Screening of Substrates and Optimization of Formulations for Exogenous Nutrient Bags of Morchella sextelata (Black Morel)

Abstract

In the artificial cultivation of Morchella sextelata (Black Morel), exogenous nutrient bags (ENBs) commonly employ wheat grains as the primary substrate raw material. However, this approach is costly and runs counter to the “non-grain” development direction advocated by the edible mushroom industry. Under controlled field conditions, twelve self-made formulations were set up and compared with a conventional market formulation to comprehensively analyze their impacts on the agronomic traits, yield, soil physicochemical properties, and economic benefits of M. sextelata fruiting bodies. The research findings indicate that the nutrient bag formulations have a significant effect on soil available nutrients. Specifically, the contents of alkali-hydrolysable nitrogen (AN) and available potassium (AK) exhibit a significantly negative correlation with M. sextelata yield (r = −0.60, p < 0.05; r = −0.72, p < 0.01, respectively). Among all the treatment groups, the KY1 formulation (comprising 30% wheat grains, 5% rice bran, 60% corncobs, 2% rice husks, 1% lime, and 1% gypsum) achieved the highest yield of 915.13 kg per 667 m², which was 16.1% higher than that of the control group. The net economic benefit per unit area (667 m²) reached CNY 75,282.15, representing a 20.7% increase compared to the traditional wheat grains-based formulation. In conclusion, partially substituting wheat grains with rice bran in ENBs can not only reduce reliance on staple food resources but also enhance yield and economic efficiency. Due to the differences in cultivated strains and environmental conditions, the impact on morel yield is substantial; therefore, the results of this study need further validation through pilot trials.

1. Introduction

True morel (Morchella spp., Morchellaceae, Ascomycota) are exquisitely flavored edible mushrooms, highly esteemed for their culinary and scientific value, and are predominantly distributed across the Northern Hemisphere [1]. Current research has confirmed that, in addition to their immunostimulatory and anti-tumor properties, morels also possess antioxidant and anti-inflammatory bioactivities [2]. Consequently, their global consumption has surged year-on-year. To meet the escalating demand for morels, diverse cultivation methods have been explored and implemented. The cultivation of morels was first developed in the early 1980s [3]. The first documented instance of outdoor morel cultivation dates back to 1882 in France [4]. China began relevant explorations in 1985, but the issue of stable production remained unresolved for a long time. It was not until 2003, with the successful development of the ENB technology, that the commercial cultivation of morels was effectively promoted [3,4]. Currently, greenhouse cultivation in China allows M. sextelata to fruit from late February to mid-April, creating an off-season market with higher selling prices [5]. However, the yield is only 150–250 kg per 667 m², which still lags behind that of high-yield regions [6].

Since the commercialization of morel (Morchella spp.) cultivation in China in 2012, the application of advanced exogenous nutrient bag (ENB) technology has played a crucial role in promoting large-scale development of the industry. Consequently, the total cultivation area expanded dramatically from approximately 200 hectares at the initial stage to 16,466 hectares during the 2021–2022 period [7] The mechanisms underlying the theory of exogenous nutrient supplementation and the theory of morel sclerotium formation exhibit notable parallels. Both theories posit that in nutrient-deficient environments, nutrients tend to be stored within the mycelium or sclerotium formation [8,9]. Research findings indicate that the ENB substrate (wheat grains and rice husks) promotes the secretion of diverse hydrolytic and redox CAZymes by morel mycelium, accelerating the degradation of polysaccharides such as starch and cellulose. This leads to a rapid increase in organic carbon content in the surface soil, providing a carbon source for fruiting [10]. Morels can actively uptake nutrients from ENB and subsequently transfer these nutrients to the surface soil. Furthermore, nitrogen -cycling processes significantly enhance morel fruiting body yields [11]. Zhang Lijuan et al. [12] conducted experiments using different types of sawdust as substrates for nutrient bags, and the results indicated that using raw apple wood sawdust as the substrate for ENB could effectively enhance the nutrient content of M. sextelata fruiting bodies. Research by Shen Tong et al. [13] has demonstrated that adding spent mushroom substrate (SMS) from Lentinus edodes cultivation to the growth substrate for morel mushroom cultivation is beneficial for enhancing both the yield and nutritional value of morel fruiting bodies.

Current commercial ENB formulations are predominantly based on wheat grains as the primary substrate, supplemented with auxiliary materials such as corncob, lime and gypsum 10. Although these formulations are effective in promoting the formation of morel fruiting bodies, they are associated with high costs, substantial consumption of grain resources, and vulnerability to contamination. In recent years, the continuous rise in wheat prices has further increased production costs, posing a serious threat to the sustainability of the industry and contradicting the fundamental principle of “not competing with humans for grain” advocated by the edible mushroom sector. Given that this study focuses on ENB formulation, the development of alternative substrates to reduce reliance on wheat grains represents an essential strategy for ensuring the long-term sustainable development of the morel cultivation industry.

