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- MATERIAL AND METHODS
Important cultivated edible and medicinal mushrooms belong to the Agaricus genus. Among them is the almond mushroom formerly known as Agaricus blazei Murrill. In the 2000s, two new species names, Agaricus subrufescens Peck and Agaricus brasiliensis Wasser et al., were proposed for this fungus, which is believed to originate from Brazil. Currently, many publications refer to the Brazilian cultivars as A. blazei or A. brasiliensis. Recently, A. subrufescens Peck was declared the correct name, but the authors excluded neither the existence of infraspecific taxa nor the fact that A. subrufescens might be a complex of species. The mushroom has been produced on a commercial scale in Brazil since the early 1990s and exported to several countries. Nowadays it is also cultivated at the industrial level in Japan, China, Taiwan and Korea. These cultures rely on local agroindustrial waste-based substrates. The majority of articles on A. subrufescens cultivation available in the literature refer to experiments in Brazil. The raw materials commonly used to prepare the substrate are sugar cane bagasse, various grasses (e.g. Brachiaria spp., Cynodon dactylon, Panicum maximum), cereal straw (Triticum aestivum, Avena sativa, Oryza sativa) and manure supplemented with nitrogen sources (soybean, wheat, corn and cotton meal, urea, ammonium sulfate) and sources of phosphorus and calcium. Experiments performed in China showed the possible use of cottonseed hulls, rice hulls, asparagus straw and soybean cake.[8, 9] Efficient A. subrufescens production was also obtained with substrate compositions closer to that used to grow the button mushroom Agaricus bisporus in temperate countries, such as cattle bedding compost/sawdust/cereal bran and chicken manure/wheat straw. Local soils, with or without the addition of vegetal charcoal, have been tested as an alternative to peat in the casing layer used for A. subrufescens cultivation.[11-14] The casing soil composition is important, regardless of the substrate formulation. The physical characteristics of the soil contribute greatly to the mushroom yield. These cultivation conditions are well adapted to tropical countries, although yields are significantly lower than those obtained with A. bisporus, but A. subrufescens might be a seasonal option for mushroom growers in western countries. They can save energy by producing the almond mushroom efficiently during summer, owing to its higher optimal temperature requirements when compared with A. bisporus. Our aim was to define cultivation conditions suitable for A. subrufescens starting from the substrate and casing mixture used for A. bisporus commercial production and making changes easy to perform by producers of button mushroom in western countries.
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- MATERIAL AND METHODS
Works aimed at comparing the productivity of different mushroom strains have generally considered total biomass obtained during the same period of time starting at casing. The eight strains studied in the present work showed very different time to fruiting, meaning harvests lasted for very different periods of time in experiments analysed 65 days after casing. Comparing biomass production during the same period of time is a better representation of the ability of strains to mobilise nutrients to fruit. Consequently, strains were compared for mushroom biomass 10, 20 and 30 days after the first picking.
Under our cultivation conditions (straw- and horse manure-based compost for A. bisporus, incubation and fructification in a climatic chamber), using a 5 cm casing layer instead of the 2.5 cm conventionally used in experiments with A. bisporus significantly improved biomass production of the strains tested. A similar effect of casing depth was obtained in Brazil with different casing mixtures and substrate compositions. Both 5 and 8 cm of casing mixture based on Brazilian soils were better than 3 cm for cultivating ABL-97/12 in a greenhouse on substrate prepared with local Brazilian wastes. Increasing casing quantity led to higher yield in strain M7700 grown in a climatic chamber on substrate made of chicken manure, wheat straw and gypsum. Besides the effect observed in a greenhouse, no improvement occurred in a bamboo-covered structure. The quantity of casing material proved important for biomass production of A. subrufescens, irrespective of the strain, casing and substrate materials, but its effect could be affected by the cultivation environment.
Peat use causes the loss of non-renewable resources and participates in the greenhouse effect by the liberation of CO2 through the aerobic decomposition of carbon, thus generating a worldwide demand for alternatives in agriculture. In Brazil the most utilised casing for both A. bisporus and A. subrufescens is subsoil or subsoil mixtures with charcoal, and several works have shown various effects of charcoal on A. subrufescens. Rhodic Hapludox/eucalyptus charcoal 4:1 (v/v) led to a better yield of strain CS1 compared with Xanthic Hapludox or Humic Haplaquox soil. In contrast, casing made of peat or shale increased the productivity of ABL-99/26 and ABL-99/29 by 10–20% compared with a mixture of 70% ravine soil/30% charcoal (v/v), and lime schist or peat led to better biological efficiency of the same strains compared with a mixture of subsol/charcoal 7:3 (v/v). In outdoor cultivation, strain BZ-04 showed better yield on a substrate covered with local soil horizon A compared with casing mixtures composed of 30% eucalyptus charcoal/70% local soil horizon B (v/v). Our aim was to replace the standard casing used for A. bisporus cultivation with a casing mixture efficient for growing A. subrufescens and easy to implement by European mushroom growers. Peat/limestone was far less efficient than peat/limestone/sand casing for time to fruiting and biomass production of CA487. Based on this result, our reference casing mixture was A. bisporus casing (peat/limestone) supplemented with fine sand. Under our cultivation conditions, not only did the partial replacement of peat with vegetal charcoal (casing C2 vs C1) cause no improvement in mushroom biomass at the end of the experiment, it also reduced the d1–d30 yield of the two wild strains.
