Method for efficiently cultivating dictyophora indusiata and adjusting phenolic acid of soil after cultivation

Abstract

The invention provides a method for efficiently cultivating dictyophora indusiata and adjusting phenolic acid of soil after cultivation. And sampling the soil sample at a fixed time every month in a period from the sowing of the bamboo fungus strains to the harvesting of the bamboo fungus sporocarp. The research analyzes soil physicochemical properties, soil microflora, phenolic acid metabolites and the like. A more suitable bamboo fungus cultivation formula of the pennisetum sinese and the hypsizygus marmoreus bacterial grains is optimized and screened, the practicability and the use form of secondary utilization of the hypsizygus marmoreus bacterial grains in the bamboo fungus cultivation process are discussed, and finally, the traditional bamboo fungus cultivation formula and mode are improved in combination with soil physicochemical properties and phenolic acid metabolite content indexes. According to the technology, the adverse effect of phenolic acid increase caused by bamboo fungus cultivation on soil can be reduced; the method for applying the bacterial agent and the hypsizygus marmoreus bacterial grains to the bamboo fungus cultivation soil after fermentation is preliminarily discussed, the yield of the bamboo fungus in the second year can be kept equal to that in the first year in the same field, and the method can provide a reference scheme for relieving continuous cropping obstacles in bamboo fungus cultivation.

Description

Technical Field

[0001] The invention belongs to the technical field of agriculture, and in particular relates to a method for efficiently cultivating bamboo fungus and regulating phenolic acid in soil after cultivation. Background Art

[0002] 1. Research significance

[0003] Dictyophora indusiate is a rare edible fungus with both medicinal and edible properties, belonging to the Phalloidaceae family, Dictyophora genus, Fungi phylum, containing a variety of enzymes and high molecular weight polysaccharides. With the increasing demand for Dictyophora, the Dictyophora cultivation industry has been rapidly developed in Fujian, Yunnan, Guizhou and other provinces, and the cultivation scale and output have been continuously improved. Dictyophora cultivation has become an important source of income for local farmers.

[0004] The traditional cultivation formula of bamboo fungus requires a large amount of bamboo chips and sawdust, which has high production costs, limited sources, and a great impact on the ecological environment. Therefore, "using grass instead of wood" to cultivate edible fungi is an important technical means to alleviate the "mushroom-forest contradiction". Shunchang County, as the main production area of ​​seafood mushrooms, produces several tons of discarded seafood mushroom waste every day. Studies have found that seafood mushroom waste contains a large amount of fiber, lignin and many trace elements, and can still be used as a substitute for the cultivation of other edible fungi. The rational use of seafood mushroom waste can not only save production costs, but also effectively reduce the environmental pollution caused by unreasonable disposal of waste. At present, in the use of seafood mushroom waste as a substitute for the cultivation of edible fungi, Mao Ling and others have successfully cultivated edible fungi such as Pleurotus eryngii, Agrocybe oleraceus, and Volvariella volvacea.

[0005] For edible fungi cultivated in soil, the study found that after the first wave of mushrooms appeared, the activity of Agaricus bisporus mycelium gradually decreased with the decrease of laccase activity, while the activity of Ascomycetes laccase increased. Studies on possible allelopathic autotoxic substances found that these chemicals generally inhibit the growth and development of crops by affecting the activity of crop soil enzymes and nutrient metabolism.

[0006] Therefore, in the present study, giant fungus grass and seafood mushroom dregs were used to partially replace the traditional cultivation materials. The effects of giant fungus grass and seafood mushroom dregs on the cultivation of bamboo fungus and its soil microecology were analyzed from the aspects of soil physical and chemical properties, soil microbial flora and phenolic acid metabolites. The purpose was to comprehensively analyze the soil conditions after bamboo fungus cultivation with soil microorganisms and phenolic acid metabolites as the key points.

[0007] 2. Current research status at home and abroad

[0008] 2.1 Juncao edible fungus cultivation technology

[0009] Giant Juncao (Cenchrus fungigraminus) is rich in nutrients required for edible fungi cultivation materials such as crude protein and crude fiber, and has the characteristics of high per-acre yield, which has the advantage of developing "grass instead of wood". In long-term exploration and practice, Juncao has been successfully used to cultivate a variety of edible and medicinal fungi including shiitake mushrooms, black fungus, bamboo fungus, and Hericium erinaceus. By cultivating edible fungi with Juncao, not only can a large number of edible fungi be produced efficiently in a short time, but also the nutritional value of the cultivated edible fungi can be improved. At the same time, the cost of cultivating edible fungi with Juncao is relatively low, which can effectively reduce production costs.

[0010] In the research on formula screening for cultivating edible fungi with giant grass, it was found that the best ratio of grass to cottonseed hull was 1:1 in the formula screening experiment of Pleurotus ostreatus. By comparing the biological conversion efficiency and input-output ratio of different formulas, it was concluded that giant grass can be used as the main raw material for cultivating Pleurotus ostreatus. When Dong Xiaona and others were screening the best formula for cultivating Ganoderma lucidum with grass, they found that although the mycelium growth rate was proportional to the giant grass content, considering comprehensive factors such as mycelium growth rate, mycelium growth potential, and mushroom bag yield, the optimal formula was when the grass content was 48%.

[0011] To sum up, cultivating edible fungi with Juncao has the advantages of protecting the ecological environment, reducing costs, realizing resource recycling, and high efficiency and yield.