To optimize the formulation of ENB for M. sextelata and investigate the effects of reducing wheat grains inclusion on fruiting body yields, this study employed a control formulation containing 40% wheat grains (by composition) as the baseline for comparison. Four different substrates (K: rice bran, D: soybean meal, M: cottonseed hulls, F: fermented maize stover) were designed to substitute for wheat grains. The aim was to evaluate their effects on the main agronomic traits, yield, soil physicochemical properties, and economic benefits of M. sextelata, thereby screening out suitable ENB formulation sustainable cultivation. While the current study provides a relatively comprehensive evaluation of ENB formulation effects on M. sextelata yield, soil nutrient dynamics, and economic returns under greenhouse conditions, it is based on a single-season, single-location trial. As such, although the results offer valuable practical insights, their generalizability and reproducibility under diverse environmental conditions remain to be further validated through multi-location and multi-year studies.

2. Materials and Methods

2.1. Experimental Materials

The M. sextelata strain used in this research was obtained from the Institute of Economic Plant Research at the Jilin Academy of Agricultural Sciences. The components of the substrate used for the ENB in the experiment, such as wheat grains, rice husks, gypsum, lime, cottonseed hulls, rice bran, soybean meal, and corncobs, were sourced from the mushroom cultivation base of the Institute of Economic Plant Research in Changchun, China. The fermented maize stover was supplied by the Institute of Rural Energy and Ecology of the Jilin Academy of Agricultural Sciences, also located in Changchun, China. It is worth noting that the fermented maize stover mentioned in this context was the end-product resulting from the crushing of raw maize stover followed by a high-temperature fermentation process. The cultivation experiment was conducted in Fanjia Tun Town, Gongzhuling City, Jilin Province. Sowing of M. sextelata began on 10 November 2024, and the first fruiting flush was harvested between 25 March and 12 April 2025.

2.2. Spawn Preparation

The primary spawn was prepared with a mixture of 85% wheat grains, 10% wheat bran, 4% cornmeal, and 1% lime. The cultivation spawn was formulated with 50% wheat grains, 20% corncobs, 19% sawdust, 10% wheat bran, and 1% lime. All materials were mixed thoroughly, sterilized, and inoculated under aseptic conditions.

2.3. Experimental Design

Using the ENB (Expanded Nutrient Bed) commonly employed by edible mushroom producers in Hebei Province as the control (CK), twelve self-formulated ENBs with distinct compositions were designated as experimental treatments. Four primary substrates, namely, rice bran (denoted as k), soybean meal (D), cottonseed hulls (M), and fermented maize stover (F), partially substituted wheat grains. The proportion of wheat grains in the mixtures was adjusted to 30%, 15%, and 0%, and these formulations were labeled Y1, Y2, and Y3, respectively.

The wheat grains were weighed according to the specified proportion and pre-moistened for 48 h. Subsequently, gypsum and lime were added at a ratio of 1.0%, followed by the addition of water to adjust the moisture content to 60%. The mixture was then thoroughly blended using standard procedures.

Polypropylene bags with dimensions of 12 cm × 24 cm and a thickness ranging from 0.04 to 0.06 mm were utilized for filling. Each bag was filled to attain a wet weight of approximately 1.0 kg. After sealing the bags, they were autoclaved at a temperature between 121 and 126 °C for 1 to 2 h. Once sterilized, the bags were cooled down and prepared for subsequent use. The detailed formulations of the ENBs are presented in

Table 1. Formulations of exogenous nutrient bags.

Substrates	Formula %
	CK	KY1	KY2	KY3	DY1	DY2	DY3	MY1	MY2	MY3	FY1	FY2	FY3
wheat grains	40	30	15	-	30	15	-	30	15	-	30	15	-
rice husk	-	3	3	3	3	3	3	3	3	3	3	3	3
rice bran (K)	-	5	20	35	-	-	-	-	-	-	-	-	-
soybean meal (D)	-	-	-	-	5	20	35	-	-	-	-	-	-
cottonseed hulls (M)	-	-	-	-	-	-	-	5	20	35	-	-	-
fermented maize stover (F)	-	-	-	-	-	-	-	-	-	-	5	20	35
corncob	50	60	60	60	60	60	60	60	60	60	60	60	60
lime	1	1	1	1	1	1	1	1	1	1	1	1	1
gypsum	1	1	1	1	1	1	1	1	1	1	1	1	1
wheat bran	8	-	-	-	-	-	-	-	-	-	-	-	-

.

2.4. Cultivation Methods

2.4.1. Land Preparation

The cultivation experiment was conducted in a greenhouse at the edible mushroom cultivation base in Fanjiatun Town, Gongzhuling City. Prior to planting in the greenhouse, weeds were thoroughly cleared. Subsequently, 100 kg of lime was applied per 667 m² of land, and the soil was tilled to a depth of approximately 25 cm using a rotary tiller to achieve a loose soil structure. Cultivation trials were implemented in a ridged cultivation pattern. The ridges were created and oriented in a north–south direction. Each ridge measured 8.0 m in length, 1.0 m in width, and 0.1 m in height, with a spacing of 0.2 m between ridges. Each replicated plot corresponded to an 8 m² (8.0 m × 1.0 m) bed. Inserts were installed at a depth of 25 cm within the beds to effectively isolate different treatment areas and prevent mutual interference. The seeding rate was set at 300 g/m². Nutrient bags were placed in the beds 10 days after sowing. The experimental design comprised 13 treatments, with three plots allocated to each treatment. In each plot, 24 nutrient bags were arranged following a randomized block design.

