Obtaining of plant material

Fresh thunder leaves (Ligustrum lucidum) were collected at the Antonio Narro Autonomous Agrarian University (3532º north latitude, 101.0332º west longitude, 1 786 masl), moringa leaves (Moringa oleifera) were obtained in Ciudad Obregón, Sonora (27° 22’ 04” north latitude, 109° 55’ 28” west longitude, 37 masl), sorghum (Sorghum bicolor) grains were obtained from the BRS-72 variety (with high tannin content) in the City of Tecomán, Colima (18° 54’ 51” north latitude, 103° 52’ 30” west longitude, 33 masl). The plant material was transferred to the laboratories of the Food Department of the Faculty of Chemical Sciences, where leaves and grains were selected based on size and without the presence of damage, to be subjected to dehydration for a period of two weeks at ambient temperature, then pulverized and sieved (RO-TAP; TYLER) using a 150 μ sieve.

Ultrasound-microwave-assisted extraction

The pulverized plant material was placed in the reactor with the quantities obtained from the ratios shown in the (Table 1) and this reactor was introduced into the ultrasound and microwave equipment (Nanjing ATPIO Instrumens Manufacture Co., Ltd Company, China) under the following conditions: Ultrasonic (VS): Power Radio 20, Ultrasonic on Relay 10, Ultrasonic off Relay 3, Amplitude off Relay 25 and Set Time 20. Microwave (MV): Power Radio 800, Display power 0, Set Temp 70 °C and Holding Time 5. After the ultrasound and microwave process, the samples were stored in a deep freezer at a temperature of -70 °C.

Amberlite column chromatography

Column chromatography was performed with the amberlite XAD-16N chromatographic filler. This includes activation of amberlite with methanol, column packing, filtration of extracts, drying of extracts and unpacking of the chromatography column (^{De Asmundis et al., 2011}).

Recovery of dry extract

Once the ethanolic fraction was obtained, it was distributed in glass containers and dried in an oven at 60 °C, without exposure to light for 24 to 48 h. Finally, the dry extract in powder form was collected in an amber jar at ambient temperature for later analysis.

Characterization of phytochemicals present in plant extracts by reverse-phase liquid chromatography (HPLC masses)

The analysis by reverse-phase high-performance liquid chromatography was performed following the methodology of ^{Ascacio et al. (2016}), which consists of using a Varian HPLC system that includes an automatic injector (Varian ProStar 410, USA), a ternary pump (Varian ProStar 2310, USA) and a PDA decanter (Varian ProStar 330, USA). A liquid-chromatography ion-trap mass spectrometer (Varian 500-MS IT Mass Spectrometer, USA) equipped with an electrospray ion source was also used.

Samples (5 μl) were injected into a Denali C18 column (150 mm x 2.1 mm, 3 μm, Grace, USA). The temperature of the furnace was maintained at 30 °C. The eluents were formic acid (0.2%, V/V; solvent A) and acetonitrile (solvent B). The following gradient was applied: initial, 3% B; 0-5 min, 9% B linear; 5-15 min, 16% B linear; 15-45 min, 50% B linear. After the column was washed and reconditioned, the flow rate was maintained at 0.2 ml min^-1 and the elution was controlled at 245, 280, 320 and 550 nm. All effluent (0.2 ml min^-1) was injected into the mass spectrometer source, without dividing.

All MS experiments were conducted in negative mode [M-H]^-1. Nitrogen was used as a nebulizer gas and helium as a buffer gas. The parameters of the ion source were: spray voltage 5 kV and capillary voltage and temperature were 90 V and 350 °C, respectively. Data was collected and processed using the MS Workstation Software (V 6.9). Samples were first analyzed in full scan mode acquired in the range m/z 50-2000.

Obtaining and identification of fungi

The strains used in the bioassays were isolated from chickpea plants with symptoms of wilting and yellowing, located in the Yaqui Valley at latitude 27° 29.185’, west longitude -109° 56.45’. Small pieces of plant roots with symptoms of the disease were disinfected with a 3% chlorine solution and rinsed with sterile distilled water. They were placed in Petri dishes with potato-dextrose-agar (PDA) culture medium and incubated for three days at 28 °C. Finally, the isolation was carried out by means of a hypha tip.