Spent mushroom substrate has also been evaluated as a substitute for peat in Agaricus cultivation. A casing mixture composed of Sphagnum peat/spent mushroom substrate (wheat straw and poultry manure ingredients) 4:1 (v/v) + CaCO3 was as efficient as a peat/lime mixture for A. bisporus productivity. When all strains were taken as a whole, we obtained the worst biomass production with the two casing mixtures (C3 and C4) containing spent compost. In contrast to C3 that reduced the yield of the four strains, the C4 effect varied with the strain. Despite the yield of CA487 being similar with C1 and C4, the casing mixture C1 was chosen because it gave the best yield for all strains. Indeed, our objective was to find the best casing mixture to grow A. subrufescens, irrespective of the strain. Besides, C4 has the same peat/spent substrate/CaCO3 proportion as the casing mixture with which Pardo-Giménez et al. obtained a good performance for an A. bisporus strain. The difference in Agaricus species, the use of several strains and possibly the peat origin and the characteristics of the spent substrate could contribute to explaining why spent compost was not a good material to prepare a standardized casing for A. subrufescens.
The C1 casing mixture used for the different experiments allowed CA487 to produce 171–228 g kg−1 substrate in the d1–d30 period, which fell between the values of 443 and 1315 g per 4 kg substrate that Mata et al. obtained for this strain 30 days after pin formation with casing made of sand/peat/limestone 2:1:1 and 1:1:1 (v/v/v) respectively and substrate based on wheat straw supplemented with sugar cane bagasse. In contrast to their observation, we did not improve the mushroom biomass by increasing the percentage of sand in the casing mixture. The substrate we used, based on wheat straw and horse manure, might explain this difference.
Most air temperatures during incubation reported for experiments performed in Brazil ranged between 25 ± 2 and 28 ± 1 °C.[5, 11, 12, 14] Incubation at a minimum temperature of 8 °C during the night and a maximum temperature of 26 °C during the day was also applied. Our incubation conditions were close to the 22 ± 1 °C used in a Slovenian facility. These temperatures are suitable for the incubation of A. subrufescens and more adapted to cultivation in temperate countries with moderate energy expenditure. The variations in temperature measured in the compost during incubation reflected neither the propensity of the strain to colonize the substrate nor its ability to produce mushroom biomass.
The mushroom can fruit at a temperature between 20 and 30 °C. Although, in contrast to A. bisporus, the almond mushroom does not need a decrease in temperature for fruiting, a reduction in air temperature to 17–20 °C to induce primordial formation followed by an increase to 22–28 °C for the development of the fruiting body was reported.[6, 12, 14, 24-26] However, various experiments performed in Brazil showed that large and uncontrolled variations in temperature also proved suitable. A mushroom yield of 9.6% (strain BZ-04) was obtained after 90 days under natural conditions in an area bordering the Guaramiranga forest, Ceará State (climate predominantly warm and wet) with minimum temperature around 20 °C and high variations in maximum temperature ranging from approximately 20 to 32 °C. Strain CS1 cultivated at Lavras (Minas Gerais State) in a room under natural conditions with the temperature ranging from 17 to 28 °C had a yield of 13.3% at 101 days after casing. More interesting are the Brazilian works showing valuable production in a greenhouse where local climatic conditions directly influenced mushroom yield. Cultivating ABL-97-12 in a plastic greenhouse where minimum, maximum and mean temperatures ranged from 13 to 21 °C, from 23 to 36 °C and from 14.3 to 25.6 °C respectively led to the same yield as in a climatic chamber set at 25 ± 2 °C. Strain ABL-04/49 cultivated in a greenhouse where the air temperature varied between 20.4 and 36.8 °C and the compost temperature between 20.3 and 30.8 °C showed 20% higher yield compared with that obtained in a climatic chamber with primordial induction.[14, 26] Starting from these observations, we decided to investigate whether variations in temperature could improve the productivity of the eight studied strains. The air temperature was regulated to mimic variations between night and day. Minimum and mean temperatures recorded were in the same range as those reported by Braga et al., but the maximum temperature did not exceed 28.4 °C, while it reached 36 °C in the Brazilian greenhouse. The lack of high temperatures during our experiment could explain the absence of yield improvement. However, the lowest temperature recorded in the compost was 17.5 °C and the highest 26.8 °C, which did not differ markedly from the 20.3 and 30.8 °C measured by Zied et al. in a greenhouse. The only notable difference related to variations in temperature was that CA565 began to fruit earlier than at constant temperature and no longer differed from the other two cultivars for this trait. The Brazilian strains remained far less productive than the European strains, regardless of the experiment.
Precocity is defined as yield at mid-cycle of harvest expressed as a percentage of total harvest and depends on the type of compost and casing. Zied et al. observed precocity ranging from 50 to 62% in a first set of crops and from 69 to 75% in a second set. We chose to assess the percentage of biomasss production during three successive periods of 10 days following the first picking of the strain. The major part of the biomass was obtained during the first 10 days, and production was very low during the last period (d20–d30). The kinetics of production differed dramatically from that known for cultivars of A. bisporus and observed under our cultivation conditions for this species. These observations suggest that the commercial compost used for A. bisporus is probably not the optimal substrate to grow A. subrufescens, but yields obtained with the wild European strains were much better than those reported for commercial strains in Brazil (e.g. 80–110 g kg−1 after 65 days for the strain ABL-97/12 and 88 g kg−1 after 70 days for the commercial strain ABL-04/49). The average production in Brazil was estimated to be 8–16% after 120 days of cultivation.
As Colauto et al. reported for two Brazilian cultivars, we observed that changes in mushroom fresh weight during harvest depended on the strain, although all strains except CA643 produced less heavy mushrooms during the d20–d30 period. As regards the whole harvest, Brazilian strains were generally more interesting than wild European strains for individual fresh weight and dry matter content, but this was offset by the far higher yield of the wild strains. In particular, the Spanish strain CA438-A proved to be a good material owing to its classification for fresh weight and dry matter, which did not differ from those of the Brazilian cultivar CA561.