Penicillium cyclopium capable of efficiently degrading straw fibers and application of penicillium cyclopium

Abstract

The invention discloses Penicillium cyclopium capable of efficiently degrading straw fibers and application of the Penicillium cyclopium. According to the method, a nitrosoguanidine method is adopted for mutagenesis of a Penicillium cyclopium strain MP-03 (2554-2) (CGMCC No.41542) to obtain a mutant strain deltaMP-03 (455-1) (CGMCC No.41541) capable of strongly degrading corn stalks, and by optimizing the nitrogen-carbon ratio, culture time, temperature and pH range conditions of an optimal enzyme-producing culture medium, compared with a non-mutant strain 2554-2, the mutant strain deltaMP-03 (455-1) capable of strongly degrading the corn stalks has the advantages that the mutant strain deltaMP-03 (455-1) capable of strongly degrading the corn stalks is obtained, and the mutant strain deltaMP-03 (455-1) capable of strongly degrading the corn stalks is obtained; the 455-1 mutant strain obviously improves the degradation rate of corn straws and the activity of endo-cellulase and exo-cellulase. According to the method disclosed by the invention, enlarged culture conditions of the 455-1 mutant strain are optimized, a novel organic coupling cultivation technology of the greenhouse vegetable 455-1 mutant strain fermentation liquor, the corn straw, the organic fertilizer and the like is researched and developed, and the growth vigor, the root development and photosynthetic efficiency and the resistance to soil-borne oomycetes diseases of greenhouse tomatoes, peppers and cucumbers are remarkably improved.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms and their applications, in particular to Penicillium circumflexum for efficiently degrading straw fibers and applications thereof. Background Art

[0002] The matrix of crop straw is mainly composed of cell walls, which contain a large amount of carbohydrates, a small amount of protein and minerals. The carbohydrates mainly include cellulose, hemicellulose and pectin polysaccharides. Returning straw to the field can increase soil organic matter, improve soil physical and chemical properties, and reduce the loss of phosphorus and potassium nutrients in straw to a certain extent. Crop straw is mainly composed of lignocellulose, which is difficult to be effectively decomposed by microorganisms under natural soil conditions, and the effect of fertilizing the soil is significantly reduced. In addition, the degradation of straw under soil conditions requires a certain carbon-nitrogen ratio, which can easily cause the straw to compete with crops for nitrogen fertilizer and affect the growth of vegetables. Therefore, based on the selection or improvement of efficient straw-degrading microbial strains, it is easy to develop a new type of organic coupling cultivation technology for microbial suspension degradation of crop straw matrix. At present, the screening and application technology of crop straw biodegradation microbial strains have been carried out, and some fungal strains that can degrade crop straw have been screened, including Pleurotus spp., Trichoderma spp., Penicillium spp., Aspergillus spp. and some bacterial complex systems, among which Aspergillus fumigatus XC6 strain has a degradation rate of 72%, 93% and 84% for straw cellulose, hemicellulose and crop straw, respectively. Thermostatic bacterial strains, mesophilic actinomycete strains and mesophilic fungal strains with strong cellulose decomposition ability have been isolated from soil, horse manure, cow manure and other matrices, and their effects on the cellulase activity of rice straw matrix have been determined. There is relatively little research on Penicillium biodegradation of crop straw, but existing studies have shown that a few Penicilliums have a strong ability to degrade lignocellulosic substances in crop straw, and actinomycetes with strong degradation ability are widely distributed under different substrate conditions such as soil, mature compost, horse manure and rotten wood. Penicillium has a simple structure and single-cell conidia, which is convenient for large-scale optimization and reproduction. Based on screening, we obtained Penicillium strains with high degradation of crop straw lignin, defined the optimal nitrogen-carbon ratio of the enzyme-producing culture medium, culture time, temperature and pH range, and ensured the effective development and application of the beneficial Penicillium. Summary of the invention

[0003] The invention aims to provide a Penicillium circumflexum for efficiently degrading straw fibers and application thereof.

[0004] The invention separates a Penicillium strain MP-03 (2554-2) for improving the cellulase activity of corn straw from corn straw, which is identified as Penicillium cyclopium, and uses the nitrosoguanidine method to induce mutation in the 2554-2 strain, and screens out a strong corn straw degradation mutant ΔMP-03 (455-1). The strains 2554-2 and 455-1 are now deposited in the General Microbiology Center of China Microbiological Culture Collection Committee, with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, the deposit number of strain 2554-2 is CGMCC NO.41542, the deposit number of strain 455-1 is CGMCC NO.41541, and the deposit date is October 12, 2024.