[0012] 2.2 Secondary Utilization of Edible Mushroom Waste

[0013] Mushroom residue, also known as mushroom bran, refers to the culture medium for growing edible fungi. It is the residue left after the fruiting bodies are picked after the mycelium secretes extracellular enzymes to degrade lignin, cellulose and other substances in the culture medium during the growth process. China is the world's largest producer, consumer and exporter of edible fungi. It is reported that 5 kg of mushroom residue is expected to be produced for every 1 kg of edible fungi produced. Mushroom residue is easily discarded or burned by farmers if it is not used properly, which not only causes waste of resources but also produces certain air pollution. Now, after relevant research and promotion, it is no longer considered a waste, but a renewable resource. It has been widely used in agriculture. The main ways of resource utilization include: making edible fungi culture medium, producing organic fertilizer, making fertilizer, absorbing soil pollutants, etc.

[0014] Due to the different cultivation types and raw materials, the physical and chemical properties and nutritional components of different mushroom residues are different. The seafood mushroom residue has the characteristics of high soluble protein, reducing sugar and starch content, and its crude polysaccharide content is as high as 33.40 mg / g. The study on the effect of seafood mushroom residue extract on the mycelium growth and laccase activity of Pleurotus eryngii found that the extract increased the mycelium growth rate, mycelium growth amount and laccase activity of Pleurotus eryngii, and is a suitable cultivation material for Pleurotus eryngii. In addition, the study on the effect of Agaricus bisporus mushroom residue compost matrix on tomato seedling cultivation found that no matter what concentration of mushroom residue was added, it had the effect of improving the quality of tomato seedlings compared with the vermiculite group, and the tomato seedling quality and cultivation matrix quality were best when the ratio of mushroom residue to vermiculite was 1:1. Therefore, mushroom residue can be used as an agricultural production resource to provide nutrients to crops and maximize the utilization of resources.

[0015] 2.3 Effects of cultivated edible fungi on soil phenolic acids

[0016] Phenolic acid is one of the most abundant secondary metabolites of higher plants and an important class of plant allelopathic substances. It can enter the soil environment through root secretion, decomposition of residues and litter, rain and fog leaching, etc. Studies have shown that when phenolic acids accumulate to a certain concentration in the soil, they will inhibit the growth of plant roots, reduce the activity of root enzymes, affect hormone synthesis and photosynthesis, and then lead to poor growth, even disease and death. Some other studies have found that phenolic acids can promote the proliferation of soil-borne pathogens, destroy the balance of soil microecology, and increase the risk of soil-borne diseases and continuous cropping disorders. Therefore, phenolic acids are considered to be the key inducement for driving continuous cropping disorders in crops. In the study of bamboo fungus cultivation, Wu Hongping and others found that continuous planting of long-skirted bamboo fungus under rubber forests affected the physical and chemical properties of the soil and the activity of soil enzymes. The accumulation of "toxic and side effects" produced by the "autolysis" of long-skirted bamboo fungus affected the number and function of cultivable microorganisms and functional microorganisms in the soil, and also affected the functional characteristics of soil microbial communities. It can be seen that the change of soil "microenvironment" may be the main reason for the continuous cropping problems of long-skirted bamboo fungus under rubber forests.

[0017] The present invention can provide a scientific basis for bamboo fungus cultivation operations, optimize bamboo fungus production management measures, improve the yield and quality of bamboo fungus, reduce the occurrence of diseases and insect pests, and protect the soil ecological environment through methods such as optimization of bamboo fungus cultivation soil microecology and culture materials, application of fungus residue, etc. Summary of the invention

[0018] The object of the present invention is to provide a method for improving the yield and quality of bamboo fungus and reducing the phenolic acid content in the soil after bamboo fungus cultivation by jointly fermenting and applying giant fungus grass and seafood mushroom residue.

[0019] In order to achieve the above object, the present invention adopts the following technical solution:

[0020] A method for efficiently cultivating bamboo fungus and reducing phenolic acid in soil after cultivation comprises the following steps:

[0021] 1. Cultivation bedding: Collect fresh, disease-free and insect-free seafood mushroom dregs (90wt%), crush them, mix them with urea (5wt%) and light calcium carbonate (5wt%), and then pile them up and ferment them according to the size of 1.4-1.6 meters in height, 1.4-1.6 meters in width, and unlimited length. Turn the pile evenly once every 10 days to make the seafood mushroom dregs ferment evenly, with a water content of 60-65tw%, and a fermentation time of 35-45 days;

[0022] 2. Cultivation formula: stack giant fungus grass and sawdust in a mass ratio of 1:1, and pile up one layer of giant fungus grass and one layer of sawdust to a height of 1.4-1.6 meters, a width of 1.4-1.6 meters, and an unlimited length for fermentation. Turn the pile evenly once every 10 days or so to make the sawdust and giant fungus grass mix evenly for fermentation, with a water content of 60-70wt% and a fermentation time of 40-45 days;

[0023] 3. Application method: In the process of cultivating bamboo fungus, fermented seafood mushroom residue is used as the bedding material with a height of about 10 cm (the amount of bedding material is 2.5 tons / mu), and fermented giant fungus grass and sawdust are used as the main culture materials. The material is laid according to the specification of 40 cm high. The bamboo fungus is divided into egg-sized pieces and inoculated at a distance of 30-50 cm each, and then covered with soil. The height of the soil is 3-5 cm. The amount of bamboo fungus is 200-230 bags / mu, about 0.9 kg / bag, and the bamboo fungus mycelium recovery and fruiting body cultivation stage are carried out. The bamboo fungus fruiting body management method and mode are consistent with the traditional method (control the relative humidity of the air to 85%-95%, and only scattered light is needed during the fruiting body growth stage, etc.);

[0024] 4. It has been determined that the yield and quality of the bamboo fungus fruiting bodies cultivated according to the method of the present invention are higher than those cultivated by the traditional method.

