Method for increasing growth speed of stock seeds and cultispecies of stropharia rugoso-annulata

Abstract

The invention discloses a method for increasing the growth speed of stock seeds and cultivated seeds of stropharia rugoso-annulata. The invention discloses a method for increasing the growth speed of a stock seed and a cultivated seed of stropharia rugosoannulata, which comprises the following steps: inoculating bacillus subtilis to a stropharia rugosoannulata culture medium, and then inoculating the stropharia rugosoannulata for culturing, so as to increase the growth speed of the stock seed and the cultivated seed of the stropharia rugosoannulata. The method for increasing the growth speed of the stropharia rugoso-annulata stock seeds and the cultivated species can be used for preparing the stropharia rugoso-annulata stock seeds and the cultivated species, pollution to the stropharia rugoso-annulata strains cannot be caused when the obtained strains are subjected to a fruiting test, and fruiting is not affected. The invention provides a theoretical basis for further development of the stropharia rugoso-annulata growth-promoting microbial inoculum, and has a good application prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and relates to a method for increasing the growth speed of original species and cultivated species of Stropharia rugosa. Background Art

[0002] Stropharia Stropharia rugosoannulata ) is a grass-decaying edible fungus with important development prospects. The scale of cultivation in China has expanded rapidly in recent years. In China, there are more than 30 species of edible fungi cultivated on a large scale, but most of them are wood-decaying fungi. The lack of wood has seriously restricted the further development of the edible fungus industry. However, China produces more than 1 billion tons of agricultural straw, such as rice straw and corn straw, every year. Except for a small amount used as feed and returned to the field, a large amount of straw is wasted and pollutes the environment (Zhang Fangfang et al. 2021; Zhao et al. 2024). As a grass-decaying fungus, Stropharia rugosa can not only make use of various straws such as rice straw, but also rotate with crops such as rice and vegetables to improve soil fertility and soil microbial flora (Hao et al. 2024; Tang et al. 2022). In addition, Stropharia rugosa is not only delicious, but also rich in a variety of active substances that are beneficial to the human body, and is very popular in the market (Chen et al. 2024; Huang et al. 2023; Jiang et al. 2023; Li et al. 2024; Li et al. 2023; Zhang et al. 2014). In recent years, Stropharia rugosa has gradually become a new hotspot in the development of the edible fungus industry.

[0003] Edible fungi are generally divided into mother species, original species and cultivated species. Mother species are strains directly isolated from fruiting bodies (tissues or spores) and mushroom wood (ear wood). The culture medium often uses sterile culture media such as potato agar medium that is rich in nutrients and easy to absorb. In order to facilitate preservation, transportation and prevent contamination by miscellaneous bacteria, it is generally cultured in a test tube, so it is also called "test tube strain". The original species is a strain obtained by inoculating the cultured mother species into culture media such as sawdust and wheat straw. Commonly used culture media include sterile culture media such as wood rot fungus culture media and grass culture media rot fungus. Due to the limited number of original species, in order to meet the needs of mass production, the original species must be expanded and cultured again, and the resulting strains are called cultivated species. The culture medium is basically the same as the original species culture medium, but the culture medium of some fungus cultivation species can be taken from a wider range of materials. The cultivation method of Stropharia is simple and easy to operate, but the original species and cultivated species grow very slowly. Stropharia is cultivated with raw materials or fermented materials, and the soil is directly covered after sowing. The culture medium and soil contain a variety of microorganisms, and the mycelium of the giant puffball mushroom grows very fast. Under the condition of 20-30℃ in autumn, the thickness of the culture medium is 20-25cm, and the seed amount per mu is 250kg (wet weight). The mycelium of the giant puffball mushroom can cover the culture medium 30-40 days after sowing, and the mushrooms will start to grow in 40-50 days (Liu Ruibi 2021). The spawn of the giant puffball mushroom is made of cooked material (that is, the culture medium after high pressure or normal pressure sterilization). After the culture medium is bottled, it is sterilized at 121℃ for 2-3h, or at 100℃ for 8-10h. The spawn bottle with a diameter of 10cm and a filling depth of 15cm, after inoculation, is placed at 25℃ for static culture, and it takes 60-70 days to grow full. When some spawn bottles grow to about 10-13cm, the mycelium growth stagnates, and even more than 100 days cannot fill the spawn bottle. In production, in order to increase the growth rate of the fungus, it is necessary to shake it regularly to mix the culture medium and mycelium. Even so, it takes 50-60 days to fill the fungus bottle. The problem of slow growth of fungus has become a bottleneck restricting the production of Stropharia rugosa.

