Effect of Cultivation Practices on the Î²-Glucan ... - ACS Publications

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Effect of Cultivation Practices on the β‑Glucan Content of Agaricus subrufescens Basidiocarps Diego Cunha Zied,*,† Arturo Pardo Giménez,‡ Jose Emilio Pardo González,§ Eustáquio Souza Dias,# Maiara Andrade Carvalho,# and Marli Teixeira de Almeida Minhoni⊥ †

Faculdades Integradas de Bauru (FIB), Rua Rodolfina Dias Domingues 11, Jardim Ferraz, 17056-100 Bauru, SP, Brazil Centro de Investigación, Experimentación y Servicios del Champiñoń (CIES), C/Peñicas s/n, Apartado 63, 16220 Quintanar del Rey, Cuenca, Spain § Escuela Técnica Superior de Ingenieros Agrónomos, Universidad de Castilla-La Mancha (UCLM), Campus Universitario s/n, 02071 Albacete, Spain # Departamento de Biologia, Universidade Federal de Lavras (UFLA), CP 3037, 37200-000 Lavras, MG, Brazil ⊥ Faculdade de Ciências Agronômicas, Universidade Estadual Paulista (FCA/UNESP), Rua José Barbosa de Barros 1780, 18610-307 Botucatu, SP, Brazil ‡

ABSTRACT: The present work aimed to assess the effect of the following treatments on the medicinal potential (β-glucan content) and agronomical performance (yield) of Agaricus subrufescens: five different fungal strains, three cultivation substrates (compost), four casing layers, and four cultivation environments. Two experiments were performed, and the results indicate that the greatest contribution to the variation in β-glucan content was the strain (35.8%), followed by the casing layer (34.5%), the cultivation environment (15.7%), and the type of compost (9.9%). On the other hand the variation in yield was affected most by the cultivation environment (82.1%), followed by the strain (81.3%), casing layer (49.1%), and compost type (15.2%). These findings underscore the importance of developing a production protocol that employs specific cultivation practices for improving mushroom yield as well as β-glucan content. KEYWORDS: almond mushroom, medicinal potential, strain, compost, yield

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INTRODUCTION

Although the scientific community is increasingly concerned with the identification of the active substances present in mushrooms with medicinal properties, important agronomical and physiological characteristics have been overlooked. These factors are critical in production processes, and they may influence the chemical characteristics of mushrooms. Eira11 highlighted the importance of using samples of registered origin for experimental procedures. The same publication further described how factors such as cultivation conditions, substrate composition, and the strain used may interfere with the results. In other words, the results obtained using generic samples of unregistered origin are not conclusive. Accordingly, factors such as these should be controlled in any experiment that aims to compare the nutritional and functional properties of A. subrufescens. In a study comparing the basidiocarp β-glucan contents of field and greenhouse mushrooms from Brazil and greenhouse mushrooms from Japan, Park et al.12 observed lower β-glucan contents for A. blazei mushrooms grown in greenhouses (7.6 ± 2.8 g 100 g−1 mushroom dry weight grown in Japan and 8.4 ± 0.9 g 100 g−1 mushroom dry weight grown in Brazil) than for those grown in the field (10.1 ± 2.1 g 100 g−1 mushroom dry

The incidence of cancer is gradually increasing, and more types of diseases associated with cancer are reported each year.1 In addition to radiotherapy and chemotherapy, immunotherapy and biotherapy are also regarded as important tools for cancer treatment.2 Immunotherapy and biotherapy include several different approaches, such as biological response modifiers, cytosines, lymphocyte transplant, and genetic therapy, along with the use of medicinal plants and alternative medicines, such as mushrooms.3 Clinical trials for these therapies continue to be widely performed, and there is increasing evidence to support the efficacy of these approaches; however, the underlying mechanisms remain poorly understood.1,4,5 Agaricus subrufescens, which is synonymous with Agaricus blazei ss. Heinemann and Agaricus braziliensis, is a basidiomycete fungus commonly referred to as “almond mushroom” or “medicinal mushroom”. Its medicinal properties (namely, tumor-inhibitory activity) have been attributed to the presence of β-glucan polysaccharides.6 There is some controversy in the literature regarding the medicinal substances present in A. subrufescens. Camelini et al.7 reported that the quality of the extracts depends on the chemical composition of the mushrooms and the β-glucan content, in particular. Other chemical components, such as steroids,8,9 lectin,10 and several polysaccharides,1 have also been extensively studied. © 2013 American Chemical Society