[0025] 5. According to high performance liquid chromatography-mass spectrometry analysis, the content of total phenolic acids (ferulic acid, konjac acid, p-coumaric acid, syringic acid, vanillic acid and p-hydroxybenzoic acid) in the soil during the cultivation of bamboo fungus according to the method of the present invention is significantly lower than that of other formulas and cultivation modes, which helps to reduce the impact of bamboo fungus cultivation on the soil.

[0026] 6. A soil conditioner prepared by fermenting Bacillus subtilis (1 kg / mu) and fermented seafood mushroom residue (6 tons / mu) was applied to the soil once in the third month before cultivating bamboo fungus. In the second year of continuous cultivation, the per-mu yield basically recovered to the same level as in the first year.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] 1. Currently, mushroom residue has been used as soil fertilizer to increase soil organic matter and total nitrogen, but there is no relevant report on whether planting bamboo fungus affects the content of phenolic acid in the soil.

[0029] 2. Traditional cultivation of bamboo fungus mainly uses wood chips and bamboo chips. The present invention uses giant fungus grass and seafood mushroom residue as main raw materials to cultivate bamboo fungus, which can obtain stable and high-yield bamboo fungus fruiting bodies.

[0030] 3. Significantly shorten the formation time of bamboo fungus eggs (shortened by 5-7 days) and prolong the fruiting time of bamboo fungus fruiting bodies (20-35 days). BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of experimental treatments in the first year of this study;

[0032] Figure 2 Phenolic acid content in soil at different stages in different treatment groups; Note: a is a stacked bar graph of phenolic acid content in soil at different stages in different treatment groups; b is a percentage bar graph of phenolic acid content in soil at different stages in different treatment groups. R-JS in the figure is the mushroom dregs base material-juncao sawdust group, R-JR is the mushroom dregs base material-juncao mushroom dregs group, JS is the mushroom grass sawdust group, and JR is the mushroom grass mushroom dregs group. CK represents the soil sample before planting, Apr, May, and Jun are the soil samples of April, May, and June, respectively.

[0033] Figure 3 Figure 2 PCoA analysis of soil microbial diversity in different experimental groups, (a) is the PCoA analysis of fungal communities; (b) is the PCoA analysis of bacterial communities; Note: In the figure, R-JS is the experimental group with 50% giant fungus grass and 50% sawdust as cultivation materials and seafood mushroom fermented dregs as bedding, R-JR is the experimental group with 50% giant fungus grass and 50% seafood mushroom fermented dregs as cultivation materials and seafood mushroom fermented dregs as bedding, JS is the experimental group with 50% giant fungus grass and 50% sawdust as cultivation materials, and JR is the experimental group with 50% giant fungus grass and 50% seafood mushroom fermented dregs as cultivation materials; CK represents the soil sample before planting. DETAILED DESCRIPTION

[0034] A method for efficiently cultivating bamboo fungus and reducing phenolic acid in soil after cultivation comprises the following steps:

[0035] 1. Cultivation bedding: Collect fresh, disease-free and insect-free seafood mushroom residue (90wt%), crush it, mix it with urea (5wt%) and light calcium carbonate (5wt%), and then pile it up to 1.5 meters high, 1.5 meters wide and 3 meters long for fermentation. Turn the pile evenly once every 10 days to make the seafood mushroom residue ferment evenly. The water content is 60wt% and the fermentation time is 40 days;

[0036] 2. Cultivation formula: stack giant fungus grass and sawdust in a mass ratio of 1:1, with one layer of giant fungus grass and one layer of sawdust cyclically stacked into a height of 1.5 meters, a width of 1.5 meters, and a length of 3 meters for pile fermentation. Turn the pile evenly once every 10 days or so to make the sawdust and giant fungus grass mix evenly for fermentation, with a water content of 65wt% and a fermentation time of 40 days;

[0037] 3. Application method: In the process of cultivating bamboo fungus, fermented seafood mushroom residue is used as the bedding material with a height of about 10 cm (the amount of bedding material is 2.5 tons / mu), fermented grass and sawdust are used as the main culture material, and the material is laid according to the specifications of 40 cm high and 60 cm wide. The bamboo fungus is divided into egg-sized pieces and inoculated at a distance of 30 cm each, and then covered with soil. The height of the soil is 3 cm. The amount of bamboo fungus is 200 bags / mu, about 0.9 kg / bag, and the bamboo fungus mycelium recovery and fruiting body cultivation stage are carried out. The bamboo fungus fruiting body management method and mode are consistent with the traditional method (the relative humidity of the air is controlled at 90%, and the light intensity is maintained at 1000 Lux during the growth of the fruiting body);

[0038] 4. A soil conditioner prepared by fermenting Bacillus subtilis (1 kg / mu) and fermented seafood mushroom residue (6 tons / mu) was applied to the soil once in the third month before cultivating bamboo fungus. In the second year of continuous cultivation, the per-mu yield basically recovered to the same level as in the first year.

