Vercochaete versicolor and application thereof

Abstract

The invention relates to a vermicochaete versicolor and an application thereof. The name of the strain is Peniphora incarnata H46, the preservation number is CGMCC No.41169, the strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on February 5, 2024, and the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No.1 Yard, West Beichen Road, Chaoyang District, Beijing, China. The vermicochaete carnosus provided by the invention has lignin and cellulose degradation capability, has good enzyme production activity, and can be used for degrading ground combustibles.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a fungus and an application thereof. Background Art

[0002] Forest combustibles are the material basis for forest fires and are also a necessary condition for forest burning. my country is one of the countries with serious forest fires, and the forest fire situation is extremely severe. The degree of harm caused by forest fires, especially high-intensity forest fires, mainly depends on the amount of forest combustibles. Therefore, how to effectively reduce the amount of forest combustibles, thereby reducing and controlling forest fires, is an important task in modern forest fire management.

[0003] Forest floor combustibles are composed of cellulose, hemicellulose, and lignin, which are tightly combined in a certain proportion. Since lignin surrounds cellulose, it is very stable and not easy to degrade. Therefore, lignin degradation is the rate-limiting process of carbon cycle on the earth, and it is also one of the key issues that should be solved for the effective utilization of plant cellulose materials. Due to the crystalline structure of cellulose and its close combination with hemicellulose and lignin, the hydrolysis rate and saccharification rate of lignin, cellulose, and hemicellulose are greatly affected. Relying solely on microorganisms in the natural environment to degrade forest floor combustibles is inefficient and takes a long time to degrade. Therefore, if a strain that can efficiently degrade forest floor combustibles can be provided, it will be of great significance to the management of forest surface combustibles, the prevention of forest fires in forest areas, and the safety of forest resources and ecology. At present, there is no report on the degradation of lignin and cellulose by the flesh-colored spore-forming fungi. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a strain of Pseudomonas carnosus and application thereof.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] The present invention provides a Peniophora incarnata strain, the strain name is Peniophora incarnata H46, the preservation number is CGMCC No.41169, the strain was deposited in the General Microbiology Center (CGMCC) of the China Culture Collection Administration on February 5, 2024, and the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0007] The present invention has found through research that the strain H46 has the ability to degrade lignin and cellulose, has good enzyme production activity, and can be used for the degradation of ground cover combustibles.

[0008] The present invention provides a fermentation method of the above-mentioned Peniophora incarnata, which comprises inoculating a culture medium with Peniophora incarnata H46 for fermentation and culture.

[0009] Furthermore, in the culture medium, the carbon source is selected from one or more of sucrose, corn flour, soluble starch and glucose.

[0010] Furthermore, in the culture medium, the nitrogen source is selected from one or more of yeast powder, tryptone, soybean powder and malt extract powder.

[0011] Furthermore, in the culture medium, the inorganic salt is selected from one or more of calcium chloride, sodium chloride, magnesium sulfate and potassium dihydrogen phosphate.

[0012] Furthermore, in the culture medium, the carbon source content is 10 g / L, the nitrogen source content is 20 g / L, and the inorganic salt content is 3 g / L.

[0013] The use of the above culture medium is beneficial to improving the enzyme production activity of strain H46.

[0014] The invention provides a bacterial agent, comprising the above-mentioned Psoralea corylifolia or a bacterial suspension of Psoralea corylifolia.

[0015] The microbial agent provided by the present invention has the ability to degrade lignin and cellulose and has good enzyme production activity, can be used for degrading ground cover combustibles, and can increase the weight loss rate of forestland combustibles.

[0016] The invention provides a method for preparing the bacterial suspension of the flesh-colored pygmy worm, comprising the following steps: inoculating the flesh-colored pygmy worm into a culture medium, culturing, centrifuging, and taking a supernatant.

[0017] The bacterial suspension prepared by the above method can be used to degrade lignin and cellulose, has good enzyme production activity, can be used to degrade ground cover combustibles, and can increase the weight loss rate of forestland combustibles.

