Trichoderma asperellum strain ZC3 and its application in the degradation of Zanthoxylum bungeanum branches

Abstract

The present invention discloses Trichoderma asperellum ZC3 and its application in the degradation of Zanthoxylum bungeanum branches. This strain has a fast growth rate and strong degradation ability. In a liquid filter paper strip culture medium, after culturing at 28 °C for 7 days, the filter paper strip completely disintegrates; in a solid culture medium of Zanthoxylum bungeanum branch powder, after culturing at 28 °C for 40 days, its lignin degradation rate for Zanthoxylum bungeanum branches reaches 78.11%, and the degradation rates of cellulose and hemicellulose reach 82.58% and 75.84% respectively. Under a scanning electron microscope, obvious fiber breakage and structural disintegration phenomena can be seen in the Zanthoxylum bungeanum branches, showing a high degradation ability. In addition, the lignin-degrading bacterium ZC3 has good tolerance to the leaching solution of Zanthoxylum bungeanum branches.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a Trichoderma aspergillus ZC3 strain and application thereof in the degradation of Zanthoxylum bungeanum branches. Background Art

[0002] Zanthoxylum bungeanum is a traditional economic tree species in my country, belonging to the genus Zanthoxylum (Zanthoxylum) in the Rutaceae family, with a cultivation history of over 2,600 years (Yao Jia et al., 2010). Its peel is a traditional food seasoning used in our daily lives, known as one of the "eight major condiments" and the primary source of numbing and aromatic substances (Li Jianhong et al., 2009; Li Hongliang et al., 2014), with significant economic value (Bi Jun et al., 2006; Cui Jun et al., 2008).

[0003] According to incomplete statistics, my country's pepper cultivation area covers 8 million mu (approximately 1.5 million hectares), with a production value exceeding 5.05 billion yuan. Chongqing's pepper cultivation area covers approximately 1.17 million mu (approximately 1.5 million hectares), with the primary variety being the nine-leaf green pepper (Zanthoxylumarmatum V. novemfolius), with the largest area cultivated in Jiangjin District. Currently, the nine-leaf green pepper cultivation area in Jiangjin District is approximately 530,000 mu (approximately 1.5 million hectares), with a total dried pepper production exceeding 40,000 tons (Bian Hao et al., 2021), an increase of nearly 63.5% from 193,000 mu (approximately 1.5 million hectares) in 2002. This has led to a surge in green pepper harvest waste.

[0004] Green peppercorn harvest waste refers to the branches, leaves, and thorns removed from pepper trees when the fruit is harvested. Statistics show that up to 30,000 tons of green peppercorn harvest waste is generated annually in Jiangjin District, Chongqing. However, due to current technological limitations, policy frameworks, and the fragmented distribution of peppercorn branch resources, a large amount of these branches are burned or discarded, resulting in both resource waste and serious environmental pollution (Wen X., 2020; Nguyen A.T., 2019; Liu T., 2020). Furthermore, local farmers annually pile large amounts of green peppercorn harvest waste in pepper gardens or along roadsides, potentially creating conditions for the breeding of pests and diseases and fires.

[0005] Currently, straw is primarily used as fertilizer (composting, mechanical return, and sloughing), feed (silage), substrate (for edible fungi production), fuel (biogas, biochar), and raw material (for industrial manufacturing). Compared to other utilization methods, composting Sichuan pepper branches not only replenishes trace elements lost from the soil in pepper gardens but also improves soil quality, enhances soil fertility, and mitigates soil acidification. Furthermore, composting is convenient, adaptable to local conditions, and requires minimal investment.

