Impact of Substrate Amount and Fruiting Induction Methods in Lentinula edodes Cultivation

Abstract

Mushroom production is a sustainable practice but requires improvements, such as in Lentinula edodes (Berk) Pegler cultivation, which has high water and labor demands. In this context, this study proposed replacing the traditional primordia induction method by submersion with a water injection method. Two primordia induction methods (submersion and injection) and two cultivation block formats were compared: rectangular cube (2 kg) and cylindrical (3.5 kg). The substrate, composed of eucalyptus sawdust (72%), wheat bran (12.5%), rice bran (12.5%), CaCO₃ (1%), and CaSO₄ (2%), was inoculated with strain LED 19/11 and incubated for 80 days at 26 ± 5 °C and 85 ± 15% humidity. After this period, the blocks were washed and transferred to the production environment. Fruiting was induced either by submersion or water injection, and production was evaluated over four harvest flushes. The 2 kg blocks had higher yields with submersion (16.62%), while the 3.5 kg blocks responded better to injection (13.01%), showing more homogeneous production. Increasing the substrate quantity contributes to greater harvest stability across production cycles. Water injections proved to be a viable alternative, reducing handling and facilitating large-scale production. The use of this technique demonstrates great importance in reducing water use and also the need for labor in cultivation.

1. Introduction

Lentinula edodes (Berk.) Pegler is a mushroom of Asian origin, with its center of origin in countries such as China, Japan, and South Korea [1]. Commonly known as black mushroom, oak mushroom, and in Brazil and worldwide as shiitake, it has attracted the attention of researchers due to its nutritional, medicinal, and therapeutic properties, as well as its excellent palatability [2,3].

Currently, shiitake is the most widely produced and consumed mushroom in the world, accounting for 22% of global production [4]. In Brazil, shiitake ranks as the third most cultivated mushroom, following white shimeji (Pleurotus ostreatus var. Florida) and button mushroom (Agaricus bisporus), with an annual production of approximately 2,178,000 kg of fresh mushrooms, generating an economic turnover of approximately 90 million reais [5].

However, this scenario is expected to change, as the main bottleneck in shiitake production is linked to the cultivation method, which requires complex physical infrastructure. These structures are influenced by the size and shape of the substrate blocks, as well as by the technique used to apply water for primordia induction and harvesting. Considering these factors, mushroom growers must carefully design their cultivation systems, taking into account the use of production chambers or greenhouses (with fruiting temperatures between 18 and 20 °C), the number of shelves (i.e., substrate density per square meter), and the proximity to immersion tanks, which are essential for inducing water stress.

Currently, the cultivation block is rectangular cube (20 cm in length × 15 cm in height × 10 cm in depth) and weighs approximately 2 kg [6]. This block design allows for easy handling during both harvesting and the application of water stress (necessary for primordia induction), considering that a medium-sized mushroom grower typically cultivates around 10,000 blocks per month.

However, in recent decades, shiitake growers have faced significant challenges regarding the labor required for cultivation, as part of operations are performed manually [7]. Therefore, the search for mechanized processes and practices that reduce labor demand is essential, given that the cultivation cycle of L. edodes lasts approximately 160 days. This cycle is divided into the vegetative phase (mycelium run and brown cap formation), which lasts 80 days, and the reproductive phase (four harvest cycles), which also lasts 80 days [8].

In Brazil, initiating harvest cycles of L. edodes requires, as a fundamental step, the submersion of cultivation blocks in water for approximately 8 to 12 h [9]. This process involves the manual transport of each block from the cultivation chamber to immersion tanks, which are typically located 100 to 300 m away. In light of this context, the present study proposes an alternative method for water-based primordia induction that eliminates the need to relocate the blocks, using portable equipment capable of injecting water directly into the substrate.

