L.) in response to water regimes

Experimental Site and Growth Conditions

A field experiment was conducted at the Agricultural University farm located in Athens (southern Greece: latitude 37°58′ N, longitude 23°32′ E, altitude 30 m above sea level), and repeated in three consecutive growing years, 2019, 2020, and 2021. The soil was clay loam (29% clay, 35.4% silt, and 35.6% sand) with pH 7.48, available phosphorus (P) 13.5 mg kg⁻¹ soil, available potassium (K) 201 mg kg⁻¹ soil, and 2.7% organic matter. The experimental field was positioned near an automatic meteorological station (Davis Vantage Pro2 Weather Station; Davis Instruments Corporation, California, USA), and the average air temperature and rainfall were recorded daily. The weather data were reported as mean monthly data for May to August during the studied years (Figure 1). The mean air temperature values did not show any significant variability between the growing seasons recording mean monthly values of 26.1 ℃, 25.9 ℃, and 26.9 ℃ for the growing periods 2019, 2020, and 2021, respectively. The long-term mean annual rainfall at the site is 435.9 mm (1989-2018), but temporal variability is high. The highest total rainfall for the periods May to August was recorded at the growing season 2020 (64 mm) followed by 2021 (34.2 mm) and 2019 (4.6 mm). Intense phenomena did not occur during the three-year study.

Experimental Set Up

The experimental design was a randomized complete block design replicated three times in a split-plot restriction with irrigation regimes as the main plots and hemp cultivars as the sub-plots. Five hemp cultivars widely grown in European environments, and often selected by Greek farmers, were used in the study. These cultivars were monoecious with THC content below 0.3% as required by EC regulation, and suitable for seed and/or dual-purpose cultivation, while differed in earliness and origin (Table 1). Moreover, cultivars were chosen based on their morphological, and maturity traits. Seeds of industrial hemp cultivars were obtained from iHempFarms EAD (Veliko Tarnovo, Bulgaria).

The irrigation regimes consisted of three different water regimes supplying 100% of the crop’s water requirement (the well-watered; IRR₁₀₀), 75% (the mild water-stressed; IRR₇₅), and 50% (severe water-stressed; IRR₅₀). A surface drip irrigation system was used for irrigation, A 16 mm diameter polyethylene pipe with inline pressure compensating drippers at 0.33 m intervals was placed on one side of each hemp row. The average discharge of emitters was 2.3 l h^-1 at the 0.1 MPa. The crop was irrigated with 80 mm for the first two weeks after sowing to achieve a successful crop establishment. The irrigation was stopped one week before harvest. The total water inputs including the precipitation amount and the irrigation water that were applied during each growing season are described in Table 2.

The plot size of each irrigation treatment was 5 m x 12 m and the spacing between each main plot was 4 m to minimize water movement among treatments. The experimental plots were 5 m x 2.4 m and consisted of 8 rows 0.30 m apart. The experimental area was plowed at a depth of 25 cm followed by secondary tillage with a disc harrow to prepare the crop seedbed. An organic fertilizer 7-4-7 (Biogen, Phytothreptiki S.A., Athens, Greece) at a rate of 140 kg N, 80 kg P₂O₅, and 140 kg K₂O ha^-1 was broadcast applied two days before seeding and incorporated into the soil using a cultivator. Hemp seeds were sown by hand at a depth of 3 cm on 9 May 2019, 14 May 2020, and 13 May 2021 to achieve the planned crop density of 120-125 plants m^-2. No pesticides were utilized during the crop establishment and growth, while the emerged weed species were hand-removed when necessary. Agronomic practices were the same during all three growing seasons.

Determination of Agronomical Traits, and Seed Yield Components

To evaluate the effect of irrigation regimes (IRR) on hemp agronomical traits, plant height, and dry above-ground biomass were documented from five randomly selected plants of the inner plant row of each plot at 61 days after sowing (DAS). Plant height was measured from the base of the plant to the growing tip. For the above-ground biomass determination, plants were cut to ground level with manual shears, and the sampled plant material was oven-dried at 80 ℃ for 72 hours until their weight remained constant and reweighted.