[0039] 1. Methods and steps

[0040] 1.1 Experimental design

[0041] The field bamboo fungus cultivation experiment was carried out from December 2022 to July 2024. The implementation site is located in the Juncao Science and Technology Courtyard Experimental Area, Xilan Village, Shunchang County, Nanping City, Fujian Province (26°45'7”N, 117°48'20”E), which is located in the mid-subtropical marine monsoon climate zone with a mild and humid climate, abundant rainfall, distinct four seasons, short winter and long summer, early spring and late autumn; the average annual temperature is 18.5℃, the average annual rainfall is 1756mm, and the average annual sunshine is 1740.7 hours. In the first year of the experiment, the plants were divided into four groups according to different cultivation material formulas, namely, mushroom dregs base material-juncao + sawdust (R-JS), mushroom dregs base material-juncao + mushroom dregs (R-JR), mushroom grass + sawdust (JS), and mushroom grass + mushroom dregs (JR). Each group was repeated three times in the field. The specific layout is as follows Figure 1As shown. In the second year of the experiment, a soil conditioner was prepared by fermenting Bacillus subtilis and fermented seafood mushroom residue, and the soil in the first year of bamboo fungus cultivation (mushroom residue base material-juncao + sawdust (R-JS) group) was applied once 3 months in advance (R-3), once 2 months in advance (R-2), once 1 month in advance (R-1), and no application (CK). The six-point sampling method was used to sample the 0-15 cm soil under the bamboo fungus cultivation substrate of different cultivation groups before bamboo fungus cultivation, in the early morning of each month from mycelium sowing to the first wave of mushroom fruiting body formation, and in the last wave of fruiting body harvest period. The samples were collected in sterile bags and stored in dry ice and brought back to the laboratory. After the samples were collected, the samples used for soil physical and chemical properties, enzyme activity and phenolic acid compound analysis were spread in a cool and ventilated place, air-dried, sieved and sealed for testing; the samples used for microbial diversity analysis were stored in a -80℃ refrigerator for testing.

[0042] The above-mentioned mushroom grass is giant mushroom grass, the mushroom dregs base material is fermented seafood mushroom dregs, and the wood chips are general commercially available miscellaneous wood chips.

[0043] 1.2 Analysis of the yield and agronomic characteristics of bamboo fungus fruiting bodies

[0044] The total yield of bamboo fungus fruiting bodies in all treatments was collected; since the formation of bamboo fungus eggs, the diameter of bamboo fungus eggs in different treatment groups, the length of fruiting body stipes, the diameter of stipes, the length of caps and other agronomic traits were analyzed. The fruiting bodies were stored at low temperature and transported back to the laboratory after picking, and the samples were dried in an oven to constant weight to determine the moisture content, and the total sugar was determined using GB / T15672-2009.

[0045] 1.3 Analysis of soil physical and chemical properties

[0046] The total nitrogen (TN) in soil was determined by the Kjeldahl method; the total phosphorus (TP) in soil was determined by the alkali fusion-molybdenum antimony counterspectrophotometry; the total potassium (TK) was determined by the NAOH fusion-flame photometry; the soil organic carbon (OC) was determined by the potassium dichromate oxidation-spectrophotometry; the soil organic matter (OM) was calculated by the conversion coefficient of organic carbon to organic matter; the soil ammonium nitrogen (AMN) was determined by the KCl extraction-indophenol blue colorimetry; the soil nitrate nitrogen (NIN) was determined by the nitrosalicylic acid method; and the soil pH was determined by the potentiometric method.

[0047] 1.4 Analysis of soil enzyme activity

[0048] The 3,5-dinitrosalicylic acid colorimetric method was used to determine soil sucrase (S-SC); the disodium nitrophenylphosphate colorimetric method was used to determine soil acid phosphatase (S-ACP); the 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) colorimetric method was used to determine soil laccase (SL); the potassium permanganate titration method was used to determine soil catalase (S-CAT); and the indophenol colorimetric method was used to determine soil urease (S-UE).

[0049] 1.5 Determination of soil phenolic acid content

[0050] Take 10g soil sample and place it in a 50mL centrifuge tube, add 25mL of 2mol / L NAOH solution, let it stand for 24h, then place it in a shaker at 200r / min and 25℃ to shake and mix for 2h and take it out. Centrifuge at room temperature at 8000r / min for 15min, take 15mL of supernatant, adjust the pH to 2.5 with 36% HCl solution to remove humic acid. Centrifuge at 8000r / min for 15min, take 15mL of supernatant, extract three times with equal amount of ethyl acetate, and combine the extracts. The extract was evaporated to dryness at 45℃, re-dissolved with 2mL of methanol, and filtered through a 0.22μm filter for testing. Use a diode array detector in a Waters high performance liquid chromatography system (E2695, Waters, Milford, USA), and use a UranusC18 column (250mm×4.6mm×5μm) and a guard column (20mm×4.6mm×5μm) for detection and analysis at 280nm. Use the following mobile phase solution to carry out high performance liquid chromatography separation: trifluoroacetic acid aqueous solution (volume fraction 0.1%) and acetonitrile mobile phase, column temperature 30 ℃, injection volume 10 μ L, flow rate 1.0 mL / min, elution gradient is 5%-10% acetonitrile (0-10min), 10%-10% acetonitrile (10-20min), 10%-35% acetonitrile (20-60min), 35%-60% acetonitrile (60-75min), 60%-60% acetonitrile (75-85min), 60%-5% acetonitrile (85-86min), 5%-5% acetonitrile (86-90min) and keep 5min. Each injection interval uses the mobile phase to make the column equilibrate for 10min.