[0006] Zanthoxylum bungeanum twigs are composed of three major parts: cellulose, hemicellulose, and lignin. Among them, the lignin content is approximately 35%, and the cellulose and hemicellulose contents are 32% and 18% respectively. Lignin has a network structure that surrounds and reinforces cellulose and hemicellulose as a supporting framework, binds tightly with cellulose and hemicellulose, and restricts the further utilization of cellulose (Wang YX., 2013; Xu Congfeng et al., 2019; Xu Ying et al., 2016). Lignin is a polymer with a basic structure of phenylpropane, having very strong intramolecular hydrogen bonds and an irregular three-dimensional network structure. It binds to hemicellulose through covalent bonds, while hemicellulose and cellulose are bound by composite hydrogen bonds. Lignin and hemicellulose are intertwined, presenting a complex and intermittent layered structure. Coupled with the interaction of ether bonds and ester bonds between them, it affects the hydrolysis of cellulose, making the degradation of Zanthoxylum bungeanum twigs relatively difficult and even more difficult to be effectively utilized.

[0007] At present, the degradation of lignin and cellulose in nature is jointly completed by fungi, bacteria, and their corresponding microbial communities. Compared with bacteria, fungi - especially white rot fungi (Phanerochaete chrysosporium) - have the strongest lignin degradation ability and can completely decompose lignin into CO2 and H2O, and are considered the most effective lignin-degrading microorganisms (Zhou Simeng., 2015; Takehito Nakazawa., 2018). However, due to the special chemical components contained in Zanthoxylum plants - mainly including alkaloids, phthalamides, lignans, coumarins, volatile oils, and fatty acids, and other components such as triterpenoids, sterols, hydrocarbons, and flavonoid glycosides, among which the volatile oil of Zanthoxylum is the main metabolite of Zanthoxylum plants - having antibacterial effects, the resource utilization of Zanthoxylum residual waste is further restricted.

[0008] Studies have shown that the volatile oil of Zanthoxylum bungeanum has inhibitory or lethal effects on bacteria such as Bacillus subtilis, Proteus vulgaris, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Clostridium tetani, Pseudomonas fluorescens, Propionibacterium acnes, Bacillus cereus, Pseudomonas aeruginosa, Salmonella, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Bacillus laterosporus, and has inhibitory or lethal effects on fungi such as Saccharomyces cerevisiae, Aspergillus oryzae, Fusarium graminearum, Aspergillus niger, Fusarium graminearum Schwabe, Phyllosticta sp., Fusarium oxysporum, Colletotrichum sp., Aspergillus oryzae, Candida albicans, Aspergillus niger, yeast, Candida sp., and Cryptococcus sp. (Tuo Congcong et al., 2020; Lou Jingrong et al., 2018; Zhao Erlao et al., 2019; Silva Fernanda Bda., 2017). Wang Shanshan et al. (2017) found through research that the volatile oil of Zanthoxylum bungeanum extracted by ether, petroleum ether, methanol, and absolute ethanol generally has antibacterial properties against bacteria, but has no obvious inhibitory effect on molds. Tuo Congcong et al. (2020) extracted the essential oil of Longnan Dahongpao Zanthoxylum bungeanum and found that it not only has inhibitory effects on Gram-positive bacteria, namely Staphylococcus aureus and Clostridium tetani, but also on Gram-negative bacteria, namely Escherichia coli and Pseudomonas fluorescens.

[0009] In addition, the aroma components of Zanthoxylum bungeanum also have certain antibacterial effects. Gao Fengjing et al. (2007) found that the aroma components of Zanthoxylum bungeanum extracted by different methods can inhibit the growth and development of Staphylococcus aureus, Candida tropicalis, Candida utilis, and Geotrichum candidum to varying degrees; Zanthoxylum armatum tablets have obvious antibacterial effects on Escherichia coli. Ding Xiong et al. (2020) found through research that the linalool component in Zanthoxylum bungeanum has good antibacterial effects on Candida albicans, drug-resistant Candida albicans, and Escherichia coli.

[0010] Due to the complex structure of Zanthoxylum bungeanum branches and the special chemical substances they contain, there are few reports on the microbial degradation of Zanthoxylum bungeanum harvesting residues. Based on this, it is urgent to screen out a strain with good degradation ability that is specific to Zanthoxylum bungeanum branches, in order to improve the current situation of difficult treatment, decomposition, and easy pollution of harvesting residues in the green Zanthoxylum bungeanum industrial chain. Summary of the Invention

[0011] In view of this, the purpose of the present invention is to provide a Trichoderma asperellum ZC3 strain and its application in the degradation of Zanthoxylum bungeanum branches.

