Method for cultivating stropharia rugoso-annulata and synchronously improving soil based on tobacco stems

Abstract

The invention discloses a method for cultivating stropharia rugoso-annulata and synchronously improving soil based on tobacco stalks, and relates to the technical field of agricultural waste resource utilization and edible fungus cultivation, and the method comprises the following steps: preparing a cultivated species culture medium, sterilizing the culture medium, inoculating stropharia rugoso-annulata strains, and culturing under a dark condition until hyphae grow fully to obtain cultivated species; a field base material is prepared, after the water content of the field base material is adjusted, the field base material and the cultivated species obtained in the S1 are spread and inoculated, and an inoculated material surface is obtained; covering the inoculated material surface with soil, and keeping the soil moist until fruiting. According to the method, a cultivated species and field base material two-stage synergistic technical system is constructed, it is found for the first time that the optimal proportions of tobacco stems in the two stages are different and functions are complementary, the pollution problem of the tobacco stems is solved, high quality and high yield of fast growth of stropharia rugoso-annulata hyphae and good mushroom shape and efficient improvement of mushroom planting soil are synergistically achieved, the rapidly available potassium is increased by more than 216%, and the yield of the stropharia rugoso-annulata hyphae is increased by more than 90%. And a circular agriculture mode of promoting production by waste and improving soil by production is realized.

Description

Technical Field

[0001] This invention relates to the fields of agricultural waste resource utilization and edible fungi cultivation technology, and in particular to a method for cultivating king oyster mushrooms based on tobacco stalks while simultaneously improving the soil. Background Technology

[0002] Stropharia rugosoannulata is an edible mushroom with high nutritional value and strong adaptability. Traditional cultivation mainly relies on raw materials such as sawdust and cottonseed hulls, which is costly and puts pressure on forest resources. Meanwhile, my country produces a large amount of tobacco straw every year; this agricultural waste is often burned or discarded, causing environmental pollution and resource waste. While some studies have attempted to use tobacco straw for edible mushroom cultivation, such as CN111802132A which discloses a method for cultivating edible mushrooms using tobacco straw, most of these studies only focus on fruiting results or treat tobacco straw as a simple supplement among various raw materials. These methods have the following shortcomings: (1) A systematic technical solution for the entire chain from strain cultivation to fruiting management was not constructed, especially the profound impact of the cultivar formula on the final fruiting body quality was ignored.

[0003] (2) The soil improvement effect of the cultivation process was not fully considered and utilized, and the design concept of integrated cultivation and improvement was lacking.

[0004] (3) There is a lack of in-depth research and precise definition on the optimal application ratio of tobacco straw and its synergistic effect with different substrates such as corn straw and rice straw, which leads to unstable cultivation results or the inability to achieve multiple goals such as high yield, high quality and soil improvement at the same time.

[0005] Therefore, there is an urgent need in this field for a method that can systematically and efficiently utilize tobacco stalks while simultaneously achieving high-quality and high-yield production of King Oyster Mushrooms and improving soil ecology to solve the above problems. Summary of the Invention

[0006] The main objective of this invention is to provide a method for cultivating king oyster mushrooms based on tobacco stalks and simultaneously improving the soil. This method forms a synergistic technical system by designing specific cultivar formulas and field substrate formulas, which not only makes efficient use of tobacco stalk resources, but also significantly improves the yield and quality of king oyster mushrooms, and simultaneously improves the nutrient status and enzyme activity of the mushroom-growing soil.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for cultivating *Agaricus bisporus* based on tobacco stalks while simultaneously improving the soil, comprising the following steps: S1. Preparation of spawn: Prepare spawn culture medium, sterilize the culture medium and inoculate with Pleurotus ostreatus spawn, and culture in the dark until the mycelium is fully grown to obtain spawn. S2, Field Cultivation: Prepare field substrate, adjust the moisture content of the field substrate, and then inoculate it with the cultivar obtained in S1 to obtain the inoculated substrate surface. S3. Covering with soil and fruiting: Cover the substrate with soil after inoculation and keep the soil moist until fruiting.