[0005] In a first aspect, the present invention provides a Penicillium cyclopium that can efficiently degrade straw fiber, which is Penicillium cyclopium 455-1, with a preservation number of CGMCC No.41541.

[0006] In a second aspect, the present invention provides a bacterial agent containing the Penicillium circumflexus 455-1.

[0007] In a third aspect, the present invention provides the use of Penicillium Circularis 455-1 in producing cellulase.

[0008] In a fourth aspect, the present invention provides the use of the Penicillium arcuate 455-1 or a bacterial agent containing the fungus that significantly improves the cellulase activity in corn stalk degradation.

[0009] In a fifth aspect, the present invention provides the use of the Penicillium arcuate 455-1 or a bacterial agent containing the same in improving the enzymatic activities of endo-cellulase and exo-cellulase in crop straw.

[0010] The crop straws described in the present invention include but are not limited to corn straws.

[0011] In a sixth aspect, the present invention provides a method for degrading corn stalks, which uses corn stalks as raw materials, is inoculated with a bacterial suspension of Penicillium arcus 455-1, and degrades the corn stalks at 20-40°C and an initial pH of 6.0-8.0 (preferably 30°C and an initial pH of 7.0).

[0012] Furthermore, a proper amount of farmyard manure or bacterial fertilizer is added to the raw materials.

[0013] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0014] The present invention uses the nitrosoguanidine method to induce the arc Penicillium MP-03 strain to obtain a strong corn stalk degradation mutant strain 455-1, and by optimizing the nitrogen-carbon ratio of the culture medium with the highest enzyme activity, the culture time, the temperature and the pH range conditions, the 455-1 mutant strain significantly improves the corn stalk degradation rate, the endocellulase and the exocellulase activities compared with the non-mutant strain MP-03. The present invention also optimizes the conditions for expanding the culture of the 455-1 mutant strain, and develops an organic coupling cultivation technology of the 455-1 mutant strain fermentation liquid + corn stalk + organic fertilizer for greenhouse vegetables, which significantly improves the growth of greenhouse tomatoes, peppers, cucumbers, root development and photosynthetic efficiency, and the performance of inhibiting the occurrence and damage of soil-borne oomycete diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Comparison of morphological characteristics of the ΔMP-03 (A) and MP-03 (B) strains of the present invention.

[0016] Figure 2 It is the agarose gel electrophoresis diagram of the complete ITS sequence of the MP-03 and ΔMP-03 strains of the present invention.

[0017] Figure 3 The degradation effects of ΔMP-03 and MP-03 strains on corn straw in the preferred embodiments of the present invention are shown in Figure 2. Different letters indicate significant differences (P>0.05), and the same letters indicate insignificant differences.

[0018] Figure 4 The effects of temperature (a), initial pH (b) and different added ingredients (c) on the degradation of corn straw by ΔMP-03 and MP-03 in the preferred embodiment of the present invention. Different asterisks in Figure c represent the significant differences in the degradation of corn straw **P<0.01, ***P<0.001, and each experiment was repeated at least three times.

[0019] Figure 5 This is the effect of ΔMP-03 and MP-03 on the activity change of corn straw endocellulase in the preferred embodiment of the present invention.

[0020] Figure 6 This is the effect of ΔMP-03 and MP-03 on the activity change of corn straw exocellulase in the preferred embodiment of the present invention. DETAILED DESCRIPTION

[0021] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0022] The data processing method in the following examples uses Office 2003 software Excel for data processing, tabulation and drawing, and PPSS 18.0 statistical software is used for data significance analysis.

[0023] Example 1 Identification and mutation of Penicillium arcus MP-03 and optimization of corn straw fermentation technology

[0024] 1. Materials and Methods

[0025] 1.1 Test materials

[0026] Several Penicillium strains were isolated from corn fields in Tai'an, Shandong Province in autumn and stored in a refrigerator at 4°C. Fungal genomic DNA rapid extraction kit: purchased from Shanghai Biotech.

[0027] The corn stalks came from the corn fields in Taishan District, Tai'an City, Shandong Province in the autumn of 2022. Fresh and mold-free stalks were selected, washed, dried, crushed, and passed through a 20-mesh sieve for later use.

