The field trial was carried out in the nursery of the Centre for Agricultural Research, Martonvásár, Central Hungary (N 47°19’, E 18°47’, 110 m asl.) over the 2018–19 and 2020–21 growing seasons (2019 and 2021 hereafter). The soil is classified by FAO-WRB (IUSS Working Group 2015) as a Haplic Chernozem (34% sand, 42% silt and 24% clay in the 0–25 cm layer), with a pHH2O of 7.59, 1.84% CaCO3, 3.39% humus, 1799/374/429 mg kg–1 total N/P/K, and 0.322 and 0.476 cm3 cm–3 water content at field capacity and saturation point, respectively.
The climate is continental with a mean (1988–2017; recorded by an on-site weather station) annual temperature of 10.9 °C (January: –1.2 °C, June: 21.2 °C) and total rainfall of 552 mm with 193 mm falling during the main winter wheat growing season (March–June; Fig. 1).
Crop cultivation and FACE system
A factorial experiment was set up in three replicates with (1) two winter wheat (Triticum aestivum L.) cultivars: the early maturing Mv Nemere and the medium-early Mv Dandár, (2) two levels of N supply: low (80 kg ha–1) and high (160 kg ha–1), and (3) two levels of [CO2]: ambient and elevated to ~600 ppm. A split-plot arrangement was used, where the [CO2] treatment was in the main plot and the wheat cultivar by N combination in the subplots. A moldboard plow was used for tillage to a depth of 25 cm. The soil was manually fertilized with ammonium nitrate a week before planting (30% of the total doses) and in early spring (70%). Wheat was sown in mid-October 2018 and 2020 at a density of 500 seedlings m–2 with a row spacing of 12.5 cm, using a standard 3 m wide seeder to ensure uniform row and plant spaces for the whole area. The crop stand was sprayed with a pesticide combination at the 3-leaf stage in April.
The FACE system was engineered by the Institute for Biometeorology, Italian National Research Council, Florence (for details, see Miglietta et al. 2001). The facility consisted of three 18-m diameter octagonal rings (~250 m2) of horizontal tubes releasing pure CO2 on the upwind side to a targeted [CO2] of 600 ppm. The fumigation of the small amount of CO2 at high velocity through a large number of small gas jets allows open-air elevation of [CO2] without altering the microclimate (Ainsworth and Long 2020). A GMP343 type sensor (Vaisala Co. Ltd., Helsinki, Finland) was installed centrally in each ring to monitor [CO2] and control the venting. The FACE rings were installed right after wheat planting. The plots were fumigated throughout the whole vegetation season (from crop emergence to the fully ripe stage) every time when the plants were photosynthetically active, i.e. during the daylight hours with ambient temperature above 0 °C. The tubes were kept at a height of 0.1–0.2 m above the wheat canopy. The average [CO2] in the FACE rings during the treatment period was 597 and 587 ppm in 2019 and 2021, respectively, and was within 600 ppm ± 10% for 76.2% and 69.5% and within 600 ppm ± 20% for 94.2% and 90.8% of the operational time in the given years. Three further rings without CO2 enrichment were established as control (ambient) plots.
Monitoring of root electrical capacitance and leaf area index
CR was measured eight times between the 2-node stage (late March) and the over-ripe stage of wheat (early July) to monitor the seasonal root dynamics. The phenology stages were documented using the BBCH scale (Meier 2001). On each occasion, twelve plants were randomly selected from various rows in the centre of each plot (36 plants per treatment, 288 plants in total), and new plants were chosen on the next sampling date. SWC was measured at a depth of 0–12 cm in the root zone of each sample plant with a calibrated CS620 portable TDR meter (Campbell Sci. Ltd., Loughborough, UK), inserting the sensor vertically in the interrow, 6 cm away from the plant.
