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)} 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.

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.

Methods

Plant collection. The plant collection was in January 2016. This collect was in General Cepeda, Coahuila, México (25°21’40.55’’ N y 101°28’08.68’’ W). Samples stored in black plastic bags for their transfer at Laboratorio de Micología y Biotecnología from Universidad Autonoma Agraria 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 (^{Castillo et al., 2010}).

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.

Phytochemical analysis of plant extracts. The phytochemicals identification used qualitative techniques, in these techniques the presence or absence of phytochemicals were determinate by colorimetry where the extract reacted and change of color; for these tests concentrated extracts prepared to 1000 mg/L; the identified phytochemicals were alkaloids (Dragendorff and Sonnenschein reaction), cyanogenic glycosides (Grignard reaction), reducing sugars (Feeling and Benedict reaction), saponins (Liberman Bouchnard reaction), tannins (Jelly and FeCl₃), quinones (ammonium hydroxide and Bontrager reaction), coumarins (Erlich reaction and ammonium hydroxide), purines and carotenoids (^{Sahgal et al., 2009}; ^{Usman et al., 2009}).

Results

Identified phytochemicals in aqueous and ethanolic plant extracts. The identified phytochemicals from ethanolic extracts are in Table 2; from A. lechuguilla both leaves and roots were identified compounds like carbohydrates, reducing sugars, saponins, and tannins; in the case of C. illinoinensis husk extracts alkaloids, flavonoids, cyanogenic glycosides, reducing sugars, saponins, tannins, quinones, coumarins, purines and carotenoids presented; for J. dioica root extract observed saponins, tannins, quinones, and purines; while L. tridentata extract presented alkaloids, flavonoids, cyanogenic glycosides, reducing sugars, saponins, tannins, quinones, coumarins, and carotenoids; finally in L. graveolens extracts both leaves and steam alkaloids, flavonoids, reducing sugars, saponins, tannins, quinones, coumarins, and carotenoids identified.

Phytochemicals detected from aqueous extracts presented in Table 3. From A. lechuguilla, both leaves and roots presented the same compounds as reducing sugars, saponins, tannins, and quinones, only carbohydrates, and purines were different compounds being present in leaves, for C. illinoinensis husk identified all the phytochemicals listed in Table 3 except alkaloids.

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.

LtLAC: L. tridentata leaves. AlLAC: A. lechuguilla leaves. AlRAC: A. lechuguilla roots. JdRAC: J. dioica roots. JdSAC: J. dioica stem. CiHAC: C. illinoinensis husk. LgSAC: L. graveolens stem and LgLAC: L. graveolens leaves.

Fig. 1 Inhibition percentage of aqueous crude extracts on F. oxysporum. 

LtLAP: L. tridentata leaves. AlLAP: A. lechuguilla leaves. AlRAP: A. lechuguilla roots. JdRAP: J. dioica roots. JdSAP: J. dioica stem. CiHAP: C. illinoinensis husk. LgLAP: L. graveolens leaves and LgSAP: L. graveolens stem.

Fig. 2 Inhibition percentage of concentrated aqueous extracts on F. oxysporum. 

Antifungal activity of plant ethanolic extracts against F. oxysporum. In the case of the ethanolic extracts, in crude extracts and concentrated extracts, it was observed an increase in antifungal activity. In Fig. 3 it is found that all ethanolic crude extracts except LtLEC extract, presented inhibition percentage higher to 90 % in the concentration of 500 mg/L, nevertheless the treatments CiHEC, LgLEC and LgSEC were the best because there was inhibition of the pathogen to 100 % in the concentration of 250 mg/L. About concentrated ethanolic extracts, in Fig. 4, it is observed that the extracts LgSEP, AlLEP, AlREP, and LgLEP inhibited the pathogen development to 100 % to concentrations of 125 mg/L, 250 mg/L and 500 mg/L respectively.

LtLEC: L. tridentata leaves. AlLEC: A. lechuguilla leaves. AlREC: A. lechuguilla roots. JdREC: J. dioica roots. JdSEC: J. dioica stem. CiHEC: C. illinoinensis husk. LgLEC: L. graveolens leaves and LgSEC: L. graveolens stem.

Fig. 3 Inhibition percentage of ethanolic crude extracts on F. oxysporum. 