Molecular identification by PCR and random amplification of polymorphic DNA (RAPD)

For the extraction of DNA from each of the isolates of the fungi, pure mycelium cultures grown in 50 mL of dextrose potato broth, incubated at 25 °C for eight days, were used. At the end of this time, the cultures were centrifuged at 10 000 rpm for 10 min at ambient temperature, the supernatant was discarded, and the cell pellet was washed with PBS. The lysis of the sample was performed with 3 freeze-heating cycles (-80 °C, 75 °C) for 15 min, maceration and incubation for 15 min in TES buffer (Tris-HCl 50 Mm pH 7.5, EDTA 20 Mm, SDS 1%).

The total DNA of the mycelium was obtained by purification with the phenol-chloroform-isoamyl alcohol (25:24:1) method and precipitated with ethanol. The concentration and quality of the genetic material was determined by spectrophotometry (EPOCH; BioTek). For the identification of M. phaseolina and F. solani, specific oligonucleotides were used, which amplify for a conserved region adjacent to the 5.8 S gene and the TEF-1α gene, respectively (^{Babu, 2007}; ^{Arif, 2012}).

Amplification was performed by PCR with the Taq DNA polymerase enzyme (qARTA Bio) and 100 ng μl^-1 of genetic material, using the amplification conditions described by these authors: 25 denaturation cycles at 95 °C for 30 s, alignment at 56 °C for 1 min, extension at 72 °C for 2 min, and a final extension step at 72 °C for 10 min for M. phaseolina and 40 denaturation cycles at 94 °C for 1 min, alignment at 58 °C for 1 min, extension at 72 °C for 2 min for F. solani.

In addition, the identification of pathogenic races of Fusarium oxysporum f. sp. ciceris was performed using the random amplification of polymorphic DNA (RAPD) technique using the markers OPF and OPI, where the races are distinguished by the absence or presence of electrophoretic patterns with the same mobility, the amplification conditions were as follows, denaturation at 94 °C for 4 min, followed by 30 cycles of 94 °C for 1 min, alignment at 40 °C for 1 min, extension at 72 °C for 3 min and final extension at 72 °C for 7 min (^{Jiménez-Gasco, 2001}). The amplified products were separated by 1% agarose gel electrophoresis (UltraPure Agarose Invitrogen), in TAE buffer. The gels were stained with nucleic acid gel stain (Invitrogen). The corresponding bands of the amplified products were observed with UV light. Images were captured with a digital camera (UVP; GDS-8000).

Antifungal activity of plant extracts against M. phaseolina by the poisoned medium method

Antifungal activity was determined by the poisoned medium method proposed by ^{Jasso de Rodríguez et al. (2011)}, the treatments of the polyphenols obtained from: Ligustrum lucidum, Moringa oleifera and Sorghum bicolor were in concentrations of 50 mg L^-1, 100 mg L^-1, 200 mg L^-1, 300 mg L^-1, 400 mg L^-1, 500 mg L^-1 and 600 mg L^-1: with four repetitions per treatment. The concentration and volume for each of the polyphenols was first determined and added to a flask with the required amount of sterile PDA. Subsequently, discs of 0.4 cm in diameter with active mycelium of the fungus of seven days of growth were placed, finally they were incubated at 28 ±2 °C until the absolute control filled the box completely. The percentage of inhibition was determined by the following formula: percentage of inhibition= (DC-DT/DC)*100. Where DC is the diameter of the control treatment, and DT is the diameter of the different concentrations.

Antifungal activity of plant extracts against F. oxysporum f. sp. ciceris and F. solani by the plate microdilution method

The plate microdilution method adapted from the techniques proposed by ^{Masoko et al. (2005}) and ^{Gabrielson et al. (2002}) was used. For this, 96-well polystyrene plates were used. The procedure started by placing 100 μl of sabouraud liquid medium in each well of the plate, then each group of polyphenols was prepared at a concentration of 2 000 mg L^-1 using ethanol as a solvent, then 100 μl of the polyphenols prepared at 2 000 mg L^-1 were placed in column four, mixed and 100 μl were retaken and transferred to the next column and so on, making 50% serial dilutions up to column 12, resulting in concentrations of 1 000 mg L^-1, 500 mg L^-1, 250 mg L^-1, 125 mg L^-1, 62.5 mg L^-1, 31.2 mg L^-1, 15.6 mg L^-1, 7.8 mg L^-1 and 3.9 mg L^-1, the next step was to add 40 μl of 2,3,5- triphenyltetrazolium chloride as a growth developer to all wells; finally from column two, a solution of spores of F. oxysporum and F. solani at a concentration of 1x10⁸ was placed.

