This study utilized the Kiskun 4291 (FAO 290) hybrid maize cultivar provided by Kiskun Kutatóközpont Kft. Kiskunhalas, Hungary. In greenhouse experiments, the seeds were sown in radio-tagged Plexiglass columns encased in polyvinyl chloride tubing. The columns were filled with a soil mixture consisting of 80% peat (Florimo, Kecel, Hungary) and 20% sand, supplemented with 6 g of Osmocote fertilizer (Substral, Evergreen Garden Care, UK). Five seeds were seeded for each combination, and 2x10 seeds for the control group. The installed columns were arranged randomly within an automated modular phenotyping system (Photon System Instruments, Drasov, Czech Republic) and irrigated to maintain 60% field capacity. The growth temperature was sustained at 24°C during the day and 20°C at night, with relative humidity ranging between 60% and 80%. The illumination level was determined by the modular phenotyping system and set to 400 μmol photons m^-² s^-¹, with minor variations detected throughout the experiment.

In the field experiments, maize seeds were sown at two different locations: our experimental garden in Szeged, Hungary (46°14’01.1”N 20°10’01.1”E) and at the breeding garden of the Kiskun Kutatóközpont in Mórahalom, Hungary (46°10’48.2”N 19°54’09.2”E). The row spacing was 70 cm, and the plant spacing was 50 cm; the rows extended 8 m in Szeged and 10 m in Mórahalom. Two rows were implemented for each combination, accompanied by an additional four rows of control plants. The maize plants were not irrigated throughout the experiment, during the growing period precipitations were the following: Szeged: 78mm, Mórahalom:57mm.

The genotypes Maros and Rába of short rotation willows were used in the extraction processes. These genotypes originated from our triploid willow breeding program (Dudits et al., 2022). The willow plants were grown at our experimental garden in Szeged, Hungary (46°14’01.1”N 20°10’01.1”E). The plant material for the extraction was collected in May 2024. Around 80 cm long shoots were cut from the plants, and the stems, leaves and meristematic tissues were collected separately. 30g of these materials were finely chopped and placed into beakers on a heated magnetic stirrer containing 750 ml 90 °C distilled water. The plant tissues were extracted at this temperature for 30 min with continuous stirring. The extracts were cooled down and filtered, then stored at 4 °C or -20 °C for prolonged preservation.

All solvents and chemicals used for sample preparation and analysis were UPLC gradient/GC pestinorm supratrace grade (VWR, Radnor, PA, USA). Willow extract samples were diluted with methanol: water (2:1 v/v%) solution to 0.5 ml/ml followed by vigorous vortex mixing and centrifugation at 16500 g (at 4°C for 10 min), and 1 ml of the methanol-water sample solution was liquid-liquid partitioned by adding 0.5 ml of n-hexane. Afterwards, centrifugation at 10000 g (at 4°C for 10 min) was addressed to collect the methanol: water phase that was finally filtered through 0.22 µm PTFE syringe filters prior to analysis. For analysis a Waters Acquity I-class UPLC system equipped with PDA detector were coupled to a Xevo TQ-XS Triple Quadrupole Mass Spectrometer with a UniSpray™ (US) ion source (Waters Corp.; Milford, MA, USA) was utilized. Separation of phenolic compounds was achieved on a HSS T3 column (1.8 μm; 100 mm × 2.1 mm) as described by Vrhovsek et al., 2012. Xevo TQ-XS MS was utilized in multiple reaction monitoring (MRM mode) according to Pál et al., 2020; UPLC and MS conditions and settings, method details, and respective MRM transitions used for quantification are listed in Supplementary material Table S1 .

In seed priming experiments, maize seeds were immersed in water or the original willow extracts for one day before sowing in either a greenhouse or a field. In foliar spray experiments, the willow extracts were diluted 1:1 with distilled water supplemented with a non-ionic wetting and sticking agent, Nonit (Agrokemia Sellye, Sellye, Hungary) at a concentration of 0.0025%. The treatment was performed once during the field experiment when the plants were 5 weeks old. The foliar spray treatment in the greenhouse was carried out twice; on the 23^rd and 30^th day.

Irrigation and digital imaging of the shoots were performed twice per week. Water consumption was continuously monitored, and irrigation was automatically adjusted by the phenotyping system. For imaging of the shoots, each column was transferred to an imaging chamber equipped with a rotatable platform, where two RGB cameras captured seven side-view and one top-view image of each maize shoot. Image segmentation and the extraction of shoot parameters were carried out automatically using the system’s software according to predefined settings.

Root imaging was conducted once per week. The Plexiglass columns were photographed from four side angles (90° apart) and from below using two Canon EOS 600D digital cameras in a separate imaging chamber located outside the PSI modular system, following the protocol described by Zombori et al. (2023). Root-associated white pixels were isolated by subtracting the background of the dark soil using custom-developed software. The visible root surface area was quantified by integrating data from the four side views and the bottom view.

The heights (cm) of maize plants developed from extracts or water-primed seeds were measured three times post-germination in the field experiment: at the developmental stage of three, five, and seven leaves. The plants subjected to foliar spray were measured prior to treatment and ten days after the treatment.