Received: Revised: Accepted: Published: 41

August 14, 2013 November 29, 2013 December 5, 2013 December 5, 2013 dx.doi.org/10.1021/jf403584g | J. Agric. Food Chem. 2014, 62, 41−49

Journal of Agricultural and Food Chemistry

42

2006 ABL 06/59

Brası ́lia, Federal District, Brazil

2004 ABL 04/49

São José do Rio Preto, São Paulo, Brazil

2003 ABL 03/44

Lençoí s Paulista, São Paulo, Brazil

1999 ABL 99/30

Piedade, São Paulo, Brazil

general characteristics collection city, state, country

1999

Botucatu, São Paulo, Brazil

collection year code

MATERIALS AND METHODS

Two experiments were performed. The first experiment tested the effect of different strains and compost formulations. The strains and formulations that yielded the best results in the first experiment were subsequently used in the second experiment, which assessed the effect of different casing layers and cultivation environments. Spawn. For the first experiment, five commercial A. subrufescens strains that have been deposited at the Mycotec of the Módulo de Cogumelo FCA/UNESP were used. The main characteristics of the strains are listed in Table 1. Strain ABL 99/30 was selected for the second experiment. The spawns used in both experiments were produced according to the methodology described by Zied et al.18 Composting (Phases I and II). The three composts used in the first experiment were produced according to the “traditional” composting method. Phase I (26 days total) consisted of 7 days of prewetting and 19 days of fermentation. The composts were turned six times. Phase II of composting lasted 9 days (8 h at 59 ± 1 °C pasteurization and 8 days at 47 ± 2 °C conditioning). The three composts were prepared simultaneously. The materials used on each compost and their chemical composition at the end of phase II are listed in Table 2. The initial C/N ratio of the compost at the moment the pile was set up was previously calculated for 33-37/1. The compost containing sugar cane bagasse and oat straw was used for the second experiment and produced in the same manner as described for the first experiment. At the end of phase II, the chemical characteristics of the compost were as follows: 1.66% N content, 0.45% P2O5, 0.26% K2O, 5.53% Ca, 0.23% Mg, 0.98% S, 65% organic matter (OM), 36.2% C, 300 mg kg−1 Na, 10 mg kg−1 Cu, 3300 mg kg−1 Fe, 128 mg kg−1 Mn, 28 mg kg−1 Zn, a C/N ratio of 22/1, and a pH of 6.83. Compost Inoculation and Spawn Run. In both experiments, the respective strains were mixed with the composts after pasteurization and conditioning and were then placed into plastic boxes containing 12 kg of compost and 120 g of spawn. The composts were subsequently incubated for 21 days in the dark in a climate-controlled chamber with a compost temperature of 28 ± 1 °C, at 75 ± 5% relative humidity, and without ventilation. The spawn growth of the strains during this period was periodically monitored, and the cultures were checked for compost contamination by mold and other microorganisms. Casing Layer. In the first experiment, a mix of soil and charcoal (4:1, v/v) was used. Calcitic lime was added for pH correction, followed by pasteurization for 12 h at 58 °C. When the mycelia were fully developed, the compost was pressed and leveled to facilitate the addition of the casing layer to a height of 5 cm. A 12.5 L volume of

ABL 99/28

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Table 1. Strain Code; Collection Year, City, State, and Country; and General Characteristics of the Strains Used in the Present Study

weight). However, the authors did not indicate whether all of the basidiocarps analyzed were of the same strain. Firenzuoli et al.13 previously noted the lack of studies with data for specific cultivation characteristics, as well as the agronomical and physiological behavior of A. blazei strains used in pharmacological, biochemical, and medicinal research. The cultivation practices used for A. subrufescens in Brazil derive from the practices used for A. bisporus production and incorporate some of the management techniques from recently published studies.14−17 At a national and international level, there is still no cultivation protocol or guide that addresses how certain practices influence the presence of active substances in harvested mushrooms and the agronomical performance of A. subrufescens as well as how the cultivation of this species may be rendered viable and sustainable. In the present study, two experiments were performed to assess how various cultivation practices (different strains, compost materials, casing layers, and cultivation environments) affect the final β-glucan content of the cultivated basidiocarps and agronomical outcomes (yield, number and weight of basidiocarps, precociousness, and time to first harvest).