To measure hemp Normalized Difference Vegetation Index (NDVI), a Trimble^® GreenSeeker^® handheld sensor (Trimble Agriculture Division, Westminster, CO, USA) was used at 61 DAS in 2020 and 2021. The sensor unit has self-contained illumination in both near-infrared (NIR) (visible 770 nm) and red (visible 660 nm) ranges (Tremblay et al., 2009Tremblay N, Wang Z, Ma BL, Belec C, Vigneault P, 2009. A comparison of crop data measured by two commercial sensors for variable-rate nitrogen application. Precision Agric 10: 145-161. https://doi.org/10.1007/s11119-008-9080-2
) corresponding to the subsequent Equation (1):

Regarding the procedure of NDVI records, the sensor was placed parallel to the direction of the crop rows and held stable at a height of 25-30 cm above the leaf area on bright days at midday. The device was slowly moved within the limits of the quadrat for 5 s as recommended by Kong et al. (2019)Kong B, Yu H, Du R, Wang Q, 2019. Quantitative estimation of biomass of alpine grasslands using hyperspectral remote sensing. Rangeland Ecol Manag 72(2): 336-346. https://doi.org/10.1016/j.rama.2018.10.005
. This procedure was repeated thrice per sub-subplot, then recorded values were averaged.

At the end of seed maturity (BBCH:2307), all plants in an area of 6 m² from the middle rows were hand-harvested above soil level, and their seeds were air-dried, threshed, cleaned, and weighed for seed yield determination, after removing the empty seeds. Among the harvested plants, samples from 10 plants were taken randomly, separated into stems, leaves, inflorescences, and seeds, weighed, and the length of main and secondary inflorescences and seed numbers were determined. The seed density index (SDI, cm⁻¹) was calculated as the quotient between seed number per plant and mean inflorescence length. Hemp plants were harvested at 112, 108, and 109 DAS in the 2019, 2020, and 2021 growing seasons respectively.

To characterize the effective water use of hemp cultivars, the Water Productivity index (WP) was selected to define the relationship between crop produced and the amount of water involved in crop production. WP was calculated as the ratio of biomass yield and the sum of all components of water inflow via air and ground that are used for crop growth and the total water amount during the total crop growth period (Prochnow et al., 2012Prochnow A, Drastig K, Klauss H, Berg W, 2012. Water use indicators at farm scale: methodology and case study. Food Energy Secur 1: 29-46. https://doi.org/10.1002/fes3.6
).

Cannabinoids Analysis

At the female flowering stage (BBCH: 2301-2302), which was determined when 50% of bracts formed, inflorescence samples were harvested from the top of uniform plants in each subplot. All plant material was air-dried at 25 ℃, powdered with a commercial mill, and sifted using a sieve shaker (BA 200 N, CISA, Spain). The sample fractions with a particle size of less than 1 mm were stored at -20℃ until analysis.

The CBD concentrations of samples were determined by the technique of Ultra Performance Liquid Chromatography with Photodiode Array Detection (UPLC – PDA) according to the methodology described by Tzimas et al. (2021)Tzimas PS, Petrakis EA, Halabalaki M, Skaltsounis LA, 2021. Effective determination of the principal non-psychoactive cannabinoids in fiber-type Cannabis sativa L. by UPLC-PDA following a comprehensive design and optimization of extraction methodology. Anal Chim Acta 1150: 338200. https://doi.org/10.1016/j.aca.2021.338200
and expressed as cannabidiol (CBD), cannabidiolic acid (CBDA), and total cannabidiol (CBD total). The CBDA concentration was determined as the acidic form which when exposed to environmental factors, such as light and heat is decarboxylated and converted into CBD. In each inflorescence sample, total CBD concentration (%) was calculated as a sum of CBD (%) and CBDA (%) multiplicated by 0.877 which accounts for the differences in molar mass between the acid and neutral forms of the cannabinoid.

Statistical Analysis

All data were subjected to a two-way analysis of variance (ANOVA) using the software STATGRAPHICS Centurion XVII Version statistical software (Statpoint Technologies Inc., The Plains, VA, USA). Differences between treatments and their interactions were compared by using Fisher’s least significant difference (LSD) test, where probabilities are equal to or less than 0.05 (a≤5%).

