Strains

Two strains of Pleurotus were studied: one wild variety collected in a pulque maguey field in the region of Alchichica, Puebla, Mexico (19° 24’ N y 97° 23’ W), isolated in the laboratory, registered as IE-771 in the Strain Collection of the Institute of Ecology, Xalapa, Mexico, and identified as P. agaves ^{(Mata et al. 2012)}. The other is a commercial P. pulmonarius strain widely used in Mexico and registered as IE-115 in the same mushroom collection.

For this study, strain IE-115 was cultured in a potato dextrose agar (PDA, Bioxon) culture medium, while IE771 was cultivated in PDA supplemented with wheat extract ^{(Mata and Salmones, 2021)}. Both strains were incubated at 28 °C in darkness.

Spawn production

The spawn was prepared using sorghum seeds (Sorghum vulgare Pers.). For strain IE-771, 50 g of sorghum seeds were placed in glass jars with 40 ml of water and sterilized for one hour at 121 °C. For strain IE-115, the sorghum grains were hydrated for 24 h, and then 200 g of seeds were placed in low-density plastic bags and sterilized for 1 h at 121 °C and 103.4 KPa ^{(Guzmán et al. 2013)}. Following sterilization, 1 cm² of the mycelium of each strain, cultivated in culture medium, was placed on the sterile sorghum, and incubated at 28 °C in darkness until the mycelium completely covered the seeds (30 d for strain IE-771 and 15 d for IE-115). This spawn was called the primary spawn. The secondary spawn was prepared under identical conditions for both strains; placing 175 g of sorghum seeds hydrated in low-density plastic bags and sterilizing for 1 h at 121 °C, then inoculating each strain with 25 g of the primary spawn.

Substrate and culture preparation

The maguey leaves were collected in Alchichica, Puebla, Mexico, and sun-dried for transportation to and storage in the laboratory. To obtain the maguey substrate, sun-dried maguey leaves were rewetted for 24 h and maintained for 7 d forming a pile of 70 cm in height covered with plastic to maintain values of temperature and humidity that favored the fermentation process. To promote homogeneous conditions in the pile, the materials were removed daily ^{(Mata and Torres-Hernández 2008)}. Once the fiber of the maguey leaves had softened, these were chopped into fragments of 5 to 10 cm in length using a fodder cutter. Barley straw was previously chopped, rewetted for 24 h, and fermented for 3 d at ambient temperature. This substrate, defined as S, presented a moisture content of 75 %.

Fermented maguey leaves and barley straw were mixed at a 1:1 proportion (w/w) to produce substrate M. This substrate reached a final moisture content of 80 %. The selection of days of fermentation and the combination ratio between maguey leaves and barley straw were determined by preliminary tests (data not presented). Both substrates were pasteurized using a steam tunnel of 3.5 m³, with a constant steam flow of 276 Kpa, maintaining a constant compost temperature of 65 °C for 6 h ^{(Velázquez-Cedeño et al. 2006)}, then left to cool to ambient temperature. Pasteurized substrates were then placed in polythene bags (60 x 40 cm), manually mixing the substrate with the secondary spawn at 5 % (w/w), until reaching a moist weight of 4 kg in each bag. Inoculated samples were incubated in darkness at 26±2 °C. On the second day of incubation, the plastic bags were perforated to allow gaseous exchange, 12 cross perforations (2 cm in length) were made per bag, 4 on each face and 2 on each side. At the end of the incubation period, which was marked by the appearance of the primordia, the samples were transferred to a production room under conditions of periods of light (350 Lux) and darkness of 12/12 h, with a temperature of 21±2 °C, following the recommendation of ^{Barrales and Mata (2016)}, and a relative humidity of 80-85 %. In the case of samples corresponding to the strain IE-771, it was necessary to carefully fragment the mycelium after the first harvest to make a hole in the center of the samples, without reaching the base of the bag, which allowed improved internal hydration of the samples to obtain the following harvests.

The harvested sporophores were weighed and measured manually. They were categorized into the following pileus diameter size groups: G1= ≤ 4.9 cm; G2 = 5.0-9.9 cm; G3 = 10.0-14.9 cm; G4 = 15.0-19.9 cm and G5 ≥ 20 cm ^{(Salmones et al. 2020)}. Percentages per size group were obtained by dividing the number of sporophores in each size group by the total number in all size groups and multiplying by 100, while the average weight of the mushrooms in each group was obtained by dividing the total weight in each group by the number of sporophores in that group. Productivity was evaluated over three flushes in each treatment and expressed as biological efficiency (BE %) = percentage ratio of the fresh weight of harvested mushrooms over the dry weight of the substrate ^{(Royse et al. 2004)}.

