Specialized Metabolites from the Allelopathic Plant Retama raetam as Potential Biopesticides

To cope with the rising food demand, modern agriculture practices are based on the indiscriminate use of agrochemicals. Although this strategy leads to a temporary solution, it also severely damages the environment, representing a risk to human health. A sustainable alternative to agrochemicals is the use of plant metabolites and plant-based pesticides, known to have minimal environmental impact compared to synthetic pesticides. Retama raetam is a shrub growing in Algeria’s desert areas, where it is commonly used in traditional medicine because of its antiseptic and antipyretic properties. Furthermore, its allelopathic features can be exploited to effectively control phytopathogens in the agricultural field. In this study, six compounds belonging to isoflavones and flavones subgroups have been isolated from the R. raetam dichloromethane extract and identified using spectroscopic and optical methods as alpinumisoflavone, hydroxyalpinumisoflavone, laburnetin, licoflavone C, retamasin B, and ephedroidin. Their antifungal activity was evaluated against the fungal phytopathogen Stemphylium vesicarium using a growth inhibition bioassay on PDA plates. Interestingly, the flavonoid laburnetin, the most active metabolite, displayed an inhibitory activity comparable to that exerted by the synthetic fungicide pentachloronitrobenzene, in a ten-fold lower concentration. The allelopathic activity of R. raetam metabolites against parasitic weeds was also investigated using two independent parasitic weed bioassays to discover potential activities on either suicidal stimulation or radicle growth inhibition of broomrapes. In this latter bioassay, ephedroidin strongly inhibited the growth of Orobanche cumana radicles and, therefore, can be proposed as a natural herbicide.


Introduction
Since the second half of the 20th century, the agricultural practices have been based on the uncontrolled use of chemical pesticides, and herbicides, to cope with the rising crop Thus, the main purpose of the present study was to further investigate the potential of the crude organic extract obtained from the R. raetam aerial part as a source of biopoesticides. Bioactivity-guided purification was performed using anti-fungal bioassays against the phytopathogenic fungus S. vesicarium. This is a traditional method used in natural product discovery that allows researchers to isolate the pure bioactive compounds from a complex mixture, such as plants' organic extracts [33][34][35][36]. In particular, following the identification of a crude extract with promising biological activity, the next step is its (often multiple) consecutive bioactivity-guided fractionation until the pure bioactive compounds are isolated [37].
This manuscript reports the isolation of six metabolites identified by spectroscopic and chemical methods. Their potential antifungal activity against the phytopathogen S. vesicarium and the herbicidal activity against broomrapes (as inductors of suicidal germination and inhibitors of radicle growth), are also discussed.

Results and Discussion
The dried aerial parts of R. raetam were extracted as detailed in the Materials and Methods Section. Bioactivity-guided purification was performed using anti-fungal bioassays against the phytopathogenic fungus S. vesicarium. The active CH 2 Cl 2 extract was fractionated by CC, yielding 13 fractions (F1-13) with variable chemical profiles, which were evaluated for their antifungal activity against S. vesicarium ( Figure 1). extremely arid zone of great importance among medicinal plants [28][29][30][31]. Recently, researchers have highlighted the effectiveness of aqueous extracts of some Saharan plant species, including R. raetam, on seed germination or seedling growth of target species [32]. This result suggests that the plant can be a source of allelochemicals able to inhibit the radicle growth of parasitic weeds [15]. Thus, the main purpose of the present study was to further investigate the potential of the crude organic extract obtained from the R. raetam aerial part as a source of biopoesticides. Bioactivity-guided purification was performed using anti-fungal bioassays against the phytopathogenic fungus S. vesicarium. This is a traditional method used in natural product discovery that allows researchers to isolate the pure bioactive compounds from a complex mixture, such as plants' organic extracts [33][34][35][36]. In particular, following the identification of a crude extract with promising biological activity, the next step is its (often multiple) consecutive bioactivity-guided fractionation until the pure bioactive compounds are isolated [37].
This manuscript reports the isolation of six metabolites identified by spectroscopic and chemical methods. Their potential antifungal activity against the phytopathogen S. vesicarium and the herbicidal activity against broomrapes (as inductors of suicidal germination and inhibitors of radicle growth), are also discussed.