2.4.2. Management During Sowing and Mycelial Growth Stage

The sowing of M. sextelata spawns was conducted on 10 November 2024. Soil temperature in the greenhouse was maintained within the range of 6 °C to 18 °C. Prior to sowing, all spawn bags, sowing equipment, and the operator’s hands were thoroughly disinfected with 75% ethanol to minimize the risk of contamination by competitive microorganisms. The M. sextelata spawn was subsequently fragmented and evenly distributed across the leveled ridge beds at an application rate of 300 g/m². Following sowing, a 2–3 cm layer of soil was applied, and thorough watering was carried out with a large volume of water on the same day. In case of any exposed spawns, a second covering of the soil layer could be applied. Post-sowing, intermittent micro-spray irrigation was employed to maintain soil moisture without causing soil waterlogging. During the planting management phase, the optimal range of ground temperature was 10 °C to 15 °C. The daytime temperature inside the greenhouse was controlled below 20 °C, while the night-time ground temperature was maintained no lower than 6 °C. The relative humidity of the air within the shed was kept at 60–70%. Meanwhile, the cultivation beds needed to be kept continuously moist to create favorable conditions for mycelial growth.

2.4.3. Placement of Exogenous Nutrient Bags

Approximately 10 days after sowing, the nutrient bags should be placed. Each nutrient bag is to be vertically incised using a sterilized blade, creating three slits with a length of 10–12 cm each. The openings of the slits should be oriented downwards and placed in close contact with the cultivation beds. The nutrient bags were then positioned parallel to the cultivation beds, with a longitudinal spacing of 40–50 cm, achieving a density of 3 nutrient bags per square meter. Upon placement of the nutrient bags, they should be immediately covered with black polyethylene mulch. The edges of the mulch are left to drape naturally without being pressed into the soil in order to ensure sufficient air permeability within the cultivation environment.

2.4.4. Fruiting Body Management and Harvesting

The soil should be thoroughly irrigated using a micro-spray system or a spray band to maintain a moist surface without waterlogging. The ground temperature should be regulated to remain within the range of 8–12 °C for a duration of 4–5 days, with a day-night temperature differential controlled at 8–10 °C. The relative humidity inside the shed should be maintained at 85–90%. A shade net with a shading rate of 60–70% should be installed on the inner side of the shed to prevent direct sunlight exposure.

Approximately 7–10 days after mushroom initiation, spherical primordia with a diameter of about 0.5–1 mm will emerge on the beds. After protocorm formation, the soil moisture should be maintained, with the soil moisture content controlled at 25–35%. The relative humidity of the air should be maintained within the range of 85–95%. Two to three days before harvesting, it is recommended to lower the air relative humidity to 70–85%. The air temperature ought to fluctuate between 10 °C and 20 °C, while the ground temperature should be kept within the span of 8 °C to 14 °C. Additionally, the light intensity should be precisely regulated at 600–800 lux.

It is advisable to adhere to the principle of “selectively harvesting mature M. sextelata and leaving immature specimens” to minimize potential harm to young mushrooms. Subsequently, following the harvesting process, the cultivation soil ought to be meticulously cleaned, and the harvested mushrooms should be promptly dried to guarantee the high quality of the final products. In this study, data collection and harvesting were conducted exclusively during the first fruiting flush, which occurred between 25 March and 12 April 2025.

2.5. Preparation of Soil Samples

In April 2025, soil samples were collected from each plot using a “Z-shaped” sampling method [14]. These samples were obtained at a distance of 5 cm from the planting furrow during the fruiting phase of the crop. Following collection, the samples within each replicate set were thoroughly homogenized and transported to the laboratory in sterile plastic bags. The fresh soil samples were subsequently air-dried indoors under controlled conditions. After drying, the samples were ground and passed through a 2 mm mesh sieve to prepare them for soil chemical analysis.

2.6. Data Determination

2.6.1. Agronomic Traits and M. sextelata Yield of Fruiting Bodies

In accordance with the Guidelines for Specificity, Consistency and Stability Testing of Plant Varieties (NY/T4211-2022) [15] applicable in China, the dimensions of stipe length, stipe diameter, pileus thickness, pileus width, and pileus length were measured using a vernier caliper (Figure 1). Furthermore, the weight of each of the randomly selected fruiting bodies was recorded to calculate the average weight, and the plot yield was determined by weighing.

