Wu et al. AMB Expr (2019) 9:184 https://doi.org/10.1186/s13568-019-0907-1

ORIGINAL ARTICLE Open Access Optimization of agro‑residues as substrates for Pleurotus pulmonarius production Nan Wu1†, Fenghua Tian1†, Odeshnee Moodley1, Bing Song1, Chuanwen Jia1, Jianqiang Ye2, Ruina Lv1, Zhi Qin3 and Changtian Li1*

Abstract The “replacing wood by grass” project can partially resolve the confict between mushroom production and balancing the ecosystem, while promoting agricultural economic sustainability. Pleurotus pulmonarius is an economically impor- tant edible and medicinal mushroom, which is traditionally produced using a substrate consisting of sawdust and cottonseed hulls, supplemented with wheat bran. A simplex lattice design was applied to systemically optimize the cultivation of P. pulmonarius using agro-residues as the main substrate to replace sawdust and cottonseed hulls. The efects of difering amounts of wheat straw, corn straw, and soybean straw on the variables of yield, mycelial growth rate, stipe length, pileus length, pileus width, and time to harvest were demonstrated. Results indicated that a mix of wheat straw, corn straw, and soybean straw may have signifcantly positive efects on each of these variables. The high yield comprehensive formula was then optimized to include 40.4% wheat straw, 20.3% corn straw, 18.3% soybean straw, combined with 20.0% wheat bran, and 1.0% light ­CaCO3 (C/N 42.50). The biological efciency was 15.2% greater than that of the control. Most encouraging was the indication= that the high yield comprehensive formula may shorten the time to reach the reproductive stage by 6 days, compared with the control. Based on the results of this study, agro-residues may be used as a suitable substitution for sawdust and cottonseed hulls as the main cultiva- tion substrates of P. pulmonarius. These results provide a theoretical basis for the “replacing wood by grass” project on edible mushroom cultivation. Keywords: Pleurotus pulmonarius, Mushroom crop, “Replacing wood by grass”, Simplex-lattice design, Cultivation, Biological efciency

Introduction production and ecosystem balance, but it may also lead Tis study describes a comprehensive program to utilize to great improvements in soil and water conservation, herbaceous plants (mainly grasses) to grow edible and ecological environmental protection, increases of food medicinal mushrooms, and produce fungi forage and resources, and the promotion of a sustainable agricul- fungi fertilizers with agricultural residues as substrates. tural economy (Lin 2009). “Replacing wood by grass” has many apparent advan- Pleurotus pulmonarius (Fr.) Quél., also known as tages, such as the high utilization of solar energy and anchovies and Indian abalone mushrooms (Li et al. 2015), resources, lower costs, and shorter production periods is most commonly known as the grey oyster mushroom, to name a few (Sanderson and Reed 2000; Springer et al. which is characterized by a grayish colored sporophore, 2003). Applying the “replacing wood by grass” system to which has a feshy texture and produces an aromatic, not develop a more ecologically conscious mushroom indus- anise-like aroma (Lechner et al. 2004). P. pulmonarius try may not only resolve the confict between mushroom is formally classifed in the following categories: Fungi, Basidiomycota, Agaricomycetes, Agaricomycetidae, Aga- *Correspondence: [email protected] ricales, Pleurotaceae, Pleurotus (http://www.index​fungo​ †Nan Wu and Fenghua Tian contributed equally to this work. rum.org/Names​/Names​.asp). P. pulmonarius has an out- 1 Engineering Research Center of Edible and Medicinal Fungi, Ministry of Education, Jilin Agricultural University, Changchun 130118, China standing reputation as an edible mushroom that has been Full list of author information is available at the end of the article used for centuries as a food and food favoring because

© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Wu et al. AMB Expr (2019) 9:184 Page 2 of 9