[0008] Preferably, the culture medium for cultivation in S1 comprises the following components by dry matter weight percentage: 10-30% sawdust, 25-35% wheat grains, 15-20% cottonseed hulls, 0.5-1.5% lime, 0.5-1.5% gypsum, and the remainder being tobacco straw.

[0009] Preferably, the culture in S1 is carried out under dark conditions at a temperature of 25°C and a humidity of 65%.

[0010] Preferably, the field substrate in S2 comprises the following components by dry matter weight percentage: 98-99% main material, with the remainder being lime; the main material is a mixture of corn stalks or rice stalks and tobacco stalks; wherein the dry weight of tobacco stalks accounts for 10-30% of the total dry weight of the main material.

[0011] Preferably, the moisture content of the field substrate in S2 is adjusted to 60-70%; the inoculation involves two layers of field substrate sandwiching one layer of cultivar.

[0012] Preferably, the soil cover thickness in S3 is 2-4 cm.

[0013] The present invention also provides a culture medium for the cultivation of *Agaricus bisporus* based on tobacco stalks and the simultaneous improvement of soil, wherein the culture medium comprises the following components by dry matter weight percentage: 10-30% sawdust, 25-35% wheat grains, 15-20% cottonseed hulls, 0.5-1.5% lime, 0.5-1.5% gypsum, and the balance being tobacco stalks.

[0014] The present invention also provides a field substrate for the method of cultivating large-cap mushrooms based on tobacco stalks and simultaneously improving the soil. The field substrate comprises the following components by dry matter weight percentage: 98-99% main material and the remainder being lime. The main material is a mixture of corn stalks or rice stalks and tobacco stalks. The dry weight of tobacco stalks accounts for 10%-30% of the total dry weight of the main material.

[0015] The beneficial effects of this invention are as follows: 1. This invention innovatively employs a two-stage precision formulation, discovering and confirming for the first time that a 20-40% tobacco stalk ratio in the cultivar stage, especially the optimal 30%, has a decisive impact on mycelial growth rate and density; while a 10-30% tobacco stalk ratio in the field substrate stage, especially the optimal 10-20%, primarily affects the agronomic traits of fruiting bodies and soil improvement effects. These two stages are interconnected yet functionally distinct, constituting a complete and efficient technical system.

[0016] 2. This invention utilizes a culture medium for cultivating spawn, resulting in mycelial growth rates as fast as 1.69 cm / d and dense growth. Field cultivation using a substrate significantly improves the fruiting body shape index to 0.58, with the cap diameter to stipe length ratio meeting market demands and achieving optimal overall agronomic traits. After cultivation, the available nitrogen, available phosphorus, and available potassium contents in the soil were significantly higher than the control, with the available potassium content of the 10% tobacco stalk and 90% corn stalk substrate formula increasing by as much as 216.62%. Simultaneously, soil sucrase and urease activities were effectively regulated, forming a healthier soil micro-ecosystem; thus, multiple objectives were synergistically achieved, resulting in not only high quality and yield but also efficient soil improvement.

[0017] 3. This invention transforms a large amount of waste tobacco stalks into valuable raw materials for cultivating edible fungi at low cost, realizing the high-value utilization of agricultural waste and meeting the requirements of circular agriculture and green development.

[0018] 4. The formula provided by this invention has a clear ratio, a clear procedure, and a stable effect, making it easy to promote and apply to the large-scale production of giant king mushrooms and the rapid fertilization of mushroom planting soil. Attached Figure Description Figure 1 The growth status of *Agaricus bisporus* mycelium under different cultivation formulas at 7 days (A), 35 days (B), and 49 days (C) after inoculation; Figure 2 Agronomic trait indicators of the fruiting bodies of *Pleurotus ostreatus* under different field substrate formulations; Figure 3 This is a graph showing the changing trends of sucrase (A) and urease (B) activities in mushroom-growing soil under different field substrate formulations. Detailed Implementation

[0019] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings.

