Disc diffusion

Petri dishes were prepared with potato dextrose agar (PDA) medium, then filter paper discs (medium filtration, 8 μm pore) with 0.5 mm diameter were cut and sterilized in autoclave at 120 lb pressure for 20 min. Subsequently, under aseptic conditions, the filter paper discs (four discs for each alcoholic extract) were impregnated with 150 μL of each alcoholic extract and left to stand until the disc completely absorbed the extract. Then the Petri dishes with PDA were inoculated with a disc of the fungus R. solani, which was placed in the center of the dish with culture medium.

Four discs impregnated with each extract were placed equidistant to the disc with mycelium of R. solani. R. solani was isolated from the rhizosphere of poblano pepper (Capsicum annuum L.) and is part of the strain collection of the Department of Phytopathology of the Colegio de Postgraduados Montecillo Campus, State of Mexico. Treatments included a control of sterile distilled water, a methanol control and an ethanol control. The dishes were incubated at 28 °C for three days, after the incubation time it was observed if there was inhibition on the growth of R. solani and the diameter of inhibition around the disc impregnated with the extract was measured in millimeters with a ruler, all treatments were carried out in triplicate.

Well diffusion

Petri dishes were prepared with PDA medium, and three holes were made in the agar with the help of a punch of 0.5 mm in diameter. One hundred microliters of each alcoholic extract (ethanol-water 1:1, methanol-water 1:1, ethanol-water 3:7 and methanol-water 3:7) were added to each well, then a disc of R. solani with fresh mycelium was placed in the center of each dish (^{Cruz et al., 2010}). Treatments included a control of sterile distilled water, a methanol control and an ethanol control (all in triplicate). Subsequently, the dishes were incubated at 28 °C for three days, after the incubation time it was observed if there was inhibition on the growth of R. solani close to the well, to measure the inhibition halo, it was measured in millimeters.

Plate dilution

Determination of the concentration of a solution of R. solani mycelium fragments

One hundred milliliters of potato dextrose broth were prepared, then the culture medium was inoculated with a 7 mm diameter disc of fresh mycelium of R. solani. It was incubated for four days at 28 °C, after this time the fungal solution was filtered through a sterile filter system (plastic column packed with cotton), to allow the passage of the smallest fragments of mycelium. The concentration of the mycelium fragments of the filtered liquid was estimated using a Neubauer chamber.

Once the concentration of the solution of fungal mycelium fragments (2 x 10⁷ mycelium fragments / mL, 1X10⁴ CFU) was known, the antifungal activity was determined by the plate microdilution method using the method described by ^{Casaril et al. (2011)}; ^{Ren et al. (2014)} with modifications. In each well of a 96-well plate, the following were added: 100 μL of potato dextrose broth, 50 μL of the respective alcoholic extract of edible fungi (ethanol-water 1:1, methanol-water 1:1, ethanol-water 3:7 and methanol-water 3:7) and 50 μL of the solution of mycelium fragments of R. solani. Treatments included a control of sterile distilled water, a methanol control, an ethanol control, a control of potato dextrose broth, and a control of potato dextrose, all with solution of R. solani mycelium fragments (all in triplicate).

Subsequently, the dishes were incubated at room temperature in a horizontal stirrer at 400 rpm for four days, after this time the absorbance was read at 595 nm with a microplate reader. Antifungal activity was measured by comparing the optical densities of wells containing the same extracts and controls. The percentage of fungal growth inhibition was calculated using the following equation:

Where: A_F is the absorbance of the fungal control without the addition of the extract; A_FE is the absorbance of the mixture (alcoholic extract and suspension of mycelium fragments), and A_E is the absorbance of the corresponding extract.

Disc diffusion

In the results obtained with the disc diffusion method, it was observed that the fungus in some treatments such as ethanolic extract of A. jacksonii in its 1:1 methanol-water dilution, but also the extract of A. rubescens in the 3:7 ethanol-water dilution and 1:1 methanol-water dilution; extract of U. maydis 3:7 methanol-water dilution and of L. Deliciosus 3:7 ethanol-water dilution; grew slower and less abundantly compared to the controls (Figure 1).

Figure 1 Alcoholic extract and dilutions of Amanita jacksonii against Rhizoctonia solani at 28 °C for three days of incubation. 

It was qualitatively observed that R. solani grew more slowly in the extract of A. rubescens in the 3:7 ethanol-water dilution, where the fungus did not grow homogeneously and in the entire surface of the PDA as in the case of the controls. While the metabolic profile of each fungus is unique (^{Shen et al., 2017}), the observation of the impact on growth and development of R. solani could be due to metabolites reported in alcoholic extracts of edible fungi, such as low molecular weight phenolic compounds that have antimicrobial activity (^{Alves et al., 2013}).