[0012] To achieve the above object, the present invention provides the following technical solutions:

[0013] 1. Trichoderma asperellum strain ZC3, classified and named as Trichoderma asperellum ZC3, has been deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. Its deposit number is CGMCC No. 40236, the deposit date is July 1, 2022, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0014] Preferably, the ITS sequence of Trichoderma asperellum strain ZC3 is as shown in SEQ ID NO.1.

[0015] gggggggctgcggagggatcattaccgagtttacaactcccaaacCCAATGTGAACGTTACCAAACTGTTGCCTCGGCGGGGTCACGCCCCGGGTGCGTCGCAGCCCCGGAACCAGGCGCCCGCCGGAGGAACCAACCAAACTCTTTCTGTAGTCCCCTCGCGGACGTATTTCTTTACAGCTCTGAGCAAAAATTCAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCGGGGGATCGGCGTTGGGGATCGGGACCCCTCACACGGGTGCCGGCCCCTAAATACAGTGGCGGTCTCGCCGCAGCCTCTCCTGCGCAGTAGTTTGCACAACTCGCACCGGGAGCGCGGCGCGTCCACGTCCGTAAAACACCCAACTTTCTGAAAtgttgACCTCGGAtcaggtaggaatacccgctgaacttaagcaa, as shown in SEQ ID NO.1.

[0016] 2. Application of Trichoderma asperellum strain ZC3 in the degradation of lignin, cellulose or hemicellulose.

[0017] 3. Application of Trichoderma asperellum strain ZC3 in the degradation of Zanthoxylum bungeanum branches.

[0018] 4. A microbial inoculant, whose active ingredient is the aforementioned Trichoderma asperellum strain ZC3, and its mass content is 5 - 10%.

[0019] The beneficial effects of the present invention are as follows:

[0020] A new strain of Trichoderma asperellum, namely Trichoderma asperellum ZC3, was isolated from the naturally decomposed Zanthoxylum bungeanum branches in the fields of Jiangjin District, Chongqing. This strain has a fast growth rate, and its colony forms concentric rings. Dense conidia are produced on the concentric rings. The conidia near the center are dark green, and those at the edge are just white when newly formed, lacking aerial hyphae. The conidiophores form cushion-shaped to hemispherical fascicles, scattered throughout the colony or aggregated into 2 - 3 concentric rings, dark green. The back of the colony does not change color. The conidiophores are slender and curved, with opposite secondary branches. The conidia are spherical or nearly spherical, with fine spines on the wall; after culturing for 3 days, the colony diameter is 5.2 cm, and after culturing for 4 days, the colony diameter can reach 6.3 cm, showing strong degradation ability. In the liquid filter paper strip medium, after culturing at 28°C for 7 days, the filter paper strip completely disintegrates; in the solid medium of Zanthoxylum bungeanum branch powder, after culturing at 28°C for 40 days, the degradation rate of lignin in Zanthoxylum bungeanum branches reaches 78.11%, and the degradation rates of cellulose and hemicellulose reach 82.58% and 75.84% respectively. Under scanning electron microscopy, obvious fiber breakage and structural disintegration can be seen in the Zanthoxylum bungeanum branches, showing a high degradation ability. In addition, the lignin-degrading bacterium ZC3 has good tolerance to the leaching solution of Zanthoxylum bungeanum branches. Brief Description of the Drawings

[0021] To make the objectives, technical solutions, and beneficial effects of the present invention clearer, the following drawings are provided for illustration.

[0022] Figure 1 For the guaiacol and aniline blue plate chromogenic reactions of strain ZC3;

[0023] Figure 2 For the cellulase and lignin enzyme activity diagrams of strain ZC3, where A is the cellulase activity and B is the lignin enzyme activity;

[0024] Figure 3 For the phylogenetic tree of strain ZC3;

[0025] Figure 4 For the electron microscope structure diagrams of Zanthoxylum bungeanum branches before and after treatment with strain ZC3, where A is the CK untreated with inoculation and B is the treatment with strain ZC3.