[0020] Example 1 Prepare the culture media (T1-T6) for the six cultivars shown in Table 1. All components are calculated as a percentage of dry matter weight. Table 1 Treatments in Cultivation Formula Experiment

[0021] Each culture medium formulation was dispensed into 300mL glass bottles, sterilized, cooled, and then inoculated with *Agaricus bisporus* spawn. The bottles were then incubated in the dark at 25°C and 65% humidity. Mycelial growth rate was measured periodically, and the results are as follows: Figure 1 As shown in Table 2.

[0022] Table 2 Mycelial growth rate

[0023] Note: Data are the average of three trials ± standard error. Lowercase letters indicate differences of P < 0.05.

[0024] The mycelial growth rate initially increased and then decreased with increasing tobacco stalk proportion (Table 3). The T4 treatment (30% tobacco stalk) showed the fastest mycelial growth rate (1.69 cm / d), significantly higher than other treatments. The T6 treatment (50% tobacco stalk) showed the lowest growth rate (1.20 cm / d). The mycelial growth rates of the treatments with added tobacco stalk (T2-T4) were all higher than that of T1 (1.36 cm / d), indicating that an appropriate amount of tobacco stalk (≤30%) can promote mycelial growth. Figure 1 However, when the proportion of tobacco stalks exceeded 30% (T5-T6), mycelial expansion was inhibited. It is noteworthy that the mycelial density of the group cultivated using tobacco stalks was higher in the later stages of cultivation than the group without tobacco stalks. Furthermore, the smaller particle size (powder-like) of the tobacco stalks increased the contact area between the mycelium and the substrate, improving aeration and further explaining the optimal mycelial density in T4. Table 2 shows that the T4 formulation (30% tobacco stalks) exhibited the fastest mycelial growth rate, reaching 1.69 cm / d, and also the optimal mycelial density. Therefore, a tobacco stalk content of 20%-40%, preferably 30%, is the optimal formulation range for the cultivar of this invention.

[0025] Example 2: Field cultivation and effect verification 2.1 Effects of different proportions of flue-cured tobacco straw formulations on the growth and development of Pleurotus ostreatus fruiting bodies Six types of field substrates (D1-D6) were prepared as shown in Table 3 below. All components were calculated as a percentage of dry matter weight, with the total amount of main materials being 100%. Tobacco straw, rice straw, and corn straw were mechanically mixed in a specific ratio, with the straw moisture content adjusted to 65% beforehand and an average thickness of 20 cm. All straw was disinfected with 1% lime water for 24 hours in advance. After adjusting the moisture content of the field substrate to approximately 65%, the strains were cultured by sandwiching a layer of inoculum between two layers of straw substrate. The surface of the inoculated substrate was covered with approximately 3 cm of soil, and the soil was kept moist until fruiting. Each treatment was repeated three times. During the fruiting period, the fruiting bodies of *Agaricus bisporus* in each treatment group were sampled once a day, and the overall height, cap diameter, cap thickness, stipe diameter, stipe length, and weight were recorded. The single mushroom shape index (cap diameter / stipe length) was also calculated.

[0026] Table 3. Experimental treatment of straw substrate formulation

[0027] All substrates were adjusted to a moisture content of 65% using 1% lime water. Then, the cultivar T4 from Example 1 was layered with one layer of substrate between two layers of substrate, and then mixed with one of the six field substrates. After fruiting, the agronomic traits of the fruiting bodies were measured, and the results are as follows: Figure 2 As shown. Among the agronomic traits of fruiting bodies, T3 (10% tobacco stalks + 90% corn stalks) had the highest stipe diameter ( Figure 2 A and 2B), cap diameter ( Figure 2 E), and weight of a single mushroom ( Figure 2 In terms of indicators such as G, it performed best. However, T4 (20% tobacco stalks + 80% corn stalks) had the best overall performance, with a mushroom shape index (cap diameter / stem length) of 0.58. Figure 2 F), significantly higher than T1 (0.45), meets market demand. It is worth noting that T6 (100% corn stalks) had the highest mushroom shape index (0.93), but the overall development of the fruiting bodies was inhibited, manifested as shortened stipes and reduced cap diameter. Figure 2 The C-2F ratio may be related to a high proportion of tobacco stalks or the accumulation of metabolites. The proportion of tobacco stalks showed a highly significant positive correlation with the fruiting body shape index (r=0.729**), indicating that the contents of the tobacco stalks may directly affect fruiting body morphology by regulating the activity of carbon and nitrogen metabolic enzymes. The results showed that formulations D3 and D4 produced better fruiting body shape indices (D4 reaching 0.58), indicating better marketability.