Agronomic Growth Traits of Hemp

Based on the combined analysis of variance, significant effects of irrigation regimes (IRR), and cultivar (C) were observed on the hemp growth traits in all growing seasons (p<0.001). Irrigation regimes significantly impacted hemp height and above-ground biomass recording the highest values in well-watered plants (IRR₁₀₀) of all the cultivars. Both treatments of water stress mild (IRR₇₅) and severe (IRR₅₀) resulted in plants of reduced growth regardless of cultivars. The water deficit (IRR₅₀) was the most limiting factor decreasing the plant height by 38.7%, 15.8%, and 18.7%, and the above-ground biomass by 71.9%, 74.6%, and 27.7% during 2019, 2020, and 2021 respectively compared to fully irrigated plants. Among the cultivars, the most compact plants with the shortest stems and minimum biomass were obtained from the early flowering Cannabis sativa ʻUso 31ʼ regardless of irrigation. The tallest plants were recorded in Cannabis sativa ‘Santhica 27’, while the highest values of dry biomass were observed in the late French cultivar Cannabis sativa ‘Futura 75’. Moreover, the factor of year impacted significantly on both plant height (p<0.001), and above-ground biomass (p<0.01). Within the growing seasons, the tallest plants including the highest biomass were performed in the second experimental year regardless of irrigation and cultivar treatments. All three factors showed no interaction for the studied plant growth traits (p>0.05, ns) (Table 3).

In addition, the effect of water deficit on hemp growth was well depicted in the vegetation index since a significant effect of irrigation regimes was recorded on NDVI (p<0.01). Index values were boosted by the maximum irrigation rate (IRR₁₀₀) in all cultivars. The highest mean value of NDVI (0.82) was recorded in fully irrigated plants, while treatments of mild (IRR₇₅) and severe (IRR₅₀) water stress reduced mean values by 2.4%, and 2.7%, respectively compared to well-watered plants. Moreover, the effect of the cultivar was of high significance for NDVI during the three growing seasons. The greatest values of NDVI were recorded from the ‘Futura 75’ with a mean value of 0.82, while the mean values of other cultivars ranged between 0.79, and 0.81, respectively. Among the growing seasons, the highest values of NDVI were recorded in the third growing year 2021 (Y3). Even if all cultivars were positively affected by the full irrigation, the two factors showed no interaction for NDVI (p>0.05, ns) as well as the three factors (Table 3).

Seed Yield, and Yield Contributing Characteristics

Regarding the yield parameters of hemp, irrigation demonstrated a significant effect on inflorescence length, seed weight per inflorescence, and seed yield in all three experimental years. Both water stress treatments reduced the studied yield characteristics without differing significantly in all cultivars. Among them, the severe water deficit (IRR₅₀) was the most damaging treatment decreasing the mean values of inflorescence length, seed weight per inflorescence, and seed yield by 19%, 27.6%, and 29.9% respectively, compared to fully irrigated plants. Even though irrigation regimes did not differ significantly for the seed density index (p>0.05, ns), water stress treatments IRR₇₅ and IRR₅₀ decreased mean values recording 6.07 cm^-1 and 6.17 cm^-1 respectively compared to 6.97 cm^-1 of well-watered plants (Table 4).

The combined analysis of variance also demonstrated the highly significance effect of cultivar on all yield parameters of hemp (p<0.001). The longest inflorescences with numerous seeds were performed in the medium-maturity cultivar ‘Fedora 17’ with mean values ranging between 35.1 cm and 36.8 cm, and between 223 and 247 seeds per inflorescence respectively during the three growing seasons. Among the cultivars, the highest mean seed yield was performed from the ‘Fedora 17’ (2.80 t ha^-1) and was twice the mean yield of the less productive ‘Santhica 27’ (1.42 t ha^-1) regardless of irrigation and year. The lower seed density index was poorly performed on average from the ‘Santhica 27’ recording 4.56 cm^-1, while the highest values of 7.49 cm^-1 were presented in the cultivar ‘Futura 75’. Between the growing seasons, plants with the most seeds, performing the highest seed yield, and the greatest seed density index were produced in the second experimental year regardless of irrigation and cultivar treatments. All three factors showed no interaction for the studied hemp yield components (p>0.05) (Table 4).

Results revealed that water productivity was affected by the irrigation and cultivar (p<0.001). Increasing irrigation water to full significantly increased water productivity compared to water-stressed plants, whereas drought stress showed just the opposite effects in most of the studied cases. Among the cultivars, ‘Futura 75’ recorded the highest mean value of 2.49 kg m^-3 followed by ‘Fedora 17’ with 1.82 kg m^-3. The lowest mean value was 0.95 kg m^-3 and was observed in ‘Uso 31’ plants. Moreover, no significant difference was noticed among years for water productivity, whereas the interactions of year with irrigation, year with cultivar, and irrigation with cultivar showed a large interaction. All three factors showed no interaction in the case of water productivity (p>0.05, ns) (Table 4).