[0026] Figure 5 For the glucose standard curve.

[0027] Biological Deposit Information

[0028] Taxonomic name: Trichoderma asperellum ZC3;

[0029] Deposit number: CGMCC No. 40236;

[0030] Deposit date: July 1, 2022;

[0031] Depositary institution: China General Microbiological Culture Collection Center;

[0032] Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Detailed implementation manners

[0033] The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings.

[0034] Example 1

[0035] Isolation and identification of ZC3 strain

[0036] (1) Test materials

[0037] Collect the naturally decomposed Chinese prickly ash branches in the fields of Jiangjin District, Chongqing. Select the completely decomposed branches with a high degree of decomposition, black-brown color, surface with bacterial plaques, moist and soft texture and no pungent smell, put them in a sealed bag, and store them in a 4°C refrigerator in the laboratory.

[0038] (2) Culture media

[0039] Enrichment medium: 200 g potato extract, 20 g glucose, 0.33 g streptomycin, 1000 mL distilled water, natural pH.

[0040] Lignin selective medium: 2 g alkaline lignin, 1.33 g ammonium sulfate, 0.5 g magnesium sulfate, 1 g potassium dihydrogen phosphate, 0.2 g disodium hydrogen phosphate, 0.33 g streptomycin, 20 g agar, and [(200 g Chinese prickly ash branches are rinsed with clean water, dried at 65°C for 12 h, cut into 2-3 cm segments, boiled with 200 g Chinese prickly ash branches: 1000 g water, then extracted at 60-80°C for 2 min, and then transferred to a shaker at 120 r / min for extraction for 48 h)] 20% Chinese prickly ash branch extract, and made up to 1000 mL with distilled water (refer to Guo Xiaowei et al., 2017; Wang Jing et al., 2020).

[0041] Gu-PDA medium: 200 g potato extract, 20 g agar, replace 6 g glucose with Chinese prickly ash branch powder, 0.4 m guaiacol, make up to 1 L with water, sterilize at 121°C for 3 min, and use for qualitative determination of the laccase / peroxidase production ability of the strain (as Figure 1 shown in the upper part).

[0042] AB-PDA medium: 200 g potato extract, 20 g agar, replace glucose with 6 g Chinese prickly ash twig powder, 0.1 g aniline blue, add water to make up to 1 L.

[0043] PDA medium: 200 g potato extract, 20 g glucose, 20 g agar (no agar is added for liquid medium, 8 g / L agar is added for semi-solid medium).

[0044] (3) Isolation and purification of ZC3 strain

[0045] Weigh 10 g of the decomposed Chinese prickly ash twig sample into 90 mL of enrichment medium, culture at 28 °C and 120 r / min for 48 h, select dilution gradients of 10 -1 、10 -3 、10 -5 、10 -7 and 10 -9 , respectively take 100 μl and spread it on the lignin selection medium, culture at 28 °C for 48 h. Pick the strains with obvious fungal morphology onto Gu-PDA and AB-PDA media, select the strains with obvious color development circles and color change circles onto the PDA medium for repeated purification until pure culture, then make glycerol bacterial liquid and store the strains at -80 °C. See Figure 1 specifically. The upper part is guaiacol color development, and the lower part is aniline blue color development.

[0046] Name the above-selected strain ZC3, and deposit it in the China General Microbiological Culture Collection Center (abbreviation: CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on July 1, 2022. The deposit number is CGMCC No. 40236, and the taxonomic name is Trichoderma asperellum ZC3.

[0047] Example 2

[0048] Identification of ZC3 strain

[0049] 1) Morphological identification

[0050] After activating the deposited strain ZC3 with PDA medium, observe its colony morphological characteristics; then use the slide culture method and observe its morphological structure under the microscope.