Experimental design

The following treatments were used: a 1:1 mixture of barley straw and maguey leaves (M) and barley straw alone (S). Both substrates were inoculated with the strains IE-115 and IE-771, with 10 replicates utilized per strain. The arrangement in the production area was a completely random design, with 40 units placed at three levels on three shelves.

Data analysis

Data related to pileus size group and treatment was converted to proportions and the values arcsin square-root transformed ^{(Zar 1999)}. These data were analyzed with a nested analysis of variance using the General Linear Model (GLM) process, the declaration LSMEANS Statement, and the PDIFF option of SAS, version 2003. The BE data obtained were subjected to an analysis of variance with a completely random design. When significant differences were detected, means were compared using Tukey´s multiple range test with a significance level of 0.05 %.

Substrate fiber analysis

Variation in the cellulose, hemicellulose, and lignin contents was determined in the substrates (S and M) used for the fructification of Pleurotus agaves before inoculation of the fungus (T1), at the end of the incubation stage (T2), and after the final flush of mushrooms (T3). Samples of approximately 250 g were extracted from each bag (T1, T2, and T3), dried at 65 °C for 24 h, and ground in a blender. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined, as well as acid detergent lignin (with sulfuric acid at 72 %), according to ^{Van Soest et al. (1991)}, using the Ankom Fiber Analyzer, model 200/220. NDF fraction was determined by adding ND solution (water/sodium lauril, sulfate/EDTA/triethylene, glycol/sodium, borate/sodium phosphate) and ADF with water/sulfuric acid/cetyltrimethylammonium bromide solution. Cellulose content was directly estimated from ADF-lignin. Hemicellulose was calculated as NDF-ADF. All samples were analyzed in triplicate.

Production of carpophores

During the incubation period, mycelia covered the entire surface of the substrates between 21 to 39 days, and the first flushes were harvested between 27.9 (IE115, S substrate) to 43.1 (IE-771, M substrate) days of cultivation. Two or three flushes were obtained from different treatments (Table 1). During the cultivation of P. agaves in both substrates and after the first flush, the samples gave no indication of a second flush. In an attempt to activate production, the mycelium in the bags was partially broken for better rehydration of the samples. ^{Rodríguez-Estrada et al. (2009)} reported that certain substrates must be fragmented following the first flush and ^{Guzmán et al. (2013)} cited the formation of a pseudosclerotium in some strains of Pleurotus. This is the case with P. agaves, after the manipulations of the mycelium and substrate carried out in this study, primordia of the second and third flushes were obtained. The development and morphology of the mature fruiting bodies produced by both strains on the different flushes were typical characteristics of the genus Pleurotus (Figure 1).

Figure 1 Sporophore of Pleurotus agaves, class G5 (more than 20 cm), obtained in agave leaves mixed with barley straw (substrate M). 

Average fresh mushroom production varied from 302.9 g (IE-115, S) to 555.6 g (IE-115, M) per sample. The strains cultivated on the mixture of maguey leaves and barley straw had the highest production, reaching BE averages of 71.3 % (IE-115) and 64.6 % (IE-771) (Table 1). The BE of the three flushes reported for P. pulmonarius in barley straw (S) is within the range of values recorded by ^{Gaitán-Hernández et al. (2009)} in four flushes, even in the supplemented substrates ^{(Vogel and Salmones 2000)} (in two flushes). The BE values found in this study did not differ from those reported for P. pulmonarius in other substrates ^{(Philippoussis 2009}, ^{De Siqueira et al. 2012}, ^{Adebayo et al. 2014}, ^{Vega and Franco 2013}, ^{Abdulgani et al. 2017}, ^{Picornell et al. 2017)}. ^{Sánchez et al. (2002)} reported a BE of 38.3 for the strain IE-115 (one flush) in grape skins, which is comparable to our finding in barley straw for the same strain. Nevertheless, our results are lower than those reported by ^{Salmones et al. (2020)} in barley straw and ^{Bernabé-González et al. (2004)}, who reported three flushes using maguey bagasse from Agave cupreata (sourced from the mezcal industry) with and without rice straw as a substrate. Regarding the cultivation of P. agaves, ^{Barrales and Mata (2016)} reported biological efficiencies of P. agaves (~ P. opuntiae) strains in barley straw combined with sugarcane bagasse, with and without a 7-day fermentation process, ranging from 86.1 to 105.2 %, and of P. pulmonarius at greater than 230 %. ^{Portilla-Segura et al. (2019)} cultivated P. agaves (cited as P. opuntiae) in maguey leaves alone or combined with wheat straw, corn cobs, and bean straw, reaching biological efficiencies of between 47.92 to 89.81 % and observing that formulations containing maguey residues favored higher yields. This has been reported for other commercial species of the genus. Pleurotus ostreatus cultivated in A. salmiana residues has presented biological efficiencies from 11 to 70 % ^{(Heredia Solís et al. 2014}, ^{España Rodríguez et al. 2021)} and P. djamor, a pantropical species, from 58.3 to 70.56 % ^{(Velázquez de Lucio et al. 2022)}.