Results and Discussion
The dried aerial parts of R. raetam were extracted as detailed in the Materials and Methods Section. Bioactivity-guided purification was performed using anti-fungal bioassays against the phytopathogenic fungus S. vesicarium. The active CH2Cl2 extract was fractionated by CC, yielding 13 fractions (F1-13) with variable chemical profiles, which were evaluated for their antifungal activity against S. vesicarium ( Figure 1).

Figure 1.
Inhibitory effect of the CH2Cl2 crude extract (RR) and the thirteen fractions obtained from the preliminary purification of RR, on the mycelium of S. vesicarium, at a concentration of 2 mg/mL (crude extract) and 250 μg/mL (fractions), respectively. The negative control was absolute methanol (C−), and the positive control was 200 μg/mL of PCNB. The fungal growth inhibition is represented as the percentage reduction of the fungal mycelia diameter in the treated plate compared to that in the control plate. All experiments were performed in triplicate with three independent trials. Data are presented as means ± standard deviation (n = 3) compared to the control. *** p < 0.0001 and * p < 0.05. Figure 1. Inhibitory effect of the CH 2 Cl 2 crude extract (RR) and the thirteen fractions obtained from the preliminary purification of RR, on the mycelium of S. vesicarium, at a concentration of 2 mg/mL (crude extract) and 250 µg/mL (fractions), respectively. The negative control was absolute methanol (C−), and the positive control was 200 µg/mL of PCNB. The fungal growth inhibition is represented as the percentage reduction of the fungal mycelia diameter in the treated plate compared to that in the control plate. All experiments were performed in triplicate with three independent trials. Data are presented as means ± standard deviation (n = 3) compared to the control. *** p < 0.0001 and * p < 0.05.
Interestingly, the activity exhibited by the fractions F2-F5 (~60%) was stronger than that displayed by the total crude extract (~43%) and comparable to the commercial fungicide pentachloronitrobenzene (PCNB), used as a positive control ( Figure 1). These latter fractions were further purified by combined column and TLCs (Scheme S1) to afford six pure metabolites (1-6, Figure 2), as described in the Materials and Methods Section. The first investigation of their 1 H NMR and ESI MS spectra (Figures S1-S14) showed that they are isoflavones and flavones with different substitutions. They were identified by comparing their 1 H NMR and MS data with those reported in the literature as alpinumisoflavone (1) [38][39][40], hydroxyalpinumisoflavone (2) [41], laburnetin (3) [42], licoflavone C (4) [43], retamasin B (5) [44], and ephedroidin (6) [41]. The structure of alpinumisoflavone (1) was confirmed by X-ray diffractometric analysis. An ORTEP view is reported in Figure 3. The X-ray crystal structure is reported here to undoubtedly identify the compound 1 as alpinumisoflavone [45]. Alpinumisoflavone consists of three nearly coplanar fused rings, and one attached out-ofplane twisted phenyl ring. In the tricyclic ring system, a liner junction of A/B/C rings is observed. It should be noted that compound 1 showed a different junction between the dimethylpyran ring (A) and the chromenone moiety (B/C), with respect to the analog derrone ( Figure 4) previously isolated from R. raetam flowers [46].  The structure of alpinumisoflavone (1) was confirmed by X-ray diffractometric analysis. An ORTEP view is reported in Figure 3. The X-ray crystal structure is reported here to undoubtedly identify the compound 1 as alpinumisoflavone [45]. Alpinumisoflavone consists of three nearly coplanar fused rings, and one attached out-of-plane twisted phenyl ring. In the tricyclic ring system, a liner junction of A/B/C rings is observed. It should be noted that compound 1 showed a different junction between the dimethylpyran ring (A) and the chromenone moiety (B/C), with respect to the analog derrone ( Figure 4) previously isolated from R. raetam flowers [46].
Interestingly, the activity exhibited by the fractions F2-F5 (~60%) was stronger than that displayed by the total crude extract (~43%) and comparable to the commercial fungicide pentachloronitrobenzene (PCNB), used as a positive control ( Figure 1). These latter fractions were further purified by combined column and TLCs (Scheme S1) to afford six pure metabolites (1-6, Figure 2), as described in the Materials and Methods Section. The first investigation of their 1 H NMR and ESI MS spectra (Figures S1-S14) showed that they are isoflavones and flavones with different substitutions. They were identified by comparing their 1 H NMR and MS data with those reported in the literature as alpinumisoflavone (1) [38][39][40], hydroxyalpinumisoflavone (2) [41], laburnetin (3) [42], licoflavone C (4) [43], retamasin B (5) [44], and ephedroidin (6) [41]. The structure of alpinumisoflavone (1) was confirmed by X-ray diffractometric analysis. An ORTEP view is reported in Figure 3. The X-ray crystal structure is reported here to undoubtedly identify the compound 1 as alpinumisoflavone [45]. Alpinumisoflavone consists of three nearly coplanar fused rings, and one attached out-ofplane twisted phenyl ring. In the tricyclic ring system, a liner junction of A/B/C rings is observed. It should be noted that compound 1 showed a different junction between the dimethylpyran ring (A) and the chromenone moiety (B/C), with respect to the analog derrone ( Figure 4) previously isolated from R. raetam flowers [46].   