strain deposited at the Mycotec of the Mushroom Module; characterized by its medium size, strong texture, and average to low yield commercial mushroom strain from the Atushi group; characterized by its medium to small size, strong texture, high yield and precociousness, reduced time to first harvest (±38 days), and slightly low fructification temperature (±25 °C) commercial mushroom strain from Fazenda Santa Fé; characterized by its medium size, strong texture, reduced time to first harvest (±40 days), and average to high yield commercial mushroom strain from the producer Goto; characterized by its medium to large size, strong texture, average to high yield, and high production temperature (±28 °C) commercial mushroom strain from the Brasmicel Co.; characterized by its large size, moderate texture, high precociousness, reduced time to first harvest (±39 days), and low yield; it is highly susceptible to attacks by Verticillium f ungicola and Mycogone perniciosa

Article

dx.doi.org/10.1021/jf403584g | J. Agric. Food Chem. 2014, 62, 41−49


Article

Table 2. Materials Used in the Formulation of Composts 1, 2, and 3 and Their Respective Chemical Characteristics at the End of Phase II of Compostinga dry weight (kg) compost

sugar cane bagasse

oat

Massai

Aruana

1 2 3

141 141 141

0 130 0

130 0 0

0 0 131

soybean meal

urea

9.2 9.2 9.2 chemical

ammonium sulfate

simple superphosphate

gypsum

calcitic lime

4 4 4

15 15 15

25 25 25

2 1.5 2.5 2.4 1 1 characteristics

mg kg−1 dry matter

% compost

N

P2O5

K2O

Ca

Mg

S

OM

C

Na

Cu

Fe

Mn

Zn

C/N

pH

1 2 3

1.81 1.76 1.6

1 0.66 0.64

1.09 1.04 1.31

5.15 5.44 5.20

0.48 0.48 1.68

1.36 1.26 1.40

64 68 65

35.6 37.8 36.2

440 440 1160

0 0 172

500 400 400

260 160 306

20 18 212

20:1 21:1 23:1

6.25 7.04 6.94

a

Composts: substrates prepared with 1, sugar cane bagasse (Saccharum of f icinarum) and Massai straw (Panicum maximum cv. Massai); 2, sugar cane bagasse (S. of f icinarum) and oat straw (Avena sativa); and 3, sugar cane bagasse (S. of f icinarum) and Aruana straw (P. maximum cv. Aruana).

Table 3. Physicochemical Properties of the Casing Layers Used in the Present Study chemical characteristicsa mg kg−1 dry matter μs cm−1

% casing layer S S S S

+ + + +

b

CH CF PB PM

μs cm−1

g cm−3

WHC

pH

OM

P

H + Al

K

Ca

Mg

SB

CEC

V

Fe

Mn

EC

De

saturated

0.006 MPa

6.8 6.7 7.1 7.3

9 52 31 45

4 19 6 22

10 11 9 10

1.1 1.7 2.1 3.2

64 135 163 183

3 17 8 10

68 154 173 196

78 164 182 206

88 94 95 96

5 13 5 10

0.6 2.5 0.6 0.8

190.7 492.0 104.5 367.8

1.12 0.86 0.84 0.81

0.48 0.58 0.62 0.72

0.20 0.22 0.25 0.26

a

OM, organic matter; SB, sum of bases; CEC, cationic exchange capacity; EC, electrical conductivity; De, total density; WHC, water-holding capacity. bS, soil; CH, charcoal; CF, coconut fiber; PM, peat moss; PB, pine bark.

Table 4. Performance of Environmental Variables under the Different Cultivations air temperature (°C)

compost temperature (°C)

relative humidity (%)

light intensity (20000 lx)

cultivation environmenta

max

min

av

max

min

av

max

min

av

max

min

av

CC GDF GMF GTF av

30.1 41.2 46.9 55.3 43.3

16.3 16.7 17.2 17.0 16.8

25.9 24.2 27.6 30.6 27.0

29.0 30.5 33.4 38.3 32.8

17.5 17.7 18.2 17.9 17.8

25.0 21.0 24.3 29.6 24.9

100 100 100 100 100

90 35 37 32 48.5

97 60.8 54.3 48.7 65.2

35 23 1390 2990 1110

35 0 0 0 0

35 11.5 695 1495 560

CC, climate-controlled chamber; GDF, greenhouse with Duplalon film; GMF, greenhouse with milky film; GTF, greenhouse with transparent film; av, average. a

