Penicillium cyclopium for efficiently degrading straw fibers and its application

Abstract

The present invention discloses Penicillium cyclopium with high efficiency in degrading straw fiber and its application. The present invention uses nitrosoguanidine method to mutagenize the Penicillium cyclopium strain MP-03(2554-2) (CGMCC No. 41542) to obtain a strong corn straw-degrading mutant strain ΔMP-03(455-1) (CGMCC No. 41541). By optimizing the nitrogen-carbon ratio, culture time, temperature and pH range conditions of the optimal enzyme-producing medium, compared with the non-mutant strain 2554-2, the 455-1 mutant strain significantly improves the degradation rate of corn straw, the activities of endo-cellulase and exo-cellulase. The present invention optimizes the scale-up culture conditions of the 455-1 mutant strain, and develops a new type of organic coupling cultivation technology such as the fermentation broth of the 455-1 mutant strain + corn straw + organic fertilizer for protected vegetables, which significantly improves the growth, root development and photosynthetic efficiency of protected tomatoes, peppers and cucumbers, as well as the resistance to soil-borne oomycete diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms and their applications, and specifically, to a Penicillium cyclopium with high efficiency in degrading straw fibers and its applications. Background Art

[0002] Crop straw substrates are mainly composed of cell walls, which contain a large amount of carbohydrates, a small amount of proteins and minerals. The carbohydrates mainly include cellulose, hemicellulose and pectin polysaccharides. Returning straw to the field can, to a certain extent, increase soil organic matter, improve the physical and chemical properties of the soil, and reduce the loss of phosphorus and potassium nutrients in the straw. Crop straw is mainly composed of lignocellulose and is difficult to be effectively decomposed by microorganisms under natural soil conditions, resulting in a significant reduction in the effect of improving soil fertility. In addition, the degradation of straw under soil conditions requires a certain carbon-nitrogen ratio, which is likely to cause competitive absorption of nitrogen fertilizer between straw and crops and affect the growth of vegetables. Therefore, based on the research and selection or improvement of highly efficient straw-degrading microbial strains, it is easy to develop a new microbial suspension degradation technology for crop straw substrates through organic coupling. Currently, research on the screening and application technology of crop straw biodegradation microbial strains has been carried out, and some fungal strains capable of degrading crop straw have been screened, mainly including Pleurotus spp., Trichoderma spp., Penicillium spp., Aspergillus spp. and some bacterial complex systems. Among them, the Aspergillus fumigatus XC6 strain has degradation rates of 72%, 93% and 84% for straw cellulose, hemicellulose and crop straw, respectively. High-temperature bacterial strains, mesophilic actinomycete strains and mesophilic fungal strains with strong cellulose-decomposing ability have been isolated from substrates such as soil, horse manure and cow manure, and the effects on the cellulase activity of rice straw substrates have been measured respectively. There is relatively little research on the biodegradation of crop straw by Penicillium, but existing research shows that a few Penicillium have a strong ability to degrade lignocellulose 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-celled conidia, which is convenient for large-scale optimization culture and reproduction. Based on the screening of high-degrading crop straw lignin-degrading Penicillium strains, the optimal nitrogen-carbon ratio, culture time, temperature and pH range of the enzyme-producing medium are defined to ensure the effective development and application of the beneficial Penicillium collected. Summary of the Invention

[0003] The purpose of the present invention is to provide a Penicillium cyclopium with high efficiency in degrading straw fibers and its applications.

[0004] A Penicillium strain MP-03(2554-2) that can improve the cellulase activity of corn straw was isolated from corn straw in the present invention. It was identified as Penicillium cyclopium, and the nitrosoguanidine method was used to induce mutation of strain 2554-2, and a strong corn straw-degrading mutant strain ΔMP-03(455-1) was screened. Strains 2554-2 and 455-1 have now been deposited in the China General Microbiological Culture Collection Center, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a postcode of 100101. The deposit number of strain 2554-2 is CGMCC NO.41542, and the deposit number of strain 455-1 is CGMCC NO.41541. The deposit date is October 12, 2024.

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

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

[0007] In the third aspect, the present invention provides the application of the Penicillium cyclopium 455-1 in cellulase production.

[0008] In the fourth aspect, the present invention provides the application of the Penicillium cyclopium 455-1 or a bacterial agent containing the same, which can significantly improve cellulase activity, in the degradation of corn straw.