[0004] The fact that the mycelium of Stropharia rugosodium grows fast on raw materials (i.e. culture materials that have not been heated and sterilized) or fermented materials (culture materials that have been composted and fermented) but grows slowly on cooked materials may be related to the microorganisms in the culture materials. Edible fungi, as a kind of fungus, have a very complex relationship with environmental microorganisms. According to their relationship with edible fungi, environmental microorganisms are often divided into three categories: one is miscellaneous bacteria, which compete with edible fungi mycelium for common nutrients and living space. Some miscellaneous bacteria can produce metabolites that inhibit or kill edible fungi mycelium. Common ones include Trichoderma ( Trichoderma spp.), Aspergillus ( Aspergillus spp.), Neurospora ( Neurospora spp.), Penicillium ( Penicillium spp.), Mucor ( I am sick.spp.) etc. (Bian Yinbing 2013); the second is the companion bacteria, which live together with certain edible fungi. The companion bacteria often have a strong ability to degrade lignocellulose, providing lignocellulose degradation products for edible fungi and helping them complete their sexual life history, such as the incense ash mushroom ( Hypoxylonstygium ) and Steremphirus sutum, which are Tremella fuciformis ( Tremella fusiform ) and golden ear ( Orange Tremella ) (Huang Nianlai 2007; Tian Yunxia et al. 2019); the third is pathogenic bacteria, which infect the vegetative mycelium or fruiting body of certain edible fungi and cause diseases, such as Trichoderma harzianum, which can infect a variety of edible fungi ( Trichoderma harzianum ), Pantotrichum eryngii and Stropharia officinalis soft rot ( Pantoea sp.), Pseudomonas tolas that causes brown spot disease of Pleurotus ostreatus ( Pseudomonas tolaasii )(Jia Jiao et al. 2021; Zhang Ruiying et al. 2013; Allaga et al. 2021; Zhang et al. 2013); Fourth, beneficial microorganisms, which promote the growth of edible fungi vegetative mycelium and the differentiation and development of fruiting bodies, such as Pseudomonas putida ( Pseudomonas putida ), which not only promotes Agaricus bisporus ( Agaricus bitorquis ) fruiting body formation, and can also promote the growth of Morchella ( Morel spp.) mycelium growth (Lei et al. 2024; Chen et al. 2023; Colauto et al. 2016; Pion et al. 2013; Rainey 1991).

[0005] At present, most artificially cultivated edible fungi use completely sterilized culture media when making strains to avoid contamination by foreign bacteria. Strains contaminated by foreign bacteria are often easily distinguished from their morphology, color, and smell. For example, bacteria will form water-stained colony spots, emit a sour and smelly odor, and inhibit the growth of edible fungi hyphae.

[0006] Since the growth of Stropharia cap mushrooms on a thoroughly sterilized sawdust culture medium is very slow, the production of strains has become a bottleneck problem in the production of Stropharia cap mushrooms. In actual production, the growth rate of Stropharia cap mushroom mycelium is often increased by reducing the sterilization temperature and sterilization time of the sawdust culture medium. For example, for a mushroom bag with a diameter of 11cm and a height of 20cm, it is sterilized at 100℃ for only 4-6h. Through sampling and testing of strain companies, it was found that a large number of bacteria remained in the culture medium using this sterilization method. The quality of strains prepared by this method is often unstable. On the one hand, the rate of contamination symptoms is often relatively high; on the other hand, some strains without contamination symptoms still grow slowly.

[0007] Regarding the production of Stropharia, Chinese patent application (201710060165.1) discloses a Stropharia cultivation matrix, which is composed of pine hair, livestock and poultry manure, composite bacterial agent, urea, superphosphate, and auxiliary materials. The mass percentage of each component is: pine hair 70%, livestock and poultry manure 20%, urea 0.1%, superphosphate 1%, auxiliary materials 8.9%, composite bacterial agent accounts for 5% of the total mass of the cultivation matrix, and the composite bacterial agent contains Bacillus subtilis strains and Pichia pastoris engineered strains. At present, no one has disclosed how to improve the preparation efficiency of Stropharia strains. Summary of the invention

[0008] The technical problem to be solved by the present invention is how to improve the production efficiency of original species and cultivated species of Stropharia rugosa under aseptic conditions.

[0009] In order to solve the above technical problems, the present invention first provides a method for preparing Stropharia officinalis ( Stropharia rugose-annulated ) strains, the method comprising: introducing Bacillus subtilis ( Bacillus subtilis ) was inoculated onto the culture medium of Stropharia rugosa, and then inoculated with Stropharia rugosa ( Stropharia rugosoannulata ) to obtain Stropharia rugosotis ( Stropharia rugosoannulata ) strains.