Cannabidiol Yield Characteristics

Water regimes noticed a significant impact on cannabidiol yield in both the 2019 and 2020 growing seasons (p<0.001). In 2019, the highest CBD concentration was recorded in the inflorescences of fully irrigated plants without differing meaningfully from the water-stressed plants of IRR₅₀. As for CBDA, the severe water stress increased the concentration by 27% compared to well-watered plants regardless of cultivar. Thus, the total CBD concentration was observed in the fully irrigated plants recording 1.25 % w/w followed by the 1.03% w/w of severe water-stressed plants (IRR₅₀) without varying consistently. In the next growing season, the effect of different water regimes on cannabidiol yield was evident, and severe water stress seemed the most enhancing treatment compared to full irrigation and mild water stress. By increasing drought stress to IRR₅₀, the CBD and CBD total concentrations were almost duplicated, and CBDA concentration increased thrice (Table 5).

Furthermore, the analysis of variance revealed the significant effect of cultivar on CBD concentration (p<0.001). ‘Ferimon’ inflorescences produced the highest CBD concentration recording 1.59% w/w and 0.92% w/w in 2019 and 2020, respectively. As for CBDA and CBD total, ‘Ferimon’ showed the greatest concentrations marginally higher than ‘Futura 75’. Among the cultivars, the early maturity ‘Uso 31’ produced inflorescences with the lowest CBD Total concentration. For all the cannabidiol yield components, the year effect was significant, and the highest values were recorded in the second experimental year. All three factors also showed a large interaction for cannabidiol yield components (p<0.01) (Table 5).

Agronomic Traits of Hemp

By reducing the water supplied the crop decreased significantly the performed plant height and biomass. Among the irrigation regimes, the severe water stress treatment (IRR₅₀) was the most damaging factor in hemp growth reducing the plant height and biomass up to 24.1% and 62.6%, respectively, regardless of cultivars and years. These findings could be attributed to the long-term effects of water stress that negatively affects plant morphology (Tang et al., 2018Tang K, Fracasso A, Struik PC, Yin X, Amaducci S, 2018. Water- and nitrogen-use efficiencies of hemp (Cannabis sativa L.) based on whole-canopy measurements and modeling. Front Plant Sci 9: 951. https://doi.org/10.3389/fpls.2018.00951
) through the decreased stomatal conductance, transpiration rate, and passage of CO₂ in plants. Previous research confirmed the reduction of hemp height due to water deficit, as obtained from field studies in Spain (García-Tejero et al., 2014García-Tejero IF, Durán-Zuazo VH, Pérez-Álvarez R, Hernández A, Casano S, Morón M, Muriel-Fernández ML, 2014. Impact of plant density and irrigation on yield of hemp (Cannabis Sativa L.) in a Mediterranean semi-arid environment. J Agr Sci Tech 16:887-895. http://jast.modares.ac.ir/article-23-9987-en.html
), USA (Campbell et al., 2019Campbell BJ, Berrada AF, Hudalla C, Amaducci S, McKay JK, 2019. Genotype × environment interactions of industrial hemp cultivars highlight diverse responses to environmental factors. Agrosystems Geosci Environ 2(1): 1-11. http://dx.doi.org/10.2134/age2018.11.0057
), and Greece (Scordia et al., 2022Scordia D, Papazoglou EG, Kotoula D, Sanz M, Ciria CS, Pérez J, Maliarenko O, Prysiazhniuk O, von Cossel M, Greiner BE, Lazdina D, Makovskis K, Lamy I, Ciadamidaro L, Grezeriat LP, Corinzia SA, 2022. Towards identifying industrial crop types and associated agronomies to improve biomass production from marginal lands in Europe. GCB Bioenergy 14(7): 710-734. https://doi.org/10.1111/gcbb.12935
). Similar negative effects on hemp biomass were recorded in the literature (Cosentino et al., 2013Cosentino SL, Riggi E, Testa G, Scordia D, Copani V, 2013. Evaluation of European developed fibre hemp genotypes (Cannabis sativa L.) in semi-arid Mediterranean environment. Ind Crop Prod 50: 312-324. https://doi.org/10.1016/j.indcrop.2013.07.059
; Tang et al., 2018Tang K, Fracasso A, Struik PC, Yin X, Amaducci S, 2018. Water- and nitrogen-use efficiencies of hemp (Cannabis sativa L.) based on whole-canopy measurements and modeling. Front Plant Sci 9: 951. https://doi.org/10.3389/fpls.2018.00951
; Herppich et al., 2020Herppich WB, Gusovius HJ, Flemming I, Drastig K, 2020. Effects of drought and heat on photosynthetic performance, water use and yield of two selected fiber hemp cultivars at a poor-soil site in Brandenburg (Germany). Agronomy 10(9): 1361. https://doi.org/10.3390/agronomy10091361
; Gill et al., 2022Gill AR, Loveys BR, Cowley JM, Hall T, Cavagnaro TR, Burton RA, 2022. Physiological and morphological responses of industrial hemp (Cannabis sativa L.) to water deficit. Ind Crop Prod 187: 115331. https://doi.org/10.1016/j.indcrop.2022.115331
).