[0028] Solid slant medium: Potato dextrose agar medium PDA: 15g agar, 20g glucose, 200g potato, 1000mL water. Liquid medium PD: 20g glucose, 200g potato, 1000mL water.

[0029] 1.2 Enrichment, isolation and screening of corn straw-degrading strains

[0030] Enrichment culture: Take 10g corn stalk pellet sample and add it to 90mL sterile water for full shaking, mix well and let it stand for 10min; then take 1mL suspension and inoculate it into 9mL PD culture medium, add filter paper strip as the appearance indicator of decomposition, and let it stand at 30℃ for culture. When the filter paper strip is completely decomposed, take 1mL culture medium and transfer it to 9mL fresh PD culture medium. Continue to pass the culture medium that has lost the ability to decompose the filter paper, and keep the culture medium with strong decomposition ability for single bacteria isolation.

[0031] Single colony isolation: dilute the selected enrichment solution in gradients and evenly apply it on a PDA solid plate, then place the plate in a 28°C constant temperature incubator for 2-3 days; pick single colonies with different morphologies and perform multiple single plate culture. Finally, separate and obtain single colonies and transfer them to PDA slant test tubes and store them at 4°C. Inoculate the obtained multiple single bacteria into PD liquid culture medium respectively, and use the complete disintegration of filter paper as an indicator to screen out candidate strains that may have the ability to degrade corn stalks.

[0032] 1.3 Methods for identification of bacteria

[0033] Morphological observation: Observation of morphological characteristics of the inoculum: Take the activated inoculum and streak it on the PDA plate, culture it in a 28℃ incubator for 24 hours, insert a sterilized slide on the plate with the grown inoculum at an angle of 45 degrees, continue to culture for 48 hours, then gently take it out and observe the morphological characteristics such as mycelium, conidia and conidiophores under a microscope, and take photos of the group plates.

[0034] Molecular biological identification: For the extraction of mycelial DNA, a 250 mL conical flask containing 100 mL of PD medium was sterilized with high pressure steam at 121°C for 20 min, cooled to room temperature, inoculated with the fungus species Penicillium circumflexum MP-03, and cultured in a shaking table at 28°C and 150 r / min for 2 days. The mycelium was collected by centrifugation, ground with liquid nitrogen, and then DNA was extracted using a fungal genomic DNA rapid extraction kit and stored in a -80°C refrigerator for later use.

[0035] PCR amplification: The extracted DNA was pre-denatured at 94°C for 5 min using the ITS forward primer (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and reverse primer (5′-TCCTCCGCTTATTGATATGC-3′) for identifying Penicillium circumflexus; denatured at 95°C for 1 min, annealed at 55°C for 1 min, and extended at 72°C for 1 min, and amplified for 35 cycles.

[0036] Purification of PCR amplification products: Detect the DNA amplification products by 5% agarose gel electrophoresis, cut out the gel fragments, and purify the target DNA fragments using an agarose gel DNA recovery kit.

[0037] DNA sequencing and Blast analysis: The gel-cutting products were sent to Shanghai Biotechnology Co., Ltd. for sequencing. The sequencing results were analyzed by Blast software for homologous sequence comparison of the target gene sequence.

[0038] Sequence analysis: The gene sequences of the corresponding strains with high homology to the tested Penicillium strains were downloaded from the GenBank database using the Blast software for comparison analysis. The MP-03 strain isolated was identified by combining the morphological characteristics and molecular DNA sequence comparison results.

[0039] 1.4 Mutation Induced by Nitrosoguanidine Method in MP-03 Strain

[0040] Nitrosoguanidine (NTG, N-methyl-N'-nitro-N-nitrosoguanidine) is an effective chemical mutagen, which has a strong mutagenic effect at extremely low concentrations that kill bacteria and is known as a super mutagen. The mutagenic effect of nitrosoguanidine is mainly to cause the conversion of GC→AT in the DNA chain.

[0041] The mutagenesis steps are as follows:

[0042] 1) Pick a loop of the MP-03 strain from the slant and inoculate it into a test tube containing 5 mL of PD culture medium, incubate it at 25°C with shaking for 48 hours, take 1.0 mL of overnight culture medium into another test tube containing 5 mL of PD culture medium, incubate it at 25°C with shaking for 12 hours. Melt the PDA culture medium and lower it to 45°C, invert it onto 10-20 sets of plates, invert about 20-25 mL on each plate, and coat the plates with the bacteria after solidification. Take 0.2 mL of the above Penicillium solution and place it on the surface of a single set of PDA plate culture medium, and use a sterile glass coating rod to evenly coat the entire surface of the plate with the solution.

