Actividad antifúngica de compuestos fitoquímicos de extractos de plantas del semidesierto mexicano contra Fusarium oxysporum del tomate por el método de micro dilución en placa Antifungal activity of phytochemical compounds of extracts from Mexican semi-desert plants against Fusarium oxysporum from tomato by microdilution in plate method

Introduction: In several regions of the world, Fusarium oxysporum causes losses on tomato crops; for control it, chemical fungicides are used. Nevertheless, these fungicides causing environmental and resistance problems; therefore, ecological alternatives as plant extracts have been developed. Due to the aim of this work, identify phytochemicals present in ethanolic and aqueous extracts from Agave lechuguilla qualitatively, Carya illinoinensis, Jatropha dioica, Larrea tridentata, and Lippia graveolens and determine their antifungal activity against F. oxysporum. Method: The plants collected from the northeast of Mexico; crudes and concentrated plant extracts obtained; the inhibition percentage and inhibitory concentration to 50 % (IC50) of F. oxysporum for each plant extract were determinate trough microdilution in the plate method Results: The essential phytochemicals were flavonoids, saponins, tannins, and quinones. The antifungal activity showed at 1000 mg/L inhibition around 40 to 60% by aqueous crude extracts from leaves of L. graveolens and concentrated aqueous extracts from the stem of L. graveolens, respectively. The ethanolic extracts presented 100 % of inhibition for crude extracts of husk from C. illinoinensis; in leaves and stem from L. graveolens the inhibition started from 250 mg/L; for resuspended extracts, the inhibition started from 125 mg/L with L. graveolens stem and leaves; and finally in roots of A. lechuguilla and leaves from L. graveolens the inhibition started from to 250 and 500 mg/L respectively. The best IC50 was of 8.02 mg/L from the ethanolic resuspended extract of L. graveolens stem. Conclusion: The ethanolic plant extracts from L. graveolens, A. lechuguilla, and C. illinoinensis, showed 100 % of inhibiting activity against the development of F. oxysporum, representing an alternative for control of F. oxysporum. Tucuch-Pérez, M. A. et al. No 25, Vol. 12 (2), 2020. ISSN 2007 – 0705, pp.: 1 19 3 Introduction Tomato is the fourth vegetable more cultivated in the world with three millions of hectares, just behind rice, wheat, and soy (FAOSTAT, 2018). Nevertheless, the tomato crop is affected by several fungal diseases as Botrytis cinerea, Leveillula taurica, Alternaria solani, and F. oxysporum (Villasanti & Pantoja, 2013). F. oxysporum causes vascular wilt that is one of the most destructive diseases on tomato crop, and it can cause yield losses until of the 80 % (Marlatt et al., 1996; HernándezMartínez et al., 2014; González et al., 2012). At present exist several methods to control the F. oxysporum; chemical control is the most used; nevertheless, several reports indicate that farmers use intensive applications of synthetics products, triggering on phytopathogenic microorganisms resistance (Bautista-Baños, 2006). For these reasons, at present, there is a great necessity to develop alternatives methods for the control of plant diseases (Jeong et al., 2017). The plants from the Mexican semi-desert present a large number of phytochemicals with antifungal activity. Some of these plants are A. lechuguilla that exhibit the presence of steroidal saponins; C. illinoinensis with a high content of total phenolics compounds; that causes enzymatic inhibition by compound oxidation; J. dioica with a considerable amount of phytochemicals; L. tridentata with flavonoids, triterpenes, and triterpenoids; and L. graveolens that present phytochemical compounds like essential oils, iridoids, flavonoids and naphthoquinones (Blunden et al., 1980; Do Prado et al., 2009; García-Bores et al., 2017; Martínez et al., 2014; Martins et al. 2013). The action mode of the phytochemical compounds present in plants are diverse, in case of terpenes and essential oils there is a membrane rupture by lipophilic compounds, the alkaloids intercalate their self with DNA, and the lectins and polypeptides create Ion channels in the microbial membrane or cause the competitive inhibition by adhesion of microbial proteins to the polysaccharide receptors from the host. (Masson, 1987; Cowan, 1999; Hernández–Lauzardo et al., 2007). In this sense, Jasso de Rodríguez et al. (2011) reported 100 % of inhibition with extracts from L. graveolens and A. lechuguilla on Rhizopus stolonifer. In the same way, the antifungal activity of L. tridentata was reported by Osorio et al. (2009) with extracts which presented fungicidal effect on the growth of Phytium sp., Colletotrichum coccodes, Colletotrichum truncatum, Alternaria alternata, Fusarium solani, and Rhizoctonia solani; and fungistatic impact on Fusarium verticilloides. Actividad antifúngica de compuestos fitoquímicos de extractos de plantas del semidesierto mexicano contra Fusarium oxysporum del tomate por el método de micro dilución en placa No 25, Vol. 12 (2), 2020. ISSN 2007 – 0705, pp.: 1 19 4 The objectives of this study were identified some phytochemicals compounds present in plant extracts from A. lechuguilla, C. illinoinensis, J. dioica, L. tridentata, and L. graveolens; and determinate the antifungal activity against F. oxysporum and their IC50 concentration of each plant.