[0018] The present invention provides a method for preparing the bacterial agent, comprising the following steps: inoculating the above-mentioned fungus into a fermentation medium and performing fermentation culture.

[0019] Furthermore, the method may further include the step of centrifuging and taking out the supernatant.

[0020] The fermentation medium can refer to the medium used in the fermentation method of H46.

[0021] The present invention provides the use of the above-mentioned Mycosporum fruticosa in any one or more of (1) to (7),

[0022] (1) Degradation of lignin;

[0023] (2) preparing a preparation for degrading lignin;

[0024] (3) Degradation of combustible materials;

[0025] (4) Improving enzyme activity; for example, improving the activity of lignin-degrading enzymes and / or cellulose-degrading enzymes;

[0026] (5) Increase the weight loss rate of combustible materials in forest land;

[0027] (6) Degradation of cellulose;

[0028] (7) Preparation of cellulose-degradable preparations.

[0029] The present invention provides the use of the above-mentioned bacterial agent in any one of (1) to (5),

[0030] (1) Degradation of lignin;

[0031] (2) Degradation of combustible materials on the ground;

[0032] (3) Improving enzyme activity, for example, improving the activity of lignin-degrading enzymes and / or cellulose-degrading enzymes;

[0033] (4) Increase the weight loss rate of combustible materials in forest land;

[0034] (5) Degradation of cellulose.

[0035] The strain and the bacterial agent provided by the invention have the ability to degrade lignin and cellulose and have good enzyme production activity, and can be used for degrading ground cover combustibles.

[0036] The present invention provides a method for using the strain or the bacterial agent, comprising the following steps: using the fungus or the bacterial agent to treat lignin or a substance containing lignin.

[0037] The above-mentioned Mycoplasma fusca or the above-mentioned bacterial agent can also be used to treat cellulose or a substance containing cellulose.

[0038] The above method can also be used as a method for degrading lignin or cellulose, a method for degrading ground combustibles, a method for increasing the activity of lignin-degrading enzymes, and a method for increasing the weight loss rate of forest combustibles. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The proportion of each fungal genus.

[0040] Figure 2 Changes in lignin content in indoor degradable ground cover combustibles.

[0041] Figure 3 Changes in the indoor degradable cellulose content of ground cover combustibles.

[0042] Figure 4 This is the colony growth status of strain Red 1-3-4-⑥.

[0043] Figure 5 This is a phylogenetic tree based on rDNA-I TS sequences.

[0044] Figure 6 Changes in lignin degradation rate of ground cover combustibles under different bacterial agent treatments.

[0045] Figure 7 Changes in cellulose degradation rate of ground cover combustibles under different bacterial agent treatments. DETAILED DESCRIPTION

[0046] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0047] The present invention isolates 62 fungi from ground cover combustible samples of four forest stands, namely broad-leaved Korean pine forest, poplar-birch mixed forest, birch-larch mixed forest, and red spruce pure forest. The fungi are identified as 21 species belonging to 18 genera, among which Pseudocephalotaxus is the dominant genus, accounting for 55% of the total number of isolated strains. After qualitative screening, lignin-degrading fungi red 1-3-4-⑥ are obtained.

[0048] In order to clarify the degradation ability of the above-mentioned degradation strains on ground cover combustibles, indoor degradation tests were carried out with combustibles from spruce, poplar-birch, larch-birch, and broad-leaved Korean pine forests as degradation substrates. The results of the indoor degradation test showed that after 50 days of degradation by the strain Red 1-3-4-⑥, the mass loss rates of combustibles from larch-birch, spruce, and broad-leaved Korean pine forests reached 14.17%, 19.83%, and 17.67%, respectively, showing stronger degradation ability than other strains.

[0049] Based on the degradation results of lignin and cellulose in ground cover combustibles of different forest types, the strain Red 1-3-4-⑥ has a strong ability to degrade lignin and degrade cellulose, and can be used to prepare degradation agents.