[0051] 1.6 Analysis of soil microbial diversity in Dictyophora cultivation

[0052] The soil microbial DNA in each experimental group in the field experiment was extracted using the MagBeads FastDNAKit for Soil kit from MP Biomedicals, USA. After the DNA concentration and quality reached the standard, PCR (2720, ABI, USA) was performed for amplification. Diluted genomic DNA was used as a template, and primers carrying barcodes were used for amplification and quantification. The primers for bacterial genome (16SrDNA V3-V4 region) were 338F (5'-ACTCCTACGGGAGGCAGCA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The primers for fungal genome (ITS1) were ITS1F (5'-GGAAGTAAAAGTCGTAACAAGG-3') and ITS2R (5'-GCTGCGTTCTTCATCGATGC-3'). The purified product was connected to the sequencing adapter to construct a sequencing library, and the community DNA fragments were sequenced with double ends using the Illumina platform.

[0053] After splicing, filtering, and removing chimeras, the primer fragments of the excision sequence were trimmed using the Qiime2 software (Version 2019.4) and the sequences without matching primers were discarded; then DADA2 was called through qiimedada2denoise-paired for quality control, denoising, splicing, and chimera removal. The Qiime2 software (Version 2019.4) was used to annotate species using the classify-sklearn algorithm with reference to the Unite database. In the Alpha diversity analysis, Chao1 and Observed species indices were used to characterize richness, Shannon and Simpson indices were used to characterize diversity, Faith's PD index was used to characterize evolution-based diversity, Pielou's evenness index was used to characterize uniformity, and Good's coverage index was used to characterize coverage. In the Beta diversity analysis, principal coordinate analysis (PCoA) was used for dimensionality reduction sorting analysis.

[0054] 2. Results and Analysis

[0055] 2.1 Effects of different treatments on the yield and quality of Dictyophora fruiting bodies

[0056] As shown in Table 1, there was no significant difference in the moisture content of bamboo fungus fruiting bodies in different treatment groups (P<0.05). The weight of bamboo fungus fruiting bodies was ranked from large to small, R-JR>JS>JR>R-JS, which were 24.57g, 21.33g, 21.15g, and 12.88g, respectively. The fresh weight of bamboo fungus fruiting bodies per mu was ranked from large to small, R-JR>JS>R-JR>JR, which were 1406.25kg / mu, respectively. -1 , 1271.25kg·mu -1 、896.25kg·mu -1 、708.75kg·mu -1 There were no significant differences in the diameter of the mushroom eggs and the diameter of the stipes among the different treatment groups. In terms of the length of the mushroom cap and the stipe, there were significant differences in the presence or absence of bedding in the JS group, but no significant differences in the JR group. In the two formulas that both used the bedding material, there was no significant difference in the length of the mushroom cap, and the difference in the length of the stipe was not significant. In the two formulas that both did not use the bedding material, there was a significant difference in the length of the mushroom cap, and the difference in the length of the stipe was not significant.

[0057] As shown in Table 2, the total sugar content of bamboo fungus in different treatment groups was ranked from high to low as JS>R-JS>JR>R-JR, which were 61.72%, 58.31%, 50.93%, and 37.19%, respectively. Among them, there was no significant difference in the JS group with or without sterile dregs, but there was a significant difference in the JR group; there were significant differences between the different treatment groups with or without sterile dregs, that is, there was a significant difference between R-JS and R-JR, and there was a significant difference between JS and JR groups.

[0058] Table 1 Agronomic characteristics of Dictyophora

[0059]

[0060] Note: R-JS in the table is the mushroom dregs base material-juncao sawdust group, R-JR is the mushroom dregs base material-juncao mushroom dregs group, JS is the mushroom grass sawdust group, and JR is the mushroom grass mushroom dregs group. The content of each index is expressed as mean ± standard deviation. According to the ANOVA one-way analysis of variance, different lowercase letters after the same column of data indicate significant differences (p<0.05).

[0061] Table 2 Nutritional composition of bamboo fungus

[0062]

[0063]

[0064] Note: R-JS in the table is the mushroom dregs base material-juncao sawdust group, R-JR is the mushroom dregs base material-juncao mushroom dregs group, JS is the mushroom grass sawdust group, and JR is the mushroom grass mushroom dregs group. The content of each index is expressed as mean ± standard deviation. According to the ANOVA one-way analysis of variance, different lowercase letters after the same column of data indicate significant differences (p<0.05).