[0010] In the above method, the stropharia seed culture medium is a sterile culture medium. The stropharia seed culture medium can be a culture medium made from sawdust, rice husks or corn cobs. The sawdust, rice husks or corn cobs are mixed, bagged, sterilized and cooled in sequence to obtain the stropharia seed culture medium.

[0011] In one embodiment of the present invention, the Stropharia cap mushroom culture medium is a poplar wood chip culture medium with a water content of 60%.

[0012] In the above method, the Stropharia officinalis ( Stropharia rugosoannulata ) The fungus species may be Stropharia rugosa ( Stropharia rugosoannulata ) original species or cultivar.

[0013] In the above method, the Bacillus subtilis ( Bacillus subtilis ) may be Bacillus subtilis ( Bacillus subtilis ) T2-19 or Bacillus subtilis ( Bacillus subtilis ) T3-7, the Bacillus subtilis ( Bacillus subtilis ) T2-19 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with a deposit number of CGMCC No. 31410. The Bacillus subtilis ( Bacillus subtilis )The deposit number of T3-7 in the General Microbiology Center of China Microorganism Culture Collection Administration is CGMCC No.31966.

[0014] In one embodiment of the present invention, the Stropharia capitis is the Stropharia capitis strain ACCC 51711.

[0015] The present invention also provides a method for improving the performance of Stropharia officinalis ( Stropharia rugosoannulata ) (original species or cultivated species) growth rate, the method comprising: Bacillus subtilis ( Bacillus subtilis ) was inoculated onto the Stropharia culture medium, and then inoculated with Stropharia ( Stropharia rugosoannulata ) to increase the growth rate of Stropharia rugosotis ( Stropharia rugosoannulata ) growth rate.

[0016] In the above method, the stropharia culture medium can be a sterile culture medium. The stropharia culture medium can be a culture medium made from sawdust, rice husks or corn cobs. The sawdust, rice husks or corn cobs are mixed, bagged, sterilized and cooled in sequence to obtain the stropharia culture medium.

[0017] In one embodiment of the present invention, the Stropharia rugosa culture medium is a poplar wood chips culture medium with a water content of 60%.

[0018] In the above method, the Bacillus subtilis ( Bacillus subtilis ) may be Bacillus subtilis ( Bacillus subtilis ) T2-19 or Bacillus subtilis ( Bacillus subtilis ) T3-7, the Bacillus subtilis ( Bacillus subtilis ) T2-19 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with a deposit number of CGMCC No. 31410. The Bacillus subtilis ( Bacillus subtilis )The deposit number of T3-7 in the General Microbiology Center of China Microorganism Culture Collection Administration is CGMCC No.31966.

[0019] The present invention also provides Bacillus subtilis ( Bacillus subtilis ) T2-19, whose deposit number in the General Microbiological Center of China Culture Collection Administration is CGMCC No. 31410.

[0020] The present invention also provides Bacillus subtilis ( Bacillus subtilis ) T3-7, whose deposit number in the General Microbiological Center of China Culture Collection Administration is CGMCC No.31966.

[0021] The present invention also provides a bacterial agent, the active ingredient of which is the Bacillus subtilis ( Bacillus subtle ) T2-19 or the Bacillus subtilis ( Bacillus subtilis )T3-7.

[0022] In the above-mentioned bacterial agent, the bacterial agent may further include a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier may be a mineral material, a plant material or a polymer compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the plant material may be at least one of corn flour, soybean flour and starch; the polymer compound may be polyvinyl alcohol and / or polyglycol. The liquid carrier may be an organic solvent, vegetable oil, mineral oil or water; the organic solvent may be decane and / or dodecane. In the bacterial agent, the active ingredient may be present in the form of cultured living cells, fermentation broth of living cells, filtrate of cell culture or a mixture of cells and filtrate. The dosage form of the composition may be a variety of dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water-dispersible granule.

[0023] As required, a surfactant (such as Tween 20, Tween 80, etc.), a binder, a stabilizer (such as an antioxidant), a pH adjuster, etc. may also be added to the bacterial agent.

[0024] The present invention also provides any of the following applications: X1, the Bacillus subtilis ( Bacillus subtilis ) T2-19 or the Bacillus subtilis ( Bacillus subtilis ) T3-7 as a large mushroom ( Stropharia rugosoannulata ) Application of growth-promoting bacteria; X2, the Bacillus subtilis ( Bacillus subtilis ) T2-19 or the Bacillus subtilis ( Bacillus subtilis )T3-7 in Stropharia officinalis ( Stropharia rugosoannulata ) Application in production; X3, the bacterial agent is used in the preparation of Stropharia officinalis ( Stropharia rugosoannulata ) Application of bacterial strains; X4, the bacterial agent is agaricus ( Stropharia rugosoannulata ) application in production.