[0050] After morphological and molecular biological identification, the strain Red 1-3-4-⑥ was identified as Peniophora incarnata, and was named H46. The strain was deposited in the General Microbiology Center (CGMCC) of the China Culture Collection on February 5, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, and the deposit number is CGMCC No.41169.

[0051] In order to further increase the degradation effect of the strains, the nutritional components of the two strains were optimized. According to the results of enzyme activity determination, the best carbon source was determined to be sucrose, the best nitrogen source was soybean powder, and the best inorganic salt was calcium chloride. The fermentation medium formula (g / L) was determined to include: sucrose 10.0, soybean powder 20.0, calcium chloride 3.0, Tween 80 0.5.

[0052] In order to verify the degradation effect of the degradation agent prepared by the strains with strong lignin and cellulose degradation ability under laboratory conditions under natural conditions, field degradation tests of ground cover fuels were carried out in birch, larch and Mongolian oak-Mongol pine forests. The results of the field degradation test showed that the degradation effect of lignin and cellulose in ground cover fuels of different forest types with the application of the agent was better than that of the control group. Among different doses, the degradation rate of lignin and cellulose in ground cover fuels was ranked as high dose > medium dose > low dose > control group.

[0053] Unless otherwise specified, the experimental methods used in the present invention are all conventional methods in the art; the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art, and those skilled in the art can obtain them through commercial channels or prepare them by conventional methods.

[0054] The research area of ​​the present invention is located in the Yitong Forestry Pest Natural Enemy Breeding Demonstration Base of Jilin Academy of Forestry Sciences (124°50′-125°46′E, 43°03′-43°38′N), with an altitude of about 300m. The area belongs to the mid-temperate continental monsoon climate, with an average annual temperature of 2.8°C and an average annual precipitation of 723mm, mainly concentrated in June-August, and a frost-free period of 120-140d. The soil is mainly dark brown forest soil, with an average soil layer thickness of 40-45cm and an average humus layer thickness of 10cm. The main forests in the research area are Fraxinus mandshurica forest, Querus mongolica forest, Betula platyphylla forest, broad-leaved Korean pine forest, mixed wood forest, etc.

[0055] PDA medium was prepared according to the following proportions: 200 g potato, 20 g glucose, 20 g agar, 3 g KH2PO4, 1.5 g MgSO4·7H2O, 0.01 g VB1, and 1000 mL distilled water.

[0056] The PDA-guaiacol medium was prepared according to the following ratio: guaiacol was added to the PDA medium, and the final concentration of guaiacol was 0.02% (volume percentage).

[0057] The PDA-aniline blue medium was prepared according to the following ratio: aniline blue was added to the PDA medium, and the final concentration of aniline blue was 0.1 g / L.

[0058] PDB medium was prepared according to the following proportions: 200 g potato, 20 g glucose, 3 g KH2PO4, 1.5 g MgSO4·7H2O, 0.01 g VB1, and 1000 mL distilled water.

[0059] The malt extract powder medium was prepared according to the following proportions: glucose 10.0 g, malt extract powder 20.0 g, Tween 80 (Tween80) 0.5 g, KH2PO4 3.0 g, and distilled water 1000 mL.

[0060] The basal culture medium was prepared in the following proportions: glucose 10.0 g, malt extract powder 20.0 g, potassium dihydrogen phosphate 3.0 g, Tween 80 0.5 g, distilled water 1000 mL, natural pH.

[0061] In the embodiment, primer I TS1 and primer I TS4 were both synthesized by Shanghai Bioengineering.

[0062] In the embodiments, if data statistics and analysis are involved, Excel and Organization software are used to perform statistical analysis on the data.

[0063] The following is an introduction through specific embodiments.