2.6.2. Determination of Soil Physicochemical Properties

The content of soil alkaline nitrogen (AN) was determined following the protocol outlined by Prasad et al. [16]. Specifically, the determination was carried out using a Kjeldahl apparatus. First, 1 g of air-dried soil was treated with 10 mL of 4 M NaOH and 0.1 g of FeSO₄. Following distillation, the ammonium nitrogen was absorbed by a boric acid solution. Subsequently, the AN content was calculated through titration with 0.005 M sulfuric acid. The soil’s available phosphorus (AP) content was assessed according to the method proposed by Olsen et al. [17]. A total of 50 mL of 0.5 mol/L NaHCO₃ (adjusted to pH = 8.5) was added to 2.5 g of air-dried soil. The resulting suspension was shaken on a shaker for 30 min to facilitate extraction. Then, 10 mL of the filtrate was mixed with 5 mL of a molybdenum –antimony solution and allowed to stand at room temperature for 30 min. The AP content was finally determined using a spectrophotometer. The available potassium (AK) content in the soil was measured following the approach of Leaf et al. [18]. In this process, 50 mL of 1 mol/L NH₄OAc (adjusted to pH = 7) was added to 5 g of air-dried soil. After shaking for 30 min, the filtrate was collected. The excitation intensity of the filtrate was measured with a flame photometer, and the AK content was calculated based on the measurement results. The pH value of the soil was measured through potentiometric titration, with a soil-to-water ratio of 1:2.5 [19]. Soil organic matter (SOM) was oxidized using the potassium dichromate and concentrated sulfuric acid method [20]. The soil moisture content was quantified through the drying-based approach [21].

2.6.3. Economic Benefit Analysis

In the context of M. sextelata cultivation, economic benefits can be rigorously quantified through the analysis of the input–output relationship.

The total output value is as follows $\left(\mathrm{T}\mathrm{V}\right)$:

$$[\mathrm{T}\mathrm{V}=\mathrm{P}\times \mathrm{Q}=100\times \mathrm{Q}]$$

Here, $\left(\mathrm{P}\right)$ represents the market price per kilogram of fresh morel mushrooms, and $\left(\mathrm{Q}\right)$ represents the actual mass of fresh morel mushrooms harvested. Currently, the market price for fresh M. sextelata stands at CNY 100 per kilogram, that is, $\mathrm{P}=100 \mathrm{C}\mathrm{N}\mathrm{Y}/\mathrm{k}\mathrm{g}$.

The total input cost is as follows $\left(\mathrm{T}\mathrm{C}\right)$:

$$[\mathrm{T}\mathrm{C}={\mathrm{C}}_{\mathrm{E}\mathrm{M}\mathrm{B}}+{\mathrm{C}}_{\mathrm{s}\mathrm{p}\mathrm{a}\mathrm{w}\mathrm{n}}+{\mathrm{C}}_{\mathrm{r}\mathrm{a}\mathrm{w}-\mathrm{m}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{s}}+{\mathrm{C}}_{\mathrm{f}\mathrm{u}\mathrm{e}\mathrm{l}-\mathrm{p}\mathrm{o}\mathrm{w}\mathrm{e}\mathrm{r}}+{\mathrm{C}}_{\mathrm{l}\mathrm{a}\mathrm{b}\mathrm{o}\mathrm{r}}+{\mathrm{C}}_{\mathrm{f}\mathrm{a}\mathrm{c}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}-\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{l}}+{\mathrm{C}}_{\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{d}-\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{l}}+{\mathrm{C}}_{\mathrm{s}\mathrm{h}\mathrm{e}\mathrm{d}}]$$

The variables are defined as follows: $\left({\mathrm{C}}_{\mathrm{E}\mathrm{N}\mathrm{B}}\right)$ represents the expenditure incurred on exogenous nutrient bag. Other costs include $\left({\mathrm{C}}_{\mathrm{s}\mathrm{p}\mathrm{a}\mathrm{w}\mathrm{n}}\right),$ which denotes the cost associated with the spawn; $\left({\mathrm{C}}_{\mathrm{r}\mathrm{a}\mathrm{w}-\mathrm{m}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{s}}\right)$, which accounts for the cost of raw materials; $\left({\mathrm{C}}_{\mathrm{f}\mathrm{u}\mathrm{e}\mathrm{l}-\mathrm{p}\mathrm{o}\mathrm{w}\mathrm{e}\mathrm{r}}\right)$, which encompasses the expenses related to fuel, water, and electricity; $\left({\mathrm{C}}_{\mathrm{l}\mathrm{a}\mathrm{b}\mathrm{o}\mathrm{r}}\right)$, which signifies the labor cost; $\left({\mathrm{C}}_{\mathrm{f}\mathrm{a}\mathrm{c}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}-\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{l}}\right)$, which pertains to the cost of renting facilities and equipment; $\left({\mathrm{C}}_{\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{d}-\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{l}}\right)$, which indicates the cost of land rental; and $\left({\mathrm{C}}_{\mathrm{s}\mathrm{h}\mathrm{e}\mathrm{d}}\right)$, which corresponds to the cost of constructing shed facilities.

The input –output ratio is as follows $\left(\mathrm{I}\mathrm{O}\mathrm{R}\right)$:

$$[\mathrm{I}\mathrm{O}\mathrm{R}={\displaystyle \frac{\mathrm{T}\mathrm{V}}{\mathrm{T}\mathrm{C}}}]$$

Here, $\left(\mathrm{T}\mathrm{V}\right)$ represents the total output values; $\left(\mathrm{T}\mathrm{C}\right)$ denotes the total output values.