[0043] 2) Place a small amount of nitrosoguanidine crystals on a spot slightly to the side of each PDA plate coated with bacterial solution, then carefully invert the plate in a 25°C incubator and incubate for 36 hours. An inhibition zone will appear around the location of the nitrosoguanidine on the plate coated with bacteria.

[0044] 3) For the mutant bacteria culture, pick a small amount of mycelium close to the outside of the inhibition zone and place it in a triangular flask containing 20 mL of PD liquid culture medium, shake it well to make a bacterial suspension, and at the same time pick a small amount of mycelium far from the inhibition zone and place it in another triangular flask containing 20 mL of PD liquid culture medium, shake it well to make a control bacterial suspension. Place the above two triangular flasks at 25°C for shaking culture overnight.

[0045] 4) Spread the plates. Take 0.1 mL of the above two overnight culture suspensions and spread them on agar medium plates. Spread 6 sets of plates with each overnight culture suspension and 3 sets of plates with the control culture suspension. The coated plates are placed in a 25°C incubator for 48 hours. In actual operation, the two bacterial solutions can be appropriately diluted with sterile saline according to their concentrations. Note that each set of plates should be marked on the back to distinguish between the treated and the control.

[0046] 5) Add a few drops of iodine solution to the 5 to 6 colonies formed by mutation. A transparent circle will appear around the colony. Measure the diameter of the transparent circle and the colony diameter respectively, and calculate their ratio (HC ratio). Compared with the control plate, analyze the mutagenic effect, select colonies with a large HC ratio and transfer them to the PDA test tube slope or PDA plate, and compare the characteristics of the mutant strain colony, conidia and conidiophores.

[0047] 1.5 Enrichment and fermentation of corn straw mutant 455-1 and non-mutant MP-03

[0048] 1) Preparation of fermentation bacterial suspension of 455-1 and MP-03 strains: The mutant and non-mutant strains were inoculated into a sterilized 2000 mL Erlenmeyer flask containing 1000 mL PD liquid culture medium, and cultured in a shaking incubator at 25°C and 150 rpm for 3 days to obtain a liquid strain containing sufficient conidia, which was enriched to 200 million conidia / mL.

[0049] 2) The 455-1 and MP-03 strains were used to ferment corn stalks. A small bowl with a diameter of 120 mm and a depth of 80 mm was used as a fermentation container. 20 g of crushed corn stalks was added to each bowl, and other substances were added according to the experimental design method. The bowl was sterilized with high-pressure steam at 121°C for 20 min. After cooling, the liquid strain was inoculated with 10% of the inoculum, sealed with a sterile fresh-keeping bag, and fermented in an incubator at 25°C. Samples were taken once after 5 days of fermentation. The fermentation period was 50 days, and samples were taken separately for determination of various enzyme activities and the degree of straw reduction.

[0050] Determination of the effect of 1.6455-1 on corn straw degradation

[0051] The mutant suspension was inoculated into several flasks filled with corn straw pellet culture medium (corn straw pellet 50%, corn cob pellet 36%, wheat bran 6%, corn flour 3%, salt 1%, diammonium phosphate 1%, lime 3%) at a 10% inoculation amount and cultured at 30°C. The flasks were taken out at 1, 3, 5, 7, 9 and 11 days, and the weight loss rate of corn straw pellets was measured. The samples that reached the degradation peak were selected to determine the degradation rate of the three elements, and the non-mutant Penicillium suspension was used as a control.

[0052] 1.7 Factors affecting the degradation effect of 455-1 on corn straw

[0053] 1) Cultivation temperature: A fresh mutant Penicillium suspension was inoculated into the corn straw pellet culture medium at a 10% inoculation rate and cultured at 20, 25, 30, 35 and 40°C, respectively. The weight loss rate of the corn straw pellets was measured after 7 days.

[0054] 2) Initial pH: corn straw pellet culture media with initial pH of 4, 5, 6, 7, 8, 9 and 10 were prepared, and fresh bacterial suspension was inoculated at a 10% inoculation rate. The culture was statically cultured at 30°C, and the straw weight loss rate was measured after 7 days.