Thereafter, CR was measured for each plant in a parallel circuit at 1 kHz, 1 V AC with a U1733C handheld LCR instrument (Agilent Co. Ltd., Penang, Malaysia). One kHz is widely considered to be the optimum operating frequency, which induces an efficient electrostatic energy storage, and eliminates electrode polarization effects and stray capacitances (Ozier-Lafontaine and Bajazet 2005; Středa et al. 2020). A stainless steel rod, 15 cm in length and 6 mm in diameter, was used as a ground electrode, pushed vertically into the soil to a depth of 12 cm, 6 cm distance from the plant (in the place of the TDR probe). The plant electrode was clamped to all basal parts of the plant 15 mm above the soil (Svačina et al. 2014), after smearing them with conductivity gel to ensure good electric contact. The measured volumetric SWC values were divided by the saturation water content to obtain the relative water saturation (θrel). The saturation capacitance was calculated for each of the 288 θrel–CR data pairs as CR* = CR × 5.807e–1.115θrel to eliminate the SWC effect. The procedure for calculating the exponential function was previously reported in detail by Cseresnyés et al. (2018).
LAI was measured non-destructively in each plot five times in each growing season, on the same dates as the CR measurements (or 1–2 days later due for weather reasons). The monitoring terminated at the wheat flowering stage (mid-May), when LAI peaked but the reading was not yet affected by leaf senescence (Pokovai and Fodor 2019). LAI was detected during clear midday hours (from 10 a.m. to 2 p.m.) using an LP-80 handheld ceptometer (Meter Group Inc., Pullmann, WA, USA). Each LAI value, calculated as a mean of 22 readings, represented a ~0.6 m2 area under the canopy. The 80 cm long probe was placed parallel and perpendicular to the crop rows, and was read twice in each position.
Plant sampling and harvest
In 2019 destructive plant sampling had to be postponed from anthesis to the end of flowering owing to bad weather conditions, so sampling was also carried out in this stage in 2021 to ensure comparability. A 0.5 m long row was manually cut just above the soil surface in each plot. After drying the samples at 70 °C, SDM was determined (±0.01 g) and expressed as t ha–1 values.
Plants from the central 2 m × 6 m area of each plot were harvested and threshed at grain maturity (mid-July 2019 and 2021) using a plot combine. GY was determined in t ha–1 on a dry weight basis.
Considering that only one GY value was obtained per plot (though they represented a relatively large area), a plant scale investigation was also carried out to confirm the relationship of CR* to TAB and GM. On the day in 2019 when CR* measurements were taken at the flowering stage (day of year, DOY 140), the 36 sample plants in the “NL(–)” and “DL(–)” treatments were individually tagged at the stem base. These plants were cut separately at maturity, after which TAB was weighed (±0.001 g) after drying at 70 °C. The spikes were hand threshed to record GM.
The data were analysed in R programming language (R Core Team 2021). At first, two-way ANOVA was used to examine the effect of N (two levels: low and high) and CO2 (two levels: ambient and elevated), as categorical variables, and their interactions N × CO2 on quantitative variables of CR*, LAI, SDM and GY within each year for both cultivars. Thereafter, multivariate ANOVA was performed to evaluate the effect of year, N, CO2, as categorical variables, and their interactions on the maximum CR*, maximum LAI, SDM and GY, as quantitative variables for both cultivars. The normality, and the equality of variances in the data groups were examined with the Shapiro–Wilk test and Levene test, respectively. Statistical significance was assessed at p < 0.05 in each case.
Linear regression models were compiled: (i) to evaluate the relationships between CR* and LAI (across the N and CO2 treatments), and between the maximum CR*, SDM and GY (across the N and CO2 treatments and years) at a stand scale; and (ii) to relate CR* to TAB and GM at a plant scale for the cultivars. The line slopes and y-intercepts were compared using ANOVA. The reason for applying the maximum CR* (measured at flowering) was that it previously proved to be the best predictor of yield (Chloupek et al. 2006).