of its highly desirable taste qualities and unique aroma Fungi of Jilin Agricultural University, China. Mycelia (Oliveira et al. 2002; Zhang et al. 2013). It also contains a were cultured on potato dextrose agar (PDA) in a Petri large range of proteins, polysaccharides, essential amino dish for 6 days at 24 °C in darkness. When mycelia had acids, a high potassium to sodium ratio, and multivita- grown to completely cover the media, mycelia with a mins, including niacin and ribofavin (Velázquez-Cedeño diameter of 5 mm were collected for use as future inocu- et al. 2002; Stanley et al. 2011). P. pulmonarius is widely lum sources. marketable and sells well in southeast Asia, Japan, and Liquid spawn was prepared by culturing mycelial plugs several other countries. However, it is more expensive (2 mm in diameter, 10 plugs/fask) in 1 L fasks contain- than other mushrooms, such as Flammulina velutipes ing 400 mL of liquid media, while shaking (150 rpm/min) and P. ostreatus. Recently, many studies have confrmed in a rotatory incubator at 24 °C for 9 days, in darkness. that P. pulmonarius has pharmacological properties, Liquid media consisted of maize meal (40 g/L), glucose such as antioxidants, anti-cholinesterase, antitumor, anti- (20 g/L), peptone (1 g/L), ­KH2PO4 (10 g/L), and ­MgSO4 bacterial, immuno-modulating, anti-infammatory, aids (5 g/L) at a of pH 6.5 (Wu 2011). in resisting potential vascular complications, and may reduce blood sugar levels (Olufemi et al. 2012; Smiderle et al. 2012; Xu et al. 2012; Wahab et al. 2014; Ge 2015; Substrate preparation Nguyen et al. 2016; Ni 2016; Zhang et al. 2016). Various agro-residues, including wheat straw, rice straw, Sawdust and cottonseed hulls are the main materials soybean straw, corn straw, corn cobs, peanut straw, rape for the traditional cultivation of P. pulmonarius. How- straw, and supplements, including wheat bran and light ever, the price of cottonseed shells has risen to 282 US ­CaCO3, for the cultivation of P. pulmonarius were pur- dollars per ton (Liu et al. 2011). Complicating the matter, chased from the mushroom base of Jilin Agricultural the logging of trees has been declared as a social, envi- University, Changchun, China. All agro-residues were ronmental, and global issue. In China, there are abundant chopped, air dried, and ground into a fne powder. agro-residue resources, such as wheat straw, corn cobs, rice straw, soybean straw, rice straw, peanut straw, rape straw, and corn straw. Substrates play an indispensable Screening of appropriate agro‑residue substrates in Petri role in the nutritional composition of oyster mushroom dishes and previous studies have found astounding diferences Te CK (control) substrate, or standard formulation, for in both proximate and mineral composition of Pleurotus screening and comparing additional substrates was com- when these mushrooms are raised in diferent substrates prised of 40.0% sawdust, 39.0% cottonseed hulls, 20.0% (Sarker et al. 2007; Bhattacharjya et al. 2015). Te great wheat bran, and 1.0% calcium carbonate (Fan et al. 2016). potential of this industry is derived from its promise of In the additional substrate formulations, or the treatment saving land and labor, its eco-friendly nature, and its low groups, one of seven kinds of agro-residues (wheat straw, required startup capital. For these reasons, it is urgent rice straw, soybean straw, corn straw, corn cobs, peanut to fnd new substrates for mushroom crop cultivation in straw, rape straw) was used to make up 79.0% of the total relation to the “replacing wood by grass” project. substrate by replacing sawdust and cottonseed hulls. All Te objectives of this study were to evaluate diferent the substrates were pressed into separate Petri dishes agro-residues as potential substrates for P. pulmonarius (90 mm in diameter) to a height of 5 mm and autoclaved cultivation using a simplex-lattice design method, opti- at 121 °C for 1 h. mize substrate composition, and evaluate the combined Mycelial plugs, 5 mm in diameter, were cut from the efects of the substrate on mycelial growth rates, yield, leading edge of actively growing cultures on PDA plates. and fruiting body traits of P. pulmonarius. Tis study One plug was inoculated into the center of each of the was also intended to provide information for the further Petri dishes containing the various agro-residue sub- development of the “replacing wood by grass” project strates. Te Petri dishes were incubated in darkness at in China by investigating a variety of agro-residues that 24 °C for 7 days. Each treatment was replicated fve times. could replace sawdust and cottonseed hulls in P. pulmo- Mycelial growth rates were measured as subsequently narius cultivation. described. Every day, the radial growth of each treatment was observed. Te diameter of each colony was meas- Materials and methods ured on two perpendicular axes bisecting the center of Microorganism and spawn preparation the colony, then the mean value over time was calculated Mycelia of P. pulmonarius CCMJAU 201708 were to refect the growth rate of the mycelia. Te three agro- obtained from the Engineering Research Center of the residues associated with the fastest mycelial growth rates Chinese Ministry of Education for Edible and Medicinal were then considered in further analyses. Wu et al. AMB Expr (2019) 9:184 Page 3 of 9