[0055] 3) In the corn straw pellets used for the experiment, the proportions of various ingredients were: corn straw 50% (crushed or not), peanut cake 8.2%, wheat bran 8.2%, farmyard manure 9.7% (the main ingredients include organic matter, nitrogen, phosphorus, potassium and other nutrients, Shandong Green Protection Biotechnology Co., Ltd.), bacterial fertilizer 6.8% (Jinzhengda Qintu No. 1), compound fertilizer (Anyang Zhongsheng Fertilizer Industry) 5.2% (nitrogen, phosphorus and potassium content 45%-55%), water 6.8%, selected mutant Penicillium mutant strain suspension (200 million spores / mL) was added at an inoculation rate of 5%, and vegetable virus vaccine (attenuated strain vaccine or double-linked small RNA vaccine to prevent the occurrence and harm of vegetable viruses in the later stage) (Nanjing Biotechnology Co., Ltd.) was added at 0.1%, and cultured at 30°C. After 7 days, the straw weight loss rate and cellulase activity changes were measured, and the non-mutant Penicillium suspension was used as a control at an inoculation rate of 5% (200 million spores / mL).

[0056] 1.8 Determination of weight loss rate of corn straw

[0057] The culture solution was filtered with filter paper, and the corn stalk particle residue was repeatedly washed with a mixture of hydrochloric acid and nitric acid to eliminate the bacteria, and then weighed after drying at 105°C to constant weight. The weight loss rate of the stalk was calculated using the weight loss method. The calculation formula is as follows:

[0058] Straw weight loss rate = (m0-m) / m0×100% Formula (1)

[0059] In formula (1), m0 is the dry mass of corn straw pellet residue in the control group (g); m is the dry mass of corn straw pellet residue in the treatment group (g).

[0060] 1.9 Determination of the degradation rate of three elements in corn straw

[0061] The Van Soest method was used to determine the content of cellulose, hemicellulose and lignin in corn stalk particles. The sample was first boiled with a neutral detergent, the filtrate was discarded, and the residue was treated with an acidic detergent. The acidic detergent soluble product was hemicellulose; the acidic washing residue was then treated with 72% sulfuric acid, and the soluble product was cellulose; the residue was dried and burned to ashing, and the part lost by ashing was lignin.

[0062] The degradation rates of cellulose, hemicellulose and lignin were calculated as follows:

[0063] Degradation rate = (c o ×m o -c×m) / (c o ×m o )×100% Formula (2)

[0064] In formula (2): c o is the cellulose, hemicellulose or lignin content of the control group g; m o is the dry mass of corn straw pellet residue in the control group (g); c is the cellulose, hemicellulose or lignin content in the treatment group (g); m is the dry mass of corn straw residue in the treatment group (g), and the non-mutant Penicillium suspension was used as the control.

[0065] 1.10 Determination of cellulase activity in corn straw

[0066] The glucose standard curve was drawn according to the experimental steps shown in Table 1. The light absorption value of each test tube sample at 540 nm was measured, and the glucose standard curve was drawn with the glucose concentration as the horizontal axis and the optical density as the vertical axis.

[0067] Table 1 Experimental steps for drawing a glucose standard curve

[0068]

[0069] OD measured by cellulase activity assay540 , converted into the corresponding amount of glucose.

[0070] The mutant Penicillium solution and the non-mutant Penicillium solution were inoculated into 50 mL of enzyme-producing culture medium at a 10% inoculation rate and cultured at 30°C; samples were taken at 0, 2, 4, 6, 8 and 10 days, respectively, and centrifuged at 4°C and 10000r / min for 5 min. The supernatant crude enzyme solution was taken for subsequent cellulase activity determination.