[0009] In the fifth aspect, the present invention provides the application of the Penicillium cyclopium 455-1 or a bacterial agent containing the same in improving the enzyme activities of endocellulase and exocellulase in crop straw.

[0010] The crop straw described in the present invention includes but is not limited to corn straw.

[0011] In the sixth aspect, the present invention provides a method for degrading corn straw. Using corn straw as the raw material, a bacterial suspension of the Penicillium cyclopium 455-1 is inoculated, and the degradation of corn straw is carried out under the conditions of 20-40°C and an initial pH of 6.0-8.0 (preferably 30°C and an initial pH of 7.0).

[0012] Furthermore, an appropriate amount of farmyard manure or bacterial fertilizer is added to the raw material.

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

[0014] The present invention uses the nitrosoguanidine method to mutagenize Penicillium cyclopium MP-03 strain to obtain a strong corn straw-degrading mutant strain 455-1. By optimizing the nitrogen-carbon ratio, culture time, temperature, and pH range conditions of the medium with the highest enzyme activity, compared with the non-mutant strain MP-03, the 455-1 mutant strain significantly improves the corn straw degradation rate, endocellulase and exocellulase activities. The present invention also optimizes the enlarged culture conditions of the 455-1 mutant strain, and develops an organic coupling cultivation technology of the fermentation broth of the 455-1 mutant strain + corn straw + organic fertilizer for protected vegetables, which significantly improves the growth, root development, photosynthetic efficiency of protected tomatoes, peppers, and cucumbers, and the performance of inhibiting the occurrence and harm of soil-borne oomycete diseases. Brief Description of the Drawings

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

[0016] Figure 2 Agarose gel electrophoresis diagram of the ITS full sequences of the MP-03 and ΔMP-03 strains of the present invention.

[0017] Figure 3 Degradation effects of the ΔMP-03 and MP-03 strains on corn straw in the preferred embodiment of the present invention. Different letters indicate significant differences (P>0.05), and the same letters indicate no significant differences.

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

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

[0020] Figure 6 Effects of the ΔMP-03 and MP-03 strains on the change of exocellulase activity of corn straw in the preferred embodiment of the present invention. Detailed Embodiments

[0021] The following examples are used to illustrate the present invention, but are not used 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 the morphological characteristics of the inoculum: Take the activated inoculum and streak it on a PDA plate, and culture it in an incubator at 28 °C for 24 h. Insert a sterilized glass slide into the plate where the inoculum has grown at an angle of 45 degrees. After continuing to culture for 48 h, gently take it out and observe the morphological characteristics such as mycelium, conidia, and conidiophores under a microscope, and take pictures and compose them into a plate.

[0034] Molecular biology identification: Extraction of mycelial DNA. A 250 mL conical flask containing 100 mL of PD medium is sterilized at 121 °C under high-pressure steam for 20 min. After cooling to room temperature, inoculate the Penicillium cyclopium MP-03 strain and culture it on a shaker at 28 °C and 150 r / min for 2 days. Centrifuge to collect the thalli, grind them with liquid nitrogen to break the cell wall, and then extract DNA using a rapid fungal genomic DNA extraction kit. Store it at -80 °C in a refrigerator for standby.

[0035] PCR amplification: Use the forward primer (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and reverse primer (5′-TCCTCCGCTTATTGATATGC-3′) for identifying Penicillium cyclopium ITS to pre-denature the extracted DNA at 94 °C for 5 min; denature at 95 °C for 1 min, anneal at 55 °C for 1 min, extend at 72 °C for 1 min, and perform 35 amplification cycles.

[0036] Purification of PCR amplification products: Use 5% agarose gel electrophoresis to detect the DNA amplification products, cut off the gel fragment, and purify the target DNA fragment using an agarose gel DNA recovery kit.

[0037] DNA sequencing and Blast analysis: Send the gel-cut and recovered products to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and use the Blast software to perform a homologous sequence alignment analysis on the target gene sequence of the sequencing results.

[0038] Sequence analysis: Download the corresponding inoculum gene sequences with high homology to the gene sequence of the tested Penicillium strain from the GenBank database with the help of the Blast analysis software for alignment analysis. Identify the isolated MP-03 strain by comprehensively combining the morphological characteristics and the molecular DNA sequence alignment results.