[0028] 2.2 The impact of cultivating *Schefflera heptaphylla* on soil nutrients Sixty days after planting, the surface soil was loosened, and samples were taken (3-5 cm deep) using a five-point sampling method. The samples were mixed thoroughly, air-dried indoors for 14 days, then ground and sieved through 2 mm and 0.25 mm sieves for soil nutrient testing. Soil from uncultivated areas served as the control (CK). Organic matter content was determined using the potassium dichromate oxidation-colorimetric method, alkaline nitrogen using the diffusion absorption method, available phosphorus using the sodium bicarbonate extraction-molybdenum antimony colorimetric method, and available potassium using the ammonium acetate extraction-colorimetric method (Nannipieri). et al. (2011). Soil sucrase activity was determined using the 3,5-dinitrosalicylic acid assay, and soil urease activity was determined using the indophenol blue colorimetric method, according to the instructions of the assay kits. Enzyme activity was expressed as the amount of glucose produced after 1 g of soil was incubated at 37°C for 24 h (mg·g). -1 ·d -1 The amount of ammonia produced after 1g of soil is cultured at 37℃ for 24h (μg·g) -1 ·d -1 The reagent kits are manufactured by Beijing Solarbio Science & Technology Co., Ltd., and their catalog numbers are Cat#BC0245 and Cat#BC0125, respectively.

[0029] Sixty days after cultivation, samples were taken to analyze the nutrients and enzyme activity of the mushroom-growing soil. The effects of growing *Schefflera heptaphylla* on the nutrients in the mushroom-growing soil are shown in Table 4.

[0030] Table 4. Effects of growing *Schefflera heptaphylla* on soil nutrients.

[0031] Table 4 shows that the contents of available nitrogen, available potassium, and available phosphorus in each treatment group were significantly higher than those in the control group (CK) (Table 4). Among them, D3 had the highest available potassium content (543.29 mg / kg), an increase of 216.62% compared to CK. D6 had the highest available phosphorus content (6.38 mg / kg), but its organic matter content (29.01 g / kg) was abnormally high, possibly due to undecomposed tobacco residue increasing the soil carbon pool. D4 had the highest available nitrogen content (118.77 mg / kg), indicating that the combination of tobacco straw and corn stalks can balance carbon and nitrogen release, promoting the secretion of extracellular enzymes by mycelia to decompose the culture medium and release nitrogen. Overall, when the proportion of tobacco straw was ≤20% (D1-D4), the improvement in soil nutrients matched the growth requirements of fruiting bodies well. Therefore, it can be seen that using tobacco straw in the cultivation of *Agaricus bisporus* can increase the contents of available nitrogen, available phosphorus, and available potassium in the mushroom-growing soil to varying degrees.

[0032] 2.3 Activity of sucrase and urease in mushroom-growing soil under different cultivation formulas Trends in sucrase and urease levels in soil from different treatments for mushroom cultivation are as follows: Figure 3 As shown. By Figure 3It can be seen that the sucrase activity was highest on day 6 (153.12 mg / g). -1 ·d -1 This indicates that group D6, using corn stalks as the sole substrate, significantly increased sucrase activity in the soil. This may be because certain components of corn stalks are more conducive to microbial growth and metabolism, thereby promoting the production and activity of sucrase in the soil. The sucrase activity in group D2 (80% rice straw + 20% tobacco straw) was lower than that in group D6. This may be related to the fact that the combination of rice straw and tobacco straw provided richer nutrients for soil microorganisms, resulting in the production of more sucrase during microbial metabolism. Urease activity was highest in group D5 (1.75 μg·g⁻¹). -1 ·d -1 D6 was the lowest (0.43 μg·g). -1 ·d -1 This indicates that rice straw as a substrate can effectively increase urease activity in the soil, while the imbalance of carbon-nitrogen ratio in corn straw inhibits urease production. Therefore, different formulations have different effects on sucrase and urease activities, and can be selected according to the target.