[0025] The Stropharia officinalis ( Stropharia rugosoannulata ) The fungus species is Stropharia rugosotis ( Stropharia rugose-annulated ) original species or cultivar.

[0026] In the process of producing the original and cultivated species of Pleurotus eryngii, if the culture medium is not sterilized or not sterilized thoroughly, although the mycelium growth rate can be increased, the bacterial contamination rate of the strain is very high and cannot be directly used for later production. This is mainly because the culture medium contains various molds and other bacteria in addition to Bacillus subtilis, resulting in serious bacterial contamination and cannot be used for later field cultivation. The present invention separates Bacillus subtilis ( Bacillus subtilis ), on sterilized culture medium, Bacillus subtilis can increase the mycelial growth rate of Pleurotus ostreatus by 8.63%-46.82%. The use of this strain can significantly increase the growth rate of the original species and cultivated species, shorten the production cycle, and improve the efficiency of seed production. This shows that Bacillus subtilis has a growth-promoting effect on Pleurotus ostreatus. Preparing Bacillus subtilis to form a bacterial agent and inoculating it with Pleurotus ostreatus can not only solve the problem of slow growth of Pleurotus ostreatus on sterile culture medium, but also solve the problems of high contamination rate and poor stability on incompletely sterilized culture medium, and can be used to prepare Pleurotus ostreatus cultivated species. Using Bacillus subtilis ( Bacillus subtilis When the original species prepared by the method and the cultivated species are used for fruiting, the contamination of the stropharia species will not be caused, and the fruiting of the stropharia will not be affected. The present invention provides a theoretical basis for the development of the stropharia growth-promoting agent in the next step, and has a good application prospect.

[0027] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0028] Description of biological material deposit Abbreviation of depository unit: CGMCC Name of depository: China National Microbiological Culture Collection Administration General Microbiology Center Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101 Deposit date: August 1, 2024 CGMCC registration number: CGMCC NO. 31410 Classification name: Bacillus subtilis ( Bacillus subtilis ) Strain ID: T2-19 Description of biological material deposit Abbreviation of depository unit: CGMCC Name of depository: China National Microbiological Culture Collection Administration General Microbiology Center Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101 Deposit date: September 13, 2024 CGMCC Registration Number: CGMCC No.31966 Classification name: Bacillus subtilis ( Bacillus subtilis ) Strain number: T3-7 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The mycelial growth rate of Stropharia rugosotis on different sterilized culture media.

[0029] Figure 2 The mycelial morphology of Stropharia rugosodium on different sterilized culture media after 16 days of cultivation.

[0030] Figure 3 This is the mycelial morphology of Stropharia capillaris after 16 days of culture for the purpose of inoculating bacteria before inoculating Stropharia capillaris.

[0031] Figure 4 For the phylogenetic tree.

[0032] Figure 5 This is the effect of the inoculation concentration of strain T2-19 on the mycelial growth of Stropharia rugosotis.

[0033] Figure 6 This is the effect of the inoculation concentration of strain T3-7 on the mycelial growth of Stropharia rugosotis.

[0034] Figure 7 This is the effect of the inoculation concentration of strain T2-19 on the mycelial growth of Stropharia rugosotis.

[0035] in, Figure 1 , 5 , , and 6, the data marked with different letters are significantly different. DETAILED DESCRIPTION

[0036] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples are all commercially available unless otherwise specified. The quantitative tests in the following examples are all repeated at least three times, and the results are averaged.

[0037] The Stropharia capitis strain ACCC 51711 comes from the China Agricultural Microbiological Culture Collection Center (ACCC). The strain ACCC 51711 was preserved using liquid nitrogen preservation technology, inoculated on PDA culture medium before use, and cultured and activated at 25°C to obtain the mycelial disk of the Stropharia capitis strain ACCC 51711.

[0038] PDA medium: (Difco, USA), autoclaved at 121°C for 20 minutes.

[0039] Sawdust medium: It is composed of poplar wood chips with a water content of 60%.

[0040] Example 1, Stropharia officinalis growth-promoting bacteria Bacillus subtilis ( Bacillus subtilis ) Isolation and identification of T2-19 and T3-7 1. The influence of sterilization degree of culture medium on Stropharia rugosa The sawdust medium was filled into a test tube or a spawn bottle. In order to determine the effect of the sterilization method on the mycelial growth of Stropharia rugosa, it was divided into 4 treatments, namely ① sterilization at 100℃ for 20 minutes (treatment I), ② sterilization at 100℃ for 60 minutes (treatment II), ③ sterilization at 121℃ for 60 minutes (treatment III), and ④ no sterilization (treatment IV). Then, a mycelial disk of Stropharia rugosa strain ACCC 51711 with a diameter of 0.8 cm was inoculated into these sawdust medium and cultured at 25℃ in the dark. The height of the substrate colonized by mycelium was measured on the 40th day after inoculation, and the mycelial growth rate (mm / d) was calculated.