[0064] Example 1 Screening of efficient forest fuel degradation bacteria

[0065] Sample plot setting and sample collection: In 2022, representative sites were selected as test plots from four forest stands, including broad-leaved Korean pine (Pinus koraiensis) forest, poplar-birch mixed forest, birch-larch (Larix gmelinii) mixed forest, and red spruce (Picea koraiensis) pure forest, at the Yitong Forest Pest Natural Enemy Breeding Demonstration Base of Jilin Academy of Forestry Sciences. The forest stands were divided into four types, namely broad-leaved Korean pine (Pinus koraiensis) forest, poplar-birch mixed forest, birch-larch (Larix gmelinii) mixed forest, and red spruce (Picea koraiensis) pure forest. Five 1m×1m small sample plots were randomly set up in each forest type sample plot, and combustibles in the undecomposed layer (A01) and the decomposed layer (A02) were collected as the source of degradation bacteria and degradation test samples.

[0066] Isolation and identification of strains: After the collected samples of different decomposition layers were crushed appropriately, they were placed in sterile water for oscillation and mixing, and the mother liquor was diluted into a series of concentration gradients (i.e., the mother liquor was diluted into a series of concentration gradients by 10, 20, and 30 times), and the dilutions were spread on PDA medium plates containing streptomycin (streptomycin concentration was 200 μg / L), and placed in a 28°C constant temperature incubator for 5 days. Single colonies with significant differences in morphological characteristics were picked, and pure strains were obtained after repeated separation and purification and numbered and preserved. Fungal genomic DNA was extracted, and PCR amplification was performed using primers I TS1 (TCCGTAGGTGAACCTGCGG, shown in SEQ ID NO: 1) and I TS4 (TCCTCCGCTTATGATATGC, shown in SEQ ID NO: 2). After 1% (mass volume percentage) agarose gel electrophoresis, the unpurified product was entrusted to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing. According to the I TS sequencing results, Blast was used for sequence alignment at NCBI, and a phylogenetic tree was constructed using MEGA5.1 software in combination with the existing related I TS sequences in GenBank.

[0067] A total of 62 fungal strains were isolated by the above method, and the isolated strains were classified and identified by combining morphological observation and IT-rDNA sequencing. After identification, a total of 21 species were classified into 18 genera: Aspergillus, Cerrena, Coniochaeta, Coniothyrium, Discosia, Fusarium, Phoma, Paraconiothyrium, Paramyrothecium, Peniophora, Pestalotiopsis, Phlebiopsis, Plectosphaerella, Trichoderma, Microdiplodia, Myrothecium, Schizophyllum and Letendraea. Among them, Pseudocephalosporium was the dominant genus, accounting for 55% of the total number of isolated strains, followed by Pseudocephalosporium and Pseudocephalosporium, accounting for 11% and 6% of the total number of isolated strains, respectively. Figure 1 ).

[0068] Screening of lignin-degrading fungi: One strain was selected from each of the 21 identified species as a representative strain for screening of lignin-degrading fungi. The guaiacol plate colorimetric method and the aniline blue plate decolorization method were used to evaluate whether the strain had the ability to secrete extracellular lignin-degrading enzymes.

[0069] The guaiacol plate colorimetric method comprises the following steps: preparing a bacterial cake at the edge of a colony of a pure strain, inoculating it in the center of a PDA-guaiacol plate, and culturing it at a constant temperature of 28°C. After the strain has grown for 5 days, picking a colony with an obvious color circle formed around it, measuring the diameter of the colony and the color circle on the PDA-guaiacol medium with a vernier caliper, and screening out a strain with a larger ratio of the color circle to the colony diameter.

[0070] The aniline blue plate decolorization method includes the following steps: inoculating the screened bacterial beads onto a PDA-aniline blue plate, culturing at a constant temperature of 28°C, observing and recording the formation time of the decolorization zone, and measuring the colony and decolorization zone diameter on the PDA-aniline blue culture medium with a vernier caliper.