Each plate was considered a repetition, and three repetitions were performed per treatment, incubated at 28 °C for 48 h and finally an absorbance reading was performed at 490 nm in spectrophotometer. Growth of the fungus was considered positive in the treatments where the well presented a pink color and negative in those that did not present any color, in addition to their respective absorbance values. The percentage of inhibition was calculated by adapting the formula proposed by ^{Moreno-Limón et al. (2011}), considering that the percentage of inhibition is the inverse of the percentage of growth. Growth percentage= (A-B) /C (100). Where: A= treatment absorbance, B= negative control absorbance, C= positive control absorbance. Inhibition percentage= 100 - growth percentage.

Statistical analysis

The experiments were established under a completely randomized block design with three repetitions. A Probit analysis was performed to determine the inhibitory concentration (IC₅₀) of each polyphenol group, then an analysis of variance was performed with inhibitory concentrations (IC₅₀) and the means of the treatments were compared using the Tukey multiple range test (α< 0.05).

Phytochemicals present in plant extracts, characterized by reverse-phase liquid chromatography (HPLC)

Among the results obtained by HPLC, the presence of compounds from the family of flavones, hydroxycinnamic acid and anthocyanins can be observed more frequently (Table 2).

Identification and molecular characterization of phytopathogenic fungal isolates

The strain of Macrophomina developed colonies with the following characteristics: dense mycelial growth, hairy in appearance, at first dark gray and subsequently it turned black. The colony developed microsclerotia, these characteristics were similar to those of M. phaseolina (Santos-Mier et al., 2015). For the fungal isolate analyzed by PCR, amplification of fragments of a size of 350 bp was achieved, this confirms that the strain belongs to the species M. phaseolina, since the indicators MpKF1 and MpKR1 are specific for this species of fungus.

According to ^{Babu et al. (2007}), DNA sequences obtained with the specific primers MpKFI and MpKRI can be used to quickly, selectively and specifically identify the fungus M. phaseolina. Similarly, ^{Zhang et al. (2011}) and ^{Sánchez et al. (2013}) used the specific primers MpKFI and MpKRI for the identification and detection of M. phaseolina isolates in strawberry and mung bean (Vigna radiata L.), respectively (Figure 1a). For the identification and discrimination of F. solani, the TEFs4 primer set was used, with which the TEF-1α gene was amplified, showing a product of 658 bp (Figure 1b), as reported by ^{Arif et al. (2012}).

Finally, for the identification of pathogenic races 0, 1B/C, 5 and 6 of Fusarium oxysporum f. sp. Ciceris by the RAPD technique, seven specific oligonucleotides called OPF and OPI were used (^{Jiménez-Gasco, 2001}). All isolated samples were examined, and the banding pattern was evaluated. Only in one sample an amplification product at 900 bp with the primer OPF-10 was observed (Figure 1c), this product corresponds to race 5 according to what was previously reported by Del Mar Jiménez et al. (2001).

Figure 1 Identification of pathogens: a) M. phaseolina (350 bp); b) F. solani (658 bp); and c) F. oxysporum f. sp. ciceris (900 bp). Amplification products separated in a 1% agarose gel and stained with Nucleic acid gel stain (Invitrogen). 

The results of other studies have shown that, in the place where the isolates were carried out, different races have been identified in different sampling areas (^{Arvayo-Ortiz et al., 2011}). Therefore, the results obtained by our work team are consistent with these findings.

Antifungal activity of plant extracts against M. phaseolina by the poisoned medium method

The effect of polyphenols from different plant sources on the growth of the fungus M. phaseolina is shown in Figure 2. As can be seen, the polyphenols of L. lucidum and S. bicolor showed total inhibition on the mycelial growth of the fungus from 300 ppm, while the polyphenols of M. oleifera at the same concentration managed to inhibit 94.5% of the mycelial growth. IC₅₀ ranged from 53.65 ppm M. oleifera to 78.09 ppm S. bicolor.

Figure 2 Percentage of inhibition of polyphenols obtained by ethanolic extracts from plant sources on M. phaseolina. 