casing was added to each plastic box containing 12 kg of compost. The boxes were placed in a greenhouse (with a low-density polyethylene milky plastic film 150 μm thick; E.L.V. Leitoso, Electro Plastic, São Paulo, Brazil) with partial control of temperature, relative humidity, and aeration. During the cultivation, the air temperature ranged from 10 to 40 °C, the compost temperature ranged from 18 to 32 °C, and the relative humidity was between 35 and 100%. In the second experiment, four casing layers were used: soil + charcoal (4:1, v/v), soil + coconut fiber (4:1, v/v), soil + composted pine bark (4:1, v/v), and soil + sphagnum peat moss. The soil used was the same as in the first experiment. Prior to the addition of the casing layer, calcitic lime was added to the soil, which was subsequently wetted and mixed with the organic materials (charcoal, coconut fiber, composted pine bark, and sphagnum peat). The main physicochemical properties of the casing layers are listed in Table 3. After homogenization, the casing layers were pasteurized and placed over the compost as described for the first experiment. The boxes containing the four casing layers were then placed under four different cultivation environments: a climate-controlled chamber (length × width × height: 12 × 2.3 × 2.3 m) (Model Dalsem Mushrooms Container, Horst, The Netherlands); an experimental greenhouse (length × width × height: 8 × 4 × 2.5 m) with a low-density polyethylene Duplalon double-sided (black/white) plastic film 120 μm

thick (E.P.B. Estufa Preto e Branco, Electro Plastic, São Paulo, Brazil); an experimental greenhouse (length × width × height: 8 × 4 × 2.5 m) with a low-density polyethylene milky plastic film 150 μm thick (E.L.V. Leitoso, Electro Plastic); and an experimental greenhouse (length × width × height: 8 × 4 × 2.5 m) with a low-density polyethylene transparent plastic film 150 μm thick (E.L.V. Difusor M36, Electro Plastic). Primordial Induction. In the first experiment, the primordial induction occurred naturally, according to the weather conditions of the day or week. The total production time was 120 days, with some primordial growth observed after 21 days. Depending on the strain and compost used, the number of harvest flushes during cultivation ranged from two (strain ABL 99/30 inoculated on compost 1) to seven (strain ABL 03/44 inoculated on compost 3). In the second experiment, the environmental conditions varied for each cultivation environment (compost and air temperatures, relative humidity, and light intensity). The environmental conditions during production for the different cultivation environments are listed in Table 4. In all three greenhouse environments (Duplalon, milky, and transparent plastic film), the primordial induction occurred naturally, according to the weather conditions of the day or week. In the climatecontrolled chamber, the environmental variables (temperature, relative 43



Article

Table 5. Agronomical Performance of A. subrufescens and the Basidiocarp β-Glucan Content According to the Type of Compost and Fungal Strain Used strain composta

ABL 99/28

ABL 99/30

ABL 03/44

ABL 04/49

ABL 06/59

Yield (%) 1 2 3 CVe (%) MSDf avg (%)

10.76 a ABb 7.97 a B 5.45 a B

1 2 3 CV (%) MSD av (u)

74.62 a AB 61.01 a B 47.87 a AB

1 2 3 CV (%) MSD av (g)

17.72 a A 18.02 a A 19.54 a A

1 2 3 CV (%) MSD av (%)

33.32 a B 29.69 a B 28.89 a B

1 2 3 CV (%) MSD av (days)

42.19 a A 40.80 a AB 55.60 b B

1 2 3 CV (%) MSD av (g 100−1)

15.34 a A 20.37 a A 16.13 a A

12.89 a A 9.83 a B 9.01 a AB

11.59 a AB 10.09 a B 10.18 a AB

2.9 a B 3.66 a B 1.9 a B

64 7.46c

4.73 a A 3.45 a A 3.04 a B

8.71d

9.81 Number of Basidiocarps (u) 119.07 a A 99.08 a AB 154.92 a A 74.67 a AB 128.71 a A 73.66 a AB 80.47 70.13c 73.35 Basidiocarp Weight (g) 18.48 a A 14.95 a A 16.78 a A 19.48 a A 13.91 a A 14.84 a A 37.65 8.08c 18.07 Precociousness (%) 57.71 a AB 33.06 a B 63.68 a AB 40.11 a B 59.86 a AB 24.65 a B 56.84 36.37c 53.85 Time to First Harvest (Days) 39.60 a A 46.18 b A 36.92 a A 34.67 a A 39.54 a A 39.22 a A 10.9 6.94c 41.91 β-Glucan (g 100 g−1 Mushroom Dry Weight) 4.63 a A 4.01 b A 5.10 a A 4.86 b A 5.46 a AB 9.67 a A 44.37 4.64c 5.19