[0071] The guaiacol colorimetric method can better reflect the laccase activity of the strain, while the decolorization of aniline blue is related to the production of lignin peroxidase and manganese peroxidase. Through observation and calculation, only strains of Phlebiopsis, Cerrena, Paraconiothyrium, Coniothyrium, Letendraea, and Peniophora among the 18 genera can produce color circles by oxidizing guaiacol. Some experimental data are shown in Table 1. Among them, the φC / φF of the strain Red 1-3-4-⑥ is greater than 1 and the diameter of the fungal color circle is larger. It is speculated that it may be a lignin-degrading fungus with high laccase activity. The strain Red 1-3-4-⑥ can decolorize aniline blue, indicating that it can produce peroxidase. It is preliminarily judged that the strain is a lignin-degrading fungus.

[0072] Table 1 Guaiacol plate color development and aniline blue plate decolorization results

[0073]

[0074]

[0075] Note: φF: colony diameter; φC: color development zone diameter; φD: decolorization zone diameter

[0076] Example 2 Indoor Degradation Test of Forest Floor Combustibles

[0077] The fungi screened in Example 1 were inoculated onto a PDA plate and cultured at 28°C for 7 days. A fungus cake was prepared in the area where mycelium was growing vigorously using a hole puncher (φ=6 mm). The fungus cake was inoculated into a conical flask containing 100 mL of PDB culture medium and cultured at 28°C and 160 rpm for 7 days. The culture was centrifuged at 4000 rpm for 10 min, and the supernatant was taken as the fungus suspension.

[0078] The surface combustible sample collected from the undecomposed layer in Example 1 was dried to constant weight and cut into pieces. 2 g of the pieces were weighed and placed in a flask (the volume of the flask was 50 mL) containing 20 mL of malt extract powder culture medium, and inoculated with 600 μL of bacterial solution (i.e., the bacterial suspension prepared by the above method) for the determination of the mass loss of combustibles and the lignin content during the degradation process.

[0079] Prepare another triangular flask containing combustible samples of different forest types and add sterile water equal to the amount of bacterial solution as a control.

[0080] The above triangular flasks were placed in an artificial incubator and cultured at 25°C and 80% humidity. Sampling began on the 10th day after inoculation, and three samples from triangular flasks treated with different bacterial solutions were taken for testing each time. Sampling was performed once every 10 days, and the last sampling was performed on the 60th day.

[0081] The mass loss rate detection method comprises the following steps: taking out the degraded ground cover combustible material sample from a triangular flask, removing the surface hyphae with tweezers, drying to constant weight, and calculating the mass loss rate of the sample after degradation.

[0082]

[0083] The changes in the mass loss of ground cover fuels during indoor degradation are shown in Table 2. During the indoor degradation process, the mass loss of ground cover fuels in each forest type showed an overall fluctuating upward trend, with a more obvious weight loss in the first 10 days. After 10 days of degradation by strain Hong 1-3-4-⑥, the mass loss rate of larch-birch fuels reached 16.33%. There were differences in the mass loss rate of fuels in different forest types. After 50 days of degradation by strain Hong 1-3-4-⑥, the mass loss rates of larch-birch, spruce and broad-leaved Korean pine forests reached 14.17%, 19.83% and 17.67%, respectively, showing a stronger degradation ability than other strains.

[0084] Table 2 Changes in mass loss of indoor degradation of ground cover combustibles

[0085]

[0086] Qingdao Stand Testing Group Co., Ltd. used the ELISA double antibody sandwich method to determine the lignin content and cellulose content of ground cover combustible samples during the degradation process, and calculated the lignin degradation rate and cellulose degradation rate.