Chlorophyll fluorescence was measured using a Pocket PEA portable fluorimeter (Hansatech Instruments, King’s Lynn, UK). After a 20-min dark adaptation period, the minimal fluorescence yield (F₀) was recorded. Leaves were subsequently exposed to a saturating light pulse of 3500 μmol m^-² s^-¹ for 1 s to determine the maximal fluorescence yield (F_m) and the variable fluorescence (F_v). Additionally, several chlorophyll fluorescence parameters were calculated using appropriate formulas and equations of the JIP-test, which yields multiple parameters quantifying the photochemistry of Photosystem II (Strasser et al., 2004).

The data are shown as the mean of a minimum five replicates ± standard error of the mean. The statistical significance of the observed variations in different parameters was determined by using Student’s T-test via the online software GraphPad (www.graphpad.com). Statistical significance was defined at p ≤ 0.001 ***, p ≤ 0.01 **, and p ≤ 0.05 *.

Metabolites in water extracts from the leaves (L), stems (S), and meristems (M) of Rába (R) and Maros (M) genotypes were identified and quantified by LC-MS/MS in the following categories: 14 compounds in phenylpropanoid biosynthesis; 5 compounds in flavonoids; 4 compounds in flavones; 18 compounds in flavonols; 6 in anthocyanins; 10 in aminobenzoate degradation; 6 in plant hormones; 2 in stilbenes; and 1 in coumarins (see Supplementary Table 1 ). Here we give quantitative data about the identified compounds in different categories with potential bioactivity. Furthermore, we provide literature references discussing biostimulatory and therapeutic role of defined compounds detected in short rotation willow extracts. Table 1 . presents selected compounds with considerable amount (ng/ml) in various willow extracts.

Studies involving diverse plant species (Golzarian et al., 2011; Fehér-Juhász et al., 2014) indicate that green pixel values, which represent leaf and stem surface area are assumed to be directly proportional to the green biomass of plants. Consequently, this method was employed for comparing stimulatory effects of extracts derived from different organs of Rába and Maros willow genotypes on growth of maize plants. The phenotyping system employed in this work facilitated the cultivation of maize plants for 29 days under controlled optimal conditions ( Figures 1A, B ). Priming commercial maize hybrid seeds with water extracts from stems or meristems of willow plants can enhance shoot surface area and plant height. The stimulatory effect varied between extracts from the two genotypes and their respective organs. The mean shoot surface areas of maize plants were significantly greater following seed priming with Rába stem extract, in comparison to the plants treated with water or leaf extract ( Figures 1B ). The stimulatory effects of Maros and Rába stem extracts are expressed in the final growing period, as indicated by pixel values ( Figures 1A, B ). Extracts from Rába meristem tissues showed positive effect that did not attain statistical significance.

Physiological effects of willow extracts were also analyzed by determination of pixel-based plant height and stem width after spraying of maize plants at two time points: day 22nd and day 29th. As shown by Figure 2A seed priming with Maros stem and Rába meristem extracts significantly increased plant height. Spraying with Rába stem extract had a small but not significant positive effect on this parameter ( Figure 2A ). Interestingly, both priming seed and foliar spraying stimulated the width of maize stems ( Figure 2B ). Presently, we cannot explain the reason for differential responses of these two traits to the foliar treatment since the used extracts represent a complex chemical composition.

This study utilized a digital phenotyping system to ascertain the growth dynamics of the root system of the maize plants across various treatments. The growth diagrams ( Figures 3A–D ) indicate that alterations in root development are detectable after the third week. The seed priming treatment induced more significant changes, particularly with Rába meristem extract; however, the highest root pixel values were measured following the foliar spray treatment with Rába stem extract (7789.9 kilopixel, B). The stem and meristem extracts from shoots were generally more effective, regardless of genotype, except when using the Maros meristem extract in foliar spray treatment. Figure 4 presents examples of characteristics of maize root systems under various treatments with extracts from Rába and Maros genotypes. Positive effects of biostimulators on root systems have been documented in several experiments (see review by Han et al., 2024).

In photosynthesis, a portion of the solar energy absorbed by plants is re-emitted as chlorophyll fluorescence (ChlF), which servs as an effective indication of the plant´s physiological condition in assessing plant growth responses (see review by Rinmawii et al., 2024). From the large set of ChlF parameters (Makhtoum et al., 2023), Table 2 illustrates selective instances showing elevated values in maize leaves treated with various short rotation willow extracts. The examination of maize plants derived from treated seeds reveals a lasting impact, whereas ChlF markers detected in leaves one-week post-spraying signify an initial reaction. This difference may elucidate the finding that the set of responding individuals is fundamentally distinct between the two treatment methods. Moreover, the enhanced efficacy of Rába stem extract was detectable following seed treatment, although a comparable trend could not be seen after spraying.