85.53 a AB 77.50 a AB 67.37 a AB

18.51 a B 15.75 a B 8.87 a B

81.86d

17.62 a A 16.60 a A 18.63 a A

19.55 a A 22.25 a A 22.55 a A 9.43d

46.94 a B 37.95 a B 57.86 a AB

100 a A 100 a A 98.52 a A

42.46d

47.15 a A 45.26 a B 44.29 a A

39.16 a A 39.15 a AB 38.85 a A

8.12d

5.95 a A 3.62 a A 4.83 a AB

7.79 a A 6.81 a A 3.90 a B

5.45d

a

Composts: substrates prepared with 1, sugar cane bagasse (Saccharum of f icinarum) and Massai straw (Panicum maximum cv. Massai); 2, sugar cane bagasse (S. of f icinarum) and oat straw (Avena sativa); and 3, sugar cane bagasse (S. of f icinarum) and Aruana straw (P. maximum cv. Aruana). bLower case letters compare results within the same column, and upper case letters compare results within the same row according to Tukey’s test (P ≤ 0.05). cMinimum significant difference for values within the same column (compost comparison). dMinimum significant difference for values within the same row (strain comparison). eCV, coefficient of variation. fMSD, minimum significant difference. gav, average. Harvest. The basidiocarps were harvested manually, followed by the scraping of the bottom of the stalk to remove casing layer residues. The analysis was subsequently performed to quantify yield, time to first harvest, precociousness, basidiocarp weight, and the number of basidiocarps. Experimental Design. For the first experiment, a double-factorial design was used, with five A. subrufescens strains (ABL 99/28, 99/30, 03/44, 04/49, and 06/59) and three composts (Massai and sugar cane bagasse, oat straw and sugar cane bagasse, and Aruana straw and sugar cane bagasse) in a completely randomized design. A double-factorial design was also used for the second experiment, with four casing layers

humidity, and aeration) were manipulated according to the methodology described by Zied and Minhoni19 to control the production and to obtain five flushes. The total production time was 120 days, with some primordial growth observed after 21 days, in the climate-controlled chamber and in the greenhouses with Duplalon or milky plastic film. Depending on the casing layer and cultivation environment used, the number of harvest flushes during cultivation ranged from two (casing layer with charcoal grown in a greenhouse with Duplalon plastic film) to five (casing layer with charcoal grown in the climate-controlled chamber). 44



Article

Figure 1. Yield (%) and β-glucan content (g 100 g−1 mushroom dry weight) for the five fungal strains, three composts, four casing layers, and four cultivation environments tested.

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(soil + charcoal, soil + coconut fiber, soil + composted pine bark, and soil + sphagnum peat moss) and four cultivation environments (climate-controlled chamber, experimental greenhouse with Duplalon plastic film, experimental greenhouse with milky plastic film, and experimental greenhouse with transparent plastic film) in a completely randomized design. For both experiments, eight replicates were performed for each treatment, with each replicate consisting of a box containing 12 kg of compost. Analyzed Variables. The following data were measured and analyzed as previously described by Pardo et al.,20 and Zied et al.:16 yield (basidiocarp fresh weight divided by the compost fresh weight multiplied by 100 and expressed as a percentage); the number of basidiocarps (count of the harvested basidiocarps); basidiocarp weight (basidiocarp fresh weight divided by the number of basidiocarps, expressed in grams); precociousness (yield over the first half of the total harvest time divided by the yield over the total harvest time multiplied by 100 and expressed as a percentage); and time to first harvest (number of days between casing and harvesting of the first flush). The β-glucan content of the basidiocarps was determined using the methodology of the Foundation of Japanese Food Analysis Center21 and the modifications proposed by Prosky et al.22 as described by Park et al.12 Statistical Analyses. The means of each variable analyzed were compared using Tukey’s test (p ≤ 0.05) and SAS JMP software. Sigma Stat 3.5 software was used to calculate linear correlations among the values for yield, number, and weight of basidiocarps, precociousness, time to first harvest, and basidiocarp β-glucan content.