[0087]

[0088] During the indoor degradation process, the lignin content in the ground cover combustibles showed obvious differences under different bacterial species treatments ( Figure 2). In the first 10 days, except for the broad-leaved Korean pine forest fuel, the lignin content in the fuels of other forest types decreased rapidly, which was consistent with the results of fuel mass loss in the same stage. The weight loss of fuel samples may be partly attributed to the degradation of lignin; among them, in the 50d-60d stage, the lignin content in the broad-leaved Korean pine forest, larch-birch and spruce fuels degraded by the strain red 1-3-4-⑥ decreased most significantly, and the broad-leaved Korean pine, larch-birch and spruce fuels treated with a few strains such as YY-3-3-⑥ and 1-2-1 decreased significantly. The lignin content in the combustibles of Korean pine and larch-birch increased to varying degrees, showing an enrichment phenomenon; considering the entire degradation process, the degradation effect of lignin in spruce combustibles and larch-birch combustibles was better than that in poplar-birch and broad-leaved Korean pine combustibles. After 60 days of degradation by the strain Red 1-3-4-⑥, the degradation rates of spruce and larch-birch combustibles were as high as 23.42% and 15.72%, respectively. Compared with other strains, the strain Red 1-3-4-⑥ showed a stronger degradation ability.

[0089] Changes in cellulose content in ground cover fuels of different forest types Figure 3 Except for spruce, the cellulose content of red 1-3-4-⑥ in larch-birch, poplar-birch and broad-leaved Korean pine decreased significantly during 0-10 days, among which the largest decrease was in the fuel of broad-leaved Korean pine forest.

[0090] Based on the results of the indoor degradation test of ground cover combustibles, the strain Red 1-3-4-⑥ showed a relatively high ability to degrade lignin. Although the strain 2-1-24 screened in the early stage has the ability to degrade both lignin and cellulose, its growth rate is slow during the cultivation process. Therefore, Red 1-3-4-⑥ was selected as the source strain for the preparation of the microbial agent and applied to the field degradation test.

[0091] Example 3 Bacterial species identification

[0092] The strain Red 1-3-4-⑥ was inoculated on PDA medium and cultured at 28℃ for 7 days. The surface of the colony of the strain Red 1-3-4-⑥ was white cotton-like ( Figure 4 ), the hyphae in the new growth area are uniform and sparse, the culture medium turns dark brown in the later growth stage, and the colony grows slowly.

[0093] The ITS sequence of strain Hong 1-3-4-⑥ was compared with the model strain and closely related strains in GenBank, and the strain was identified as Peniophora incarnata. The results of phylogenetic tree analysis are shown in Figure 5 .

[0094] The strain Red 1-3-4-⑥ was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on February 5, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China. The strain name is Peniophora incarnata H46, and the deposit number is CGMCC No. 41169.

[0095] Example 4 Optimization of culture medium components

[0096] The following ingredients were selected as the carbon source, nitrogen source, and inorganic salt to replace the glucose, malt extract powder, and potassium dihydrogen phosphate in the basal culture medium, respectively.

[0097] Carbon source: Sucrose, corn flour, and soluble starch were used to replace glucose in the basal medium;

[0098] Nitrogen source: yeast powder, tryptone, and soybean powder were used to replace the malt extract powder in the basal medium;

[0099] Inorganic salts: Calcium chloride, sodium chloride, and magnesium sulfate replace the potassium dihydrogen phosphate in the basal culture medium.

[0100] The preparation of H46 seed solution includes the following steps: preparing a bacterial cake at the edge of the colony of the strain, inoculating it into PDB medium, culturing it at 28°C and 160rpm for 5 days, centrifuging it at 4000rpm for 10 minutes, taking the supernatant, and obtaining the strain seed solution.

[0101] The seed liquid of H46 was inoculated into a triangular flask (volume of the triangular flask was 250 mL) containing 50 mL of the corresponding culture medium, with an inoculation amount of 3% (volume percentage), and cultured at 28°C and 180 rpm for 3 days. After centrifugation, the supernatant was taken to determine the enzyme activities of lignin peroxidase, manganese peroxidase, laccase, carboxymethyl cellulase, filter paper enzyme and β-glucosidase. The enzyme activity detection was carried out by Qingdao Kechuang Quality Inspection Co., Ltd. Based on the results of multiple enzyme activity determinations, the best carbon source, nitrogen source and inorganic salt were selected as the new fermentation medium formula to provide a source of nutrients for the preparation of field degradation agents.