Substrate formulations Lundstedt et al. 1998; Yang et al. 2016; Song et al. 2018) A one-factor-at-a-time (OFAT) method (Singh et al. (Table 1). 2011) was used to determine the initial range of the three independent variables. Based on results from the Petri Cultivation method dishes, the variables X­ 1 = wheat straw, X­ 2 = corn straw, Substrates were prepared from the various agro-residue and X­ 3 = soybean straw were selected for a simplex-lattice components as 79.0% of the main substrate, supplements design. Tese were then mixed in various combinations to (wheat bran and light ­CaCO3) as 21.0% of the substrate determine the optimal substrate composition for the culti- mixed with sterilized water was added to ± 72.5%. A total vation of P. pulmonarius. of 500.0 g of each of the substrates (dry weight of 150.0 g) was used to fll a 20 cm 17.5 cm 0.05 cm polypropyl- 0% ≤ X1 ≤ 100%, wheat straw × × ene bag, with a 2.5 cm collar at the bag mouth. Te flled 0% ≤ X2 ≤ 100%, corn straw bags were then autoclaved at 121 °C for 1 h. After cooling below 20 °C, each bag was then inoculated with 15 mL of 0% ≤ X3 ≤ 100%, soybean straw liquid spawn of P. pulmonarius. After inoculation, all bags were incubated in the dark at X1 + X2 + X3 = 100% 22–24 °C, at a carbon dioxide (CO­ 2) concentration below Ten possible mixture formulations were designed with the 2500 ppm. After it mycelia flled the bags, each formu- main ingredient accounting for 79.0% of the total substrate lation was cultivated for four additional days to ensure (Table 1). Te substituting ingredients were wheat straw, physiological maturity. Subsequently, each bag was cut to corn straw, and soybean straw, as previously described. Te create two “V” shaped holes on the surface using a steri- main ingredient of the control substrate mixture (CK) was lized knife. Te bags were then transferred to a cultiva- composed of 40.0% sawdust and 39.0% cottonseed hulls. tion room. All treatments were maintained in the same Te remainder of all formulations consisted of 20.0% wheat room under the same conditions (15–17 °C, 90–95% rela- bran, 1.0% light CaCO­ 3, and water at a pH of 7.0. Environ- tive humidity, 400–700 ppm ­CO2 concentration, under mental conditions were kept constant across all treatments a 200 LUX light source for 10 h/day) to facilitate basidi- and each treatment was replicated three times. ocarp growth. Mushroom harvest times were recorded as they occurred. After harvest, the mushroom yield corre- Model analysis sponding to each bag was weighed. Te harvest included Te Schefé model was ftted using a polynomial quadratic 20 bags from each treatment with three replicates; all equation in order to correlate the response variable (Y) data were used for statistical analyses. with the independent variables (X). Te Eq. was as follows: Data collection Y= β X + β X X i i i i j 1≤i≤q 1≤i≤j≤q Te radial growth of each formula in Petri dishes, time to bud formation, growth parameters pertaining to a single Te mixture experiment and optimization design fol- fruiting body (length and width of the stipe, and length, lowed previously described methods (Schefe 1963; thickness, and width of the pileus measured using a Ver- nier caliper), time to harvest, and reproductive stage and Table 1 Experimental design and model formulations yield parameters including total fresh weight (g) were for cultivation materials collected. Te biological efciency (BE) for each substrate Formulation Substrate mixture ratio formulation was calculated as follows: X (%) X (%) X (%) Fresh weight of mushroom 1 2 3 BE(%) = × 100 Dry weight of substrate 1 100 0 0 2 0 100 0 = − 3 0 0 100 Reproductive stage(d) Harvest time Bud time 4 50 50 0 Statistical analyses 5 50 0 50 Design expert statistical software package V 8.0.6.1 was 6 0 50 50 used to construct a regression analysis. A quadratic mul- 7 33.3 33.3 33.3 tiple regression ftting of yield, mycelial growth rate, stipe 8 16.7 16.7 66.7 length, pileus length, pileus width, and time to harvest 9 16.7 66.7 16.7 against the substrate formula ingredients was performed 10 66.7 16.7 16.7 using the Schefé quadratic polynomial regression model. X1-wheat straw; ­X2-corn straw; ­X3-soybean straw Te regression equation for each evaluation index was Wu et al. AMB Expr (2019) 9:184 Page 4 of 9