Although compounds 2, 3, and 6 have chiral centers, their absolute configurations were not determined so far. Due to the limited available amounts of these compounds, a modified Mosher method [47] was applied only to compound 6 to determine the absolute configuration of its secondary hydroxylated carbon (C-2"). When compound 6 was treated with S-MTPA chloride, its ester derivative showed two sets of signals with an enantiomeric ratio of 50:50, indication that 6 is an enantiomeric mixture of 2"(S)-6 and 2"(R)-6. The same result was obtained when 6 was treated with R-MTPA chloride. An optical rotation value of zero [α]D 25 0 (c 0.4, MeOH) was also obtained. This result was not unexpected because a similar prenylated xantone, named (±)-graciesculenxanthone C, isolated from Garcinia esculenta showed an enantiomeric ratio of 60:40 when it was treated with S-and R-MTPA chloride [48].
Thus, the six compounds (1-6) isolated from R. raetam were spot-inoculated on PDA plates to test their antifungal activity against the fungal phytopathogen S. vesicarium. As shown in Figure 5, only laburnetin (3) exhibited quite a strong activity when spotinoculated (50 µg/mL), inhibiting the growth of S. vesicarium by around 55%, confirming the antagonistic effect displayed by the most active fractions, which inhibited S. vesicarium Although compounds 2, 3, and 6 have chiral centers, their absolute configurations were not determined so far. Due to the limited available amounts of these compounds, a modified Mosher method [47] was applied only to compound 6 to determine the absolute configuration of its secondary hydroxylated carbon (C-2"). When compound 6 was treated with S-MTPA chloride, its ester derivative showed two sets of signals with an enantiomeric ratio of 50:50, indication that 6 is an enantiomeric mixture of 2"(S)-6 and 2"(R)-6. The same result was obtained when 6 was treated with R-MTPA chloride. An optical rotation value of zero [α] D 25 0 (c 0.4, MeOH) was also obtained. This result was not unexpected because a similar prenylated xantone, named (±)-graciesculenxanthone C, isolated from Garcinia esculenta showed an enantiomeric ratio of 60:40 when it was treated with Sand R-MTPA chloride [48].
Thus, the six compounds (1-6) isolated from R. raetam were spot-inoculated on PDA plates to test their antifungal activity against the fungal phytopathogen S. vesicarium. As shown in Figure 5, only laburnetin (3) exhibited quite a strong activity when spotinoculated (50 µg/mL), inhibiting the growth of S. vesicarium by around 55%, confirming the antagonistic effect displayed by the most active fractions, which inhibited S. vesicarium mycelium's growth by around 60% (Figure 1). Interestingly, the commercial fungicide PCNB, used at a concentration of 0.5 mg/mL, exhibited an antagonistic effect comparable to the one exerted by laburnetin used at a 10-fold lower concentration (50 µg/mL) (Figure 5b), demonstrating how natural compounds could represent an effective alternative to chemicals in the agricultural field. The other compounds instead displayed a fungal inhibition of around 25% (1, 6), 20% (2, 4), and 17% (5) (Figure 5b). Considering the results obtained, it is possible to ascribe to laburnetin the main role in the antagonistic effect exhibited by R. raetam extract against the fungus S. vesicarium. Although compounds 2, 3, and 6 have chiral centers, their absolute configurations were not determined so far. Due to the limited available amounts of these compounds, a modified Mosher method [47] was applied only to compound 6 to determine the absolute configuration of its secondary hydroxylated carbon (C-2"). When compound 6 was treated with S-MTPA chloride, its ester derivative showed two sets of signals with an enantiomeric ratio of 50:50, indication that 6 is an enantiomeric mixture of 2"(S)-6 and 2"(R)-6. The same result was obtained when 6 was treated with R-MTPA chloride. An optical rotation value of zero [α]D 25 0 (c 0.4, MeOH) was also obtained. This result was not unexpected because a similar prenylated xantone, named (±)-graciesculenxanthone C, isolated from Garcinia esculenta showed an enantiomeric ratio of 60:40 when it was treated with S-and R-MTPA chloride [48].