[0041] The results showed that the degree of sterilization of sawdust culture medium had a significant effect on the growth of Stropharia mycelium. Figure 1 As shown, the mycelial growth rate of Stropharia rugosa on the culture medium of treatments I, II and III was significantly higher than that of treatment IV, among which the mycelial growth rate of treatment I was the fastest, followed by treatment II, and then treatment III. The mycelial growth rate of treatment I (4.92 mm / d) was 122.62% faster than that of treatment 4 (2.21 mm / d).

[0042] Due to incomplete sterilization, some test tubes in both treatments I and II were contaminated with bacteria, with the highest contamination rate in treatment I at 30%. Figure 2 As shown in the figure, in the test tube without foreign bacteria contamination, the mycelium of Stropharia capitis is pure white. In addition, the mycelium morphology of Stropharia capitis on completely sterilized and non-completely sterilized sawdust medium is also different. For example, in treatment I, the mycelium is cord-like and grows vigorously; in treatment IV, the mycelium is fluffy and grows relatively weakly.

[0043] 2. Screening of Growth-Promoting Bacteria for Stropharia rugosodium In order to study the relationship between the growth rate of Stropharia capillaris mycelium and the bacteria in the sawdust culture medium, the strains without contamination symptoms in the four treatments were used as materials, and the matrix covered with Stropharia capillaris mycelium was taken for bacterial isolation and detection. 1 g of culture was transferred to a flask containing 9 mL of sterile water and homogenized. The suspension was diluted with sterile water in 10-fold increments. 100 microliters of the dilution was inoculated on nutrient agar (Huankai Microbial, China). Cultured in the dark at 30°C for 24 hours. All colonies were re-streaked and purified to obtain pure culture. The purity of the colonies was detected by Gram staining. The results showed that different numbers of bacteria were found in treatments I, II, and III, while no bacteria were detected in treatment IV. It is speculated that the fast growth of Stropharia capillaris mycelium in treatments I, II, and III may be related to the bacteria in the culture medium that were not sterilized and killed.

[0044] From the detected bacteria, 50 strains were randomly isolated and purified, and the bacterial suspension was prepared with sterile water. The suspension was inoculated with Stropharia rugosa on a completely sterilized sawdust medium to detect its effect on the growth of Stropharia rugosa hyphae. The results after 16 days of culture were as follows: Figure 3 As shown, on these culture media inoculated with bacteria, the mycelium of Pseudomonas aeruginosa was white and grew vigorously, without any symptoms of bacterial contamination. Compared with the control without bacterial inoculation, 13 of the 50 bacterial strains had an inhibitory effect on the growth of Pseudomonas aeruginosa mycelium, 27 strains had no significant effect, and 10 strains had a significant promoting effect on the growth of Pseudomonas aeruginosa mycelium.

[0045] 3. Identification of the growth-promoting bacteria of Stropharia rugosodium The 16S rRNA gene of the 10 bacterial strains with a promoting effect was further amplified and sequenced, and the genomic DNA of the bacterial strains was extracted using a bacterial genomic DNA extraction kit (Takara Bio Inc.). The 16S rRNA gene was amplified by PCR using primer sets 27F (AGAGTTTGATCMTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT) and a DNA polymerase kit (Takara Bio Inc.). Among them, the 16S rRNA sequence of strain T2-19 is as follows:

[0046] The 16S rRNA sequence of T2-19 was aligned with the model strains of bacteria on the Ez-Taxon-e service, and the 15 model strains with the highest similarity (98.68-99.30) were selected. The phylogenetic tree was constructed using Mega11 using the Neighbor-Joining method. A validation analysis of the maximum likelihood data was performed using 1000 resamplings to determine the confidence of the branch points (Felsenstein 1985). Results Figure 4 As shown, strain T2-19 may be Bacillus subtilis ( Bacillus subtilis ).

[0047] The total DNA sample of strain T2-19 was sent to Annoroad for whole genome sequencing. The whole genome of T2-19 was sequenced by combining third-generation sequencing (PacBio sequel II) and second-generation sequencing (Illumina NovaSeq 6000). Then the data was spliced ​​using spades software to obtain the genome sequence data and uploaded to the NCBI (National Center for Biotechnology Information) database (SRX24429325). The ANI calculator was used to calculate the nucleic acid sequence consistency characteristics between T2-19 and the reference strain genome (https: / / www.ezbiocloud.net / tools / ani), the AAI calculator was used to calculate the amino acid sequence consistency characteristics between T2-19 and the reference strain (http: / / enve-omics.ce.gatech.edu / aai / ), and the GGDC 3.0 was used to calculate the dDDH value between strain T2-19 and the reference strain (http: / / ggdc.dsmz.de / ggdc.php).