[0071] Cellulase activity was determined by referring to the method reported by Kazeem et al. (2017) (Applied Biochemistry and Biotechnology. 182: 1318-1340). The enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol of reducing sugar from corn stalk particle matrix in 1 min under specific conditions. Reducing sugar was determined by 3,5-dinitrosalicylic acid (DNS) colorimetric method according to the method reported by Miller (1959) (Analytical Chemistry. 31: 426-428). The substrate for endocellulase activity determination was 1% CMC-Na (sodium carboxymethyl cellulose) solution prepared in 0.1 mol / L phosphate buffer (pH 6.0); the substrate for exocellulase activity determination was 1% trace cellulose solution prepared in the same buffer. Add 0.5 mL of the crude enzyme solution to be tested to 1.5 mL of the substrate solution, incubate in a 60°C water bath for 30 min, immediately add 1.5 mL of DNS, boil in a boiling water bath for 5 min, take out and immediately cool, shake well and measure the absorbance at a wavelength of 540 nm. Use the absorbance value to determine the amount of reducing sugar released against the glucose standard curve and calculate the corresponding enzyme activity, using the non-mutant Penicillium suspension as the control.

[0072] 2. Results and Analysis

[0073] 2.1 Screening of candidate fungal strains for corn straw degradation

[0074] A fungal strain MP-03 with strong destructive power on filter paper was screened out from several fungal strains isolated, and it was preliminarily defined as a candidate strain with the ability to degrade corn stalks. Subsequently, the strain was induced to mutate by the nitrosoguanidine method, and several suspected mutant strains were obtained. The bacterial suspensions were transferred to PD culture medium and cultured at 28°C to obtain bacterial suspensions. According to the above-mentioned degradation filter paper strip detection method, the bacterial suspension with the strongest decomposition ability was screened. The bacterial suspension was evenly spread on a PDA solid plate and cultured at 28°C for 2 to 3 days. A single colony was picked and stored in a PDA slant test tube at 4°C, and the candidate mutant fungal strain ΔMP-03 with the strongest ability to degrade corn stalks was screened out.

[0075] 2.2 Morphological characteristics of non-mutant and mutant fungal strains

[0076] (1) Morphological identification

[0077] The strain MP-03 with corn straw degradation ability and the mutant strain ΔMP-03 with strong degradation ability obtained by screening were cultured in PDA medium for 3-4 days. Figure 1 The colony color of MP-03 (A) shown in the figure changed from white to gray-green, the hyphae had colorless septa, the conidiophores had septa, produced asymmetric broom bodies and single whorled secondary conidiophores, formed a swollen apical capsule at the top, the conidia were spherical, and formed unbranched spore chains. The fungus strain was preliminarily identified as Penicillium cyclopium. The colony color of the mutant strain ΔMP-03 (B) changed from white to dark gray, the conidiophores had septa, produced densely clustered brown-red broom-shaped conidiophores, the apical swelling became smaller, the branch chain characteristics of the conidia became weaker, and the conidia produced were small and the number increased significantly.

[0078] (2) Molecular biological identification of MP-03 and ΔMP-03 strains

[0079] Agarose gel electrophoresis of PCR products of MP-03 and ΔMP-03 strains. The genomic DNA of mutant and non-mutant strains was amplified by PCR with ITS primers, and single DNA electrophoresis fragments were obtained. The sizes of the DNA fragments were basically consistent with the sizes of Penicillium DNA fragments predicted by https: / / web.expasy.org / protparam / . Figure 2 shown.

[0080] DNA sequencing results and homology comparison analysis, the DNA of MP-03 and ΔMP-03 strains were excised and recovered and sent to Sangon Biotech Co., Ltd. (Shanghai) for sequencing. The sequencing results were analyzed by Blast software for homology sequence comparison of MP-03 and ΔMP-03 target gene sequences. Comparison analysis with the Penicillium gene sequence in the GenBank database showed that both MP-03 and ΔMP-03 strains were Penicillium cyclopium. The ITS sequences of strains MP-03 and ΔMP-03 are shown in SEQ ID NO: 1 and 2, respectively.

[0081] 2.3 Corn straw degradation effect of ΔMP-03 and MP-03 strains

[0082] The weight loss rate of corn straw particles was measured by continuous sampling to detect the difference in the degradation ability of corn straw between ΔMP-03 and MP-03 strains. Figure 3As shown in the figure, the weight loss rate of corn straw after treatment with ΔMP-03 and MP-03 strains showed an upward trend with the increase of time, until the degradation peak was reached on the 7th day, and the weight loss rate of corn straw by ΔMP-03 and MP-03 strains was 37.56% and 29.45%, respectively. Therefore, the degradation rate of corn straw by ΔMP-03 was significantly higher than that of MP-03, and the degradation rate of corn straw by ΔMP-03 showed a stable increasing trend compared with MP-03 with the extension of treatment time.

