Total production of CP-50

The production of the P. ostreatus CP-50 strain in the treatments evaluated was done according to the methodology described previously and lasted 121 days, since sowing of the mycelium in Petri dishes until obtaining the third harvest. ^{Cedano et al. (1993)} reported that P. ostreatoroseus began fructification after 31 days of incubation and 60 days of production; similar results to this research, where the CP-50 strain presented its first fructification between 38 days in most of the treatments and up until 73 days of production. Regarding the harvest period, ^{Mora (2004)} reported a harvest period of five weeks, using the strain (HEMIM-50) of P. ostreatus on wheat hay; ^{Gómez (2004)} mentions an average period of six weeks, with the same strain and in the same substrate. The harvesting period for the CP-50 strain in this study was between six and seven weeks, which agrees with what was reported by ^{Gómez (2004)}.

The cultivation cycle of the P. ostreatus CP-50 strain ranged between 100 days and 121 days, with a mean of 38 days of colonization, plus 45 days of fructification. In his study, ^{Aguirre (2000)} reported a cultivation cycle of 119 days (32 days of colonization, plus 84 days of fructification); ^{Gómez (2004)} reported a cultivation cycle of 88 days for the strain (HEMIM-50) of P. ostreatus, 53 days of colonization, plus 35 days of fructification. Vernero et al. (2010) reported a cultivation cycle of 104 days for P. ostreatus in wheat hay substrate, similar results to those obtained in this research study (Table 2).

To quantify the fresh weight of the sporocarps produced, three harvests were carried out in a lapse of 45 days. The highest yield in fresh weight was obtained in the first harvest and decreased in the next ones. In average, the first harvest obtained between 46 and 60 %; in the second cut, it was 15 to 36 %; lastly, 8 to 26 % of the total production obtained.

The total production in the control treatments is shown in Figure 1, where the wheat hay substrate TP reached the highest production, with a total of 12.87 kg, counting the three harvests carried out. It should be mentioned that wheat hay is the traditional substrate, on which this productive activity is carried out (^{Kumari and Achal, 2008}). Barley hay was the one in second place with 10.22 kg, followed by maize stubble, with 6.43 kg; bean straw obtained 4.68 kg, and, lastly, dehydrated alfalfa, showing highly significant differences with the multiple range Tukey test (p≤0.05).

Different letters indicate significant differences with the multiple range Tukey test (p≤0.05).

Figure 1 Total production of the CP-50 strain of P. ostreatus in control treatments without dehydrated alfalfa. 

The total production obtained from the P. ostreatus CP-50 strain in barley hay supplemented with dehydrated alfalfa in different concentrations makes a comparison between the treatments and the traditional control (wheat hay). In this case, the highest production in fresh weight was obtained with the PC-3Al treatment that corresponds to barley hay supplemented with 3 kg of dehydrated alfalfa, giving us a total of 13.32 kg (Figure 2); the second best production presented the treatment PC-2.5Al barley supplemented with 2.5 kg of dehydrated alfalfa, with 13.11 kg. The treatment that presented the lowest production was barley without supplementation (PC), with a total production of 10.22 kg, reporting significant differences with the multiple range Tukey test (p≤0.05).

Different letters indicate significant differences with the multiple range Tukey test (p≤0.05).

Figure 2 Total production of the CP-50 strain of P. ostreatus in barley haw substrate supplemented with dehydrated alfalfa. 

The total production obtained in the maize treatment supplemented with dehydrated alfalfa in different concentrations reported highly significant differences with the multiple range Tukey test (p≤0.05). The highest production in fresh weight was obtained with the RM-3Al treatment, which corresponds to maize stubble supplemented with 3okg of dehydrated alfalfa, obtaining a total production of 12.09 kg (Figure 3). The second best production was found with the “Wheat hay” control treatment, with 12.87 kg. The treatment that presented a lower production was maize stubble “RM”, with 6.43 kg. It should be mentioned that the treatments supplemented with dehydrated alfalfa outperformed maize stubble; in addition, it showed a similarity with wheat hay. A study performed by ^{Flores (2012)} mentions that the combination of yucca husk and wheat hay had a higher yield in comparison to the yucca husk alone. Similarly, ^{Rajak et al. (2011)} reported that with the combination of rice hay (substrate) and wild weeds from India (co-substrate), they obtained the highest yield in comparison to the wild weeds alone. This could be explained by the fact that the mixture of typical substrates contributes the nutrients necessary for the incubation and development of primordia and the formation of sporophores during colonization, while the co-substrates, whose decomposition is slower than that of substrates, provide the nutritional requirements necessary for the later phases of growth.

