Introduction

Sclerotium rolfsii, which causes root and stem rot, is a polyphagous pathogen reported in tropical and subtropical areas (^{Gholami et al., 2019}). Sclerotinia sclerotiorum is a widely distributed and non-specific pathogen (^{Ordóñez-Valencia et al., 2018}). Colletotrichum gloeosporioides causes anthracnose in leaves, flowers and citrus buds (^{Guarnaccia et al., 2017}). The three phytopathogens are associated with significant losses in economically important crops, and their management is based on the use of chemical treatments.

The use of plant extracts with antimicrobial activity is of interest as part of the sustainable strategies to manage diseases in agricultural crops (^{Verdugo-Contreras et al., 2022}). Methanolic, ethanolic and aqueous Datura discolor extracts have been evaluated in vitro against Aspergillus flavus, A. niger, Penicillium chrysogenum, P. expansum, Fusarium moniliforme, F. poae (^{Tequida-Meneses et al., 2002}) and C. gloeosporioides (Verdugo-Contreras et al., 2022).

The traditional methods for the extraction of phytochemical compounds are based mainly on the use of organic solvents and thermal treatments in which the plant material is exposed to high temperatures for extended periods, which may cause the loss and degradation of thermolabile compounds with biological activity (^{Zhang et al., 2018}). Strategies for the aqueous extraction of plant compounds through non-thermal High Pressure Processing (HPP) have gained attention in the pharmaceutical and food industries due to their high impact on the modification of the cell structure and the recovery of bioactive compounds, associated to the destruction of cell walls and other structural barriers (^{Le-Tan et al., 2022}). However, no evaluations have been carried out on the effect of plant extracts obtained by HPP against plant pathogens.

The objective of this work was to determine the in vitro effect of aqueous Datura discolor extracts obtained by HPP against Sclerotium rolfsii, Sclerotinia sclerotiorum and Colletotrichum gloeosporioides, causal agents of soft rot, white mold and anthracnose, respectively.

Obtaining the extracts. The experiment used D. discolor roots, seeds and leaves, with identity confirmed molecularly by ^{Verdugo-Contreras et al. (2022}). The plant material was disinfested with a 1% v/v sodium hypochlorite solution, rinsed three times with distilled water and dried at 60 °C during 19 hours for its subsequent pulverization. The extracts were prepared from each anatomical part, the mixture of the plant material was prepared at a proportion of 1:10 p/v in distilled water, poured into polyethylene terephthalate (PET) with a high-density polyethylene (HDPE), double seal cap and exposed to a high-pressure treatment at room temperature. Two continuous, high-pressure treatments were considered; one at 600 MPa maintaining this pressure for 3 min, and another at 600 MPa with the pressure maintained for 6 min. For HPP, the Hiperbaric 55 equipment with a 55-liter basket (14.5 gal) with a diameter of 200 mm (7.9 in) was used, with an integrated intensifier and a power of 62 KW. Subsequently, the samples submitted to HPP were centrifuged for 10 min at 4000 rpm and the supernatant was extracted. The extracts were stored at 4 °C until use.

Antifungal in vitro evaluation. The effect of the aqueous extracts of D. discolor leaves was evaluated one day after the processing against Sclerotium rolfsii, Sclerotinia sclerotiorum and Colletotrichum gloeosporioides, which were previously molecularly identified, and its pathogenicity was confirmed (Martínez- Álvarez et al., 2021; ^{Martínez-Ereva, 2022}; ^{Pérez-Mora et al., 2021}). The experiments were carried out in Petri dishes with PDA (Bioxon, Cuautitlán Izcalli, State of Mexico, Mexico); the extracts were diluted to a concentration of 2, 4 and 6% v/v in the culture medium. In the center, a 5 mm PDA plug with mycelium from the fungus with 5 days old was placed. An additional treatment was included, which consisted of the fungicide at 1 ppm (tebuconazole for S. rolfsii and S. sclerotiorum and carbendazim for C. gloeosporioides), Petri dishes containing PDA without any plant extracts or fungicide were included as a control. The petri dishes were incubated at 25 °C, the growth of the pathogens was recorded, the experiments were concluded once mycelial growth in the control plates reached the edge. The percentage of inhibition (PI%) was calculated as

PI % =C-TC x 100, where C is the radius of the fungus in the control dish and T is the radius of the fungus in the presence of the extract or the fungicide (^{Paneerselvam et al., 2012}). The experiments were conducted in a completely randomized arrangement with four repetitions per treatment; the experiments were conducted twice.

The shelf life of the extract with the most efficient concentration observed in the in vitro tests was evaluated (6% leaf extract). The extract was kept at 4 °C and the evaluation of biological effectiveness was carried out six months after it was obtained. These experiments were carried out in the same way as the in vitro test described earlier.