[0048] The results showed that the ANI, AAI and dDDH values ​​of strain T2-19 and the reference bacterium Bacillus subtilis ATCC 6051 (GCA_000344745.1) were 98.80%, 98.69% and 89.40%, respectively, and the ANI, AAI and dDDH values ​​of strain T2-19 and the reference bacterium Bacillus subtilis ATCC 6051 (GCA_000344745.1) were 90.09%-93.06%, 92.24%-95.01% and 39.70%-50.30%, respectively. Compared with the recommended thresholds for describing the species level (ANI≤95-96%, AAI≤95-96%, dDDH≤70%), the ANI, AAI and dDDH values ​​of strain T2-19 and the reference bacterium Bacillus subtilis ATCC 6051 (GCA_000344745.1) were higher than the thresholds, indicating that strain T2-19 is Bacillus subtilis ( Bacillus subtilis ), hereinafter referred to as Bacillus subtilis ( Bacillus subtilis )T2-19.

[0049] Bacillus subtilis ( Bacillus subtilis ) T2-19 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 1, 2024, with the deposit number CGMCC NO. 31410.

[0050] Similarly, the 16S rRNA sequence of strain T3-7 is as follows:

[0051] Strain T3-7 was also identified as Bacillus subtilis ( Bacillus subtilis ), hereinafter referred to as Bacillus subtilis ( Bacillus subtilis ) T3-7, the strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on September 13, 2024, with the deposit number CGMCC NO. 31966.

[0052] Example 2, Bacillus subtilis ( Bacillus subtilis )The promoting effect of T2-19 and T3-7 on the mycelial growth of Stropharia rugosodium 1. Detection of the promoting effect of bacteria on the growth of Stropharia rugosotis hyphae Bacillus subtilis ( Bacillus subtilis ) T2-19 was inoculated into a nutrient medium (i.e., nutrient broth agar (Huankai Microbial, China)) and cultured at 30°C for 48 h. The cells were collected by centrifugation at 12,000 rpm for 1 min. The cells were washed twice with sterile water and then suspended in sterile water. The suspension was serially diluted with sterile water to obtain bacterial suspensions of different concentrations.

[0053] Take 100 μl of bacterial suspension (the bacterial concentration is 1.2 × 10 6 cfu / ml, 1.2×10 7 cfu / ml, 1.2×10 8 cfu / ml, 1.2×10 9 cfu / ml or 1.2×10 10 cfu / ml) was inoculated onto a completely sterilized sawdust medium (sterilized at 121°C for 60 minutes), and then a 0.8 cm diameter mycelial disk of the Stropharia capitis strain ACCC 51711 was inoculated at the same location. Sterile water was used instead of the bacterial suspension as a blank control for bacteria, and an agar disk was used instead of the mycelial disk for the blank control of Stropharia capitis. After inoculation, the cells were cultured in the dark at 25°C for 30 days. The mycelial growth rate was then detected at 16 days of culture. All experiments were performed at least in triplicate, and the results are presented as mean ± standard deviation.

[0054] After inoculation of Stropharia rugosodium, the mycelium growth rate was measured for 16 days. The results are as follows Figure 5 As shown in the figure, the five concentrations of Bacillus subtilis in the experiment all promoted the mycelium of Pleurotus eryngii. 9cfu / ml, the mycelium growth rate was the fastest, at 3.23mm / d, which was 46% higher than the control without Bacillus subtilis inoculation (2.21mm / d). When the inoculation concentration of Bacillus subtilis was reduced or increased, the promotion effect gradually weakened. This shows that in addition to the difference in strains, the inoculation concentration has a significant effect on the promotion effect of the strain, and the optimal inoculation concentration is very high.

[0055] According to the above method, Bacillus subtilis ( Bacillus subtilis )T2-19 replaced with Bacillus subtilis ( Bacillus subtilis )T3-7, other steps remain unchanged, the result is ( Figure 6 ) showed that at appropriate concentrations, Bacillus subtilis ( Bacillus subtilis ) T3-7 can also promote the growth of mycelium of Stropharia rugosotis.

[0056] 2. fhw detection In this experiment, 100 μl of the T2-19 bacterial suspension (bacterial concentration 1.2×10 9 cfu / ml) was inoculated on a completely sterilized sawdust medium, and then a 0.8 cm diameter mycelial disk of the Stropharia capitis strain ACCC 51711 was inoculated at the same location. An agar disk was used instead of the Stropharia capitis mycelial disk as a blank control of Stropharia capitis. After inoculation, the plate was cultured in the dark at 25°C. After the Stropharia capitis mycelium grew to the bottom of the sawdust medium, the bottom was tested for the presence of Bacillus subtilis.