[0083] Corn straw samples treated with fermentation broth (200 million spores / mL) of ΔMP-03 and MP-03 strains were selected to determine the degradation of wood fiber components in corn straw treated with the strains on the 7th day. As shown in Table 2, ΔMP-03 significantly improved the degradation capacity of corn straw cellulose, hemicellulose and lignin compared with the fermentation broth (200 million spores / mL) of MP-03 strains. The degradation rates of ΔMP-03 and MP-03 strains for corn straw cellulose, hemicellulose and lignin were 23.47%, 21.51% and 12.18%, respectively. This shows that the ΔMP-03 strain has a good decomposition capacity for the three main components of corn straw, while lignin is more difficult to decompose than the other two components, resulting in a relatively stable content change under the condition of rapid loss of corn straw mass. Figure 3 The results in Table 2 show that after the mutation of the selected MP-03 strain, the weight of corn straw was significantly reduced and the degradation rate of the three major components of corn straw cellulose was significantly improved starting from the 7th day of treatment.

[0084] Table 2 Degradation effect of ΔMP-03 and MP-03 strains on lignocellulosic components of corn straw

[0085]

[0086] Note: The same letters in the same column of the table indicate no significant difference, and different letters indicate significant difference (P>0.05).

[0087] 2.4 Determination of the effect of different culture conditions on the degradation of corn straw by ΔMP-03 and MP-03

[0088] The effect of temperature on the degradation of corn straw treated with ΔMP-03 and MP-03 fermentation broth (200 million spores / mL) on the 7th day was determined. Corn straw samples treated with ΔMP-03 and MP-03 fermentation broth (200 million spores / mL) on the 7th day were obtained. Figure 4a shows the effect of temperature on the degradation of corn straw by ΔMP-03 and MP-03 fermentation liquid (200 million spores / mL). With the increase of temperature, the weight loss rate of corn straw showed a trend of first rising and then falling. When the temperature was 30°C, ΔMP-03 and MP-03 fermentation liquid (200 million spores / mL) had the best degradation effect on corn straw, and ΔMP-03 was significantly higher than MP-03 on corn straw, which were 40.35% and 30.65%, respectively. When the treatment temperature was increased to 40°C, both ΔMP-03 and MP-03 still showed a high degradation effect on corn straw, and the former was better than the latter, with the weight loss rates of corn straw being 27.35% and 20.45%, respectively. It is inferred from this that the addition of the organic coupling cultivation technology of Penicillium arcuate ΔMP-03 to vegetable greenhouses in autumn will provide suitable soil temperature conditions for the effective degradation of corn stalks by Penicillium arcuate ΔMP-03, and the degradation of corn stalks will help increase the soil temperature by 4-5°C, which is more beneficial to the degradation of corn stalks and the effectiveness of the organic coupling cultivation technology.

[0089] The effects of different initial pH conditions, ΔMP-03 and MP-03 fermentation broth (200 million spores / mL) treatment on the degradation of corn straw on the 7th day were determined. Corn straw samples were treated with ΔMP-03 and MP-03 bacterial suspensions under different initial pH conditions on the 7th day. Figure 4 (b) shows the effect of different initial pH on the degradation of corn straw by ΔMP-03 and MP-03. The results show that under different pH conditions, ΔMP-03 has a higher degradation effect on corn straw than MP-03. When the initial pH is 7.0, ΔMP-03 and MP-03 cause corn straw weight loss rates of 34.56% and 29.95%, respectively. However, too low or too high pH significantly reduces the degradation effect of ΔMP-03 and MP-03 on corn straw. When the pH is lower than 6.0 or higher than 8.0, the degradation effect of ΔMP-03 and MP-03 on corn straw decreases sharply. When the pH rises to 10.0, the degradation effect of ΔMP-03 and MP-03 on corn straw is the worst. Therefore, the initial pH should be controlled between 6.0 and 8.0, and both ΔMP-03 and MP-03 have a higher degradation effect on corn straw, and ensure that the degradation effect of ΔMP-03 on corn straw is always significantly higher than that of MP-03.