Different letters indicate significant differences with the multiple range Tukey test (p≤0.05).

Figure 3 Total production of the CP-50 strain of P. ostreatus in maize stubble supplemented with dehydrated alfalfa. 

The total production obtained in the bean straw treatment supplemented with dehydrated alfalfa presented the highest production in fresh weight with the treatment (PT) that corresponds to wheat hay without supplementation, giving us a total of 12.87 kg (Figure 4); the second best production was presented by treatment PF-3Al, bean straw supplemented with three kilograms of dehydrated alfalfa with 8.41 kg. The treatment that presented the lowest production was the control bean straw (PF), with 4.68 kg, reporting significant differences (p≤0.05).

Different letters indicate significant differences with the multiple range Tukey test (p≤0.05).

Figure 4 Total production of the CP-50 strain of P. ostreatus in bean straw substrate supplemented with dehydrated alfalfa. 

Quantification of BE, PR and BR for CP-50

The biological efficiency (BE) not only depends on the nutritional balance attained, but also on other environmental aspects, such as the capacity for water retention of the substrate, aeriation and relative moisture in several stages of cultivation (^{Mane et al., 2007}). ^{Mora and Martínez-Carrera (2007)} reported biological efficiencies of 39 % to 162 % in wheat hay, with commercial strains of Pleurotus spp., results similar to this study. The PT-3Al treatment presented a BE of 179.40 %, similar to that found by ^{Salmones et al. (1997)} where they reported a BE of 75.6 to 68o% in 19 strains of Pleurotus spp., in a substrate of barley hay, lower values than those from this study (133.23 %). ^{López et al. (2005)} cite that maize stubble substrate presents a BE of 97 %, higher than those from the control group RM and RM-2Al in this research (64.30 and 91.85 %, respectively), although lower when being supplemented with dehydrated alfalfa at 2.5 and 3.0 kg, obtaining BE of 114.50 and 120.91 % (Table 3). The BE for bean straw residue without supplementing with dehydrated alfalfa is 46.84 %; when complementing this agricultural residue with dehydrated alfalfa, an increase of 60 % of its BE can be observed, higher than maize stubble (RM), but lower than the wheat hay (PT).

The highest production rate (PR) was obtained in wheat hay supplemented with 3.0 kg of dehydrated alfalfa (1.48 %); the lowest PR, in the dehydrated alfalfa treatment (0.29 %). ^{Mora (2004)} reported a PR of 1.57 % in wheat hay; ^{Gaitán-Hernández et al. (2009)} reported a PR of 0.76 % in barley hay, similar results to those obtained in this study (Table 4).

Likewise, the biodegradation rate (BR) presented by the P. ostreatus CP-50 strain showed that it is capable of converting up to 70 % of the substrate in food for human consumption, particularly in that of wheat hay supplemented at 3.0 kg of dehydrated alfalfa, which was the highest BR in comparison to the dehydrated alfalfa substrate, with 32 % and bean straw with 46 % (Table 5).

The mean temperature inside the incubation area of the production units was 22.1 °C (minimum) and 28.16 °C (maximum) during the months of February to May, which was the period in which the production units remained inside the incubation area. ^{Mora (2004)} reported a weekly mean temperature of 20.3 °C to 27.7 °C, with a maximum of 27.2 and a minimum of 20.0 °C, and ^{Gómez (2004)} reported values that range from 23°C to 26.7 °C for the months of April to May, similar to those reported in this study. Regarding the fructification area, the mean temperature ranged between 18.4 °C (minimum) and 25.5 °C (maximum) during the months of June to September, where the last harvests were obtained. ^{Gómez (2004)} reported a mean temperature between 21 and 23.9 °C for the weeks of June to July; in addition, a relative humidity that ranged between 76.5 to 87.3 % for the months of May to June, similar results to those of this study, where the average relative humidity in the rustic module of production within the fructification area was 78.3 %. It should be mentioned that the temperatures of incubation and fructification that were present in the production module had higher values than those reported by ^{Mora (2004)} and ^{Gómez (2004)}, due to the environmental conditions of location.