[0065] 2.2 Analysis of physical and chemical properties of soil for bamboo fungus cultivation

[0066] The trends of soil physical and chemical properties during the growth period of bamboo fungus in each experimental group are shown in Table 3. The six indicators of the R-JS group, except pH and AMN, showed a trend of first increasing, then decreasing, and then increasing again. The September samples of TN, NIN, OC, and OM increased by 36.81%, 22.50%, 12.05%, 62.97%, 44.53%, and 44.53% respectively compared with CK, while pH and AMN decreased by 5.68% and 23.22%, respectively. In the R-JR group, TN, OC, and OM all showed a trend of first increasing, then decreasing, and then increasing again. In September, the above four indicators increased by 26.82%, 10.29%, 36.63%, and 36.62% respectively compared with CK; AMN showed a trend of first increasing and then decreasing, and in September, it decreased by 18.52% and 9.24% respectively compared with CK; NIN showed a trend of first decreasing and then increasing, and showed a significant decrease in May, from 2.288μg / mL in CK to 0.684μg / mL; pH decreased from 6.07 to 6.01. In the JR group, the six indicators of TN, AMN, NIN, OC, OM, and pH showed a trend of first rising, then falling, and then rising. The above six indicators increased by 4.56%, 28.30%, 48.79%, 20.84%, 20.84%, and 14.58% respectively compared with CK in September, and NIN increased significantly to 5.443μg / mL in May; pH ​​increased significantly from 6.07 to 7.11. In the JS group, the five indicators of TN, NIN, OC, OM, and pH showed a trend of first rising, then falling, and then rising. The above five indicators increased by 17.87%, 48.79%, 29.73%, 29.73%, and 1.93% respectively compared with CK in September.

[0067] By comparing only the two bamboo fungus growth periods of CK and the last mushroom picking period in September, it was found that the total nitrogen in the soil increased significantly except for the JS group, and the total nitrogen content in the soil of the litter groups was higher than that of the non-litter groups in the last mushroom picking period in September, and the content was ranked from large to small as R-JS>R-JR>JR>JS; the ammonium nitrogen in the soil decreased except for the JS group, and the content was ranked from large to small as JS>R-JR>R-JS>JR; the nitrate nitrogen in the soil increased, and the content was ranked from large to small as R-JS>JS>JR>R-JR; the soil pH of the litter groups, i.e. R-JS and R-JR, decreased, and the pH of the non-litter groups, i.e. JS and JR, increased significantly, and in the same formula, the non-litter groups were higher than the litter groups, and the content was ranked from large to small as JS>JR>R-JR>R-JS; the contents of soil organic matter and organic carbon increased significantly, and in the same formula, the litter groups were higher than the non-litter groups.

[0068] Table 3 Soil physical and chemical properties of each experimental group at different periods

[0069]

[0070] Note: R-JS in the table is the group of mushroom dregs base material-juncao sawdust, R-JR is the group of mushroom dregs base material-juncao mushroom dregs, JS is the group of mushroom grass sawdust, and JR is the group of mushroom grass mushroom dregs; CK represents the soil sample before planting, and Apr, May, Jun, Sept represent the soil samples in April, May, June, and September. The contents of various indicators are expressed as mean ± standard deviation. According to the ANOVA one-way analysis of variance, different lowercase letters after the same column of data represent significant differences (p<0.05).

[0071] 2.3 Enzyme activity in soil of Dictyophora cultivation

[0072] The trends of soil physical and chemical properties during the growth period of bamboo fungus in each experimental group are shown in Table 4. The activities of S-ACP, S-SC, and SL in the R-JS group showed a trend of increasing first, then decreasing, and then increasing again during different growth periods. The activities of these three enzymes in the soil in the last mushroom harvest period, i.e., September, were significantly higher than those in the CK soil sample before planting, increasing by 29.25%, 90.12%, and 10.03%, respectively. S-CAT and S-UE showed a trend of increasing first and then decreasing, and the activities of these two enzymes in the soil in September were lower than those in CK, decreasing by 65.28% and 25.71%, respectively. Comparing the soil samples in the first mushroom harvest period, i.e., June, and the last mushroom harvest period, i.e., September, it was found that the S-ACP and SL in the September soil sample were significantly higher than those in the June soil sample, and the S-CAT and S-UE were significantly lower than those in the June soil sample. The results showed that planting bamboo fungus in the R-JS group promoted soil S-CAP and SL, and inhibited S-CAT and S-UE. The activity of S-ACP in the soil of the R-JR group at the last mushroom picking period, i.e. September, was significantly higher than that of the CK soil sample before planting, increasing by 30.73%; S-CAT and SL were significantly lower than those of the CK soil sample, decreasing by 39.57% and 13.48%, respectively. Comparison of the soil samples of the first mushroom harvest period, i.e. June, and the last mushroom picking period, i.e. September, showed that the S-ACP in the September soil sample was significantly higher than that in the June soil sample, while the S-CAT and SL were significantly lower than those in the June soil sample. The planting results showed that planting bamboo fungus in the R-JR group promoted the S-ACP and S-SC in the soil, and inhibited the S-CAT, SL, and S-UE. The activity of S-ACP in the soil of the JS group at the last mushroom picking period, i.e. September, was higher than that of the CK soil sample before planting, increasing by 33.01%; S-CAT, S-SC, SL, and S-UE were all lower than those of the CK soil sample, decreasing by 15.70%, 51.48%, 41.53%, and 28.24%, respectively. Comparison of soil samples from the first mushroom harvest period, i.e., June, and the last mushroom harvest period, i.e., September, showed that the S-ACP in the September soil sample was significantly higher than that in the June soil sample, and the S-CAT was significantly lower than that in the June soil sample. The planting results showed that planting bamboo fungus in the JS group promoted soil S-ACP and inhibited S-CAT, S-SC, SL, and S-UE. The S-ACP activity in the soil of the JR group, i.e., September, the last mushroom harvest period, was significantly higher than that of the CK soil sample before planting, increasing by 32.36%; S-UE was significantly lower than that of the CK soil sample, decreasing by 20.90%. Comparison of soil samples from the first mushroom harvest period, i.e., June, and the last mushroom harvest period, i.e., September, showed that S-CAT and S-UE were significantly lower than those in the June soil sample. The planting results showed that planting bamboo fungus in the JR group promoted soil S-ACP and inhibited S-CAT, S-SC, SL, and S-UE.