[0051] The morphology of ZC3 strain is as shown in Figure 1As shown, the colonies form concentric rings, on which dense conidia are produced. The conidia near the center are dark green, and those at the edge are just formed and white. There is a lack of aerial hyphae. The conidiophores form cushion-shaped to hemispherical fascicles, scattered throughout the colony or aggregated into 2 - 3 concentric rings, dark green. The back of the colony does not change color. The conidiophores are slender and curved, with opposite secondary branches. The conidia are spherical or nearly spherical, with fine spines on the wall. After culturing for 3 days, the colony diameter is 5.2 cm, and after culturing for 4 days, the colony diameter is 6.3 cm.

[0052] 2) Physiological and biochemical identification

[0053] a. Determination of cellulase activity:

[0054] ① Seed medium: 5 g of peptone, 10 g of beef extract, 5 g of yeast powder, 5 g of glucose, 5 g of sodium chloride, 1000 mL of distilled water, pH 7.0.

[0055] Fermentation medium for enzyme production: 3 g of peptone, 0.5 g of yeast extract powder, 2.0 g of ammonium sulfate, 4 g of potassium dihydrogen phosphate, 0.3 g of sodium chloride, 0.3 g of magnesium sulfate, 20 g of CMC - Na, 1000 mL of distilled water.

[0056] ② Take 5 fungal blocks with a diameter of 1 cm and inoculate them into the seed medium, and culture at 36 °C and 160 r / min for 18 h to prepare the seed solution. Add 3% (volume) of the seed solution to 200 mL of the fermentation medium for enzyme production and culture at 36 °C and 160 r / min for 24 h, centrifuge at 4 °C and 5000 r / min for 10 min, and take the supernatant to measure the enzyme activity.

[0057] ③ Filter paper enzyme activity assay method: Add 5 mL of the fermentation broth extracted from the sample to each centrifuge tube. Place it in a centrifuge and run at 5000 r / min for 10 min. The supernatant obtained is the crude enzyme solution required for the experiment. From the crude enzyme solution, extract 0.5 mL to measure the cellulase activity. Use 50 mg of Whatman filter paper as the substrate and place it in 4 stoppered test tubes of 20 mL. At the same time, add 0.5 mL of the enzyme solution and 1.5 mL of citrate buffer (0.05 mol / L citrate buffer, the preparation method refers to Liu Di, 2008) to each of the 4 test tubes. Add 1.5 mL of DNS reagent to one of the test tubes as a control group. Preheat the 4 test tubes in a water bath at 50 °C. After 10 min, add 50 mg of filter paper and react at this temperature for 60 min. After taking out the samples, immediately add 2.0 mL of DNS reagent to each sample, and place it in a water bath at 100 °C for another 5 min of reaction. After taking out and cooling, make up the volume to 15 mL, and measure the absorbance of each sample with a spectrophotometer. The measured OD value is compared with the glucose standard curve plotted, and the OD value is converted into glucose concentration. The amount of enzyme required to produce 1 μmoL of glucose per hour from the substrate is one enzyme activity unit (U). The FPA formula is as follows:

[0058]

[0059] Where: G is the glucose content; V is the made-up volume; t is the reaction time; A is the enzyme addition amount; B is the substrate mass; 5.56 is the amount of substance (μmol) of 1 mg of glucose substrate.

[0060] ④ Preparation of glucose standard curve: Respectively pipette 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.5 mL of 0.1 mg / mL glucose standard solution into colorimetric tubes, make up to 1.5 mL with distilled water, then add 2 mL of DNS reagent, mix well and boil in boiling water for 5 min. Cool and make up the volume to 15 mL. Measure the OD value at 540 nm with a spectrophotometer. Use the glucose content (mg) as the abscissa and the OD value as the ordinate to plot the glucose standard curve and fit the regression equation. As Figure 5 shown.

[0061] The results of cellulase activity are as Figure 2 shown in A.