[0090] The effects of different added ingredients, ΔMP-03 and MP-03 fermentation broth (200 million spores / mL) on the degradation of corn straw on the 7th day after treatment were determined. Corn straw samples treated with ΔMP-03 and MP-03 fermentation broth with different added ingredients on the 7th day were taken respectively. Figure 4(c) The effect of different added ingredients on the degradation of corn straw by ΔMP-03 and MP-03 is shown. The results show that different added ingredients, including virus vaccine, peanut cake, wheat bran, farmyard manure, bacterial fertilizer or compound fertilizer, all lead to a higher degradation effect of ΔMP-03 on corn straw than MP-03, and farmyard manure and bacterial fertilizer induce the best degradation of corn straw by ΔMP-03 and MP-03, with the weight loss rate of corn straw being almost 30.54%. The order of the degradation effect of different added ingredients on ΔMP-03 and MP-03 on corn straw is: farmyard manure ≥ bacterial fertilizer > peanut cake > compound fertilizer > wheat bran > virus vaccine.

[0091] The above research results show that different culture conditions induce ΔMP-03 to have a higher degradation effect on corn straw than MP-03. Under the conditions of temperature of 30℃, initial pH of 7.0 and addition of farmyard manure or bacterial fertilizer, ΔMP-03 and MP-03 have the best degradation effect on corn straw, and ΔMP-03 has a significantly higher degradation effect on corn straw than MP-03.

[0092] 2.5 Effects of ΔMP-03 and MP-03 bacterial suspensions on the activity of corn straw cellulase

[0093] Endocellulase mainly acts on the amorphous region of cellulose, cutting the long chain of cellulose molecules into short chains, while exocellulase mainly acts on the crystalline region of cellulose, hydrolyzing cellulose from the end to produce cellobiose. The activities of endocellulase and exocellulase in corn straw treated with ΔMP-03 and MP-03 bacterial suspensions (200 million spores / mL) were measured for 0, 2, 4, 6, 8, and 10 days. Figure 5 The results showed that the activity of corn straw endocellulase in the 4th day after treatment with ΔMP-03 and MP-03 was the highest (0.2U / mL), indicating that the selected strains can effectively activate the activity of corn straw exocellulase in the 4th day, which is conducive to the effective function of exocellulase. Moreover, at different treatment times, ΔMP-03 significantly improved the activity of corn straw endocellulase compared with MP-03, indicating that ΔMP-03 has a stronger ability to activate the activity of corn straw endocellulase, more effectively decompose the amorphous region of cellulose, provide appropriate raw materials for the effective function of exocellulase, and accelerate the degradation of cellulose into disaccharides. On this basis, the activity of corn straw exocellulase in ΔMP-03 and MP-03 treated for 0, 2, 4, 6, 8, and 10 days was measured respectively. Figure 6The results showed that the activity of exocellulase in corn straw treated with ΔMP-03 and MP-03 bacterial suspensions was the highest on the 6th day, and the activity of exocellulase in corn straw treated with ΔMP-03 was significantly higher than that in MP-03 at different treatment times. This showed that ΔMP-03 had higher activation activity of endocellulase and exocellulase in corn straw than MP-03, significantly accelerated the yield of corn straw fiber and the decomposition and utilization of corn straw, and effectively increased the organic matter content of soil in organic coupling cultivation of corn straw, which was beneficial to improve the content and composition of organic nutrients in soil of facility vegetable cultivation.

[0094] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A Penicillium cyclopium that can efficiently degrade straw fiber, which is Penicillium cyclopium 455-1, with a preservation number of CGMCC No.41541.

2. A bacterial agent containing the Penicillium circumflexus according to claim 1.

3. Use of the Penicillium arcuate according to claim 1 in producing cellulase.

4. Use of the Penicillium arcuate according to claim 1 or the cellulase produced thereby or the microbial agent according to claim 2 in the degradation of crop straw.

5. Use of the Penicillium arcuate described in claim 1 or the bacterial agent described in claim 2 in improving the enzymatic activity of endo-cellulase and exo-cellulase in crop straw.

6. The use according to claim 4 or 5, characterized in that: The crop straw includes corn straw.

7. A method for degrading corn stalks, characterized in that: Corn stalks are used as raw materials, and the bacterial suspension of Penicillium arcuate described in claim 1 is inoculated, and the corn stalks are degraded under the conditions of 20-40° C. and initial pH 6.0-8.

0.

8. The method according to claim 7, characterized in that Add appropriate amount of farmyard manure or bacterial fertilizer to the raw materials.

9. The method according to claim 7 or 8, characterized in that: The degradation of corn stover was carried out at 30°C and an initial pH of 7.0.

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