established. Te infuence of the interaction between these results, soybean straw, wheat straw, and corn straw the three main ingredients on the evaluation index was were chosen as the appropriate agro-residue substrates analyzed. Te optimization function was used to set the for the subsequent D-optimum experiment. variation range and the expected response value for each ingredient. Beginning with a random combination, the Efects of diferent agro‑residue formulations on time steepest slope prediction was performed until the target to harvest response value was reached. Te optimized yield for- With the statistical analysis of the time to harvest for mula derived from the regression analysis was verifed, each substrate formula (Additional fle 1: Table S1), the an analysis of variance (ANOVA) analysis was performed following regression model equation between the time to to estimate the statistical parameters using SPSS Statis- harvest and each ingredient was developed: tics 17, and fnally, the signifcant diferences between the Y = 42.74X1 + 42.41X2 + 36.08X3 + 4.97X1X2 means were determined by a Student–Newman–Keuls 2 test (Yang et al. 2016). Unless otherwise stated, the difer- − 5.02X1X3 + 11.64X2X3 − 29.33X1 X2X3 2 2 ences were considered statistically signifcant at P < 0.05. − 304.75X1X2 X3 − 17.31X1X2X3

Verifcation test 2 = R 0.8597 Te substrate formula, which was determined to be opti- mal according to the previous experiment, was tested Using the regression model, the predicted time to harvest against the CK substrate formula at the mushroom base was calculated for each substrate formula. Te linear cor- at Jilin Agricultural University. Te results were analyzed relation coefcient results for time to harvest was relatively to determine the feasibility of using the treatment sub- 2 high at ­R = 0.8597. Te statistical analysis showed that the strate formula in place of the CK. P values of both the mixed linear model and the quadratic regression model were < 0.0001, indicating that these two Results models were very signifcant and well ftted to the relation- Screening results of agro‑residue substrates ship between ingredients and time to harvest. Addition- Based on the results obtained from screening the Petri ally, the correlation coefcient indicated that the equation dishes, there were signifcant (P < 0.05) diferences model had an 85.97% goodness-of-ft to the experimental between the various agro-residues (Fig. 1). Soybean straw data. Te analysis of variance (ANOVA) for Y was shown in was determined to be the best for the mycelial growth of Additional fle 1: Table S1. Te P value showed the proba- P. pulmonarius. Te mycelial growth rate in the soybean bility of whether the time to harvest was afected by the dif- straw was signifcantly higher at 3.50 mm/day than in ferent agro-residues. Te terms which had P values < 0.05 the CK, which was 3.39 mm/day. Wheat straw and corn had an important efect on the time to harvest. Te coef- straw followed, with mycelial growth rates of 3.27 mm/ fcients of the independent variables in the equation day and 3.00 mm/day, respectively. Te mycelial growth refect the degree of infuence of the independent variable rate in the rice straw was slowest at 2.30 mm/day. From in the equation, i.e., the degree of contribution. As seen

4 a ab 3.5 b c d mm/ d 3 d 2.5 e e

2

1.5

1

0.5 Mycelium growth rate 0 wheat strawcorn straw rape straw corn cobpeanut strawrice straw soybean strawCK Main ingredient Fig. 1 Efect of diferent agro-residue substrates on mycelial growth rate in Petri dishes. Mean values followed by no letters are not signifcantly diferent at a level of 5% (P < 0.05) Wu et al. AMB Expr (2019) 9:184 Page 5 of 9