Table 1 shows that BE values were greater in the 1:1 maguey leaves-barley straw mixture than those reported in barley straw alone, in both P. pulmonarius and P. agaves, and that no significant differences exist between the BE values of these species in substrate M. Several studies have demonstrated that BE is directly associated with the strain used, the species, substrates, and environmental conditions of the cultivation ^{(Lechner and Albertó 2011}, ^{Dundar et al. 2009}, ^{Philippoussis 2009)}.

In addition to the BE, time of production is an important parameter for the producers since they face problems such as limited space in cultivation chambers and the risk of pests and contaminants for prolonged periods ^{(Gaitán-Hernández et al. 2009)}. The time from inoculation to the first flush was 27.9-30.6 d for P. pulmonarius and there was approximately a twoweek period between the first and second flushes and between the second and third flushes, which is within the range reported for this species ^{(Salmones et al. 2020}, ^{Bernabé-González et al. 2004)} (Table 1). More time was required to produce P. agaves than for the commercial strain. From inoculation to the first flush took 43.1-44.5 d (~13 d more than in the commercial strain), and the second flush was produced 80 d after the first flush, a period that was sufficient to produce three flushes in P. ostreatus. Between the first and second flush, to activate the production of P. agaves, the substrate and mycelium were manipulated once, as described previously, thereafter the third flush occurred after 11 d in S and 51 d in M.

The combination and/or supplementation of substrates with C and N sources is recommended to increase productivity in the Pleurotus spp. cultivation process ^{(Picornell et al. 2017)}. The mixture of maguey leaves and barley straw may cause an increased C/N ratio on the substrate, and this increases the BEs ^{(Dundar et al. 2009)}. In addition to its nutritional contribution, adding barley straw improves the rheological characteristics of the maguey leaves, avoiding compression and bonding. Barley straw also increases water retention and provides enough space for the spawn.

Pileus size is a quality parameter used by mushroom growers to classify their products. Figure 2 shows the percentage per size category recorded in the two strains used in this study. The best-represented group was G2, corresponding to between 49.4 (IE-771, M substrate) and 58.7 % (IE-115, S substrate) of the total harvested production. The second position corresponded to G1 for IE-115 and to G3 for IE-771. The latter strain also produced basidiomes of groups 4 (S and M) and 5 (S). In general, both strains cultivated on M substrate developed the largest pileus sizes. These results are comparable to those of ^{Lechner and Albertó (2011)}, who studied the pileus diameter of two species of Pleurotus (P. pulmonarius and P. albidus), using the same classification criteria utilized in this study. Moreover, ^{Gaitán-Hernández et al. (2009)} reported that the most frequent size in P. pulmonarius cultivated in fermented and non-fermented barley straw was G2, which agrees with the findings of this study, and reported that G3 was the second most frequent size class. These results also coincide with ^{Vega and Franco (2013)}, who cultivated P. pulmonarius in corn stover and corncob. On the other hand, ^{Salmones et al. (2020)}, who cultivated three parental strains and forty-eight progeny strains of P. pulmonarius in barley straw, reported that the largest group was G1 (62.5 and 58.3 %, respectively), followed by G2 (22.6. and 23.1 %, respectively) and G3 (9.9 and 15.4 %, respectively). In the present study, the second most frequent size class was G1 in P. pulmonarius and G3 in P. agaves, and this is the first time that cap diameter sizes have been reported for a cultivated strain of this species.

Figure 2 Percentage of sporophores by size category obtained in both formulations, with the strains IE-115 (Pleurotus pulmonarius) and IE-771 (P. agaves). 

Regarding the analysis of the weight of the mushrooms grouped by pileus size, it was observed that the variation between the values depended more on the strain than on the substrate (Table 2). In general, the strain IE-771 presented fruiting bodies with a higher average weight than IE-115 in the different culture conditions, although only the samples of P. agaves (G3) cultivated in the substrate M were statistically different from those of P. pulmonarius (G3), in both substrates. It is important to note that the recorded weights of the fructifications included the stipe so the variability of the weight also depended on robustness. This characteristic of IE-771 of presenting a higher average weight of basidiomes in the different size groups should be corroborated with greater variability of strains and/or substrates since it could be of interest for its commercial cultivation.