2. Materials and Methods

The experiment was conducted at the Centro de Estudo em Cogumelos, Faculdade de Ciências Agrárias e Tecnológicas (FCAT), Universidade Estadual Paulista (UNESP), Dracena campus, Brazil. The study was designed to evaluate the effectiveness of an alternative injection method for inducing shiitake fruiting in comparison to the conventional water submersion method.

2.1. Substrate Production

The substrate was prepared using a formulation commonly used for the production of L. edodes: 72% eucalyptus sawdust, 12.5% wheat bran, 12.5% rice bran, 1% calcium carbonate (CaCO₃), and 2% calcium sulfate (CaSO₄). The chemical analysis of the substrate is presented in

Table 1. Chemical analysis of the substrate for Lentinula edodes cultivation.

Variables Analyzed	Unit of Measurement	Results
N		0.703 ± 0.01
Ca	%	0.629 ± 0.01
Mg		0.321 ± 0.01
S		0 ± 0.01
Na	mg/kg	226.124 ± 3.85
C/N ratio		75 ± 1.37
Electrical conductivity	mS/cm	1.768 ± 0.06
pH		5.89 ± 0.09

, for which its determination was carried out using the methods proposed by the Brazilian Ministry of Agriculture and Livestock [10,11]. The raw materials were homogenized and mixed with water until reaching 60% moisture. The substrates were then packed in high-density polyethylene (HDPE) autoclave-resistant plastic bags in two distinct formats (rectangular cube and cylindrical).

The substrate was packed in two different container formats. The rectangular format (20 cm length × 15 cm height × 10 cm depth), currently used in experiments conducted in some countries, was filled with 2 kg of moist substrate, representing the standard quantity adopted in experimental trials. The cylindrical format (25 cm diameter × 50 cm length) was filled with 3.5 kg of moist substrate, new methodologies that are arriving in Europe and America (substrate sold by China and South Korea). The substrates were autoclaved at 121 °C for 4 h. After cooling, inoculation was carried out with 2% spawn under aseptic conditions, followed by incubation at 25 ± 2 °C and 80 ± 15% relative humidity for 80 days, until complete mycelial colonization and substrate browning. The chemical characteristics of the substrate are presented in Table 1.

2.2. Spawn Production

LED 19/11 strain was used, isolated from commercial shiitake cultivations named FUNGIBRAS^® (22°51′01″ S, 48°29′26″ W), in Botucatu, SP, Brazil. Inoculum production followed the stages: production of subculture, parent spawn, as well as the spawn, according to the methodology described by Moreaux [12]. The production of subculture was carried out in Petri dishes containing potato dextrose agar (PDA) medium, while the parent spawn and spawn were grown in a sawdust substrate with the same formulation used in the substrate production.

2.3. Production and Harvesting

At the end of the 80-day substrate browning period by the fungus, the substrate was removed from the plastic bags, washed with running water, and then transferred to the cultivation chamber with temperature of 19 ± 4 °C, relative humidity of 85 ± 15%, and a CO₂ concentration of 850 ± 500 ppm. Harvesting was carried out manually, two to four times a day, always before the full opening of the pileus, which is considered the ideal harvest point.

At the end of each production cycle—characterized by a reduction in primordia formation and, in this study, standardized as occurring after 20 days—the blocks were subjected to primordia induction, using either the immersion technique or the injection method. A total of four production flushes were carried out, resulting in a cultivation period of 160 days, which included 80 days allocated to substrate colonization by the fungus. At the end of each flush, the blocks were subjected to a water shock to induce primordia formation. Subsequently, they were reconditioned, when necessary, in the cultivation chambers, where environmental conditions—temperature, relative humidity, and CO₂ concentration—were kept constant and unaltered throughout the period.

2.4. Fruiting Induction

For primordia induction, at the end of each harvested flush, two methods were employed: submersion (traditional method) and injection (Figure 1).