Another possibility is that a minimal percentage of polar metabolites of phenolic nature were released in the alcoholic extracts of some macromycetes and could diffuse on the agar showing a slower growth in R. solani with respect to the controls (^{Naczk and Shahidi, 2006}). In addition, disc diffusion depends directly on the diffusion capacity of the active substances present in the extracts, which can cause low mobility of metabolites in the agar if they have low solubility or high molecular mass.

Well diffusion

No effect on R. solani growth was observed with the alcoholic extracts and their dilutions, Figure 2, as R. solani grew in and out of the wells with extracts. These results could be related to the molecular weight of the compounds present in the extract, since if the molecular weight of the compounds is very high or if they have low solubility, their mobility through the agar will be difficult (non-diffusible), this could be a reason why no inhibitory effect from the alcoholic extracts and their dilutions was observed (^{Ren et al., 2014}; ^{Martínez et al., 2021}).

Figure 2 Dilutions of alcoholic extracts of Lactarius deliciosus, Ustilago maydis, Amanita jacksonii and Amanita rubescens against Rhizoctonia solani at 28 °C for three days of incubation. 

Although it is not known what type of metabolites are present in the extracts of the edible fungi tested, it should be noted that the fungal material was used fresh and due to the type of solvents used (methanol, ethanol and water), it was sought to drag, in the extracts, polar metabolites such as polyphenols, sugars, alkaloids, saponins, glycosides and some proteins from macromycete fungi (^{Naczk and Shahidi, 2006}; ^{Akpi et al., 2017}). The presence or absence of these metabolites in the extracts will depend on the environment in which the fungus develops and the different sources of nutrients available during its growth (^{Shen et al., 2017}).

There may also be the possibility that the polar compounds present in the alcoholic extracts of edible fungi did not have inhibitory activity against R. solani in the well diffusion technique. On the other hand, it has been reported that, for the genus Amanita, peptides are the main constituents of polar extracts, these metabolites have greater antimicrobial activity in liquid medium or broth than in solid media such as agar, because they have low solubility (^{Li and Oberlies, 2005}).

Plate dilution

The results indicate that the greatest inhibitory activity was shown by the 1:1 ethanol-water dilution of L. deliciosus (88%), while the lowest inhibitory activity was presented by the ethanolic extract of A. rubescens (0.5%), Figure 3. These results differ from those found in well and disc diffusion techniques. This behavior can be explained by what is reported in the literature (^{Ren et al., 2014}; ^{Balouiri et al., 2016}), as they mention that, in the plate dilution technique, the metabolites present in the extracts tend to diffuse better in liquid media than in solid media, such is the case of the potato dextrose broth that was used in this technique.

Figure 3 Alcoholic extracts and dilutions of edible fungi with inhibitory activity on the growth of Rhizoctonia solani. Mean ± standard error, n= 3. 

One of the advantages of using this technique is its ability to identify antimicrobial activity and quantify it accurately, since the results are not affected by the growth rate of different microorganisms (Morales et al., 2013). This is a possible reason that there are stronger inhibitions with greater effectiveness. The inhibitory activity shown by the ethanolic extract and the 1:1 ethanol-water dilution of L. deliciosus in the growth of R. solani can be attributed to the presence of phenolic compounds contained in the ethanolic extract of this edible fungus (^{Zavastin et al., 2015}).

L. deliciosus has proven antifungal activity as reported by ^{Volcão et al. (2021)}, who evaluated the antifungal activity of the hydroalcoholic extract obtained from L. deliciosus against the phytopathogen Monilinia fructicola (Wint.), they report that the extract inhibited fungal growth and reduced mycelial development of the phytopathogen at a concentration of 1.25 mg L^-1. On the other hand, the processing of the fungal material (fresh or dried), the extraction time and the type of solvent should also be considered, which will influence the antimicrobial activity of the extracts (^{Adhikari et al., 2018}).

Among the most used solvents to perform the extraction of metabolites in fungi are water, ethanol, methanol, acetone and ethyl acetate (^{Elbatrawy et al., 2015}). In the results found in this study, a 1:1 ethanol-water dilution of L. deliciosus was the one that presented greater antifungal activity, even greater than those extracts that did not have any dilution and were only the solvent, a possible explanation for this behavior is reported by ^{Yim et al. (2012)}, who point out that the best solvent for the extraction of phenolic compounds from fungi is water since it does not break glycosidic bonds, which indicates the presence of free phenols. In the 1:1 dilution, the presence of water favored the extraction of free phenols and therefore it was possible to quantify the highest antifungal activity of this extract.