Statistical analysis. The data were subjected to the Shapiro-Wilk normality test and the non-parametric Kruskal-Wallis and Mann-Whitney, with a value of p<0.05. To allow values of zero in some treatments, the data were transformed with √x + 1 (^{Gomez and Gomez, 1984}).

The results of the in vitro tests displayed variable percentages of mycelial growth inhibition of in Sclerotium rolfsii, Sclerotinia sclerotiorum and Colletotrichum gloeosporioides, by aqueous extracts of D. discolor obtained by (HPP) from the different anatomical parts of the plants and concentrations. Datura species have been documented to have antifungal activity. However, this effect varies according to the concentration and types of solvents, as well as to the part of the plant from which they are obtained (^{Öz, 2017}).

In S. rolfsii, inhibition fluctuated between 25 and 53%, 25 and 45% and between 77 and 100% with the root, seed and leaf extracts, respectively, in comparison with the control with no extract, with differences between treatments (p < 0.001). The efficiency of the 4 and 6% leaf extracts, regardless of the time of extraction, was not significantly different to the treatment with tebuconazole at 1 ppm which inhibited the growth of S. rolfsii by 100% (Table 1; Figure 1 A-D).

Figure 1 Effect of Datura discolor aqueous leaf extract obtained High Pressure Processing for 6 minutes. A-D Sclerotium rolfsii, E-H Sclerotinia sclerotiorum, I-L Colletotrichum gloeosporioides. Chemical control = tebuconazole or carbendazim 1 ppm). 

An in vitro study carried out with a D. metel fruit methanolic extract at different concentrations (0.5 to 4.0%) displayed a significant reduction of the biomass of S. rofsii from 69 to 94% (^{Jabeen et al., 2014}). In addition, Jabeen et al. (2022) evaluated the efficiency of the methanolic extract of D. metel leaves for the in vitro growth control of S. rolfii. The extract concentrations that fluctuated between 0.5 and 4.0% significantly controlled the growth of the pathogen from 29 to 88 % over the control. The results with aqueous leaf extracts obtained by HPP in this study surpass those reported with methanolic extracts from D. metel fruits and leaves.

No reduction was observed in the mycelial growth of S. sclerotiorum in any of the treatments with root and seed extracts, nor with 2% leaf extracts, whereas in the 4 and 6% leaf extracts, inhibition fluctuated between 15 and 56%. The efficiency of the leaf extracts at 4% (with 6 min) and 6% (in 3 and 6 min) was not significantly different to the treatment with tebuconazole at 1 ppm that inhibited 72% of the growth of S. sclerotiorum (Table 1; Figure 1 E-H).

Previous studies indicate that the aqueous D. metel extract inhibited S. sclerotiorum in comparison with the control (^{Sharma et al., 2015}). The ethanolic D. inoxia leaf extract, which has a higher concentration of secondary phytochemicals and total flavonoids in comparison with the aqueous extracts displayed an inhibition of 100% of the mycelial growth of S. sclerotiorum in all the concentrations evaluated in comparison with the control, whereas the aqueous extract displayed inhibition from 29 to 94%, with the highest inhibition shown in the concentrations of 1600 and 2000 μg 100 mL^-1 (^{Matías et al., 2020}). Roy et al. (2021) evaluated three concentrations (5, 10 and 15%) of aqueous D. stramonium extracts in vitro against S. sclerotiorum and they reported percentages of inhibition of 28, 31 and 37%, respectively. Additionally, ^{Kewate et al. (2020}) reported a 28% inhibition of S. sclerotiorum with powdered extracts of D. stramonium at 10% in an in vitro test. The results reported by Matías et al. (2020) surpass the inhibition reported in our study for aqueous D. discolor leaf extracts obtained by HPP. However, the latter are higher than those reported for D. stramonium against S. sclerotiorum.

In the present study, the root extracts displayed no mycelial growth inhibition in C. gloeosporioides, whereas the seed extracts inhibited it by 50 to 64% and the leaf extracts, by 36 to 56%. The efficiency of the seed extracts was not significantly different to the treatment with carbendazim 1 ppm, which inhibited the growth of C. gloeosporioides by 78%. Although less efficient than the treatment with carbendazim 1 ppm, the leaf extracts significantly inhibited the pathogen regarding the control without an aqueous extract (Table 1, Figure 1 I-L).