Regarding the growth traits, five cultivars showed significant differences between them (p<0.001) since their growth was determined by genotype and environment. The important effect of variety on hemp growth has been confirmed by many authors (Cosentino et al., 2013Cosentino SL, Riggi E, Testa G, Scordia D, Copani V, 2013. Evaluation of European developed fibre hemp genotypes (Cannabis sativa L.) in semi-arid Mediterranean environment. Ind Crop Prod 50: 312-324. https://doi.org/10.1016/j.indcrop.2013.07.059
; Baldini et al., 2018Baldini M, Ferfuia C, Piani B, Sepulcri A, Dorigo G, Zuliani F, Danuso F, Cattivello C, 2018. The Performance and Potentiality of Monoecious Hemp (Cannabis Sativa l.) Cultivars as a Multipurpose Crop. Agronomy 8(9): 162. https://doi.org/10.3390/agronomy8090162
). Field experiments demonstrated the superiority in biomass growth of the cultivars ‘Futura 75’, ‘Santhica 27’, and ‘Fedora 17’ compared to ‘Uso 31’ (Flajšman & Kocjan Ačko, 2020Flajšman M, Kocjan Ačko D, 2020. Influence of edaphoclimatic conditions on stem production and stem morphological characteristics of 10 European hemp (Cannabis Sativa L.) varieties. Acta Agric Slov 115(2): 399-407. https://doi.org/10.14720/aas.2020.115.2.1528
). As for the impact of the environment on hemp cultivars, it has been mentioned that a more extended photoperiod with longer days, increases plant height and biomass since it delays flowering, and increases the duration of the vegetative stage. In countries geographically close to Greece, long-duration days favor the production of fiber and biomass. That fact justified the superiority of late flowering ‘Futura 75’ over ‘Fedora 17’ and ‘Ferimon’ cultivars in dry biomass (Cosentino et al., 2013Cosentino SL, Riggi E, Testa G, Scordia D, Copani V, 2013. Evaluation of European developed fibre hemp genotypes (Cannabis sativa L.) in semi-arid Mediterranean environment. Ind Crop Prod 50: 312-324. https://doi.org/10.1016/j.indcrop.2013.07.059
). Similar results were recorded in Italy according to which ‘Futura 75’ scored higher stem biomass than ‘Fedora 17’, ‘Uso 31’ (Baldini et al., 2018Baldini M, Ferfuia C, Piani B, Sepulcri A, Dorigo G, Zuliani F, Danuso F, Cattivello C, 2018. The Performance and Potentiality of Monoecious Hemp (Cannabis Sativa l.) Cultivars as a Multipurpose Crop. Agronomy 8(9): 162. https://doi.org/10.3390/agronomy8090162
), and ‘Ferimon’ (Ferfuia et al., 2021Ferfuia C, Zuliani F, Danuso F, Piani B, Cattivello C, Dorigo G, Baldini M, 2021. Performance and stability of different monoecious hemp cultivars in a multi-environments trial in North-Eastern Italy. Agronomy 11(7): 1424. https://doi.org/10.3390/agronomy11071424
).

Irrigation contributed positively to hemp height and biomass of all cultivars. Comparable results were obtained in field studies (Campbell et al., 2019Campbell BJ, Berrada AF, Hudalla C, Amaducci S, McKay JK, 2019. Genotype × environment interactions of industrial hemp cultivars highlight diverse responses to environmental factors. Agrosystems Geosci Environ 2(1): 1-11. http://dx.doi.org/10.2134/age2018.11.0057
; Scordia et al., 2022Scordia D, Papazoglou EG, Kotoula D, Sanz M, Ciria CS, Pérez J, Maliarenko O, Prysiazhniuk O, von Cossel M, Greiner BE, Lazdina D, Makovskis K, Lamy I, Ciadamidaro L, Grezeriat LP, Corinzia SA, 2022. Towards identifying industrial crop types and associated agronomies to improve biomass production from marginal lands in Europe. GCB Bioenergy 14(7): 710-734. https://doi.org/10.1111/gcbb.12935
). In addition, ‘Futura 75’ and ‘Santhica 27’ were more resistant to severe water stress (IRR₅₀). The negative effects of deficit irrigation on leaf and stem dry weight of the ‘Futura 75’ have been reported by Cosentino et al. (2013)Cosentino SL, Riggi E, Testa G, Scordia D, Copani V, 2013. Evaluation of European developed fibre hemp genotypes (Cannabis sativa L.) in semi-arid Mediterranean environment. Ind Crop Prod 50: 312-324. https://doi.org/10.1016/j.indcrop.2013.07.059
. The sensitivity of ‘Ferimon’ to water stress conditions has also been shown by Kumar (2021)Kumar IMKV, 2021. Production and quality of industrial hemp (Cannabis sativa L.) in response to water regimes. Doctoral thesis. University of Tasmania, Australia.
. However, the present literature about the resistance and susceptibility of hemp cultivars to water stress is in its infancy.