[0033] Specifically, the data on mycelial growth rate, fruiting body agronomic traits, soil nutrient indicators, and enzyme activity were processed and plotted using Microsoft 365 and Origin 2018, and the experimental data were subjected to significance tests and Spearman correlation analysis using R software v 4.3.2.

[0034] In summary, the two-stage cultivation system of this invention (preferably T4 cultivar + D3 / D4 field substrate) can synergistically achieve high-quality and high-yield production of *Schefflera heptaphylla* and effective improvement of the mushroom-growing soil.

[0035] The specific embodiments of the invention have been described in detail above, but these are merely examples, and the invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications or substitutions to the invention are also within the scope of this invention. Therefore, all equivalent transformations, modifications, and improvements made without departing from the spirit and principles of this invention should be included within the scope of this invention.

Claims

1. A method for cultivating *Agaricus macrocarpa* based on tobacco stalks while simultaneously improving the soil, characterized in that... Includes the following steps: S1. Preparation of spawn: Prepare spawn culture medium, sterilize the culture medium and inoculate with Pleurotus ostreatus spawn, and culture in the dark until the mycelium is fully grown to obtain spawn. S2, Field Cultivation: Prepare field substrate, adjust the moisture content of the field substrate, and then inoculate it with the cultivar obtained in S1 to obtain the inoculated substrate surface. S3. Covering with soil and fruiting: Cover the substrate with soil after inoculation and keep the soil moist until fruiting.

2. The method for cultivating *Agaricus macrocarpa* based on tobacco stalks and simultaneously improving the soil according to claim 1, characterized in that, The culture medium for the cultivation of seedlings described in S1, by dry matter weight percentage, comprises the following components: 10-30% sawdust, 25-35% wheat grains, 15-20% cottonseed hulls, 0.5-1.5% lime, 0.5-1.5% gypsum, with the remainder being tobacco straw.

3. The method for cultivating *Agaricus macrocarpa* based on tobacco stalks and simultaneously improving the soil according to claim 2, characterized in that, S1 was cultured in the dark at a temperature of 25°C and a humidity of 65%.

4. The method for cultivating *Agaricus macrocarpa* based on tobacco stalks and simultaneously improving the soil according to claim 1, characterized in that, The field substrate described in S2, by dry matter weight percentage, includes the following components: 98-99% main material, with the remainder being lime; the main material is a mixture of corn stalks or rice stalks and tobacco stalks; wherein the dry weight of tobacco stalks accounts for 10-30% of the total dry weight of the main material.

5. The method for cultivating *Agaricus macrocarpa* based on tobacco stalks and simultaneously improving the soil according to claim 1, characterized in that, The moisture content of the S2 field substrate was adjusted to 60-70%; the inoculation consisted of two layers of field substrate sandwiching one layer of cultivar.

6. The method for cultivating *Agaricus macrocarpa* based on tobacco stalks and simultaneously improving the soil according to claim 1, characterized in that, The soil cover thickness in S3 is 2-4 cm.

7. A culture medium for cultivating *Agaricus macrocarpa* based on tobacco stalks and simultaneously improving the soil, as described in any one of claims 1-6, characterized in that... The culture medium for the cultivation of seedlings comprises the following components by dry matter weight percentage: 10-30% sawdust, 25-35% wheat grains, 15-20% cottonseed hulls, 0.5-1.5% lime, 0.5-1.5% gypsum, and the remainder being tobacco straw.

8. A field substrate for the method of cultivating *Agaricus macrocarpa* based on tobacco stalks and simultaneously improving soil as described in any one of claims 1-6, characterized in that, The field substrate, by dry matter weight percentage, includes the following components: 98-99% main material, with the remainder being lime; the main material is a mixture of corn stalks or rice stalks and tobacco stalks; wherein the dry weight of tobacco stalks accounts for 10%-30% of the total dry weight of the main material.

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