[0102] After testing, using basic culture medium, the lignin peroxidase activity of strain H46 was 449.93U / L, the manganese peroxidase activity was 46.98U / L, and the laccase activity was 111.57U / L.

[0103] By replacing the carbon source, nitrogen source and inorganic salt in the basic culture medium, the effects of different components on the ability of strain H46 to produce lignin and cellulose degrading enzymes were analyzed, and the results are shown in Table 3. When sucrose was selected as the carbon source, the MnP of strain H46 reached the maximum, which was 42.39U / L; when soybean powder was used as the nitrogen source, the Li P of strain H46 reached the maximum, which was 580.24U / L; when calcium chloride was used as the inorganic salt, the CMCase and MnP of strain H46 had the highest activity. In summary, the carbon source, soybean powder and calcium chloride are the most suitable carbon source, nitrogen source and inorganic salt for enzyme production of strain H46, respectively, so as to determine the optimized fermentation medium (g / L): sucrose 10.0, soybean powder 20.0, calcium chloride 3.0, Tween 80 0.5. The above formula will be used as the source of nutrients for the preparation of bacterial agents. When sucrose was used to replace the carbon source, soybean meal to replace the nitrogen source, and calcium chloride to replace the inorganic salt, the lignin peroxidase activity of strain H46 was 534.73, the manganese peroxidase activity was 61.27 U / L, and the laccase activity was 139.54 U / L.

[0104] Table 3 Enzyme activity of strain H46 in different culture medium components

[0105]

[0106] Note: The units of lignin peroxidase, manganese peroxidase, laccase and filter paper enzyme are U / L, and the units of carboxymethyl cellulase and β-glucosidase are IU / L.

[0107] Example 5 Field Degradation Test of Forest Ground Combustibles

[0108] The field degradation test was conducted in the pure forests of birch and larch and the mixed forest of Quercus mongolica and Pinus sylvestris at the Yitong Base of Jilin Academy of Forestry Sciences. A 10m×10m standard plot was set for each forest type, and three 1m×1m small plots were set in each standard plot. Before the start of field degradation, the ground cover combustibles in the plot were collected, dried to constant weight, and then put into nylon mesh bags, 10.0g per bag, and the forest type and sample number were marked on the bag. The initial mass was weighed and recorded, and then put back into the plot and fixed with a metal mesh.

[0109] The screened strain H46 was selected as the source strain for preparing the bacterial agent.

[0110] The bacterial agent prepared by strain H46 can be called H46 bacterial agent. The preparation method of H46 bacterial agent includes the following steps: opening a plate of pure cultured strain H46 in a sterile operating table, using a sterilized puncher (outer diameter 6mm) to prepare bacterial cakes in the vigorous mycelium growth area, inoculating 40 bacterial cakes into a 1000mL triangular flask containing 400mL of the optimized fermentation medium, and culturing at 28°C and 160rpm for 5 days, centrifuging at 4000rpm for 10min, and obtaining a supernatant, which can be used as a bacterial suspension of the degradation strain, and bottled separately for later use. Three doses (low dose of 50mL, medium dose of 100mL, and high dose of 150mL) are divided into three doses (low dose of 50mL, medium dose of 100mL, and high dose of 150mL) and spraying different amounts of bacterial agents on the nylon mesh bags in the small sample square by manual spraying. The spraying should be as uniform as possible so that the combustibles in the bag can contact the bacterial agent. Each standard plot of the three forest types is treated in the same way. Combustible samples were collected once every 14 days, with 5 bags taken each time, for a total of 6 samplings, and the mass loss rate, lignin and cellulose content of the samples were determined.

[0111] The detection methods of mass loss, lignin degradation rate and cellulose degradation rate can all refer to Example 2.