in the regression model, the equation coefcients were K Te statistical analysis showed that the P values of both (X­ 1) = 42.74 > K ­(X2) = 42.41 > K ­(X3) = 36.08. Combined the mixed linear model and the quadratic regression model with the interaction analysis, and based on the f-value and were < 0.0001, which indicated that these two models were 2 P value, the interaction between X­ 2X3 and X­ 1X2X3 signif- very signifcant and well ftted to the relationship between cantly afected the time to harvest. It could be concluded ingredients and the pileus length. Additionally, the corre- that the time to harvest was changed by the appropriate lation coefcient indicated that the equation model had a selection of the ­X1, X­ 2, and ­X3 levels. Interactions between 95.81% goodness-of-ft with the experimental data. As seen 2 ­X1X2X3 may signifcantly reduce the time until harvest and in the regression model, the equation coefcients were K interactions between ­X2X3 may signifcantly increase the (X­ 3) = 50.20 > K ­(X2) = 48.00 > K ­(X1) = 35.06. Tis result time until harvest. indicated that soybean straw had the greatest degree of contribution to the pileus length of P. pulmonarius. Efects of diferent agro‑residue substrate formulations Combined with the interaction analysis, based on the on stipe length f-value and P value, the interactions between X­ 1X2, X­ 2X3, 2 2 2 With the statistical analysis of the stipe length for each sub- X1X2X3, X­ 1X2X3, and ­X1X2X3 were shown to have sig- strate formula (Additional fle 1: Table S2), the following nifcantly afected the pileus length (Additional fle 1: 2 regression model equation between the stipe length and Table S3). Interactions between X­ 2X3 and X­ 1X2X3 may each ingredient was developed: signifcantly reduce the pileus length, and the interac- tions between X­ X , X2X X and ­X X X2 may signifcantly Y = 32.83X1 + 34.94X2 + 42.84X3 − 3.92X1X2 1 2 1 2 3 1 2 3 2 increase the pileus length. + 23.33X1X3 − 28.19X2X3 + 743.05X1 X2X3 2 2 Efects of diferent agro‑residue substrate formulations + 401.26X1X2 X3 − 306.44X1X2X3 on pileus width With the statistical analysis of the pileus width for each R2 = 0.9241  substrate formula (Additional fle 1: Table S4), the follow- ing regression model between the pileus width and each Te linear correlation coefcient for stipe length was 2 ingredient was developed: relatively high at R­ = 0.9241. Additionally, the correla- tion coefcient indicated that the equation model had a Y = 41.10X1 + 46.77X2 + 53.17X3 + 10.40X1X2 2 92.41% goodness-of-ft with the experimental data. As seen + 4.52X1X3 − 40.41X2X3 + 889.89X1 X2X3 in the regression model, the equation coefcients were K 2 2 − 573.34X1X2 X3 + 86.31X1X2X3 (X­ 3) = 42.84 > K ­(X2) = 34.94 > K ­(X1) = 32.83. Tese results indicated that soybean straw had the greatest degree of 2 = contribution to the stipe length of P. pulmonarius. R 0.9663 Combined with the interaction analysis, and based on the f-value and P value, the interactions between X­ 1X3, X­ 2X3, Te ANOVA for Y is shown in Additional fle 1: Table S4. 2 2 2 X1X2X3, X­ 1X2X3, and ­X1X2X3, were shown to have signif- Te linear correlation coefcient for the pileus width was cantly afected stipe length (Additional fle 1: Table S2). relatively high at R­ 2 0.9241. As seen in the regression 2 = Interactions between X­ 2X3 and ­X1X2X3 may signifcantly model, the equation coefcients were K ­(X3) 53.17 > K 2 = reduce stipe length. Interactions between ­X1X3, X1X2X3, (X­ 2) 46.77 > K ­(X1) 41.10, which indicated that the 2 = = and X­ 1X2X3 may signifcantly increase stipe length. degree of contribution of each ingredient to the pileus width was as follows: ­X3 (soybean straw) > X2 (corn Efects of diferent agro‑residue formulations on pileus straw) > X1 (wheat straw). Tis result indicated that soy- length bean straw had the greatest degree of contribution to the With the statistical analysis of the pileus length for each pileus width of P. pulmonarius. substrate formula (Additional fle 1: Table S3), the follow- Combined with the interaction analysis, based on the ing regression model equation between the pileus length f-value and P value, the interactions between X­ 1X2, X­ 2X3, 2 2 and each ingredient was developed: X1X2X3, and X­ 1X2X3 were shown to have signifcantly Y X X X X X afected the pileus width (Additional fle 1: Table S4). Inter- = 35.06 1 + 48.00 2 + 50.20 3 + 6.65 1 2 2 2 actions between ­X2X3 and ­X1X2X3 may signifcantly reduce − 0.021X1X3 − 17.22X2X3 + 768.97X X2X3 2 1 the pileus width. Interactions between ­X1X2, X1X2X3 may 2 2 − 422.50X1X2 X3 + 389.72X1X2X3 signifcantly increase the pileus width. 2 = R 0.9581 Wu et al. AMB Expr (2019) 9:184 Page 6 of 9