Concerning NDVI, the results showed that deficit irrigation regimes were restrictive on the leaves' photosynthetic surface and normal plant growth. Severe drought (IRR₅₀) reduced the NDVI values by 2.7% compared with full irrigation. The lowest values caused by inadequate water availability are also in line with the formation of reduced plant biomass. In addition, decreased values are justified by the fact that water-stressed plants can lead to rapid aging of the leaves, limited photosynthetic rate, and effectiveness (Cosentino et al., 2013Cosentino SL, Riggi E, Testa G, Scordia D, Copani V, 2013. Evaluation of European developed fibre hemp genotypes (Cannabis sativa L.) in semi-arid Mediterranean environment. Ind Crop Prod 50: 312-324. https://doi.org/10.1016/j.indcrop.2013.07.059
). The cultivar effect was of high significance for NDVI during the three growing seasons. The greatest values were recorded by ‘Futura 75’, a result that agrees with the greatest biomass that was performed. In contrast, the ‘Uso 31’ formed plants with the lowest biomass, leaf surface, and soil cover, and thus the lowest values of NDVI. Similar results for the NDVI of ‘Futura 75’ and ‘Ferimon’ cultivars have been reported by Wulff (2022)Wulff HM, 2022. Growth and development of fibre hemp (Cannabis sativa L.). Master’s thesis. Lincoln University, New Zealand. 146 pp.
.

Seed Yield, and Yield Contributing Characteristics

Since a significant part of global hemp production is intended for seed, the effect of irrigation on the number of seeds per inflorescence, and seed yield was evaluated. The results demonstrated the negative effects of deficit irrigation on both studied characteristics for all experimental years. Plants subjected to the IRR₇₅ and IRR₅₀ treatments caused the formation of inflorescences with fewer and lighter seeds compared to fully irrigated plants. The reduction in seed yield can be attributed primarily to the reduced number of seeds per plant rather than reduced individual seed weight. These results probably indicate that, under conditions of intense water stress, hemp prioritizes the production of filled seeds over the number of them. This is a possible hedging strategy to ensure the successful reproduction of the plant under stress conditions, as also reported by Gill et al. (2022)Gill AR, Loveys BR, Cowley JM, Hall T, Cavagnaro TR, Burton RA, 2022. Physiological and morphological responses of industrial hemp (Cannabis sativa L.) to water deficit. Ind Crop Prod 187: 115331. https://doi.org/10.1016/j.indcrop.2022.115331
. Comparable results have been reported in the existing literature (Kumar et al., 2021Kumar IMKV, 2021. Production and quality of industrial hemp (Cannabis sativa L.) in response to water regimes. Doctoral thesis. University of Tasmania, Australia.
; Gill et al., 2022Gill AR, Loveys BR, Cowley JM, Hall T, Cavagnaro TR, Burton RA, 2022. Physiological and morphological responses of industrial hemp (Cannabis sativa L.) to water deficit. Ind Crop Prod 187: 115331. https://doi.org/10.1016/j.indcrop.2022.115331
). In addition, the inflorescence length was measured, and the seed density index was calculated to identify differences among cultivars of contrasting yield potential. Both traits have been proposed promising in breeding programs for other crops such as wheat (Ferrante et al., 2020Ferrante A, Savin R, Slafer GA, 2020. Floret development and spike fertility in wheat: Differences between cultivars of contrasting yield potential and their sensitivity to photoperiod and soil N. Field Crops Res 256: 107908. https://doi.org/10.1016/j.fcr.2020.107908
). The results revealed that water stress treatments reduced the SDI, but the differences were insignificant among irrigation regimes.