• Submersion method: The substrate (rectangular cube and cylindrical blocks) was transferred to tanks filled with water until they were completely submerged, for 8 h. The tank has a volume measurement of 2.05 × 0.57 × 0.84 m. After this period, the blocks were removed and relocated to the cultivation chamber, maintaining the previous environmental conditions.
• Injection method: A device with a fine tip, approximately 2 cm in diameter, was introduced into the center of the substrate (rectangular and cylindrical blocks) to a depth of 19 cm. This water injection equipment was designed to facilitate the process of water stress induction. During the procedure, water was injected at a flow rate of 0.045 L per second. The injection rod remained inserted in the block for a duration proportional to the block’s weight: 34 s for 2 kg blocks and 60 s for 3.5 kg blocks. To assemble the water injection system for the cultivation blocks, the following materials and configurations were employed, as exemplified in Figure 2:
  • The perforation rod was made of stainless steel with a diameter of 6 mm, featuring a 3 cm pointed tip at its end. A depth limiter, also made of stainless steel, was installed to regulate the insertion length of the rod into the cultivation block. This limiter had a circular shape with a 3 cm diameter. A ½” threaded fitting was adapted at the opposite end to connect the rod to the elbow joint.
  • The 90° elbow joint was made of galvanized steel with a ½” thickness. One end received the perforation rod, and the other was fitted with a straight male quick-connect fitting (6 mm x ½” threaded) to allow the attachment of the water supply hose.
  • A 6 mm hose (3 m in length) was connected through a hose reducer fitting (6 mm to ½”), enabling the transition to a larger diameter hose.
  • The ½” hose (10 m in length) was connected to a water tap using a quick-connect coupling system with a ¾” threaded fitting, which served as the water source.
  • The average pressure exerted by the injection rod during the water application process was 70 kPa.
  • The water used was untreated and sourced directly from an artesian well. It was stored in a conical water tank with a total capacity of 10,000 L, with the following specifications: column height of 6.0 m, cone height of 0.8 m, upper section (“bowl”) height of 3.2 m, total height of 10.0 m, column diameter of 0.95 m, and bowl diameter of 1.91 m.

2.5. Analyzed Variables and Experimental Design

The experiment was evaluated based on agronomic parameter, as yield (weight of harvested mushrooms × 100/initial wet substrate weight), biological efficiency (weight of harvested mushrooms × 100/initial weight of dry substrate, carried out by dehydration in a forced air oven at a temperature of 105 °C for 24 h), number (counted unit) and weight of mushrooms (fresh mushroom weight/number of mushrooms), Weight gain by water, where for its determination the blocks were weighed before and immediately after carrying out the method, be it submersion or injection, and the calculation was carried out through: difference between mass after induction and the initial mass × 100/initial mass (values expressed as a percentage). The experiment followed a completely randomized design in a two-factor factorial scheme. The first factor corresponded to different primordia induction methods (submersion and injection), while the second consisted of different block formats: rectangular cube (2 kg) and cylindrical (3.5 kg). Each treatment had 20 replicates, totaling 80 experimental units.

At the end of the study, data were subjected to statistical analysis and Tukey’s mean comparison test at a 5% significance. The statistical analyses were performed using R software 4.3.3 [13], while graphical representations were generated using Flourish and Canva (Canva Pty Ltd., London, UK) [14].

3. Results

The primordia induction method showed differences in substrate block format/weight. For blocks with an initial weight of 2 kg, the traditional submersion method proved to be more effective in inducing L. edodes fruiting. However, in heavier blocks (3.5 kg), the alternative injection technique demonstrated greater efficiency, leading to higher yields (

Table 2. Yield of Lentinula edodes according to different water induction methods for fruiting and varying block weights.