The profit is equal to the total output value $\left(\mathrm{T}\mathrm{V}\right)$ minus the total input cost $\left(\mathrm{T}\mathrm{C}\right)$.

2.7. Data Analysis

SPSS 21.0 software was employed for conducting data analysis, while Origin 2022 software was utilized for generating graphical plots. All presented data are expressed as the mean ± standard deviation. For single-mushroom agronomic traits, the sample size (n) was 30, whereas for other traits, n = 3. In all graphs and tables, an analysis of variance (ANOVA) was performed, and significant differences (p < 0.05) are indicated using different lowercase English letters.

3. Results

3.1. Effects of Different Nutrient Bag Formulations on the Agronomic Traits of Fruiting Bodies

Based on the data presented in

Table 2. Effects of different nutrient bag formulations on the agronomic traits of fruiting bodies.

Formula	Stipe Length (mm)	Stipe Diameter (mm)	Pileus Thickness (mm)	Pileus Width (mm)	Pileus Length (mm)	Weight of a Single Fruiting Body (g)
CK	47.44 ± 0.52 ab	20.00 ± 2.28 ab	6.50 ± 0.73 b	30.26 ± 3.04 ab	78.90 ± 5.97 a	21.87 ± 2.26 e
KY1	47.22 ± 1.90 ab	19.39 ± 2.41 ab	8.62 ± 0.79 a	28.72 ± 2.73 b	79.01 ± 9.89 a	31.42 ± 3.95 bcd
KY2	46.62 ± 1.15 b	16.69 ± 2.27 ab	8.23 ± 0.67 a	28.93 ± 3.58 ab	82.84 ± 13.42 a	28.89 ± 2.33 cd
KY3	47.00 ± 0.83 ab	19.28 ± 2.83 ab	8.68 ± 1.11 a	27.94 ± 2.08 b	86.11 ± 3.73 a	30.50 ± 2.72 cd
DY1	46.86 ± 1.56 ab	18.04 ± 3.91 ab	7.70 ± 1.33 ab	32.01 ± 3.48 ab	82.53 ± 4.88 a	31.46 ± 3.65 bcd
DY2	48.12 ± 1.21 ab	21.29 ± 2.43 ab	7.65 ± 0.29 ab	33.51 ± 2.03 ab	86.32 ± 3.48 a	31.70 ± 4.75 bcd
DY3	48.01 ± 1.14 ab	20.39 ± 3.46 ab	8.57 ± 0.62 a	33.31 ± 3.65 ab	82.42 ± 11.58 a	27.90 ± 6.04 d
MY1	47.23 ± 1.96 ab	22.55 ± 4.86 a	8.27 ± 0.66 a	29.17 ± 7.15 ab	82.45 ± 8.33 a	35.00 ± 2.03 abc
MY2	47.58 ± 1.58 ab	17.81 ± 0.96 ab	8.41 ± 0.73 a	34.72 ± 5.01 a	88.98 ± 11.26 a	38.46 ± 5.45 a
MY3	46.54 ± 0.95 b	18.90 ± 1.28 ab	8.37 ± 0.84 a	32.47 ± 3.84 ab	82.06 ± 8.34 a	36.74 ± 6.74 ab
FY1	46.97 ± 1.00 ab	21.08 ± 3.35 ab	8.12 ± 1.08 a	30.02 ± 4.64 ab	76.17 ± 5.09 a	30.97 ± 4.89 bcd
FY2	47.75 ± 1.46 ab	19.15 ± 2.73 b	8.49 ± 0.77 a	29.55 ± 2.33 ab	88.63 ± 3.65 a	30.76 ± 2.95 bcd
FY3	48.73 ± 1.36 a	20.50 ± 6.11 ab	9.05 ± 1.93 a	32.46 ± 4.50 ab	84.11 ± 16.34 a	38.53 ± 4.02 a

Note: All data are presented as mean ± standard deviation (n = 30 individual fruiting bodies for each agronomic trait). Different lowercase letters indicate statistically significant differences between treatments (p < 0.05).

, under the cultivation conditions of 13 different ENBs formulations, the agronomic traits of M. sextelata fruiting bodies exhibited a certain range of variation. Specifically, the stipe length exhibited a variation spanning from 46.54 to 48.73 mm, the stipe diameter fluctuated within the range of 16.69 to 22.55 mm, the pileus thickness oscillated between 6.50 and 9.05 mm, the pileus width ranged from 27.94 to 34.72 mm, the pileus length varied from 76.17 to 88.98 mm, and the weight of an individual fruiting body ranged from 21.87 to 38.53 g. In this research, rice bran (k), soybean meal (D), cottonseed hulls (M), and fermented maize stover (F) were chosen as exogenous nutrients. The results of the difference analysis revealed that, compared with the control group (CK), the exogenous nutrients derived from the above-mentioned four agricultural by-products resulted in a statistically significant increase (p < 0.05) in the pileus thickness and the weight of a single fruiting body. Among all the treatment groups, the FY3 treatment stood out prominently with respect to pileus thickness and the weight of a single fruiting body. It achieved the maximum pileus thickness, with a value of 9.05 ± 1.93a, and the highest weight of a single fruiting body, at 38.53 ± 4.02a. However, no statistically significant differences (p > 0.05) were observed in other agronomic traits, including stipe length, stipe diameter, pileus width, and pileus length, between the treatment groups and the control group.