^{Karim et al. (2017}) evaluated the antifungal activity of methanolic D. metel leaf, seed and root extracts at concentrations of 1.0, 1.5, 2.0, 2.5 and 3.0%. All concentrations significantly reduced the radial growth of C. gloeosporioides. The 1.5% seed extract displayed the highest inhibition rate (80%). Similarly, D. stramonium leaf extracts obtained with methyl acetate and with methanol significantly inhibited the mycelial growth of C. gloeosporioides (^{Alemu et al., 2014}). It was previously reported that aqueous D. discolor leaf extracts (at 1, 2 and 4% p/v) inhibited the mycelial growth of C. gloeosporioides by 52-73%, without significant differences with the treatment with carbendazim at 1 ppm (^{Verdugo-Contreras et al., 2022}). The aqueous extracts of D. discolor leaves at 2, 4 and 6% obtained at high pressures inhibited the pathogen by 36 to 56%, whereas the seed extracts inhibited it by 50 to 64 % (Table 1). That is, the percentages of inhibition observed for C. gloeosporioides are greater with seed extracts, as reported by Karim et al. (2017). The percentages of inhibition with aqueous D. discolor leaf extracts against C. gloeosporioides report by Verdugo-Contreras et al. (2023) are greater than those reported in this study, which may be due to differences in the concentrations and types of bioactive compounds in the extraction processes, with the ability to inhibit C. gloeosporioides.

Methanolic and ethanolic extracts from the stem and leaves of D. discolor inhibited the mycelial growth of Aspergillus flavus, A. niger, Penicillium chrysogenum, P. expansum, Fusarium moniliforme and F. poae (^{Tequida-Meneses et al., 2002}). The extracts with solvents such as ethanol, methanol and methyl acetate may, in some cases, display greater effectiveness against pathogens. Although it may be interesting to evaluate the effect of D. discolor extracts with solvents such as ethanol and methanol against S. sclerotiorum and C. gloeosporioides, alternatives are sought with the use of clean technologies.

^{Sharma et al. (2015}) reported that the aqueous D. metel extract was effective against S. sclerotiorum, but not against Rhizoctonia solani or Fusarium oxysporum. The D. inoxia aqueous leaf extract inhibited S. sclerotiorum but did not affect the growth of Fusarium solani (^{Matias et al., 2020}). D. discolor aqueous leaf extracts obtained by HPP inhibited S. rolfsii, S. sclerotiorum and C. gloeosporioides, displaying an antifungal ability of the extract with wide spectrum.

On the other hand, except for the 4% leaf extract treatment against S. sclerotiorum, no differences were observed in the inhibition of the different pathogens related to the extraction times, that is, extracts obtained by high pressures with a duration of 3 or 6 minutes of the process. This may be considered in subsequent evaluations against other pathogens, considering the 6% leaf extract, subjected to HPP for 3 minutes, which helps to streamline the process.

Despite the scarcity of D. discolor phytochemical studies, whithanolides datudiscolides A (8) and B (9) (^{González et al., 2023}) have recently been isolated from this species. Additionally, the presence of whithanolides with antifungal activity has been confirmed in D. ferox, D. metel, D. quercifolia and D. stramonium (^{Siddiqui et al., 1987}; ^{Kagale et al., 2004}), therefore the inhibition in the growth of the evaluated phytopathogens could be attributed to whithanolides present in D. discolor, although additional studies are required to confirm this hypothesis. Phytochemical studies performed on other species report that the main secondary metabolites for the Datura genus are terpenoids, flavonoids, whithanolides, tannins, phenolic compounds, steroids and fatty acids (^{Céspedes-Méndez et al., 2021}). The antifungal potential of D. inoxia leaf extracts against S. sclerotiorum have been related to the abundance of alkaloids and phenolic compounds (^{Matías et al., 2020}). Conducting a phytochemical screening of D. discolor to complement research on its antifungal activity is relevant.

The 6% leaf extract evaluated six months after its processing reduced the in vitro growth of S. rolfsii, S. sclerotiorum and C. gloeosporioides by 99, 54 and 39%, respectively. The percentages observed are similar to those found in the evaluations with the fresh extract. ^{Verdugo-Contreras et al. (2022}) reported that the aqueous extract of the D. discolor leaf at 4% obtained by maceration with heat managed to reduce the in vitro growth of C. gloeosporioides up to 3 months after its preparation, only less effectively. This suggests greater stability of the bioactive compounds obtained through HPP processes with effectiveness against the pathogens evaluated.

The 6% leaf extracts, regardless of the extraction time, show antifungal activity against the three fungi and their effectiveness remains active for at least 6 months. Future lines of research should focus on characterizing the bioactive compounds implied in the antifungal activity of the D. discolor aqueous extracts obtained through HPP, as well as in greenhouses and field studies to determine the potential of such extracts for the control of the phytopathogens evaluated in vitro.