RESULTS AND DISCUSSION Experiment 1. The ABL 99/30 strain had the highest yield and number of mushrooms, whereas the ABL 06/59 strain had the lowest yield and number (Table 5). However, the mushrooms of the ABL 06/59 strain had greater weights (not statistically significant). One possible explanation for this finding is that yield was positively correlated with the number of basidiocarps (r = 0.993) and negatively correlated with basidiocarp weight (r = −0.742). When analyzed separately, the type of compost used had no effect on the yield or the number of mushrooms produced. These results are in contrast with the findings of González Matute et al.,23 who observed an effect of substrate composition on yield. The authors used sunflower seed hulls, spent compost, vermiculite compost, peat moss, and brewery waste, with C/N ratios between 40:1 and 71:1. Although correlations between the yield and the number and weight of basidiocarps were observed, basidiocarp weight was not significantly different among the various strains or composts. The lack of statistical significance was due to the high coefficients of variation obtained for yield (64%), the number of basidiocarps (80.47%), and basidiocarp weight (37.65%). The average values of the yield and number of basidiocarps for strains ABL 99/30, 03/44, and 04/49 were higher than the 45



Article

Table 6. Agronomical Performance of A. subrufescens and the Basidiocarp β-Glucan Content According to the Type of Casing Layer and Cultivation Environment Used cultivation environment casing layer (4:1, v/v)

climate-controlled chamber

greenhouse with Duplalon film

greenhouse with milky film

greenhouse with transparent film

Yield (%) soil + charcoal soil + coconut fiber soil + pine bark soil + peat moss CVd (%) MSDe avf (%)

14.72 12.71 15.37 13.60

A A A A

aa a a a

Ab Bb Ab AB b

6.10 0.87 4.32 2.11

Ab Cb BA b CB b

5.03 1.45 5.53 1.04

A B A B

b b b b

73 3.25b

soil + charcoal soil + coconut fiber soil + pine bark soil + peat moss CV (%) MSD av (u)

137.28 174.54 97.85 120.14

soil + charcoal soil + coconut fiber soil + pine bark soil + peat moss CV (%) MSD av (g)

13.71 11.77 15.96 14.11

3.1c Number of 34.14 A 1.75 A 37.85 A 21.14 A

AB a Aa Ba Ba

5.7 Basidiocarps (u) b b b b 91.3

68. 57 A b 35.85 AB b 4.15 B b 13.85 B b

43.37b

A A A A

43.66 52.14 8.65 9.20

AB b Ab Bb Bb

15.70 15.23 24.25 21.74

A A A A

a ab ab a

65.58 64.36 69.73 66.21

a a a a

A A A A

58.23c 53.19 Basidiocarp Weight (g) 13.62 B a 27.85 A a 13.33 B b 17.38 AB a 64.21

a b b a

11.61 16.02 33.52 19.91

B B A B

a a a a

13.31b

12.72c 17.85

soil + charcoal soil + coconut fiber soil + pine bark soil + peat moss CV (%) MSD av (%)

72.30 42.71 61.75 56.94

soil + charcoal soil + coconut fiber soil + pine bark soil + peat moss CV (%) MSD av (days)

37.1 B a 38.49 B a 23.4 A a 37.6 B a

soil + charcoal soil + coconut fiber soil + pine bark soil + peat moss CV (%) MSD av (g 100 g−1)

3.63 0.35 3.94 2.00

a a a a

Precociousness (%) 84.29 a A 71.00 a A 93.00 a A 68.78 a A

A A A A

67.87 63.61 75.03 67.75

a a a a

A A A A

36.07 36.98b

34.18c 71.74 Time to First Harvest (Days) 34.7 A a 40.84 A a 36.4 A b 34.7 A a 17.34

30.2 A a 38.36 AB a 42.9 B b 34.7 AB a

33.5 A a 41.34 A a 38.7 A b 41.2 A a

10.54b

5.17 2.51 4.86 9.07

A A A A

a a a a

10.51c

36.53 β-Glucan (g 100 g−1 Mushroom Dry Weight) 3.97 A a 3.75 6.01 A a 6.65 4.20 A a 3.68 7.01 A a 5.37 71.28 7.3b 4.99

A A A A

a a a a

6.62 2.03 4.71 4.38

A A A A

a a a a

5.2c

a

Upper case letters compare results within the same column, and lower case letters compare results within the same row according to Tukey’s test (P ≤ 0.05). bMinimum significant difference for values within the same column (casing layer comparison). cMinimum significant difference for values within the same row (cultivation environment comparison). dCV, coefficient of variation. eMSD, minimum significant difference. fav, average.

for the interaction of the two factors (i.e., compost versus strain). However, when the two factors were analyzed separately, ABL 99/30 was found to have a higher yield than ABL 04/49, and the use of organic compost was found to have a higher yield than synthetic and semisynthetic composts.

overall average for all of the treatments (9.81% and 73.35 u). The average basidiocarp weight values for strains ABL 99/29 and 06/59 were higher than the overall average for all of the treatments (18.07 g). In a study analyzing three different composts and two strains, Kopytowski Filho24 observed no significant differences in yield 46