LtLEP: L. tridentata leaves. AlLEP: A. lechuguilla leaves. AlREP: A. lechuguilla roots. JdREP: J. dioica roots. CiHEP: C. illinoinensis husk. LgLEP: L. graveolens leaves and LgSEP: L. graveolens stem.

Fig. 4 Inhibition percentage of concentrated ethanolic extracts on F. oxysporum. 

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.

Table 4 Inhibitory concentration to 50 % (IC₅₀) of aqueous and ethanolic extracts for inhibition of F. oxysporum. 

Extract	IC50*	Extract	IC50	Extract	IC50	Extract
AILAC	535.30±0.06g	A1LAP	13109.00±0.06c	A1LEC	115.00±0.12a	AILEP
A1RAC	253613034.00 ± 0.03a	A1RAP	2()3.74±0.06h	A1REC	113.72±0.16b	AlREP
CiHAC	9508417.00±0.03b	CiHAP	983.5 l±0.09e	CiHEC	18.03±0.19g	CiHEP
JdSAC	7875.00±0.03f	JdSAP	83361.00±0.14a	JdSEC	92.00±0.20d	**
JdRAC	69721.00±0.04e	JdRAP	1821.00±0.06d	JdREC	75.00±0.20e	JdREP
LtLAC	88075.00±0.02d	LtLAP	673.55±0.09f	LtLEC	108.50±0.21c	LtLEP
LgLAC	535.19±0.03h	LgLAP	53047.00±0.02b	LgLEC	23.09±0.22f	LgLEP
LgSAC	195805.00±0.07c	LgSAP	218.91±0.31g	LgSEC	16.24±0.15h	LgSEP

Discussion

Because all the plants are natives from the Mexican semi-desert, the phytochemicals identified are similar; thus identified compounds in leaves and roots from A. lechuguilla coincide with the reported by ^{Sidana et al. (2016)} who reported 141 steroidal saponins of pharmacologic interest; about the tannins, ^{Castillo et al. (2010)} mentioned them in his work. C. illinoinensis husk, according to ^{Bottari et al. (2017)}, contains compounds like tannins and flavonoids. Nevertheless, it also has been identified as the presence of quinones by ^{Xiang-ming (2010)}, which is similar to the compounds identified in this work. ^{Martínez et al. (2014)} mention quinones, coumarins, flavonoids, saponins, tannins, carbohydrates, and reducing sugars for extracts from the root of J. dioica. The extracts from leaves of L. tridentata coinciding with ^{Martins et al. (2012)}, who identified flavonoids, saponins, and cyanogenic glycosides. Finally in extracts from L. graveolens was identify tannins, coinciding with the report of ^{Hernandez-Castillo et al. (2010)}; coumarins similar to the mentioned by ^{Cruz et al. (2011)}; and flavonoids as flavonols and flavons being this results analogous to the obtained by ^{Güereca et al. (2007)}. Many studies have revealed the efficacy of Mexican semi-desert plant extracts to inhibit the growth of phytopathogenic fungi; previously, several works have been reported these plants as a source of compounds with antifungal activity. ^{Carvalho et al. (2011)} tested aqueous extracts of Jatropha curcas, against Alternaria alternata and presented 46 % of control; ^{Mendez et al. (2012)} mentioned inhibition percentage of 60 % with aqueous extracts of L. graveolens and A. lechuguilla over different food bacteria. Also, these results are similar to obtained by ^{Hernandez-Castillo et al. (2010)}, who reported 100 % of inhibition in R. solani with ethanolic extracts of C. illinoinensis, and coincide with the results of ^{Jasso de Rodriguez et al. (2011)} who used ethanolic extracts of A. lechuguilla and they observed 100 % of inhibition on C. gloeosporioides. Nevertheless, our results differ with the results reported by ^{Mendez et al. (2012)}, who observed an inhibition percentage of 60 % on food bacteria using ethanolic extracts of L. graveolens and A. lechuguilla.

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-Rueda de Leon et al. (2013)} who observed an IC₅₀ of 200 mg/L with aqueous extracts of L. graveolens on F. oxysporum, but differ with the report of Hernandez-^{Castillo et al. (2010)} who reported IC₅₀ of 1930 and 4340 mg/L with ethanolic extracts of L. graveolens and C. illinoinensis on R. solani.

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 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.