Significant differences were noted between cultivars for inflorescence length, seed number per inflorescence, seed yield, and SDI (p<0.001). The longest inflorescences with the most seeds, the highest seed yield the greatest SDI values were performed in the medium-maturity cultivar ‘Fedora 17’. The least productive cultivar in terms of the seed studied traits appeared to be the ‘Santhica 27’. Similar results have been reported for ‘Futura 75’, ‘Santhica 27’, and ‘Fedora 17’ by Tsaliki et al. (2021)Tsaliki E, Kalivas A, Jankauskiene Z, Irakli M, Cook C, Grigoriadis I, Panoras I, Vasilakoglou I, Dhima K, 2021. Fibre and seed productivity of industrial hemp (Cannabis sativa L.) varieties under Mediterranean conditions. Agronomy 11(1): 171. https://doi.org/10.3390/agronomy11010171
. This superiority of ‘Fedora 17’ over the other cultivars has also been reported by Tang et al. (2016)Tang K, Struik PC, Yin X, Thouminot C, Bjelková M, Stramkale V, Amaducci S, 2016. Comparing hemp (Cannabis sativa L.) cultivars for dual-purpose production under contrasting environments. Ind Crop Prod 87: 33-44. https://doi.org/10.1016/j.indcrop.2016.04.026
, and Baldini et al. (2018)Baldini M, Ferfuia C, Piani B, Sepulcri A, Dorigo G, Zuliani F, Danuso F, Cattivello C, 2018. The Performance and Potentiality of Monoecious Hemp (Cannabis Sativa l.) Cultivars as a Multipurpose Crop. Agronomy 8(9): 162. https://doi.org/10.3390/agronomy8090162
.

Water Productivity

Hemp water productivity is a quantitative index used to determine the relationship between crop production and the total amount of water needed in crop production. It was selected to quantify the effect of irrigation and schedule the appropriate decisions regarding water management. In this study, water productivity was affected by both irrigation and cultivar. Plants of all cultivars reduced their water productivity as the amount of irrigated water decreased, while the maximum values were recorded when they were fully irrigated. This finding demonstrated that additional irrigation water was required in the water-stressed plants of all cultivars. Moreover, all five studied cultivars showed sensitivity to water deficit reducing their yield and water productivity. Among the cultivars, ‘Futura 75’ recorded the highest water productivity under full irrigation and water stress conditions. The shortest values were shown by ‘Uso 31’, especially under severe water stress conditions (IRR₅₀). The values of the present study varied between 0.49 and 3.63 kg m^-3 as in the study of Drastig et al. (2020)Drastig K, Flemming I, Gusovius HJ, Herppich WB, 2020. Study of water productivity of industrial hemp under hot and dry conditions in Brandenburg (Germany) in the year 2018. Water 12(11): 2982. https://doi.org/10.3390/w12112982
.

Cannabidiol Yield Characteristics

The results showed that irrigation significantly affected the concentration of inflorescences in CBD, CBDA, and CBD Total in the two growing seasons (p<0.05). During the first growing season, no significant differences were shown in CBD, CBDA, and CBD total concentrations between adequately and poorly irrigated hemp cultivars. Similar results were recorded by Garcia-Tejero et al. (2019)García-Tejero IF, Zuazo VD, Sánchez-Carnenero C, Hernández A, Ferreiro-Vera C, Casano S, 2019. Seeking suitable agronomical practices for industrial hemp (Cannabis sativa L.) cultivation for biomedical applications. Ind Crop Prod 139: 111524. https://doi.org/10.1016/j.indcrop.2019.111524
in a field experiment and Duong et al. (2023)Duong H, Pearson B, Anderson S, Berthold E, Kjelgren R, 2023. Variation in hydric response of two industrial hemp varieties (Cannabis Sativa) to induced water stress. Horticulturae 9(4): 431. https://doi.org/10.3390/horticulturae9040431
in greenhouse conditions. During 2020, both deficit irrigation regimes led to higher concentrated inflorescences in CBD, CBDA, and CBD total compared to fully irrigated plants. It was observed that the maximum water stress level (IRR₅₀) was the most favorable to produce higher concentrations of the specific cannabinoids. Similar results have been reported in the existing literature (Caplan et al., 2019Caplan D, Dixon M, Zheng Y, 2019. Increasing inflorescence dry weight and cannabinoid content in medical cannabis using controlled drought stress. HortScience 54(5): 964-969. https://doi.org/10.21273/HORTSCI13510-18
; García-Tejero et al., 2019García-Tejero IF, Zuazo VD, Sánchez-Carnenero C, Hernández A, Ferreiro-Vera C, Casano S, 2019. Seeking suitable agronomical practices for industrial hemp (Cannabis sativa L.) cultivation for biomedical applications. Ind Crop Prod 139: 111524. https://doi.org/10.1016/j.indcrop.2019.111524
; Morgan et al., 2024Morgan W, Singh J, Kesheimer K, Davis J, Sanz-Saez A, (2024). Severe drought significantly reduces floral hemp (Cannabis sativa L.) yield and cannabinoid content but moderate drought does not. Environ Exp Bot 219: 105649. https://doi.org/10.1016/j.envexpbot.2024.105649
), while some researchers recommend controlled drought as a means of increasing cannabinoid yield (Caplan et al., 2019Caplan D, Dixon M, Zheng Y, 2019. Increasing inflorescence dry weight and cannabinoid content in medical cannabis using controlled drought stress. HortScience 54(5): 964-969. https://doi.org/10.21273/HORTSCI13510-18
; Park et al., 2022Park SH, Pauli CS, Gostin EL, Staples SK, Seifried D, Kinney C, Vanden Heuvel BD, 2020. Effects of short-term environmental stresses on the onset of cannabinoid production in young immature flowers of industrial hemp (Cannabis sativa L.). J Cannabis Res 4: 1-13. https://doi.org/10.1186/s42238-021-00111-y
).