Thus, the six compounds (1-6) isolated from R. raetam were spot-inoculated on PDA plates to test their antifungal activity against the fungal phytopathogen S. vesicarium. As shown in Figure 5, only laburnetin (3) exhibited quite a strong activity when spotinoculated (50 µg/mL), inhibiting the growth of S. vesicarium by around 55%, confirming the antagonistic effect displayed by the most active fractions, which inhibited S. vesicarium mycelium's growth by around 60% (Figure 1). Interestingly, the commercial fungicide PCNB, used at a concentration of 0.5 mg/mL, exhibited an antagonistic effect comparable to the one exerted by laburnetin used at a 10-fold lower concentration (50 µg/mL) ( Figure  5b), demonstrating how natural compounds could represent an effective alternative to chemicals in the agricultural field. The other compounds instead displayed a fungal inhibition of around 25% (1, 6), 20% (2, 4), and 17% (5) (Figure 5b). Considering the results obtained, it is possible to ascribe to laburnetin the main role in the antagonistic effect exhibited by R. raetam extract against the fungus S. vesicarium. The isoflavonoid laburnetin (3) has already been isolated from the Genista genus [41], as well as from other plants, and its antimicrobial activity was demonstrated [49]. To the best of our knowledge, here, the antifungal activity of this compound against S. vesicarium is being reported for the first time, showing that laburnetin could be proposed as a natural antagonist for the control of this phytopathogen that infests several important cultivated species.
R. raetam is an allelopathic plant species collected in the Saharan ecosystem from the Souf region in southeastern Algeria. Allelochemicals involved in plant-plant interactions are a potential source for alternative agrochemicals to solve the negative effects caused by synthetic herbicides. Thus, the six metabolites (1-6) were tested on two independent parasitic weed bioassays to discover potential activities of R. raetam metabolites on either suicidal stimulation or radicle growth inhibition of broomrapes. First, the germination induction effect of alpinumisoflavone, ephedroidin, hydroxyalpinoisolflavone, laburnetin, licoflavone C, and retamasin B was tested on seeds of four parasitic weed species, Orobanche crenata, O. cumana, O. minor, and Phelipanche ramosa, using in vitro germination bioassays. The synthetic germination stimulant GR24 used as a positive control induced germination levels of 53.2% ± 1.7%, 64.6% ± 1.8%, 91.7% ± 1.7%, and 94.2% ± 0.4% in O. crenata, O. cumana, O. minor, and P. ramosa, respectively. Null germination was observed when seeds of the broomrape species were treated with the negative control (distilled water) or with compounds 1-6. The results obtained in the germination bioassay indicate that none of the metabolites isolated from the stem of R. raetam act as suicidal germination inducers of the broomrape species studied. Field application of inductors of suicidal germination of broomrape seeds in the absence of a specific host is a control strategy for obligate root parasitic weeds. In fact, the subsequent parasitic growth after germination leads to the death of the parasite due to nutrient starvation in the absence of host-derived nutrients [50].
In a second parasitic weed bioassay, the six purified compounds (1-6) were tested at 100 µM as potential inhibitors of radicle growth of O. crenata, O. cumana, O. minor, and P. ramosa. Among the compounds tested, low to negligible activity was found in all compounds tested except for ephedroidin (6), which strongly inhibited the normal development of O. cumana radicles in comparison with O. cumana control radicles ( Figure 6A). Ephedroidin induced a strong toxic effect, observed as darkening in the O. cumana radicle and an average length inhibition of 80.8% ± 1.6% in comparison with radicles control ( Figure 6B,C). Similar toxic effects on broomrape radicles were previously described for cytochalasans [51]. Ephedroidin (6) is a flavanoid previously isolated together with laburnetin (3) from the Genista ephedroides [41] and from R. raetam [52]. Recently, ephedroidin resulted to be the most active in inhibiting nitric oxide synthase (iNOS) and nuclear factor kappa B (NF-κB), as well as in decreasing oxidative stress, when compared with other flavonoids isolated from the same source [44]. Our results demonstrated that ephedroidin strongly Ephedroidin (6) is a flavanoid previously isolated together with laburnetin (3) from the Genista ephedroides [41] and from R. raetam [52]. Recently, ephedroidin resulted to be the most active in inhibiting nitric oxide synthase (iNOS) and nuclear factor kappa B (NF-κB), as well as in decreasing oxidative stress, when compared with other flavonoids isolated from the same source [44]. Our results demonstrated that ephedroidin strongly inhibited the radical development of O. cumana seeds and can be proposed as a natural herbicide against this dangerous parasitic weed.