Efects of diferent agro‑residue formulations on mycelial 2 Te interactions between ­X1X2, ­X1X3, X1X2X3, and growth rate 2 ­X1X2X3 were shown to have signifcantly afected the With the statistical analysis of the mycelial growth rate for yield (Additional fle 1: Table S6). Interactions between 2 each substrate formula (Additional fle 1: Table S5), the fol- ­X1X3 and ­X1X2X3 may signifcantly reduce the yield, and 2 lowing regression model between the mycelial growth rate interactions between ­X1X2 and X1X2X3 may signifcantly and each ingredient was developed: increase the yield. Y = 0.39X + 0.38X + 0.33X − 0.18X X 1 2 3 1 2 High‑yielding formula and verifcation test X X X X X2X X − 0.16 1 3 − 0.004568 2 3 + 0.18 1 2 3 Using the yield, mycelial growth rate, stipe length, pileus 2 2 − 9.22X1X2 X3 + 7.34X1X2X3 length, pileus width, and the time to harvest as the evaluation indices, and setting the variation range and expected response value of each ingredient, a steepest R2 = 0.9906  slope prediction was carried out based on the regression equation beginning from a random combination, and a Te linear correlation coefcient for the pileus width 2 high yield comprehensive formula with the agro-residues was relatively high at R­ = 0.9906. As seen in the regres- was generated. Te resulting substrate formula was as fol- sion model, the equation coefcients were K ­(X1) 0.39 > K = lows: 51.1% wheat straw ­(X1), 25.7% corn straw ­(X2), and (X­ 2) = 0.38 > K ­(X3) = 0.33. Tis indicated that wheat straw 23.2% soybean straw. Tese values were then multiplied had the greatest degree of contribution to the mycelial by the coefcient of 79.0% and thus converted to the fnal growth rate of P. pulmonarius. 2 values of 40.4% wheat straw, 20.3% corn straw, 18.3% soy- Te interactions between ­X1X2, X­ 1X3, X­ 1X2X3, and 2 bean straw, and 20.0% wheat bran and 1.0% light CaCO­ 3 ­X1X2X3 were shown to have signifcantly afected the myce- combined, which is the high yield comprehensive (HC). lial growth rate. It was concluded that the mycelial growth Using this substrate formula, the measured yield (frst rate may be changed by the appropriate selection of the ­X1, mushroom) reached 82.30 g/bag, which was similar to the ­X2, and X­ 3 levels (Additional fle 1: Table S5). Interactions 2 predicted ftted value. Tese results showed that by using between X­ 1X2, X­ 1X3, and ­X1X2X3 may signifcantly reduce 2 this formula, the yield (frst mushroom) was increased the mycelial growth rate, and ­X1X2X3 may signifcantly by 21.8% compared to the CK (67.67 g/bag), and the increase the mycelial growth rate. BE was 15.2% greater than that of the CK (HC:118.6%; CK:103.4%). Te mycelial growth rate, single-fruiting- Efects of diferent agro‑residue substrate formulations body stipe length, stipe width, pileus length, pileus width, on yield pileus thickness, time to bud formation, time to harvest, With the statistical analysis of the yield for each substrate and the reproductive stage, as they pertain to the high formula (Additional fle 1: Table S6), the following regres- yield comprehensive formula, were also examined. Te sion model between the yield and each ingredient was single-fruiting-body pileus length (69 mm), pileus width developed: (87 mm), and pileus thickness (23 mm) of P. pulmonarius Y = 2037.37X1 + 2498.48X2 + 2916.14X3 + 1256.89X1X2 for the high yield comprehensive formula were greater 2 than those of the CK, which were 68 mm, 66 mm, and − 1163.46X1X3 − 74.92X2X3 + 80178.75X1 X2X3 20 mm, respectively. However, the time to bud formation 11917.38X X2X 20445.90X X X2 − 1 2 3 − 1 2 3 of the high yield comprehensive formula was delayed by 4 days compared to the CK, even when the growth cycle 2 = R 0.8747 was started 2 days in advance. Given this delay, the repro- ductive stage of the high yield comprehensive formula Using the regression model, the predicted yield was was 6 days shorter than that of the CK (Additional fle 1: calculated for each substrate formula, and the measured Table S7). yield and the predicted yield were found to be very simi- Discussion lar. As seen in the regression model, the equation coef- Several edible fungi have traditionally been cultivated on fcients were K ­(X3) = 2916.14 > K ­(X2) = 2498.48 > K forest wood (sawdust); however, due to the recent eforts ­(X1) = 2037.37, which indicated that the degree of con- tribution of each ingredient to the yield was as follows: to manage forest resources, the use of agro-forestry products for its cultivation is no longer considered to ­X3 (soybean straw) > X2 (corn straw) > X1 (wheat straw). Tis result indicated that soybean straw had the greatest be sustainable (Song et al. 2018). Wood rot fungus straw degree of contribution to the yield of P. pulmonarius. is being utilized for the production of Pleurotus ostrea- tus, Pleurotus eryngii, Pleurotus cystidiosus, Lentinula Wu et al. AMB Expr (2019) 9:184 Page 7 of 9