3.2. Effects of Different Nutrient Bag Formulations on the Yield of M. sextelata

Sowing of M. sextelata was completed on November 10th. Throughout the entire experimental period, all samples were maintained under the same management conditions, and the number of ENB placed remained consistent. After the overwintering stage, mushrooms emerged. Observation and analysis of the mushroom emergence situation under treatments with different formulations of ENBs revealed differences among the treatments. In the treatment with ENB containing 30% wheat grains, the number of fruiting bodies of M. sextelata was generally higher than that in other formulation groups. When agricultural by-product substrates completely replaced wheat grains, the number of fruiting bodies was significantly lower than that in the treatment groups containing wheat grains.

According to the data presented in Figure 2, among the treatments in which wheat grains were replaced, the rice bran (K) treatment achieved the highest unit yield (kg/m²), followed by the cottonseed hulls (M) treatment. The production of treatments with soybean meal (D) and fermented maize stover (F) substitutions was relatively low. Specifically, when the proportion of wheat grains was 30%, the yields of the treatments were as follows: KY1 > MY1 > CK > FY1 > DY1. The yield of the KY1 treatment was significantly higher than that of the CK group (p < 0.05), while there was no significant difference in yield between the MY1 treatment and the CK group. When the proportion of wheat grains was reduced to 15%, the yields were as follows: CK > MY2 > KY2 > DY2 > FY2. There were no significant differences in yield between the KY2 and MY2 treatments and the CK group, indicating that rice bran and cottonseed hulls could effectively provide nutrients at a 15% wheat grains proportion. When the substrates completely replaced wheat grains, the yields were as follows: CK > KY3 > MY3 > DY3 > FY3. Among them, the mushroom emergence density in the DY3 and FY3 treatments was significantly sparse, and the growth rate of fruiting bodies was slow. Although the FY3 treatment had significantly higher pileus thickness and weight of a single fruiting body than the CK group, its overall yield was significantly lower than that of other treatment groups.

This study demonstrated that when the wheat grain content in the nutrient bags reached 30%, it could provide nutrients for the growth and development of M. sextelata and ensure stable yields. Using rice bran (K) and cottonseed hulls (M) to partially replace wheat grains in the nutrient bags could reduce dependence on staple grains and effectively mitigate cost risks caused by wheat grain price fluctuations.

3.3. Effects of Different Nutrient Bag Formulations on Soil Physicochemical Properties

As illustrated in Figure 3, the soil physicochemical properties during the fruiting body stage of M. sextelata under 13 different ENBs treatments exhibited the following ranges: available nitrogen (AN) ranged from 175 to 525 mg/kg, available phosphorus (AP) from 16.37 to 67.04 mg/kg, available potassium (AK) from 244.71 to 567.64 mg/kg, organic matter content from 1.23% to 3.47%, pH values from 7.35 to 7.97, and soil moisture content from 24.04% to 27.19%. Among these treatments, DY3 had the highest AK and AN contents, reaching 567.64 mg/kg and 525 mg/kg, respectively, which were significantly higher than those in other treatments (p < 0.05). The FY2 treatment significantly increased the AP content, with the highest value of 67.04 mg/kg. The KY2 treatment significantly elevated the soil moisture content to 27.19% (p < 0.05). The FY3 treatment had the highest organic matter content of 3.47%, but there was no significant difference compared with the control group (CK) (p > 0.05). There were no significant differences in soil pH values among the different ENBs treatments, all maintained within a neutral to slightly alkaline range (7.35–7.97), which is conducive to the growth and development of mycelium. The results indicate that substituting wheat grain substrates with rice bran (K), cottonseed hulls (M), soybean meal (D), and fermented maize stover (F) can effectively provide nutrient support for the growth and development of M. sextelata to varying degrees.

3.4. Correlation Between Soil Physicochemical Properties and Yield

Analysis of Pearson’s correlation heatmap (Figure 4) reveals a certain degree of correlation between the soil physicochemical properties and the yield of M. sextelata under different ENBs formulations. There is a significant negative correlation between the contents of alkali-hydrolysable nitrogen (AN) and available potassium (AK) in the soil and the yield, with correlation coefficients of r = −0.60 (p < 0.05) and r = −0.72 (p < 0.01), respectively, and the correlation with AK is the strongest. This indicates that an excessively high AK content in the soil may have an inhibitory effect on the yield of Morchella. It is speculated that a high-potassium environment may suppress the growth of M. sextelata mycelium. In addition, there is an extremely significant positive correlation between AK and AN (p < 0.01), suggesting that these two soil nutrient indicators have a synergistic change trend under exogenous nutrient regulation.