Introduction
Tomato is the fourth vegetable more cultivated in the world with three millions of hectares, just behind rice, wheat, and soy (FAOSTAT, 2018). Nevertheless, the tomato crop is affected by several fungal diseases as Botrytis cinerea, Leveillula taurica, Alternaria solani, and F. oxysporum (Villasanti & Pantoja, 2013). F. oxysporum causes vascular wilt that is one of the most destructive diseases on tomato crop, and it can cause yield losses until of the 80 % (Marlatt et al., 1996;Hernández-Martínez et al., 2014;González et al., 2012). At present exist several methods to control the F. oxysporum; chemical control is the most used; nevertheless, several reports indicate that farmers use intensive applications of synthetics products, triggering on phytopathogenic microorganisms resistance (Bautista-Baños, 2006). For these reasons, at present, there is a great necessity to develop alternatives methods for the control of plant diseases (Jeong et al., 2017).
The plants from the Mexican semi-desert present a large number of phytochemicals with antifungal activity. Some of these plants are A. lechuguilla that exhibit the presence of steroidal saponins; C. illinoinensis with a high content of total phenolics compounds; that causes enzymatic inhibition by compound oxidation; J. dioica with a considerable amount of phytochemicals; L. tridentata with flavonoids, triterpenes, and triterpenoids; and L. graveolens that present phytochemical compounds like essential oils, iridoids, flavonoids and naphthoquinones (Blunden et al., 1980;Do Prado et al., 2009;García-Bores et al., 2017;Martínez et al., 2014;Martins et al. 2013).
The action mode of the phytochemical compounds present in plants are diverse, in case of terpenes and essential oils there is a membrane rupture by lipophilic compounds, the alkaloids intercalate their self with DNA, and the lectins and polypeptides create Ion channels in the microbial membrane or cause the competitive inhibition by adhesion of microbial proteins to the polysaccharide receptors from the host. (Masson, 1987;Cowan, 1999;Hernández-Lauzardo et al., 2007). In this sense, Jasso de Rodríguez et al. (2011) reported 100 % of inhibition with extracts from L. graveolens and A. lechuguilla on Rhizopus stolonifer. In the same way, the antifungal activity of L. tridentata was reported by Osorio et al. (2009)  Antonio Narro. Once in the laboratory, the samples washed with water and let it dry, then cut in small pieces around 1 cm. Samples placed in a drying stove to 60 °C until constant weight, finally each plant pulverized and sieve with a pore of 0.2 mm, this for the particle homogenization.
Samplers stored in dark flasks to environment temperature .

Plant extracts preparation.
The plant extracts prepared following the Shami et al. (2013) methodology with some modifications. Water and ethanol used as solvents and two types of extracts developed, crudes, and concentrated. The first step was adding 14 g of the plant powder in 200 mL of solution, and then the flask was placed in a stirring grill during 72 h at 50°C (Jasso de Rodríguez et al., 2015). Lapsed 72 h, the obtained extract filtered with a filter paper Whatman No.1; after the purified extract separated in two parts, one of these parts was placed in the Eppendorf tube and stored to -20 °C, thus were obtained crude extracts. To get the concentrated extracts, the solvent was separated by rotary evaporation (IKA RV 10) to 150 RPM to 60 °C, after the rotary evaporation the extracts obtained were placed in a drying stove until the extracts presented constant weight and then were pulverized (Martins et al., 2013), the obtained powder from the pulverization stored to -20 °C. In Table 1, there are all the plant extracts.   In the case of J. dioica observed in stems and roots carbohydrates, reducing sugars, saponins, tannins, quinones, and coumarins, while cyanogenic glycosides and purines were only in stems. L. tridentata presented all the phytochemicals except alkaloids and carbohydrates; finally, in L. graveolens leaves were observed carbohydrates, flavonoids, reducing sugars, saponins, tannins, and quinones, whereas in the stems were presented the same compounds that in leaves except for purines.