edodes and Flammulina velutipes, which can be culti- of the wheat straw, corn straw, and soybean straw was vated with agro-residues, such as crop straws, either with 22.10%, 22.14%, and 32.80%, respectively (Miu 2015; Wu composting or non-composting (Moonmoon et al. 2010; et al. 2018). Te cellulose content of the soybean straw Hoa et al. 2015; Pedri et al. 2015; Rezaeian and Pourian- was the lowest, but its lignin content was the highest. It far. 2016; Papadaki et al. 2019). However, to date, there can be inferred that the substrate with higher lignin con- has been no study focused on optimizing the substrate tent could be more conducive to growth in terms of stipe formula for cultivating Pleurotus by the simple lattice length, pileus length, pileus width, yield increases, and by method. Pleurotus primarily produces laccase and is one decreasing the time to harvest of P. pulmonarius. Te dif- of the most widely cultivated, edible fungi in the world ference in the C/N ratio is also an important factor afect- (Souza et al. 2002; Zhang et al. 2011). ing the evaluation index of edible fungi (Yang et al. 2013). Te design of the simple lattice method is a basic mix- Te soybean straw had the greatest infuence the time ing design method. Trough regression analysis, the to harvest, stipe length, pileus length, pileus width, and quantitative relationship between the ratio of the main yield, indicating that the soybean straw is a suitable culti- material matrix and the evaluation index can be obtained, vation matrix. However, in addition to the soybean straw, and therefore the optimum formula can be achieved wheat straw and corn straw can also be used together to (Yang et al. 2016). Consequently, in this study, the use of maximize this impact. diferent agro-residues (wheat straw, rice straw, soybean In conclusion, this study demonstrated that the over- straw, corn straw, corn cob, peanut straw, and rape straw) all yield of P. pulmonarius could be improved by optimiz- was investigated to fnd an substrate to replace sawdust ing the substrate formula for cultivation; thus, we propose and cottonseed hulls for cultivating P. pulmonarius. A using alternatives to sawdust and cottonseed hulls. Te statistical model for optimizing production and evalu- amount of the agro-residue (wheat straw, soybean straw, ating the efects of the alternative substrate formula on and corn straw) used had a signifcant infuence on the yield, mycelial growth rate, stipe length, pileus length, dependent variables. Wheat straw had the greatest efect pileus width, and time to harvest was also described. on mycelial growth rate, followed by corn straw and soy- In the present study, three kinds of common agro-res- bean straw. Soybean straw had the greatest efect on time idues, wheat straw, corn straw, and soybean straw, were to harvest, stipe length, pileus length, pileus width, and used as the main ingredients. Te yield, mycelial growth yield, followed by corn straw and wheat straw. By verifying rate, stipe length, pileus length, pileus width, and time to the optimized high yield comprehensive substrate formula, harvest were analyzed, and then optimized by the simple it was concluded that these agro-residues (i.e., wheat straw, lattice method. In respect to the time to harvest, it was corn straw, and soybean straw) may be used to replace saw- demonstrated that when the three substrates were used dust and cottonseed hulls as the main cultivation substrates separately, the growth period associated with the soy- of P. pulmonarius. Te fndings of the present study pro- bean straw substrate was the shortest, followed by the vide a theoretical basis for the “replacing wood for grass” corn straw, and the wheat straw. It was concluded that project on edible mushroom cultivation. Future research soybean straw may be benefcial in shortening the culti- should explore the mechanisms by which wood rot fungi vation cycle of P. pulmonarius. Furthermore, the time to decompose grasses, and screen to select candidate wood harvest was shortest when 100% soybean straw was used rot fungi strains that can be cultivated with agricultural as the primary ingredient. Te mycelial growth rate was residues. the greatest on the wheat straw, followed by corn straw and soybean straw. Te mushroom yield was the great- Supplementary information est when grown on the soybean straw, followed by corn Supplementary information accompanies this paper at https​://doi. org/10.1186/s1356​8-019-0907-1. straw and wheat straw (Additional fle 1: Table S8). Tese results indicated that diferent substrate components had Additional fle 1: Table S1. Variance Analysis of the Quadratic Polynomial varying efects on several important agronomic traits. Regression Model for Time to Harvest. Table S2. Variance Analysis of Tis may be attributed to their diferent nutritional com- the Quadratic Polynomial Regression Model for Stipe Length. Table S3. ponents and structures, such as the difering contents of Variance Analysis of the Quadratic Polynomial Regression Model for Pileus Length. Table S4. Variance Analysis of the Quadratic Polynomial Regres- cellulose and lignin (Naraian et al. 2009). Te carbon to sion Model for Pileus Width. Table S5. Variance Analysis of the Quadratic nitrogen ratio (C/N) of the wheat straw, corn straw, and Polynomial Regression Model for Mycelial Growth Rate. Table S6. Variance soybean straw was 70.92, 39.30, and 75.03, respectively Analysis of the Quadratic Polynomial Regression Model for Yield. Table S7. Comparison of Main Agronomic Traits Between the HC Formula and the (Miu 2015; Wu et al. 2018). Te cellulose content of the CK Substrate Formula. Table S8. The Infuence of the Three Agro-residues wheat straw, corn straw, and soybean straw was 39.26%, as 100% of the Main Ingredient on Each Evaluation Index. 37.15%, and 30.83%, respectively, and the lignin content Wu et al. AMB Expr (2019) 9:184 Page 8 of 9