[0039] 1.4 Induced mutation of the MP-03 strain by nitrosoguanidine

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

[0041] The mutagenesis operation steps are as follows:

[0042] 1) Pick one loop of the tested MP-03 bacterial slant strain and inoculate it into a test tube containing 5 mL of PD culture medium. Incubate it at 25°C with shaking for 48 h. Take 1.0 mL of the overnight culture medium and transfer it to another test tube containing 5 mL of PD culture medium. Incubate it at 25°C with shaking for 12 h. Melt the PDA medium and let it cool to 45°C. Pour it into 10 - 20 Petri dishes, about 20 - 25 mL for each dish. After solidification, use the bacterial cells to spread on the plates. Take 0.2 mL of the above-mentioned penicillium liquid and place it on the surface of a single PDA plate medium. Use a sterile glass spreading rod to evenly spread the bacterial liquid over the entire surface of the plate.

[0043] 2) Place a little nitrosoguanidine crystal at a site slightly near the edge of each PDA plate coated with the bacterial liquid. Then carefully invert the plate and incubate it in an incubator at 25°C for 36 h. An inhibition zone will appear around the position where the nitrosoguanidine is placed on the plate coated with bacteria.

[0044] 3) Culture the suspected mutant bacteria. Pick a little 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 well to make a bacterial suspension. At the same time, pick a little mycelium far from the inhibition zone and place it in another triangular flask containing 20 mL of PD liquid culture medium. Shake well to make a control bacterial suspension. Place the above two triangular flasks in an incubator at 25°C with shaking overnight.

[0045] 4) Spread on the plate. Take 0.1 mL of each of the above two overnight cultured bacterial suspensions and spread them on the agar medium plates. Each overnight bacterial suspension is spread on 6 plates, and the control bacterial suspension is spread on 3 plates. After spreading, place the plates in an incubator at 25°C for 48 h. In actual operation, dilute appropriately with sterile physiological saline according to the concentration of the two bacterial liquids. Note that make marks on the back of each plate to distinguish the treated and the control.

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

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

[0048] 1) Preparation of fermentation bacterial suspensions of strains 455 - 1 and MP - 03. Inoculate the mutant strain and the non - mutant strain into a 2000 mL triangular flask containing 1000 mL of PD liquid culture medium that has been sterilized. Incubate it on a shaker at 25°C and 150 r / min for 3 d to obtain a liquid bacterial strain containing a sufficient amount of conidial suspension, and enrich it to 200 million conidia / mL.

[0049] 2) Fermentation treatment of corn straw with strains 455-1 and MP-03: Using small bowls with a diameter of 120 mm and a depth of 80 mm as fermentation containers, add 20 g of crushed corn straw to each bowl, and add other substances according to the experimental design method. Sterilize at 121 °C with high-pressure steam for 20 min. After cooling, inoculate with liquid strain at an inoculation amount of 10%, seal with a sterile fresh-keeping bag, and ferment and culture in an incubator at 25 °C. Take samples once after 5 days of fermentation, with a fermentation cycle of 50 days, and take samples respectively for determination of various enzyme activities and the degree of straw reduction.

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

[0051] Inoculate the mutant strain suspension at an inoculation amount of 10% into several Erlenmeyer flasks containing corn straw particle medium (50% corn straw particles, 36% corn cob particles, 6% wheat bran, 3% corn flour, 1% salt, 1% diammonium hydrogen phosphate, 3% lime), and culture statically at 30 °C. Take out the Erlenmeyer flasks at 1, 3, 5, 7, 9, and 11 days respectively, measure the weight loss rate of corn straw particles, select the samples reaching the degradation peak to measure the degradation rate of three elements, and use the non-mutant Penicillium suspension as a control.

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

[0053] 1) Cultivation temperature: Inoculate the fresh mutant Penicillium strain suspension at an inoculation amount of 10% into the corn straw particle medium, and culture statically at 20, 25, 30, 35, and 40 °C respectively. Measure the weight loss rate of corn straw particles after 7 days.

[0054] 2) Initial pH: Prepare corn straw particle media with initial pH values of 4, 5, 6, 7, 8, 9, and 10 respectively, inoculate with fresh strain suspension at an inoculation amount of 10%, culture statically at 30 °C, and measure the straw weight loss rate after 7 days.