Antifungal activity tests of the extracts against F. oxysporum in vitro
Antifungal activity of aqueous plant extracts against F. oxysporum. In general, in aqueous extracts the antifungal activity was less compared with the ethanolic extracts; in the case of crude aqueous extracts, it can see in Fig. 1, that the inhibition percentage increased when the concentration increased, even so, the higher inhibition percentage reached it did not overcome the 50 %. The treatments with the better antifungal activity were AlLAC, JdSAC, and LgLAC; these treatments presented higher antifungal activity when they achieved to the concentration of 31.2 mg/L and kept the same inhibition percentage until the strength of 1000 mg/L with 46, 37, and 45 % respectively. In the concentrated aqueous extracts, it observed a higher inhibition percentage; nevertheless, it did not overcome the 60 %, presented in Fig. 2. In this case, found that the extracts with higher antifungal activity were AlRAP with 53 and LgSAP with 60 %, both to 1000 mg/L.         IC50 of plant extracts. The variance analysis showed that existed significant differences in the IC50 (Table 4). For aqueous crude extracts, the lower IC50 was by the extract LgLAC with 535.19 mg/L, and in concentrated aqueous extracts, the lower IC50 were by the extracts LgSAP and AlRAP with 218 and 203 mg/L respectively. The ethanolic extracts had better IC50 than the aqueous extracts; this observed in Table 4. In this case, the crude extracts with the lower IC50 were the extracts CiHEC and LgSEC with 18 and 16 mg/L, respectively. By the concentrated extracts, the best IC50 was by the extracts LgLEP and LgSEP with 22 and 8 mg/L, respectively.

Discussion
Because  The phytochemical analysis showed that the extracts from these species have compounds with antifungal activity as alkaloids, flavonoids, tannins, and saponins. These compounds have different ways of effect on the pathogens; in the case of the alkaloids could be for the presence of nitrogen in their structure as amine or amide. By flavonoids presence, their composition of phenolic hydroxyls can penetrate the cellular membrane, so these hydroxyls combine, precipitate, and denature the protoplasmic proteins (Ruiton et al., 1998). From tannins form complexes with enzymes and other proteins provoking the inhibition of the enzymes, also they can inhibit the electrons transport through the membranes, and they can alter ions like iron and copper inhibiting the activity of some essential enzymes for microorganisms life (Scalbert & Williamson, 2000). The saponins form complexes with sterols, can affect proteins and membranes phospholipids (Stuardo & San Martin, 2008).
IC50 is the most widely used and informative measure of substance efficacy. It indicates how much a compound is needed to inhibit a biological process by half, thus providing a means of the potency of an antagonist substance in research. This measure is essential because a low IC50 indicates that a plant extract is an excellent candidate to control phytopathogenic fungi (Aykul & Martinez-Hackert, 2016). The results obtained in this work are comparable to reported by Caceres- The above result suggests that the low IC50 of L. graveolens could be due to the presence of compounds as thymol and carvacrol; these compounds are the most common volatile components in the Labiatae family, and distributed in plants as L. graveolens; these compounds Actividad antifúngica de compuestos fitoquímicos de extractos de plantas del semidesierto mexicano contra Fusarium oxysporum del tomate por el método de micro dilución en placa Nº 25, Vol. 12 (2), 2020. ISSN 2007 -0705, pp.: 1 -19 -14 -are capable of inhibiting the growth of many fungus species (Chen et al., 2019). In the case of the A. lechuguilla extracts, the antifungal activity attributed to compounds like flavonoids, phenylpropanoids, and polyphenols (López-Romero et al., 2018). Finally, the C. illinoinensis extract could be an essential source of non-polar compounds with antimicrobial activity, even though these extracts contain an unknown number of molecules (Cruz-Vega et al., 2008). Thus all compounds present in plant extracts award antifungal activity disrupting the cell membrane, affecting the mitochondrial function, arresting cell cycle processes at the S-phase, and provoking leakage of intercellular components due to the deterrent effect as in case of saponins (López-Romero et al., 2018).

Conclusion
The ethanolic plant extracts presented higher antifungal activity against F. oxysporum compared with the aqueous extracts, being the extracts of A. lechuguilla leaves and roots, C. illinoinensis husk and L. graveolens leaves and stem, the extracts with better antifungal activity. Therefore, it can be concluding that the solvent used to produce Mexican semi-desert plant extracts affects the phytochemical compounds presents in the extracts. For this reason, the use of plant extracts proposed as an alternative for control vascular wilt caused by F. oxysporum.