Abbreviations Lundstedt T, Seifert E, Abramo L, Thelin B, Nyströma Å, Pettersen J, Bergman BE: biological efciency; CK: control; OFAT: one-factor-at-a-time; PDA: potato R (1998) Experimental design and optimization. Chemometr Intell Lab dextrose agar; SNK: Student–Newman–Keuls. 42(1–2):3–40 Miu WJ (2015) Physicochemical composition and energy potential of main Acknowledgements crop straw and stalk. Doctoral Dissertation of China Agricultural Univer- We thank Huang Jing for her help with the experimental techniques. sity, Beijing Moonmoon M, Uddin MN, Ahmed S, Shelly NJ, Khan MA (2010) Cultivation of Authors’ contributions diferent strains of king oyster mushroom (Pleurotus eryngii) on saw dust NW and FHT conceived and designed the experiments, performed the experi- and rice straw in Bangladesh. Saudi J Biol Sci 17(4):341–345 ments, analyzed the data, wrote the paper, prepared fgures and/or tables, Naraian R, Sahu R, Kumar S, Garg S, Singh C, Kanaujia R (2009) Infuence of dif- these authors contributed equally to this work; OM, BS and CWJ conceived ferent nitrogen rich supplements during cultivation of Pleurotus forida on and designed the experiments; JQY, RNL and ZQ performed the experiments; corn cob substrate. Environmentalist 29:1–7 CTL conceived and designed the experiments, analyzed the data, contributed Nguyen TK, Im KH, Choi J, Shin PG, Lee TS (2016) Evaluation of antioxidant, reagents/materials/analysis tools, wrote the paper, prepared fgures and/or anti-cholinesterase, and anti-infammatory efects of culinary mushroom tables, reviewed drafts of the paper. All authors read and approved the fnal Pleurotus pulmonarius. Mycobiology 44(4):291–301 manuscript Ni D (2016) The biological characteristics and cultivation techniques of Pleuro- tus pulmonarius. J Changjiang Vegetables 22:70–72 Funding Oliveira SS, Da CSMG, Edmar C (2002) Chemical composition of Pleurotus This work was supported National Key Research and Development Program of pulmonarius (Fr.) Quél. substrates and residue after cultivation. Braz Arch China (2017YFD0601002), Collaborative Innovation for “Juncao” Ecology Indus- Biol Techn 45(4):531–535 try, China (K80ND8002-JCZXGGKT05), University S & T Innovation Platform of Olufemi AE, Terry AOA, Kola OJ (2012) Anti-leuCultivation of diferent strains Jilin Province for Economic Fungi (#2014B-1), Special Fund for Agro-scientifc of king oyster mushroom (Pleurotus eryngii) on saw dust and rice straw in Research in the Public Interest (201503137). Bangladeshkemic and immunomodulatory efects of fungal metabolites of Pleurotus pulmonarius and Pleurotus ostreatus on benzene-induced Availability of data and materials leukemia in Wister rats. 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