Primordia Induction	Block of 2 kg	Block of 3.5 kg
Yield, %
Submersion	16.62 ± 0.67 a A	11.30 ± 0.81 b
Injection	11.08 ± 0.70 b B	13.01 ± 0.82 a
CV, %	25.96
Biological efficiency, %
Submersion	41.55 ± 1.67 a A	28.25 ± 2.03 b
Injection	27.7 ± 1.75 b B	32.52 ± 2.05 a
CV, %	25.96
Number of mushrooms, u
Submersion	10.15 ± 0.67 a A	7.95 ± 0.68 b
Injection	6.90 ± 0.68 b B	9.20 ± 0.87 a
CV, %	38.24
Mushroom weight, g
Submersion	34.63 ± 1.83 b	51.93 ± 2.26 a
Injection	34.20 ± 2.02 b	54.66 ± 3.92 a
CV, %	26.95

Averages followed by ±the standard error value. Lowercase letters compare results between columns and uppercase letters compare results between rows. Different letters indicate statistical difference by Tukey’s test at 5% significance; absence of letters indicates that the values were not significantly different. At the end of the means obtained for the different primordia induction methods, the coefficient of variation (CV) for the evaluated trait was presented in the central row of the table.

).

Cultivation blocks of 2 kg achieved a yield of 16.6% when subjected to the submersion method. However, when using the injection method, there was a 33% reduction in yield. In contrast, for blocks weighing 3.5 kg, primordia induction via water injection resulted in an approximate 13% increase in yield. The same occurs for biological efficiency.

The highest number of mushrooms was observed in the treatments with the greatest productive yields. However, for the 3.5 kg cultivation blocks, no statistically significant differences were identified among the different induction methods. Regarding mushroom weight, the highest average individual weights were recorded in treatments using 3.5 kg cultivation blocks. Nevertheless, no statistically significant differences were detected between the induction methods evaluated.

Table 3. Weight gain of Lentinula edodes blocks with different substrate amounts after being subjected to two different fruiting induction methods.

Primordia Induction	Block of 2 kg	Block of 3.5 kg
	Weight gain before 2nd flush, %
Submersion	33.1 ± 2.20 a A	21.1 ± 1.85 b A
Injection	18.7 ± 2.11 B	13.0 ± 2.10 B
CV, %	33.12
	Weight gain before 3rd flush, %
Submersion	20.4 ± 1.43	18.5 ± 1.54 A
Injection	21.6 ± 0.92 a	9.0 ± 1.26 b B
CV, %	33.61

Averages followed by ±the standard error value. Lowercase letters compare means between columns and uppercase letters compare means between rows. Different letters indicate statistical difference by Tukey’s test at 5% significance; absence of letters indicates that the values were not significantly different.

presents the water absorption data after applying the primordia induction method before the second and third flush that was statistically significant differences. In the second flush, the 2 kg blocks absorbed more water than the 3.5 kg blocks when submerged in water. Between block sizes, the water injection demonstrated a smaller weight gain, that is, the substrate retained less water.

Unlike the second flush, in the third flush the injection method influenced the weight gain positively in the 2 kg blocks. In addition, for the third flush the weight gain was significantly equal in the 2 kg block.

Regarding production behavior across different flushes, in blocks containing 2 kg of substrate, the submersion method resulted in a production peak in the first flush, followed by a decline in the second, with subsequent recovery until the fourth flush, where the second-highest production was recorded. When using the injection method, the highest production also occurred in the first flush, and the third flush were the second-highest production (Figure 2).

For 3.5 kg blocks, the production pattern under the submersion treatment was similar to that observed in 2 kg blocks. However, in the injection treatment, production was more uniform across different harvest flushes.

The Generalized Linear Mixed Model (GLMM) analysis revealed differences in L. edodes production among the evaluated treatments. The 2 kg blocks exhibited greater data variability, evidenced by the number of outliers, indicating a less homogeneous response to fruiting induction. In contrast, the 3.5 kg blocks showed lower dispersion, suggesting a more stable and consistent process. Additionally, the submersion method exhibited a tendency toward higher yields in the first flush, while injection showed similar or superior performance in total production, especially in the larger-mass blocks. These results indicate that increasing the substrate quantity contributes to greater harvest stability across production cycles (Figure 3).