[0055] 3) For the corn straw particles used in the experiment, the proportion of various components: 50% corn straw (crushed or not), 8.2% peanut cake, 8.2% wheat bran, 9.7% farmyard manure (the main components include various nutrient elements such as organic matter, nitrogen, phosphorus, and potassium, Shandong Lubao Biotechnology Co., Ltd.), 6.8% microbial fertilizer (Kingenta Pro-Soil 1), 5.2% compound fertilizer (Anyang Zhongcheng Fertilizer Industry) (nitrogen, phosphorus, and potassium content 45%-55%), 6.8% water. Add the selected mutant Penicillium mutant strain suspension (200 million spores / mL) at an inoculation amount of 5%, add 0.1% vegetable virus vaccine (weak strain vaccine or double-stranded small RNA vaccine, to prevent the occurrence and harm of vegetable viruses in the later stage) (Nanjing Biotechnology Co., Ltd.), culture statically at 30 °C, measure the straw weight loss rate and the change in cellulase activity after 7 days, and use the non-mutant Penicillium strain suspension at an inoculation amount of 5% (200 million spores / mL) as a control.

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

[0057] Filter the culture solution with filter paper. Repeatedly rinse the filtered corn straw particle residue with a mixed solution of hydrochloric acid and nitric acid to eliminate the bacteria. After drying to a constant weight at 105 °C and weighing, calculate the straw weight loss rate by the weight loss method. The calculation formula is as follows:

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

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

[0060] 1.9 Determination of degradation rates of three components of corn straw

[0061] Use the Van Soest method to determine the contents of cellulose, hemicellulose, and lignin in corn straw particles. The sample is first treated by boiling with a neutral detergent, and the filtrate is discarded. The residue is then treated with an acidic detergent. The acidic detergent solute obtained is hemicellulose; the acidic detergent residue is further treated with 72% sulfuric acid, and the solute obtained is cellulose; the residue is dried and incinerated to ash, and the part lost during ashing is lignin.

[0062] The degradation rates of cellulose, hemicellulose, and lignin are calculated according to the following formula:

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

[0064] In Equation (2): c o is the content of cellulose, hemicellulose, or lignin in the control group (g); m o is the dry mass of the corn straw particle residue in the control group (g); c is the content of cellulose, hemicellulose, or lignin in the treatment group (g); m is the dry mass of the straw residue in the treatment group (g), with the non-mutated Penicillium mold suspension as the control.

[0065] 1.10 Determination of cellulase activity of corn straw

[0066] Draw a glucose standard curve. Operate according to the experimental steps shown in Table 1. Measure the light absorption value of each test tube sample at 540 nm. Take the glucose concentration as the abscissa and the optical density as the ordinate to draw the glucose standard curve.

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

[0068]

[0069] The OD measured for cellulase activity determination540 , 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 strains MP-03 with the ability to degrade corn straw and the mutant strain ΔMP-03 with strong degradation ability obtained by screening were cultured on PDA medium for 3 - 4 d. Figure 1 As shown, the colony color of MP-03 (A) changed from white to grayish green. The hyphae had transverse septa and were colorless. The conidiophores had transverse septa, producing asymmetrical penicilli and single-verticillate secondary conidiophores. An enlarged vesicle was formed at the top, and the conidia were spherical, forming unbranched spore chains. This fungal 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 transverse septa, producing dense clusters of brownish-red broom-shaped conidiophores. The enlarged part at the top became smaller, the characteristics of the branched spore chains of the conidia became weaker, and the conidia produced were small and the number increased significantly.

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

[0079] Agarose gel electrophoresis patterns of the PCR products of strains MP-03 and ΔMP-03. Genomic DNA of mutant and non-mutant strains was amplified by PCR with ITS primers and electrophoresed, and a single DNA electrophoretic band was obtained for each. The size of each DNA fragment was basically consistent with the size of the Penicillium DNA fragment predicted by the website https: / / web.expasy.org / protparam / , as Figure 2 shown.

[0080] DNA sequencing results and homology comparison analysis. The DNA of strains MP-03 and ΔMP-03 obtained by gel cutting and recovery was sent to Sangon Biotech Co., Ltd. (Shanghai) for sequencing. The sequencing results were used to perform homologous sequence alignment analysis of the target gene sequences of MP-03 and ΔMP-03 with the Blast software. Through comparison and analysis with the Penicillium gene sequences in the GenBank database, it was shown that both strains MP-03 and ΔMP-03 were Penicillium cyclopium. The ITS sequences of strains MP-03 and ΔMP-03 are shown as SEQ ID NO:1 and 2 respectively.