Article

the number of basidiocarps (r = 0.81) and a negative correlation between the number of harvested basidiocarps and respective unit weights (r = −0.695) were observed. No correlations were observed for precociousness, time to first harvest, or basidiocarp β-glucan content. Similar results were reported by Zied et al.,18 who observed that the number of harvested basidiocarps was negatively correlated with the water-holding capacity of the casing layer and positively correlated with the total density and porosity of the casing layer. Pardo et al.27 reported that a porous casing layer resulted in an earlier first flush for A. bisporus and an increased number of mushrooms, albeit with reduced weight. The treatments that resulted in a higher yield also resulted in a higher number of harvested basidiocarps and reduced weight. The only treatment for which this trend was not observed was the soil + pine bark casing layer in the climate-controlled chamber. In this case, a significantly higher weight was observed (Tukey’s test at a 5% significance level). These findings indicate that factors such as porosity are less important in the casing layer material in a controlled environment. The environmental variables resulted in lower yields in the greenhouses with plastic films (Table 4). Zied and Minhoni19 previously recommended 27 ± 2 °C compost temperature, 25 ± 2 °C air temperature, and 90 ± 2% relative humidity during the initial 12 days following the addition of the casing layer and during the intervals between flushes. For primordial induction, the compost temperature should be lowered to 20 ± 2 °C, the air temperature to 18 ± 2 °C, and the relative humidity to 85 ± 2%. No statistically significant differences in precociousness were observed, although the values observed were higher than those observed in the first experiment. Precociousness exceeded 50% during the first half of the harvest period in approximately 93% of the recorded cases (Table 6), which indicates a more concentrated cultivation during the first days of growth. This result may permit shorter cultivation cycles, especially for the following combinations: soil + charcoal/climate-controlled chamber, soil + charcoal/greenhouse with Duplalon film, soil + composted pine bark/greenhouse with Duplalon film, and soil + composted pine bark/greenhouse with milky film. It should be noted that casing layers with higher porosity and lower water-holding capacity (soil + charcoal and soil + pine bark) have been associated with precociousness. For instance, Colauto et al.17 observed that casing layers with high porosity (lime schist and Santa Catarina peat) had a higher biological efficiency than less porous casing layers (soil + coal and São Paulo peat). The soil + pine bark casing layer in the climate-controlled chamber and the soil + charcoal casing layer in the greenhouse with milky film resulted in the shortest times to first harvest. The only casing layer that significantly affected the time to first harvest in relation to a particular cultivation environment was soil + pine bark under greenhouse conditions, which resulted in a later first flush. The basidiocarp β-glucan content was not significantly affected by the type of casing layer or cultivation environment. This finding is in contrast with the results of Park et al.,12 for a study comparing the basidiocarp β-glucan content of mushrooms grown in Brazil and Japan. They compared field-grown and greenhouse mushrooms that were produced in Brazil with mushrooms produced in greenhouses in Japan and found that A. blazei grown in greenhouses had lower β-glucan contents (7.6 ± 2.8 g 100 g−1 mushroom dry weight grown in Japan and