Although the results of the statistical analysis do not indicate significant differences between the substrates, the values in Table 2 show an increase in the average weights of the fruiting bodies of strain IE-771 when grown in the M substrate.

Substrate fiber content

Consumption of the main fiber components of the two substrates during P. agaves cultivation is presented in Table 3. By the end of the incubation period, the polysaccharide content of the substrates had been consumed by the mycelia, and the consumption was noticeable after the third harvest. Degradation varied between times and substrates. In particular, IE771 cultivated in barley straw (S) efficiently degraded cellulose and lignin throughout the incubation period and hemicellulose after the third harvest, while the samples grown on M substrate were characterized by high consumption of all fractions (cellulose, hemicellulose and lignin) during the incubation period. In general, a greater decrease in lignocellulosic compounds was observed for barley straw samples alone than when barley straw was combined with maguey leaves.

The degradation of lignocellulosic fractions by the genus Pleurotus depends on the production and secretion of hydrolytic and oxidative enzymes such as cellulases, hemicellulases, and ligninases ^{(Mata et al. 2017)}. Comparing the initial composition (T1) of the formulations used in our study, Table 3 shows a higher proportion of cellulose and hemicellulose in substrate S, while lignin was present in a higher proportion in substrate M. We found in both substrates that the degradation of cellulose is greater during the incubation period (T2) than during fructification. This aligns with that reported by ^{Mata and Torres-Hernández (2008)} for P. ostreatus in barley straw supplemented with coffee pulp or peat. However, contrary to the findings of these authors, we found that degradation of hemicellulose was greater during fructification in fermented barley straw. This finding has also been reported by ^{Salmones et al. (2005)}, who cultivated P. ostreatus and P. pulmonarius in wheat straw and coffee pulp; among the three fractions evaluated, cellulose and hemicellulose were the most degraded during the selective growth of the fruiting bodies since they provide the necessary nutrients for their development and the variation in the decrease depends on the sugars and other carbon sources available. Regarding lignin, species of the genus Pleurotus are known to selectively degrade lignin and acquire other readily available sources of carbon ^{(Mata et al. 2017)}. The consumption of lignin recorded during the incubation period (T2) in our study, reached approximately 50 % in both substrates but was practically zero during fructification (T3). These results align with those reported by ^{Pandey and Singh (2014)}, who found a high degradation of lignin from the incubation period to the formation of primordia. However, the degradation of lignocellulose can vary during the different stages of the crop cycle since it is affected by various factors such as the enzymatic activity of the fungus, availability of O₂ in the substrate, composition, and condition of the substrate, interaction between mycelium and substrate, strain genotype, environmental conditions, and the morphogenetic status of the cultivation ^{(Baldrian et al. 2005}, ^{Sherief et al. 2010)}.

The chemical composition of a substrate can influence the nutritional and organoleptic properties of the mushroom ^{(Rodríguez-Estrada et al. 2009}; ^{Vlasenko and Kuznetsova 2020)}. Aerobic fermentation of the straw for short periods improves adequate mycelial colonization since fungistatic bacterial communities increase the competitive ability of Pleurotus against its main antagonist Trichoderma spp. ^{(Velázquez-Cedeño et al. 2006)}. Regarding the maguey leaves, fermentation allowed softening of the fiber, resulting in a better and more manageable consistency for mixing with the fungus spawn ^{(Mata and Torres-Hernández 2008)}. ^{Guzmán-Dávalos et al. (1987)} used the “piña” or heart of the Agave tequilana (a waste product of the tequila industry) to cultivate P. ostreatus, obtaining a BE of 60.2 %. This value increased to 96.4 % when the tequila industry waste was mixed with straw. These authors concluded that a more selective substrate had been obtained and that contamination by molds had been avoided ^{(Soto-Velazco et al. 1989)}. This was observed in our study since no mold contamination was detected in the samples and only one sample had to be discarded due to insect larvae contamination.

In conclusion, maguey mushrooms are highly appreciated in rural areas of central Mexico, and they have been consumed in various traditional dishes such as soups, “quesadillas” or “guisados” (Minerva Mendoza, personal communication). The P. agaves strain cultivated in this study presented size and color characteristics that made it suitable for introduction to the market.

This study demonstrated that maguey leaves and barley straw, alone or mixed, could be used as substrates in the cultivation of P. agaves. The selection of the former substrate was based on the origin of the strain studied, but the most viable option is to utilize grass straw. Given its morphological and cultivation characteristics, P. agaves could compete in the market with other commercial species from temperate environments, such as P. ostreatus and P. pulmonarius. The isolation and cultivation of wild genetic material from various regions of the country should therefore be continued and a program of selection and improvement of the commercial characteristics of the species should be initiated.