Figure 5 displays kinetic curves of OJIP, which denotes “O” (minimum fluorescence), “J” (fluorescence after short light exposure), “I” (fluorescence after a longer light exposure), and “P” (maximum fluorescence) in maize leaves treated with water or willow extracts derived from meristem and stem tissues. These O-J-I-P transients show observed variations in the efficiency of the chlorophyll antenna in harnessing light energy, with the transfer to plastoquinone A (Q_A, the primary electron acceptor) being the only limitation of photochemical conversion in PSII. Leaves treated with Maros stem extract caused a significant increase of ChlF during the “P” phase, particularly one week after foliar spraying. A slight increase is visible after one day.

As shown by Table 2 , in maize leaves derived from primed seeds the following ChlF parameters show enhanced photosynthetic efficiency: Sm (Normalised Area; Area/Fv), which is directly proportional to the number of times the single QA molecule is reduced and oxidized (the primary quinone acceptor of photosystem II) during the fast OJIP transient, or the number of electrons passing through the electron transport chain; ABS/RC, the absorption flux per reaction center exciting PSII antenna Chl a molecules; N, the turnover number representing the frequency of QA reduction and re-oxidation; PI/ABS, the performance index for energy conservation from photons absorbed by PSII to the reduction of intersystem electron acceptors; ETo/RC, the electron transport flux (beyond Q_A ⁻) per reaction center; ψo, the maximum quantum yield for primary photochemistry ( Table 2 ). Extract from Rába stems resulted in greater increase in ChlF values in maize leaves developed from primed seeds.

Different ChlF parameters showed increased chlorophyll fluorescence than in leaves after spraying with effective extract: Fo: minimum fluorescence, measured in dark adapted state, when all PSII RCs are open (≅ to the minimal reliable recorded fluorescence); Fv: maximal variable fluorescence, measured in dark adapted state; Fm: maximum fluorescence, measured in dark adapted state, when all PSII RCs are closed; RC/CSo: Density of reaction centers per excited represents cross section; Fv/Fm·(VJ/Mo)·Fo, where VJ is the relative variable fluorescence at J step, and Mo is the initial slope of the relative variable fluorescence): represents the ratio of active reaction centers to the total number of reaction centers; RC/CSm: performance index for energy conservation from photons absorbed by PSII until the reduction of intersystem electron acceptors, represents the ratio of active reaction centers to Sm related active center; PI/ABS values are increased also after spraying with Maros stem extract. The use of extract from Rába stems to maize leaves proved particularly beneficial.

Digital phenotyping indicates moderated responses of maize plants to various extracts from short rotation willow plants in greenhouse with optimal growing conditions. Plant extract gains increasing significance as biostimulants (Han et al., 2024), therefore, we have evaluated various extracts under limited water supply and various soil conditions at two field study locations (Szeged and Mórahalom). For demonstrating the stimulatory effects of different short rotation willow extracts, we present plant height data (cm) in Figure 6 . All extract treatments of maize seeds significantly stimulated plant growth at both locations. For unclear reason, extracts from Rába leaves exhibited significant activity in Szeged ( Figure 6A ), although no stimulatory effect was seen in Mórahalom location ( Figure 6B ). Conversely, leaf extracts from the Maros genotype exhibited significant activity.

Alongside seed priming, short rotation willow extracts were evaluated by spraying treatments ( Figures 7A, B ). Extracts from Rába stem tissues could induce significant growth stimulation in both locations. At Szeged experimental station, meristem and stem extracts from Maros genotype enhanced the growth of maize plants.

In addition to monitoring the stimulation of vegetative growth of maize plants, we have characterized seed yield parameters under field conditions in Szeged region. Part of the experimental field in Mórahalom was accidently irrigated, therefore we could not use these information. Primed seed-derived plants did not show significant changes in seed parameters compared to untreated plants. Spraying maize plants at the seven-leaf stage could influence seed weight (g) per ear and 1000-seed weight (g) in the selected genotype and organ extract treatments ( Table 3 ). Based on seed parameters of untreated plants, we had to realize the variations in growing conditions in various parts of experimental field. Therefore, we use two datasets from plot A and plot B. Under ideal conditions (plot A), spraying with Rába meristem extract resulted in 12,10% increase in seed weight per ear. The stimulation of 1000-grain weight was 7,00%. In plot B, the Rába leaf extract increased seed weight per year by 17,42%, the Rába meristem extract by 16,79%, and the Maros stem extract by 12,99%. The weights of 1000 seeds were also stimulated by the Maros meristem extract (21.22%) and by Rába leaf extract (17,31%). These preliminary data show the same tendency as that was published by Li et al. (2022) after meta-analysis of yield stimulation. Furthermore, both the greenhouse study conducted under optimal conditions, and the present field results across various soil conditions agree with previous observations that the biostimulant impact can be pronounced under stress situations (Li et al. (2022).

The foliar application of biostimulants is a cost-effective method strategy that is prioritized in crop production, particularly using seaweed extracts (Hariharan et al., 2024). In the present experiments, both in the greenhouse and the field, seed priming proved more effective than foliar spray for biomass production, although foliar spray was superior for yield increase. The seaweed extracts resulted in 17.1% enhancement. In our case, the overall stimulation by willow extract in yield was 13% as the maize plants productivity is concerned.