In the present study, strains ABL 06/59 and 99/30 had the highest values of precociousness, which were markedly higher than the overall average for all treatments (53.85%). These values indicate that for these two strains, most of the production occurred during the first 47 days of harvest following primordial formation. It should be noted that precociousness values were positively correlated with basidiocarp weight (0.835), indicating that under the tested conditions, the mushrooms produced during the first half of the cultivation cycle had higher weights. Zied et al.,25 studied Tifton (T)- and oat (O)-based composts and the following casing layers: acrylamide gel (gel), soil + coal (c), and soybean meal supplement (s). They reported the following precociousness values: Tgel (75%) > Oc (61%) > Ogel (58%) > Os (57%) > Ts (53%) > Tc (51%). In another study using strain ABL 04/49,25 a higher precociousness value was obtained than that observed in the present study for the same strain cultivated with oat-based compost and soil + charcoal casing (37.95%). This discrepancy may reflect differences in the environmental conditions in a given week or month. The shortest times until the first harvest were observed for strains ABL 99/30, 03/44, and 06/59 regardless of the type of compost used, with the following exception: the time until first harvest increased when strain ABL 03/44 was grown with Massai straw and sugar cane bagasse. The type of compost used also influenced the time to first harvest. In addition to reducing the values of yield and precociousness, the application of Aruana straw and sugar cane bagasse compost to strain ABL 99/28 increased the time to first harvest. The basidiocarp β-glucan content was not affected by the yield, the number of basidiocarps, basidiocarp weight, precociousness, or the time to first harvest. However, the βglucan content was affected by the strain and the type of compost used. For example, significant differences in β-glucan content were observed when strains ABL 99/30, 03/44, and 04/49 were grown with Aruana straw and sugar cane bagasse compost. The average β-glucan content observed for the tested treatments (5.19 g 100 g−1 mushroom dry weight) can be used to establish an acceptable level of β-glucan in basidiocarps for export. The strain alone is responsible for 35.8% of the variation in the β-glucan values, whereas the type of compost used is responsible for 9.9% of the variation (Figure 1). Thus, the strain used is of the utmost importance, and it should be selected in accordance with the type of raw material used in the compost. Wang et al.26 tested six different substrate formulations for the production of A. blazei, with the final N contents of the compost ranging between 1.2 and 1.55%. The authors used asparagus straw, cottonseed hull, cow manure, soybean cake, and gypsum as the raw materials for the compost production and did not find any significant differences in the final polysaccharide content of the basidiocarps.26 Experiment 2. The highest yield values were obtained in the climate-controlled chamber (Table 6) and were not affected by the type of casing layer used (Tukey, ≤0.05). However, the type of casing layer had a significant effect on yield in the greenhouses with partial climate control regardless of the type of plastic film used. The more open casings (soil + charcoal and soil + composted pine bark) resulted in a higher yield. When the different casing layers and cultivation environments were compared, a positive correlation between yield and 47



Article

8.4 ± 0.9 g 100 g−1 mushroom dry weight grown in Brazil) than A. blazei grown in the field (10.1 ± 2.1 g 100 g−1 mushroom dry weight). The differences from the present study in terms of the β-glucan content may reflect differences in the types of compost and the strains used. The average values of β-glucan content obtained in the present study for the tested treatments (4.99 g 100 g−1 mushroom dry weight) can be used to establish an acceptable level of β-glucan in basidiocarps. The casing layer and cultivation environment were responsible for 34.5 and 15.7%, respectively, of the variation in β-glucan content (Figure 1). Thus, the choice of casing layer is important for producing basidiocarps with desired physical characteristics. Production Protocol. The spatial variability of the β-glucan content and yield as affected by the tested factors (fungal strain, compost, casing layer, and cultivation environment) are presented in Figure 1. These values may be used as indicators for the adoption of production practices and the creation of a protocol of good cultivation practices that can be used by producers wishing to export A. subrufescens. The factor with the greatest contribution to the variation in β-glucan content was the strain (35.8%), followed by the casing layer (34.5%), the cultivation environment (15.7%), and the type of compost (9.9%). The variation in yield was affected most by the cultivation environment (82.1%), followed by the strain (81.3%), casing layer (49.1%), and compost type (15.2%). The present findings indicate that standard values can be set to 5 g 100 g−1 mushroom dry weight for β-glucan content and 10% for yield. The following cultivation practices are also proposed: • Strains ABL 99/30, ABL 03/44, and ABL 04/49 should be used because of their good agronomical performance and reasonable β-glucan contents. • Compost prepared from sugar cane bagasse and wheat or Massai straw can be used by Brazilian growers. An adequate formulation of the substrate is of central importance in obtaining high yields, as balanced compost (C/N ratio of 33−37:1) minimizes variation in the yield and β-glucan content. • The casing layer is determinant of the yield and β-glucan content of basidiocarps, which varied greatly in the present study depending on the treatments used. The casing layer should be selected according to the desired physical characteristics of the harvested basidiocarps (e.g., number and weight of basidiocarps). At the same time, the use of pine bark and charcoal as a supplement to the soil is recommended due to the low cost of these materials in Brazil (1:4, v/v). • The cultivation environment is another determinant of yield. The large variation in yield throughout the year caused by seasonal weather changes is one of the main reasons growers abandon commercial cultivation. Cultivation in climatecontrolled chambers is therefore recommended because it is not affected by variations in external environmental conditions and does not directly influence the β-glucan content of the harvested basidiocarps.

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Funding

We are grateful to the Foundation for Research Support of the State of Minas Gerais (FAPEMIG − CAG/BPD 00081-11) and the Foundation for Research Support of the State of São Paulo (FAPESP − 2012/15101-4) for their financial support. Notes

The authors declare no competing financial interest.

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REFERENCES

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