[0073] Comparing the groups, the S-ACP activity of the litter groups, i.e., R-JS and R-JR groups, showed a trend of first increasing, then decreasing, and then increasing, while the non-litter groups, i.e., JS and JR groups, showed a trend of first decreasing and then increasing. In April, the first month after mycelium sowing, the presence or absence of litter treatment had a greater impact on the S-ACP activity, and the litter groups were higher than the non-litter groups. Except for the JR group, the S-CAT activity in the September soil samples of the other three groups was significantly lower than that of CK, and the activity of the non-litter group was greater than that of the litter group. The activity of the JR formula was higher than that of the JS group. The results showed that the appropriate use of fungus residue can effectively promote the activity of S-CAT, while excessive use will inhibit the activity of S-CAT. Except for the JS group, the activity of S-SC increased in September compared with CK, and the order from large to small was R-JS>JR>R-JR>JS. The fungus residue showed a low-promotion and high-inhibition effect in the activity of S-SC. The SL activity showed a downward trend except for the R-JS group. In April, the SL activity of the litter group in the soil was higher than that of the no-litter group. Compared with the no-litter group, the activity of the JR group was higher than that of the JS group in both the first and last mushroom picking periods. S-UE was lower than that of CK in September. Comparing R-JS and JS, it was found that adding lees litter could increase S-UE activity in June, the first mushroom picking period. Comparing JS and JR, it was found that adding lees to the cultivation material could increase S-UE activity in June, the first mushroom picking period.

[0074] Table 4 Soil enzyme activities in each experimental group at different periods

[0075]

[0076]

[0077] Note: R-JS in the table is the group of mushroom dregs base material-juncao sawdust, R-JR is the group of mushroom dregs base material-juncao mushroom dregs, JS is the group of mushroom grass sawdust, and JR is the group of mushroom grass mushroom dregs; CK represents the soil sample before planting, and Apr, May, Jun, Sept represent the soil samples in April, May, June, and September. The contents of various indicators are expressed as mean ± standard deviation. According to the ANOVA one-way analysis of variance, different lowercase letters after the same column of data represent significant differences (p<0.05).

[0078] 2.4 Analysis of soil microbial diversity in Dictyophora serrata cultivation treated with different soil conditioners

[0079] The effects of different experimental groups on fungal diversity in the soil of cultivated bamboo fungus are shown in Table 5. The Chao1, Observed-species, Shannon and Simpson indexes in the soil before bamboo fungus cultivation were higher than those of the four experimental groups, and were significantly different from those of R-JS, R-JR and JS. However, the Chao1 and Observed-species indexes were not significantly different from those of the JR group, while the Shannon and Simpson indexes were significantly different from those of the JR group. Among the four experimental groups, the Chao1, Observed-species, Shannon and Simpson indexes of the JR group were significantly higher than those of the R-JS, R-JR and JS groups; the Chao1 and Observed-species indexes of the JS group were the lowest and significantly lower than those of the two experimental groups using fungus residue bedding. The results show that different cultivation formulas will reduce the species richness, diversity and uniformity of the community in the soil of bamboo fungus cultivation. The analysis of the four indexes showed that the four experimental groups were ranked from large to small as JR>R-JR>R-JS>JS in Chao1 index and Observed-species index; the four experimental groups were ranked from large to small as JR>R-JS>JS>R-JR in Shannon index, and from large to small as JR>R-JS>JS>R-JR in Simpson index. The results showed that the use of fungus residue in both the cultivation material and the base material could alleviate the trend of decreasing soil fungal diversity after bamboo fungus cultivation.

[0080] The effects of different experimental groups on the diversity of bacterial communities in the soil of cultivated bamboo fungus are shown in Table 5. Chao1 and Observed-species were ranked from large to small in each experimental group as R-JR>CK>R-JS>JS>JR; Shannon index was ranked from large to small in each experimental group as R-JR>JS>CK>JR>R-JS. The results showed that when "giant fungus grass + sawdust" was used as the cultivation material, the treatment method without adding the fungus dregs base material could better increase the species richness and diversity of the bacterial community; when "giant fungus grass + fungus dregs" was used as the cultivation material, the treatment method of adding the fungus dregs base material could better increase the species richness and diversity of the bacterial community. And the indexes of the R-JR group were always greater than those of the CK, indicating that adding fungus dregs to the cultivation material and using fungus dregs padding could alleviate the reduction of species diversity of bacterial communities in the soil.

[0081] The principal coordinate analysis (PCoA) of the diversity and differences in the microbial community was performed based on the Bray-Curtis distance selection algorithm. According to the principle that the closer the projection distance on the coordinate axis, the more similar the community composition of the two samples in the corresponding dimension, and the Permanova test analysis of CK and the four experimental groups, it was found that there were significant differences in the fungal community composition between CK and the four experimental groups and between the four experimental groups in the fungal community, with PCoA1 being 35.7% and PCoA2 being 14.2% ( Figure 3 a); In the bacterial community, there were significant differences in the bacterial community composition between CK and the four experimental groups, as well as between the four experimental groups, with PCoA1 being 20.8% and PCoA2 being 12.9% ( Figure 3 (b) The results showed that the diversity of soil microbial communities changed significantly after cultivating Dictyophora japonica.