Overall, the correlations between soil physicochemical properties and yield are relatively complex. Most correlations are weak and do not reach a significant level. However, as two factors with significant correlations, AK and AN may play a crucial role in the process of affecting yield changes in M. sextelata. It is worthwhile to further explore their mechanisms in subsequent studies.

3.5. Effects of Different Nutrient Bag Formulations on Economic Benefits

In this study, by comparing the economic benefits of 12 ENBs formulations with the control group CK (containing 40% wheat grains), the KY1 formulation, which utilized rice bran as the substrate and reduced the wheat grain content to 30%, demonstrated significant advantages (

Table 3. Effects of different nutrient bag formulations on economic benefits.

Formula	Cost per ENB (CNY)	Number of ENB (667 m2)	Other Costs (CNY/667 m2)	Yield (kg/667 m2)	Total Output Values (CNY/667 m2)	Total Input Cost (CNY/667 m2)	Profit (CNY/667 m2)	Input–Output Ratio
CK	0.76	1980	15,000	788.39	78,839	16,499.10	62,339.90	4.78
KY1	0.62	1980	15,000	915.13	91,513	16,230.85	75,282.15	5.64
KY2	0.43	1980	15,000	732.37	73,237	15,850.69	57,386.31	4.62
KY3	0.24	1980	15,000	468.23	46,823	15,470.53	31,352.47	3.03
DY1	0.68	1980	15,000	629.65	62,965	16,349.65	46,615.35	3.85
DY2	0.67	1980	15,000	449.56	44,956	16,325.89	28,630.11	2.75
DY3	0.66	1980	15,000	230.78	23,078	16,302.13	6775.87	1.42
MY1	0.62	1980	15,000	837.75	83,775	16,222.93	67,552.07	5.16
MY2	0.41	1980	15,000	761.71	76,171	15,819.01	60,351.99	4.82
MY3	0.21	1980	15,000	393.53	39,353	15,415.09	23,937.91	2.55
FY1	0.60	1980	15,000	668.33	66,833	16,183.33	50,649.67	4.13
FY2	0.41	1980	15,000	414.88	41,488	15,803.17	25,684.83	2.63
FY3	0.20	1980	15,000	160.08	16,008	15,387.37	620.63	1.04

Note: Chinese mu is approximately equal to 667 square meters (m²). In this table, when the area is indicated as 667 m², it refers to 1 Chinese mu. Other costs include the cost associated with the spawn, the cost of raw materials, the expenses related to fuel, water, and electricity, the labor cost, the cost of renting facilities and equipment, the cost of land rental, and the cost of constructing shed facilities.

). Data showed that the unit cost per ENB for the KY1 formulation was 18.4% lower than that of CK (CNY 0.62 per bag vs. CNY 0.76 per bag), which led to a total input reduction of CNY 268.25 per 667 m². Notably, as the wheat grain content decreased, the economic benefits generally declined under other substrate conditions, with some formulations such as DY3 and FY3 approaching a non-profitable state. The KY1 formulation achieved the highest yield increase of 16.1% (915.13 kg vs. 788.39 kg), driving an increase in total output value of CNY 12,674 per 667 m². Ultimately, the economic benefit reached CNY 75,282.15 per 667 m², an increase of 20.7% compared with CK. Its input–output ratio (5.64) was significantly higher than that of CK (4.78), indicating that for every CNY of input, an additional CNY 0.86 of output could be generated. By partially substituting wheat grains with rice bran, the KY1 formulation reduced reliance on staple grains (decreasing the wheat grains proportion by 10%), achieving the triple goals of “cost reduction-yield increase-efficiency enhancement”. Meanwhile, it saved wheat grain consumption, aligning with the original development intention of China’s edible mushroom industry to “not compete with humans for grain”.