4. Discussion

The yields observed in this study were lower than those reported by other authors [15,16,17,18], whose average yield varies around 30%, where higher values are rare and can be presented in some treatments, reaching or approaching 40% in some cases. In the present experiment, the highest yield obtained was 16.62% in the standard treatment, which used rice and wheat bran as supplements and Eucalyptus sawdust as the bulk substrate, with 2 kg blocks. These variations in productivity are considered common depending on the production period of L. edodes and may be related mainly to the season of the year, as well as its meteorological variations [19,20]. In previous experiments conducted under fully controlled conditions, only minor variations in yield were observed using the same strain applied in the present study [18,21,22]. Therefore, it is expected that under semi-controlled conditions—which are more susceptible to environmental fluctuations—a reduction in yield may occur.

During the experimental period, low winter temperatures and low relative humidity likely compromised the productive potential of L. edodes [23], as well as the constant fluctuations in temperature and environmental variables [24,25] due to the transition from summer/autumn to winter. However, due to the uniformity of the experimentation, ensured by the completely randomized design, the reliability of comparisons between the two primordia induction techniques evaluated was not compromised.

The number of mushrooms was directly related to the yields obtained, meaning higher productivity (%) resulted in a greater mushroom number. Additionally, cultivation in 3.5 kg blocks resulted in a higher mushroom mass, which may positively impact quality. Larger mushrooms are preferred both in the market, due to their greater commercial acceptability [26,27], and by growers, as harvesting fewer but larger mushrooms reduces labor demand [28].

Regarding the use of injections as an alternative method for primordia induction, it proved efficient when used in blocks with a larger substrate quantity (3.5 kg), demonstrating a reduction in the operational labor of the cultivation process as well as a more rational use of water in mushroom farming. It should be noted that when using this method, it is important to be careful with blocks contaminated with Trichoderma spp., due to the introduction of the same equipment (skewer) into different blocks.

During colonization and browning, an estimated weight loss of around 15% occurs, varying according to the substrate used and whether it is carried out in a light-exposed environment. In dark environments, this loss can be reduced to approximately 16% [29]. During the cultivation process, shiitake blocks lose weight primarily due to mushroom harvests and water evaporation. The primordia induction process through water stress also serves to rehydrate the blocks, which is crucial for mushroom development.

Before the first induction process, weight gain variation among the blocks did not result in statistically significant differences between treatments, because all blocks were washed in running water. However, before the second induction, the 3.5 kg blocks showed a lower capacity for water absorption, even with a prolonged injection time (60 s) compared to the 2 kg blocks (34 s). This phenomenon may be associated with greater fungal biomass development within the blocks, as white-rot fungi can hinder water penetration in lignocellulosic materials [30]. Given that the primary composition of the substrate used in this study was eucalyptus sawdust, this factor may have influenced the water absorption dynamics. This process occurs due to capillary forces acting on the material, and over time, as cultivation progresses, water diffusion into the wood fibers reduces, leading to a shorter water relaxation time in the logs [31].

The difference in response between 3.5 kg and 2 kg blocks may be related to the efficiency of water absorption during hydration treatments. In the injection method, the direct introduction of water into the substrate interior promotes more uniform hydration, particularly in larger blocks. Conversely, in the submersion method, the exposure time may not have been sufficient for the water to reach the center of the larger blocks, which could explain their lower performance compared to the smaller blocks, which absorb water more rapidly due to their reduced volume.

The greater variability observed in the data from the 2 kg blocks compared to the 3.5 kg blocks may be related to the fruiting induction method, as the data exhibited distinct patterns between the two procedures. In the submerged induction method, the blocks were removed from the cultivation environment and submerged in water under uncontrolled conditions for approximately 8 h. In contrast, the injection method was performed within the cultivation chamber, with minimal changes in temperature, relative humidity, and gas concentration. Furthermore, smaller blocks tend to reach water saturation more rapidly than larger ones, which may influence the physiological response of the mycelium.