[0082] Table 5 Effects of different experimental groups on soil fungal diversity

[0083]

[0084] Note: CK in the table represents soil sample before planting, R-JS represents the group of mushroom dregs-juncao sawdust, R-JR represents the group of mushroom dregs-juncao mushroom dregs, JS represents the group of mushroom dregs-juncao sawdust, and JR represents the group of mushroom dregs. The contents of various indicators are expressed as mean ± standard deviation. According to the ANOVA one-way analysis of variance, different lowercase letters after the same column of data represent significant differences (p<0.05).

[0085] 2.5 Effects of different treatments on soil phenolic acids

[0086] According to the results of HPLC determination, the standard curve was drawn with the concentration of 6 phenolic acid standards as the horizontal axis and the peak area integral value as the vertical axis. The obtained linear equation and regression coefficient R 2 As shown in Table 6.

[0087] Table 6 Linear equations for phenolic acid standards

[0088]

[0089]

[0090] According to the results of HPLC, the measured peak area was substituted into the linear equation of the standard sample to calculate the concentration of phenolic acid in different soil samples. The changes in the total content of phenolic acid in different groups before bamboo fungus cultivation, after mycelium sowing and until the first flush of mushroom fruiting body formation in the early morning of each month are as follows: Figure 2 The total phenolic acid content in the soil before cultivation of Dictyophora was 3.807 mg kg -1The total phenolic acid content of the four treatment groups showed a trend of first increasing and then decreasing. The total phenolic acid content of the end-stage mushroom in the mushroom residue-juncao sawdust group (R-JS), the mushroom residue-juncao mushroom residue group (R-JR), the mushroom grass sawdust group (JS), and the mushroom grass mushroom residue group (JR) was 3.517 mg·kg -1 4.715mg·kg -1 , 5.017mg·kg -1 , 5.998mg·kg -1 Among them, the content of JR group was the highest, and the content of R-JS group was the lowest. Compared with CK, the total phenolic acid content of R-JS group decreased by 8.22% during the first period of mushroom fruiting body formation, while R-JR, JS, and JR increased by 19.27%, 24.12%, and 36.54%, respectively.

[0091] 3. Conclusion

[0092] Using fermented seafood mushroom residue as bedding, giant fungus grass and sawdust to cultivate bamboo fungus can significantly increase the yield and variety of bamboo fungus fruiting bodies, while stabilizing the content of phenolic acid in the bamboo fungus cultivation soil and reducing the harm of bamboo fungus cultivation to the soil; at the same time, in the second year, using soil conditioner fermented with Bacillus subtilis and seafood mushroom residue to treat the soil three months before cultivation may effectively alleviate the obstacles of continuous cropping of bamboo fungus and provide scientific reference.

[0093] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for efficiently cultivating bamboo fungus and regulating phenolic acid in soil after cultivation, characterized in that: The following steps are involved: (1) Cultivation bedding: Collect fresh, disease-free and insect-free seafood mushroom residue, crush it, mix it with urea and light calcium carbonate, and then pile it for fermentation in a size of 1.4-1.6 meters in height, 1.4-1.6 meters in width, and unlimited length. Turn the pile evenly every 10 days to ensure that the seafood mushroom residue is evenly fermented, with a water content of 60-65wt% and a fermentation time of 35-45 days; (2) Cultivation formula: stack the giant fungus grass and sawdust in a cycle of 1.4-1.6 meters in height, 1.4-1.6 meters in width, and unlimited length for fermentation. Turn the pile evenly every 10 days to ensure that the sawdust and giant fungus grass are mixed and fermented evenly. The water content is 60-70wt% and the fermentation time is 40-45 days. (3) Application method: During the cultivation of bamboo fungus, fermented seafood mushroom residue is used as the bedding material with a height of 10 cm, and fermented giant fungus grass and sawdust are used as the main culture medium. The material is laid according to the specification of 40 cm in height. The bamboo fungus is divided into egg-sized pieces and inoculated at a distance of 30-50 cm each. The soil is covered with soil with a thickness of 3-5 cm. The amount of bamboo fungus is 200-230 bags / mu, 0.9 kg / bag, and the bamboo fungus mycelium recovery and fruiting body culture stage are carried out; (4) Use a soil conditioner made from a combination of Bacillus subtilis and fermented seafood mushroom residue to evenly treat the soil of cultivated bamboo fungus starting from the third month before the next year's bamboo fungus cultivation. After continuous cultivation in the second year, the per-acre yield will basically return to the same level as in the first year.

2. The method according to claim 1, characterized in that: In step (1), the mass ratio of the seafood mushroom lees, urea and light calcium carbonate is 90:5:

5.

3. The method according to claim 1, characterized in that In step (2), the mass ratio of giant fungus grass to wood chips is 1:

1.

4. The method according to claim 1, characterized in that: In step (4), the amount of Bacillus subtilis in the soil conditioner is 1 kg / mu, and the amount of fermented seafood mushroom residue is 6 tons / mu. The soil is applied once in the third month before cultivating bamboo fungus.

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