4. Discussion

Research by Chen Yuanwen et al. [22] demonstrated that the application of ENBs significantly influences the formation and yield of Morchella fruiting bodies; in the absence of such ENBs, Morchella almost fails to form fruiting bodies. This study optimized the formulation of ENBs by partially substituting wheat grains with agricultural by-products. The results indicated that the KY1 formulation exhibited the best performance in terms of both yield and economic benefits, with a 16.1% increase in fruiting body yield, a 20.7% improvement in net profit, and an input–output ratio of 5.64, which was significantly higher than the CK’s 4.78, compared to the control group (CK). These findings are consistent with those reported by Zhu Jinxia et al. [23] and Wei Zihan et al. [24], further validating the feasibility of partially replacing wheat grains with agricultural by-products as an exogenous nutrient source. Significant differences exist among various agricultural by-products in their capacity to substitute wheat grains. ENBs formulated with raw materials such as rice bran, cottonseed hulls, soybean meal, and fermented maize stover exhibited varying effects on fruiting body yield. In nutrient bag formulations containing a 30% substitution of wheat grains, the fruiting body yield of M. sextelata was generally higher than that of other treatment groups, with the rice bran treatment (KY1) showing a yield significantly greater than that of the control group (CK, 40% wheat grains) (p < 0.05). Furthermore, the cottonseed hull treatments (MY1 and MY2) did not differ significantly from CK in terms of yield, potentially due to their high crude fiber content, which may enhance substrate aeration and water retention, thereby providing a favorable microenvironment for fruiting body development. These results indicate a certain potential for cottonseed hulls as a substitute material. Cai et al. [25] conducted a comparative analysis of the physiological characteristics and secreted proteome of Morchella importuna cultivated on wheat grains versus lignocellulosic substrates, finding that the wheat grain medium significantly enhanced enzyme activity and mycelial biomass. This indicates that the species is better adapted to growth on starch-rich substrates. These findings corroborate the observations of the present study, wherein the treatment group with 30% wheat grain addition in the nutrient bags exhibited a significantly higher yield of Morchella compared to groups without wheat grain supplementation. Wheat is rich in readily degradable carbohydrates and amino acids, providing a stable and efficient source of carbon and nitrogen during the mycelial vegetative growth phase. However, when the proportion of wheat in the nutrient bags was reduced to 15% or completely replaced, the fruiting body yield decreased significantly, indicating that wheat grains play an indispensable role during the early growth stages of Morchella, particularly in sclerotium formation. Its function in maintaining carbon–nitrogen metabolic balance, promoting sclerotium induction, and facilitating energy accumulation remains difficult to fully replicate with alternative substrates. This conclusion aligns with the findings of Liu Qizheng et al. [26] regarding the mechanism of exogenous nutrition in Morchella importuna, who identified wheat grains as one of the most effective carbon sources in ENBs, significantly enhancing mycelial biomass within the main substrate during the mycelial expansion and early sclerotium stages.

Pearson’s correlation analysis showed that available nitrogen (AN) and available potassium (AK) in soil physicochemical properties were negatively correlated with the fruiting body yield of M. sextelata (r = −0.60 and −0.72, respectively), with the most pronounced effects observed in the DY3 treatment group. In this group, 35% of the wheat grains was replaced by soybean meal, resulting in a marked reduction in fruiting density and overall yield. Although this correlation trend is partially consistent with the observations of Xiang Gang et al. [27] and Ren Ziming et al. [28], which suggest that excessively high nutrient levels may interfere with fruiting body formation in Morchella, the present study only establishes a statistical association between Morchella yield and available soil nutrients. It does not provide sufficient evidence to infer underlying physiological regulatory mechanisms, and the causal relationships remain to be elucidated through further investigation. Despite the use of different substitute materials, soil pH remained within the range of 7.35–7.97, which is suitable for M. sextelata growth [29], indicating that none of the treatment formulations disrupted soil acid-base balance.

In terms of economic benefits, the KY1 formulation—achieved by partially replacing wheat with rice bran—reduced dependency on staple grains (with a 10% reduction in the proportion of wheat grains) while generating an economic return of CNY 75,282.15 per 667 m², representing a 20.7% increase compared to the control group (CK). Unit production costs decreased by 18.4%, and the input–output ratio significantly improved to 5.64. The MY1 treatment also demonstrated relatively high economic efficiency, with an input–output ratio of 5.16, indicating its potential as an alternative formulation. In contrast, although treatments such as FY3 and DY3 offered advantages in terms of raw material costs, their severely limited yields led to poor overall economic performance, with some treatments approaching break-even points, rendering them impractical for large-scale application. These findings suggest that, during efforts to reduce wheat grains usage, cost-driven substitution alone may not ensure sustainable cultivation profitability. Therefore, the selection of substitute substrates should not be based solely on price but must also consider nutrient release efficiency and synergistic interactions with Morchella growth [29,30] to achieve an optimal balance between input and output.

To further elucidate the mechanisms by which alternative materials influence M. sextelata growth and yield, future research should systematically investigate the soil microbial community structure, substrate metabolic processes, and their interactions with environmental factors. Additionally, field experiments are often affected by uncontrollable variables such as temperature and humidity, and the uniformity of nutrient bag distribution may also impact fruiting performance. Hence, future studies should enhance control over these variables to obtain more accurate and reliable experimental data.

5. Conclusions

The findings indicate that nutrient bags formulated with different compositions exert significant influences on the availability of soil nutrients. Moreover, an excessive enrichment of soil nutrients may impede the formation of fruiting bodies in M. sextelata. Notably, AN and AK exhibited a significant negative correlation with yield, suggesting that these nutrients may serve as key limiting factors for the yield of M. sextelata. Incorporating rice bran (K) and cottonseed hulls (M) as partial substitutes for wheat grains in nutrient bags presents a viable strategy to diminish reliance on staple grains. When the wheat grain content in the ENB is maintained at 30%, it provides adequate nutritional support for the growth and development of M. sextelata, thereby ensuring yield stability. Considering the aspects of yield, cost control, and economic benefits, the KY1 formulation (comprising 5% rice bran, 30% wheat grains, 60% corncob, 2% rice husk, 1% lime, and 1% gypsum) is recommended.