[0057] The results showed that the number of Bacillus subtilis at the bottom of the colonization of Stropharia rugosus mycelium reached (5.25±2.11)×10 6 cfu / g. In the treatment without inoculation of Stropharia, Bacillus subtilis was only found at the top of the medium, and no Bacillus subtilis was detected at the bottom of the medium. This indicates that Bacillus subtilis can spread Stropharia hyphae as FHW over long distances in sawdust medium, but cannot spread over long distances in sawdust medium without inoculation of Stropharia.

[0058] 3. Enzyme activity determination This experiment used sawdust culture medium, the main component of which is lignocellulose. Cellulase, laccase and peroxidase are the main enzymes for edible fungi to degrade lignocellulose, and their activities play a decisive role in the degradation of the substrate and the growth rate of mycelium. In order to detect the effect of inoculation with Bacillus subtilis on the activities of these three enzymes in the culture of Stropharia rugosa, samples were taken from the upper and lower parts of the culture medium to measure the activities of the three enzymes.

[0059] This experiment set up two treatments to determine the Bacillus subtilis ( Bacillus subtilis) Effect of T2-19 on the lignocellulase activity of Stropharia capillaris. Treatment I: Inoculate the mycelial disk of Stropharia capillaris strain ACCC 51711 with a diameter of 0.8 cm on the sterilized sawdust medium; Treatment II: Inoculate 100 μl of the bacterial suspension obtained in step 1 (bacterial concentration of 1.2×10 9 cfu / ml) was inoculated on a sterilized sawdust medium, and then a mycelial disk of the Agrocybe globosum strain ACCC 51711 with a diameter of 0.8 cm was inoculated at the same position. After inoculation, it was cultured in the dark at 25°C for 16 days, and then samples were taken from the upper and lower parts respectively. Take 2 g of the culture that was full of mycelium, add 10 mL of phosphate buffer (0.1M, pH6.5), homogenize, centrifuge at 4°C, 12000 rmp for 5 minutes, and take the supernatant as a crude enzyme solution to detect enzyme activity. The DNS method was used to determine cellulase activity. The ABTS method was used to determine laccase activity. The resveratrol method was used to determine lignin peroxidase activity. The protein content of the enzyme extract was measured using the Bradford method (Bradford, 1976).

[0060] Cellulase activity was determined using sodium salt of carboxymethyl cellulose (CMC) (Sigma-Aldrich) as substrate. 750 μl of 1% CMC (50 mM sodium acetate, pH 4.6) and 250 μl of crude enzyme solution were thoroughly mixed and incubated at 25°C for 60 min. Released glucose was determined using the 3,5-dinitrosalicylic acid reagent (DNS) method. Definition of cellulase activity: One enzyme unit (U) is defined as the amount of enzyme required to release 1 μg of glucose per minute under the assay conditions.

[0061] Laccase activity was determined using the 2,2'-azido-bis(3-ethylbenzene-6-sulfonic acid) (ABTS, Sigma-Aldrich) method. 900 μL ABTS (50 mM sodium acetate buffer, pH 3.0) and 100 μL crude enzyme solution were mixed and the resulting reaction mixture was incubated at 25°C for 5 min. The reaction was terminated by adding 500 μL TCA (5%) and the absorbance at 420 nm was measured. Laccase activity definition: One enzyme unit (U) is defined as the amount of enzyme that oxidizes 1 μmol ABTS per minute.

[0062] Peroxidase was measured using the 2,6-dimethoxyphenol (DMP) method. 700 μl of sodium tartrate buffer (50 mM, pH 4.5), 100 μL of DMP (10 mM), 100 μL of MnSO4 solution (10 mM), 70 μl of crude enzyme solution were mixed thoroughly, and the reaction was started by adding 30 μl of hydrogen peroxide (10 mM). The reaction mixture was incubated at 25 °C for 5 minutes. The reaction was terminated by adding 500 μL of TCA (5%), and the absorbance at 465 nm was measured. Peroxidase enzyme activity definition: One enzyme unit (U) is defined as the amount of enzyme that oxidizes 1 μmol of DMP per minute.

[0063] The results are as follows Figure 7 As shown in the figure, the sampling position has a significant effect on the activities of cellulase, laccase and peroxidase. For cellulase, the enzyme activity at the top is higher than that at the bottom; while for laccase and peroxidase, the enzyme activity at the bottom is higher than that at the top. In addition, inoculation with Bacillus subtilis also has an effect on the activities of the three enzymes. Among them, the activities of cellulase and manganese peroxidase decreased, while the activity of laccase increased.