[0081] 2.3 Degradation effects of strains ΔMP-03 and MP-03 on corn straw

[0082] By continuously sampling and measuring the weight loss rate of corn straw particles, the differences in the degradation ability of strains ΔMP-03 and MP-03 on corn straw were detected. As Figure 3As shown in the figure, the weight loss rate of corn straw treated with ΔMP-03 and MP-03 strains showed an upward trend with the increase of time, and reached the degradation peak until the 7th day. The weight loss rates of corn straw caused by ΔMP-03 and MP-03 strains were 37.56% and 29.45% respectively. Therefore, the degradation rate of ΔMP-03 on corn straw was significantly higher than that of MP-03, and with the extension of treatment time, the degradation rate of ΔMP-03 on corn straw showed a stable increasing trend compared with MP-03.

[0083] Corn straw samples treated with the fermentation broth (200 million spores / mL) of ΔMP-03 and MP-03 strains were selected to determine the degradation of lignocellulose components of corn straw on the 7th day after treatment with the tested strains. As shown in Table 2, the fermentation broth of ΔMP-03 (200 million spores / mL) significantly improved the degradation ability of corn straw cellulose, hemicellulose and lignin compared with that of MP-03. The degradation rates of corn straw cellulose, hemicellulose and lignin by ΔMP-03 were 23.47%, 21.51% and 12.18% higher than those by MP-03 respectively. It shows that the ΔMP-03 strain has good decomposition ability for the three main components of corn straw, while lignin is more difficult to decompose than the other two components, resulting in relatively stable content changes under the condition of rapid loss of corn straw quality. Figure 3 The results in Table 2 showed that after the selected MP-03 strain was mutated, starting from the 7th day of corn straw treatment, the weight of corn straw decreased significantly, and the degradation rates of the three major components of corn straw lignocellulose increased significantly.

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

[0085]

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

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

[0088] The effects of temperature on the degradation of corn straw treated with the fermentation broth (200 million spores / mL) of ΔMP-03 and MP-03 on the 7th day were determined. Corn straw samples treated with the fermentation broth (200 million spores / mL) of ΔMP-03 and MP-03 on the 7th day were obtained respectively. 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 6It is shown that the exocellulase activity of corn straw treated with ΔMP-03 and MP-03 bacterial suspensions for 6 days is the highest, and at different treatment times, the exocellulase activity of corn straw treated with ΔMP-03 is significantly higher than that treated with MP-03. This indicates that ΔMP-03 has higher activities in activating endocellulase and exocellulase of corn straw than MP-03, significantly accelerating the production of cellobiose from corn straw and the decomposition and utilization of corn straw, thereby effectively increasing the organic matter content in the soil of organic coupled cultivation of corn straw and facilitating the improvement of the organic nutrient content and composition in the soil for protected vegetable cultivation.

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

Claims

1. A Penicillium cyclopium for degrading straw fibers, which is Penicillium cyclopium( Penicillium cyclopium ) 455-1, and the preservation number is CGMCC No. 41541.

2. A microbial agent containing the Penicillium cyclopium described in claim 1.

3. Use of the Penicillium cyclopium described in claim 1 in producing cellulase.

4. Use of the Penicillium cyclopium described in claim 1 or the microbial agent described in claim 2 in degrading crop straw.

5. Use of the Penicillium cyclopium described in claim 1 or the microbial agent described in claim 2 in increasing the enzyme activities of endocellulase and exocellulase in crop straw.

6. The application according to claim 4 or 5, characterized in that, The crop straw is corn straw.

7. Method for degrading corn straw, characterized in that, Using corn straw as the raw material, inoculating the microbial suspension of the Penicillium cyclopium described in claim 1, and carrying out the degradation of corn straw under the conditions of 20 - 40 °C and an initial pH of 6.0 - 8.

0.

8. The method according to claim 7, wherein Adding an appropriate amount of farmyard manure or microbial fertilizer to the raw material.

9. The method according to claim 7 or 8, characterized in that, Carrying out the degradation of corn straw under the conditions of 30 °C and an initial pH of 7.0.

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