The goal of applying stress to shiitake blocks is to stimulate the development of primordia, and this stress can be administered through various methods, including mechanical and hydraulic techniques. In commercial cultivation environments, these stimuli are carefully planned and monitored, with techniques including temperature reduction, increased humidity, ventilation, and lighting adjustments. These changes alter the fungal metabolism and direct its energy toward mushroom formation [32].

Typically, primordia induction involves immersing blocks or logs in water, often chilled, to increase stress on the mycelium, thereby promoting its development [33]. Mechanical shock, in turn, is applied by subjecting logs to strong impacts at one end after immersion, which also stimulates mushroom production [30]. In general, most shiitake strains respond better to induction when submerged in water, with immersion times ranging from 8 to 72 h [34,35].

Labor shortages in mushroom production are a recurring concern, as reported in both previous and recent studies [36,37]. This factor represents one of the main costs in the production chain, and, together with current climate changes, has driven the adoption and development of new technologies in mushroom farming. These innovations aim not only to maintain good productivity levels but also to promote greater environmental balance [18,38]. Therefore, simplifying or eliminating certain processes can contribute to reducing production costs.

Another relevant issue concerns the rational use of freshwater, a resource that is becoming increasingly scarce on a global scale. Mushroom production requires a considerable amount of water throughout the entire production process. It is estimated that obtaining 1 kg of mushrooms requires approximately 9 L of water, depending on the species [39]. When considering the water demand for producing the raw materials used in cultivation, this value can reach around 4072 L of water for obtaining 100 g of protein or 282 L for generating 100 kcal, depending on the species [40]. Although these values are lower than those recorded for other production chains, such as pork, which requires approximately 12,696 L for the same amount of protein [41], optimizing water consumption in mushroom farming remains a relevant concern.

For fruiting induction in approximately 120 shiitake blocks weighing 3.5 kg, the traditional immersion method requires a total water volume of approximately 1000 L. Moreover, the blocks absorb an average of 19.82% of their initial weight, as observed in our study (Table 3). On the other hand, the injection induction method requires approximately 324 L of water (considering 60 s per block at a flow rate of 0.045 L per second) for the same number of blocks, representing a 67.6% reduction in water consumption. These results suggest that the injection technique may be an efficient alternative for reducing water waste, contributing to sustainability in shiitake cultivation. However, further studies are necessary to evaluate the efficiency of this method without compromising production yields.

From an economic perspective, the construction of a masonry tank for the submersion of approximately 120 Lentinula edodes blocks requires an area of around 3.7 m³, with an average cost of USD 60.45 per m³ (Value according to direct conversion from Real (R$) to Dollar (USD) through quotation from the Central Bank of Brazil) [42], totaling approximately USD 223.66. This estimate does not include additional expenses related to waterproofing, plumbing systems, faucets, grates, and other components necessary for complete block submersion, which may vary significantly among producers.

In comparison, the assembly of a water injection system—comprising approximately 100 m of hose and four simultaneous applicators—requires an investment of about USD 189.87. This cost includes all connectors, hoses, and the professional fabrication of the water injection needle. The system is capable of inducing up to 1900 production blocks in a single 8 h workday, operated by just one person and without the need to remove the blocks from the cultivation chamber.

Additionally, there is a notable reduction in water consumption. With the average cost of 1 m³ of water estimated at approximately USD 0.019 [43], this method results in an estimated saving of USD 107.06 per production cycle, contributing to greater process efficiency and sustainability.

5. Conclusions

The results demonstrate that the choice of primordia induction method should consider the substrate block size. For 2 kg blocks, immersion was more efficient, while for 3.5 kg blocks, water injection performed better and provided greater production stability. Additionally, the injection method reduced handling and water consumption, offering a viable alternative for large-scale cultivation. The water and operational efficiency of this method may represent a significant advancement in mushroom farming, especially in the face of resource and labor shortages.