Conclusions
Bio-guided purification of R. raetam CH 2 Cl 2 extract allowed us to isolate six metabolites, identified by spectroscopic and chemical methods as alpinumisoflavone, hydroxyalpinumisoflavone, laburnetin, licoflavone C, retamasin B, and efedroidin. In particular, the isoflavonoid laburnetin showed antifungal activity against the phytopathogen S. vesicarium 10-fold higher than that of the commercial fungicide PCNB. The flavonoid ephedroidin exhibited a strong inhibition of broomrape seed germination, suggesting their application as potential biopesticides against these noxious biotic stresses. Finally, the structure of alpinumisoflavone was confirmed by X-ray diffractometric analysis, which showed a different junction with respect to the analog derrone, previously isolated from the R. raetam plant. These data prompted further studies aimed to formulate the active compounds and test them in greenhouse and field trials. However, analyses on their ecotoxicological profile are needed before the practical application as biopesticides.

General Experimental Procedures
A JASCO P-1010 digital polarimeter was used to measure the optical rotations. A Bruker (Karlsruhe, Germany) spectrometer working at 400/100 MHz was used to record 1 H/ 13 C NMR spectra in CDCl 3 or CD 3 OD, which were also used as internal standards. The LC/MS TOF system (Agilent 6230B, HPLC 1260 Infinity) (Milan, Italy) was used to record ESI mass spectra. Analytical and preparative Thin-Layer Chromatography (TLC) was performed on silica gel plates (Kieselgel 60, F 254 , 0.25 and 0.5 mm, respectively) (Merck, Darmstadt, Germany). The spots were visualized by exposure to UV light (254 nm) and/or iodine vapors and/or by spraying first with 10% H 2 SO 4 in MeOH, and then with 5% phosphomolybdic acid in EtOH, followed by heating at 110 • C for 10 min. Column chromatography (CC) was performed using silica gel (Kieselgel 60, 0.063-0.200 mm) (Merck, Darmstadt, Germany). All the solvents were supplied by Sigma-Aldrich (Milan, Italy). The balance model used is Analytical ES 225SM-DR (Precisa, Dietikon, Switzerland).

Plant Material
Aerial parts of R. raetam were collected between December 2017 and February 2018, corresponding to the flowering phase of the plant. This study was carried out in the Souf region and is located in the north-east of Algerian Sahara, between 33 • and 34 • north latitude, and 6 • and 8 • longitude. Sea level = 40 m [32]. The plant material was then carefully rinsed with distilled water to remove dust particles and dried in the air for a few days at room temperature; finally, it was ground in a blender.
Seeds of parasitic weeds were collected from mature plants of O. crenata infecting pea in Spain, O. cumana infecting sunflower in Spain, O. minor infecting red clover in France, and Phelipanche ramosa infecting oilseed rape in France. Dry parasitic seeds were separated from capsules using winnowing combined with a sieve of 0.6 mm-mesh size and then stored dry in the dark at room temperature until use for this work.

Fungal Strain
The phytopathogen S. vesicarium was isolated from pears showing brown spot disease symptoms, sampled in Benevento, Campania, Italy, in 2019, as previously reported [1]. The

X-ray Crystal Structure Analysis of Compound 1
Single crystals of compound 1 suitable for X-ray analysis were obtained by slow evaporation from a mixture of MeOH:H 2 O (9.0:1.0). X-ray diffraction data were collected on a Bruker-Nonius KappaCCD diffractometer (Bruker-Nonius, Delft, The Netherlands) (graphite mono-chromated MoKα radiation, λ = 0.71073 Å). The structure was solved by direct methods (SIR97 program) [53] and anisotropically refined by the full-matrix least-squares method on F 2 against all independent measured reflections (SHELXL-2018/3 program) [54]. H atoms of hydroxy groups were located in different Fourier maps and freely refined. All the other hydrogen atoms were introduced in calculated positions and refined according to the riding model. Platon TwinRotMap check suggests 2-axis (0 0 1) [1 0 4] twinning with basf 0.10, and refinement was performed using the HKLF5 data file. The figure of the ORTEP view was generated using the ORTEP-3 program [55]. Crystallographic