[0112] Changes in the degradation rate of lignin in ground cover fuels under different bacterial agent treatments Figure 6 As shown in the figure, during the whole field degradation stage, the degradation rate of lignin in ground cover fuels of different forest types treated with three doses of H46 increased with the extension of degradation time, and was higher than that of the control group (the control group CK did not use the agent). By the end of the degradation experiment, the degradation rates of lignin in the fuels of birch, larch, and Mongolian oak-Mongol pine forests reached 16.62%-21.93%, 13.11%-15.81%, and 14.39%-18.52%, respectively, which were 1.15-1.84 times, 1.84-2.43 times, and 1.04-2.62 times higher than the control. For ground cover fuels of the same forest type, there were also differences between different doses of the agent H46. In the first 14 days of degradation, the lignin degradation rate increased significantly, and the highest degradation rates of the three types of forest fuels by the bacterial agent H46 reached 12.36% (birch), 13.98% (larch), and 6.95% (Quercus mongolica-Pinus sylvestris); after that, the lignin degradation rate increased and fluctuated. Overall, the lignin degradation effect between different doses was high dose > medium dose > low dose > control group. After 84 days of degradation, the degradation rates of lignin in birch fuel, larch fuel, and Quercus mongolica-Pinus sylvestris by high dose bacterial agent H46 were 21.93%, 15.81%, and 18.52%, respectively.

[0113] Changes in the degradation rate of cellulose in ground cover fuels under different bacterial agent treatments Figure 7As shown in the figure, with the extension of degradation time, the cellulose degradation rate of the combustibles in different forest types after being treated with the bacterial agent H46 showed an increasing trend, and was significantly higher than the control group. At the end of the field degradation, the cellulose degradation rates of the combustibles in the birch, larch, Mongolian oak-Mongol pine forests were 11.12%-20.59%, 7.61%-12.68%, and 12.34%-18.66%, respectively, which were 0.99-2.69 times, 1.45-3.08 times, and 3.32-5.53 times higher than the control, respectively. The cellulose degradation effect of the bacterial agent H46 at different doses was high dose > medium dose > low dose > control group.

[0114] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Psoralea corylifolia, characterized in that: The strain name is Peniophora incarnata H46, and the preservation number is CGMCC No.41169.

2. The fermentation method of the fungus according to claim 1, characterized in that: The following steps are involved: Peniophora incarnata H46 was inoculated into the culture medium for fermentation.

3. The fermentation method of the fungus according to claim 2, characterized in that: In the culture medium, the carbon source is selected from one or more of sucrose, corn flour, soluble starch and glucose.

4. The fermentation method of the fungus according to claim 2 or 3, characterized in that: In the culture medium, the nitrogen source is selected from one or more of yeast powder, tryptone, soybean powder and malt extract powder.

5. The fermentation method of Pseudomonas carnosus according to claim 2 or 3, characterized in that: In the culture medium, the inorganic salt is selected from one or more of calcium chloride, sodium chloride, magnesium sulfate and potassium dihydrogen phosphate.

6. The fermentation method of Pseudomonas carnosus according to any one of claims 2 to 5, characterized in that: In the culture medium, the carbon source content is 10 g / L, the nitrogen source content is 20 g / L, and the inorganic salt content is 3 g / L.

7. A bacterial agent, characterized in that The invention comprises the fungus suspension of the fungus fusobacterium or the fungus suspension of the fungus fusobacterium as claimed in claim 1.

8. The method for preparing the bacterial agent according to claim 7, characterized in that: The method comprises the following steps: inoculating the fungus described in claim 1 into a fermentation medium and performing fermentation culture.

9. Use of the fungus of claim 1 in any one or more of (1) to (7), (1) Degradation of lignin; (2) preparing a preparation for degrading lignin; (3) Degradation of combustible materials; (4) Improve enzyme activity; (5) Increase the weight loss rate of combustible materials in forest land; (6) Degradation of cellulose; (7) Preparation of cellulose-degradable preparations.

10. Use of the bacterial agent according to claim 7 in any one of (1) to (5), (1) degrading lignin; (2) Degradation of combustible materials on the ground; (3) Improve enzyme activity; (4) Increase the weight loss rate of combustible materials in forest land; (5) Degradation of cellulose.

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