All polyphenols showed inhibition of fungal growth, with significant differences from control treatment. The percentages of inhibition obtained in this work were higher than those reported by Abreu et al. (2015), who, in their research, managed to obtain an inhibition of 60% using ethanolic extracts from paradise (Melia azedarach L), moringa (Moringa oleifera) and neem (Azadirachta indica A. Juss) against Macrophomina phaseolina, while in the present work, an inhibition of 100% was obtained using polyphenols separated by column chromatography.

Alkaloids have an important role in the defensive strategies of plants against pathogens, such as the repair of damage by the antioxidant system (^{Matsuura et al., 2017}) and the establishment of defensive barriers of a biochemical nature (^{Zacarés, 2008}). In addition to being sometimes induced through the jasmonate signaling pathway (Matsuura et al., 2017), which would explain the positive effect of treatments on the control of Macrophomina.

The obtaining of positive results of this work is associated with the technique of obtaining polyphenols because it is a technique in which good yields are shown in short periods and at low temperatures, showing a hydration process that favors the extraction of certain substances, since ultrasound hydrates the lamella (present in the membranes of plant cells) and once the lamella is disintegrated, plant cells are exposed to the solvent extraction process (^{Toma et al., 2001}).

Antifungal activity of plant extracts against F. oxysporum f. sp. ciceris by the plate microdilution method

The antifungal activity of the selected polyphenols of Ligustrum lucidum (leaves), Moringa oleifera (leaves) and Sorghum bicolor (grains) were tested against Fusarium oxysporum f. sp. ciceris (Figure 3). The results showed that the three groups of polyphenols used inhibit the growth of the pathogen between 60 and 85% at 1 000 ppm. Similarly, the three groups of polyphenols at low concentrations did not present inhibition of the growth of the pathogen, it should be noted that the polyphenols of Ligustrum lucidum were those that showed the highest percentage of inhibition with 83.63%, followed and with a minimum difference by the group of polyphenols of Moringa oleifera with 82.86%, while Sorghum bicolor only inhibited 68.3%.

Figure 3 Percentage of inhibition of polyphenols from different plant sources against Fusarium oxysporum f. sp. Ciceris. 

The percentages of fungal inhibition obtained in the present study using groups of polyphenols were higher than those reported by ^{Dwivedi and Sangeeta (2015}), according to their results, the extract of Moringa oleifera, Tinospora cardiofolia and Cymbopogon citratus against Fusarium oxysporum f. sp. ciceris reach an inhibition of 60.29%, in the same way, the results reported by ^{Chandra and Singh (2005)} with different plant extracts with which only 65% of inhibition of Fusarium oxysporum f. sp. Ciceris was reached. The analysis of variance showed significant differences between the IC₅₀ of the three groups of polyphenols, being those obtained from Ligustrum lucidum those that promoted the lowest IC₅₀ and statistically the best of the three extracts with 492.02 ppm (Table 3).

Antifungal activity of polyphenols of different plants against F. solani by the plate microdilution method

The effect of polyphenol groups on Fusarium solani was determined by the percentage of inhibition. The analysis of variance of the inhibitory concentration shows significant differences between the treatments, being the extract of Ligustrum lucidum the one that showed the lowest IC₅₀ (Table 4). The polyphenols of ethanolic extracts of Ligustrum lucidum, Moringa oleifera and Sorghum bicolor inhibited 100% of the growth of Fusarium solani at the concentration of 1 000 ppm, the rest of the concentrations continued to show inhibition, being the polyphenols of Ligustrum lucidum the most relevant.

The above is relevant if it is considered that there are chemicals that are applied one or more times a week and do not achieve these results, even under laboratory conditions they do not manage to inhibit 100% the growth of the mycelium, as reported by ^{Yossen and Conles (2016}), who, in their work with commercial molecules, reach an inhibition between 60 and 97%. Similar results to ours on Fusarium spp. (Figure 4) were reported by ^{Duarte et al. (2013}), who, with essential oils of Piper aduncum subsp. ossanum and Piper aurintum, totally inhibit the growth of this fungus. On the other hand, ^{Zaker (2014}) concluded that the ethanolic extract of Artemisia annua leaves can inhibit the growth of F. solani.

Figure 4 Percentage of inhibition of polyphenols from different plant sources against F. solani.