Proximal chemical analysis of substrates

The substrates used in this experiment presented a different chemical composition (Table 6). These differences make the productive capacity of the P. ostreatus CP-50 strain obtain higher yields in substrates rich in dry matter, such as the case of wheat hay, which presents a concentration of 90.10o%; raw protein, 3.34 %; ethereal extract, 0.22 %; ashes, 11.34 %; NDF, 85 %; and ADF, 51.14 %. Olavarría (2000) reports values of 1.7 % of ashes for barley hay, which are lower than those found in this study. Likewise, ^{Beare et al. (2002)} found 13.58 % of ashes for every 100 grams of dry substrate in maize stubble, values higher than those reported in this study. Yumi and Duchi (2007) report 2.5 % of protein in maize stubble, results that are lower in this study (4.9 % of protein without supplementing with dehydrated alfalfa and 8.93 % of raw protein when being supplemented with dehydrated alfalfa). ^{Rivas (2005)} mentions that alfalfa contains around 50 % of raw protein in the cell wall, as well as a balanced fiber composition, 8 % of pectin, 10 % of hemicellulose, 25 % of cellulose, and 7 % of lignin, which favors the development of sporocarps of the oyster mushrooms.

Urbano and Dávila (2003) mention that the raw protein contribution is significant; alfalfa presents around 25 % of non-protein nitrogen, highly soluble and assimilable for saprophyte mushrooms, as shown by ^{Danciang (1986)}, who studied the productivity of P. ostreatus in rice hay and sawdust, where they found that the first substrate produces a higher number of carpophores and of higher diameter than the ones developed in sawdust. The difference in the productivity of these substrates can be due to the differences in raw protein (15.10 %) and fat (0.35 %) for rice hay, versus 3.2 % of raw protein and 0.14 % of fat for the sawdust. In addition, ^{Faner (2001)} mentions that alfalfa is a good source of macrominerals (calcium, phosphorus, magnesium, potassium, chlorine), microminerals (zinc, copper, iron), vitamins (liposoluble, group B), and pigments.

Oyster mushrooms require substrates with a high amount of nutrients; in this sense, ^{Amuneke et al. (2011)} obtain 95 % of fructifications in substrates rich in protein. The increase in raw protein in the substrates indicates that P. ostreatus has a high capacity for synthesis, developing better (Sudiany et al., 2012).

In this research study, maize stubble presented a dry matter concentration of 93.82 %. When complemented with dehydrated alfalfa (3.0 kg), an increase in BE, PR and BR was found, since its content of dry matter increased to 98.35 %; raw protein, from 4.90 to 8.93 %; ethereal extract, 1.23 to 1.64 %; ashes, 6.83 to 9.05 %; NDF, 72.45 to 89.02 %; and ADF, 46.75 to 49.31 %, turning it from a low-yield substrate to a high-yield substrate in the production of oyster mushrooms. In addition, its availability in rural zones of the municipality of Tetela de Ocampo, Puebla, favors its production.

It should be clarified that the figures presented before are average values of the substrates from the municipality of Tetela de Ocampo, Puebla, but there is broad variation for the proximal chemical analysis; for example, the protein in barley fiber varied from 3.9 to 8.7 %; in wheat, from 2.4 to 5.8 %; in maize stubble, from 2.0 to 7.1%; and in bean straw, from 6.0 to 7.9 %. The variation comes primarily from the type of plant, although other factors are also important such as the variety, the degree of maturity, management, soil fertility, sowing season, frost events, etc., which influence the general development of plants and, consequently, their nutritional constitution (^{Romero-Arenas et al., 2010}).