Concerning the CBD yield, the results of both growing seasons (2019, 2020) showed that the cultivars differed (p<0.001). Among the cultivars, ‘Futura 75’ and ‘Ferimon’ showed the highest content of the studied cannabinoids, while the lowest was recorded in ‘Santhica 27’ and ‘Uso 31’. Similar results were noted by Glivar et al. (2020)Glivar T, Eržen J, Kreft S, Zagožen M, Čerenak A, Čeh B, Tavčar Benković E, 2020. Cannabinoid content in industrial hemp (Cannabis sativa L.) varieties grown in Slovenia. Ind Crop Prod 145: 112082. https://doi.org/10.1016/j.indcrop.2019.112082
, where the cultivars Ferimon and ‘Fedora 17’ excelled in total cannabidiol content over ‘Santhica 27’ and ‘Uso 31’. Our results on the CBD, CBDA, and CBD Total content of the five varieties are verified by Sikora et al. (2011)Sikora V, Berenji J, Latković D, 2011. Influence of agroclimatic conditions on content of main cannabinoids in industrial hemp (Cannabis Sativa L.). Genetika 43(3): 449-456. https://doi.org/10.2298/GENSR1103449S
, and Burgel et al. (2020)Burgel L, Hartung J, Pflugfelder A, Graeff-Hönninger S, 2020. Impact of growth stage and biomass fractions on cannabinoid content and yield of different hemp (Cannabis sativa L.) genotypes. Agronomy 10(3): 372. https://doi.org/10.3390/agronomy10030372
.

Among the growing seasons, the greatest hemp growth was noticed in the second experimental year based on the plant height, and above-ground biomass values. Furthermore, the longest inflorescences with the most seeds, the greatest seed yield, and the highest SDI and WP values were also recorded in 2020. Concerning the studied CBD yield components, the second growing year was the most productive performing inflorescences with the maximum concentrations. These findings could be attributed to climatic conditions during the critical crop establishment, and the first growth months of 2020 compared with the 2019 and 2021 growing seasons. More specifically, the highest levels of precipitation and the shortest mean air temperature were recorded from May to June 2020.

In conclusion, water stress negatively affected the hemp growth and seed yield of all studied cultivars. Among irrigation deficit treatments, severe water stress (IRR₅₀) was the most limiting factor for biomass and seed yield, whereas enhanced the cannabidiol yield of inflorescences. The French cultivar of medium maturity ‘Ferimon’ was boosted the most by the severe drought recording the highest CBD Total (%w/w) concentrations. Under full irrigation conditions, all cultivars documented their greatest plant growth, seed yield, and water productivity. Results revealed that ‘Futura 75’ followed by ‘Fedora 17’ were the most suitable for biomass production as they had the highest biomass yield. Both ‘Fedora 17’ and ‘Ferimon’ showed high yields for seed production. Among the tested cultivars, ‘Fedora 17’ combined high biomass and seed yield, noted great water productivity and thus is considered suitable for dual-purpose production. Further investigation is needed to build up a database about the impact of adequate and limited water availability on the growth and productivity of hemp plants.