Antifungal Assay
The extract obtained from R. raetam aerial parts was tested against the phytopathogen S. vesicarium, as described by Yusoff et al. [56], with some modifications. The crude extract and the following fractions were dissolved in MeOH and mixed with 5 mL of cooled PDA to obtain a final concentration of 2 mg/mL and 250 µg/mL, respectively. The mix was then poured into Petri dishes and left to dry. Fungal plugs (6 × 6 mm diameter) cut from the growing edge of S. vesicarium mycelium were placed in the center of the plates and grown for 6/7 days at 28 ± 2 • C. Plates containing the fungal plugs alone were used as a control. As a positive control, fungicidal pentachloronitrobenzene ≥ 94% (PCNB) (Sigma-Aldrich, Saint-Louis, MO, USA) dissolved in toluene was used. Toluene and MeOH were used as negative controls. The in vitro antifungal bioassays of the purified metabolites, 1 laburnetin, 2 licoflavone C, 3 alpinumisoflavone, 4 hydroxyalpinumisoflavone, 5 raetamsin B, and 6 ephedroidin, were performed according to the method previously described in [57], with some modifications. The metabolites and PCNB dissolved in 8% acetone and toluene, respectively, were placed at the four opposite sides of each Petri dish, 1 cm away from the fungal plug at the center of the plate, at a final concentration of 50 µg/mL. Acetone and toluene were used as negative controls. The plates were incubated for 6/7 days at 28 ± 2 • C. The percentage of inhibition of the fungal growth was calculated using the following formula: where Rc is the radial growth of the test fungi in the control plates (mm), and Ri is the radial growth of the fungi in the presence of different compounds tested (mm). The results show the antifungal activity of different compounds analyzed by ANOVA using Tukey's test. The experiments were performed in triplicate.
Seeds of four broomrape species, Orobanche crenata, Orobanche cumana, Orobanche minor, and Phelipanche ramosa, were surface-sterilized by immersion in 0.5% (w/v) NaOCl and 0.02% (v/v) Tween 20, for 5 min, rinsed thoroughly with sterile distilled water, and dried in a laminar airflow cabinet. First, broomrape seeds were submitted to a conditioning period using a warm stratification, as follows. Approximately 100 seeds of each broomrape species were placed separately on 9 mm-diameter glass fiber filter paper disks (GFFP) (Whatman International Ltd., Maidstone, UK), moistened with 50 µL of sterile distilled water, and placed in incubators at 23 • C for 10 days inside Parafilm-sealed Petri dishes, to allow seed conditioning.
Then, GFFP disks containing conditioned broomrape seeds were transferred onto a sterile sheet of filter paper and transferred to new 9 cm sterile Petri dishes. For the assay of suicidal germination, induction stock solutions of each metabolite respectively dissolved in methanol were individually diluted in sterile distilled water up to an equivalent concentration of 100 µM. For the assay of radicle growth inhibition, stock solutions of each metabolite respectively dissolved in methanol were individually diluted to 100 µM using an aqueous solution of GR24. For each assay, triplicate aliquots of each sample were applied to GFFP discs containing conditioned broomrape seeds. Treated seeds were incubated in the dark at 23 • C for 7 days and the percent of germination and radicle growth was determined for each GFFP disc, as described previously [51], using a stereoscopic microscope (Leica S9i, Leica Microsystems GmbH, Wetzlar, Germany). For germination induction assays, the germination was determined by counting the number of germinated seeds on 100 seeds for each GFFP disk. For the characteristic of radicle growth, the value used was the average of 10 randomly selected radicles per GFFP disc [58]. The percentage of germination induction of each metabolite was then calculated relative to the average germination of control seeds (seeds treated with water), and the percentage of radicle growth inhibition of each treatment was then calculated relative to the average radicle growth of control treatment (radicles treated with GR24) [41].

Data Analysis
Statistical analyses were performed using GraphPad Prism 8 software, and data were expressed as the mean ± SD. Differences among groups were compared by the ANOVA test. Differences were considered statistically significant at p < 0.05.