[0064] Laccase is not only involved in the degradation of lignocellulose, but also in the anti-stress response of fungi. Studies have shown that some fungi can produce a variety of resistance mechanisms when subjected to biological stress, including a rapid increase in laccase activity. For example, when the mycelium of Pleurotus ostreatus encounters microorganisms such as Trichoderma, Bacillus, and Escherichia coli, the laccase activity increases. This increase in laccase activity is regulated by the ROS signaling pathway. In the present invention, the effect of inoculation with Bacillus subtilis on the laccase activity of Pleurotus ostreatus is not significant, indicating that for Pleurotus ostreatus, Bacillus subtilis is a friend rather than an enemy.

[0065] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Preparation of Stropharia rugosodium Stropharia rugosoannulata ) Methods for producing bacterial strains, comprising: Bacillus subtilis ( Bacillus subtilis ) Inoculate on the culture medium of the oxalis caissonia species, and then inoculate the oxalis caissonia species ( Stropharia rugosoannulata ) to obtain Stropharia rugosotis ( Stropharia rugosoannulata ) strains.

2. The method according to claim 1, characterized in that: The culture medium for the Stropharia rugosoannulata strain is a sterile culture medium.

3. The method according to claim 1 or 2, characterized in that: The Stropharia officinalis ( Stropharia rugosoannulata ) The fungus species is Stropharia rugosotis ( Stropharia rugosoannulata ) original species or cultivar.

4. The method according to any one of claims 1-3, characterized in that: The Bacillus subtilis ( Bacillus subtilis ) is Bacillus subtilis ( Bacillus subtilis ) T2-19 or Bacillus subtilis ( Bacillus subtilis ) T3-7, the Bacillus subtilis ( Bacillus subtilis ) T2-19 in the General Microbial Center of the China Microbial Sprout Preservation Management Committee is CGMCC No. 31410, the Bacillus subtilis ( Bacillus subtilis ) The deposit number of T3-7 in the General Microbial Center of the China Microbial Sprout Preservation Management Committee is CGMCCNo.31966.

5. Improve the growth of Stropharia officinalis ( Stropharia rugosoannulata ) Growth rate methods include: Bacillus subtilis ( Bacillus subtilis ) Inoculate on the medium of large pellet caissonia, and then inoculate the medium of large pellet caissonia ( Stropharia rugosoannulata ) to increase the growth rate of Stropharia rugosotis ( Stropharia rugosoannulata ) growth rate.

6. The method according to claim 5, characterized in that: The Stropharia rugosoannulata culture medium is a sterile culture medium.

7. The method according to claim 5 or 6, characterized in that: The Bacillus subtilis ( Bacillus subtilis ) is Bacillus subtilis ( Bacillus subtilis ) T2-19 or Bacillus subtilis ( Bacillus subtilis ) T3-7, the Bacillus subtilis ( Bacillus subtilis ) T2-19 in the General Microbial Center of the China Microbial Sprout Preservation Management Committee is CGMCC No. 31410, the Bacillus subtilis ( Bacillus subtilis ) The deposit number of T3-7 in the General Microbial Center of the China Microbial Sprout Preservation Management Committee is CGMCCNo.31966.

8. Bacillus subtilis ( Bacillus subtilis ) T2-19, its collection number in the General Microbial Center of the China Microbial Sperm Preservation Management Committee is CGMCC No. 31410; or, Bacillus subtilis ( Bacillus subtilis ) T3-7, its collection number in the General Microbial Center of the China Microbial Sperm Preservation Management Committee is CGMCC No. 31966.

9. A fungic agent, whose active ingredient is Bacillus subtilis as claimed in claim 8 ( Bacillus subtilis ) T2-19 or the Bacillus subtilis ( Bacillus subtilis )T3-7.

10. Any of the following applications: X1. The Bacillus subtilis according to claim 8 ( Bacillus subtilis ) T2-19 or the Bacillus subtilis ( Bacillus subtilis ) T3-7 as a large mushroom ( Stropharia rugosoannulata ) Application of growth-promoting bacteria; X2. The Bacillus subtilis according to claim 8 ( Bacillus subtilis ) T2-19 or the Bacillus subtilis ( Bacillus subtilis )T3-7 in Stropharia officinalis ( Stropharia rugosoannulata ) Application in production; X3. The fungus agent as claimed in claim 9 in preparation of large ball caissoni ( Stropharia rugosoannulata ) Application of bacterial strains; X4. The fungal agent of claim 9, Pleurotus eryngii ( Stropharia rugosoannulata ) application in production.

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