[0062] b. Lignin enzyme activity assay:

[0063] ①Liquid medium: 20 g of glucose, 5 g of yeast extract powder, 1 g of KH2PO4, 0.5 g of MgSO4·7H2O, 50 mg of ZnSO4·7H2O, add water to make up to 1 L, natural pH, dispense into 250 mL conical flasks, pour 100 mL of liquid medium into each flask, sterilize at 121 °C for 30 min, and then add 40 μL of vitamin B1.

[0064] Solid medium of Zanthoxylum bungeanum twigs: Dry the Zanthoxylum bungeanum twigs at 60 °C until constant weight, then crush them, pass through a 40-mesh sieve, add them to the petri dish according to the ratio of Zanthoxylum bungeanum twigs: water = 1:2.5 (mass ratio), and sterilize at 121 °C for 30 min.

[0065] ②Take 5 mycelial blocks with a diameter of 1 cm and inoculate them into the liquid medium, and culture them at 25 °C and 150 r / min with shaking for 6 d.

[0066] ③Inoculate the seed fermentation broth into the solid medium of Zanthoxylum bungeanum twigs according to the ratio of seed fermentation broth volume: Zanthoxylum bungeanum twig mass = 1 ml: 1 g, culture at 25 °C for 30 d, take out 3 pretreated Zanthoxylum bungeanum twig samples every 5 d, weigh 1 g of the above samples into centrifuge tubes, add 3 mL of distilled water, shake at 25 °C and 150 r / min for 4 h, filter with 4 layers of gauze, centrifuge the filtrate at 3000 r / min at 4 °C for 10 min, take the supernatant, and the obtained liquid is the crude extracellular enzyme solution, which is stored in a refrigerator at 4 °C (Cong Shan 2014; Ye Jianqiang et al. 2018).

[0067] Use a kit (Chongqing Amida Biotechnology Co., Ltd.) to measure the activities of laccase, manganese peroxidase (MnP), and lignin peroxidase (LiP) in the pretreated Zanthoxylum bungeanum twig samples. Definition of laccase activity unit (U): The amount of enzyme required to oxidize 1 μmol of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) substrate per minute at a wavelength of 420 nm. Definition of MnP activity unit (U): The amount of enzyme required to oxidize 1 μmol of guaiacol per minute at a wavelength of 465 nm. Definition of LiP activity unit (U): One enzyme activity unit is defined as the change in absorbance value per minute of 0.01 in a 1 mL reaction system at a wavelength of 651 nm.

[0068] The results of lignin enzyme activity are as Figure 2 shown in B.

[0069] Data analysis: Use software SPSS 22.0 to calculate the mean and standard deviation, and analyze the significance of differences between each treatment.

[0070] 3) Molecular biological identification of fungi ITS

[0071] Inoculate the purified strain on 100 mL of PDA liquid medium and culture overnight. Filter with a funnel and blot dry the residual culture solution. Transfer it to a mortar and quickly add liquid nitrogen to grind the tissue into a powder. Immediately transfer the ground powder to a 1.5 mL Eppendorf tube. Add 2 volumes of preheated 2% CTAB (containing 0.1% β-mercaptoethanol by mass concentration), mix well, incubate at 65 °C for 30 - 45 min, and mix once every 15 min. Cool to room temperature, centrifuge at 10000 rmp for 10 min, and transfer the supernatant to another clean 1.5 mL Eppendorf tube. Add an equal volume of phenol-chloroform-isoamyl alcohol mixture (the volume ratio of Tris phenol, chloroform, and isoamyl alcohol is 25:24:1) for extraction. Centrifuge at 10000 rmp for 10 min, and transfer the supernatant to another clean 1.5 mL Eppendorf tube. Extract once again with an equal volume of chloroform-isoamyl alcohol mixture (the volume ratio of chloroform to isoamyl alcohol is 24:1), invert and mix for 10 min, centrifuge at 10000 rmp for 10 min, and transfer the supernatant to another clean 1.5 mL Eppendorf tube. Add 0.6 - 0.7 volumes of pre-cooled isopropanol to precipitate DNA, invert and mix well, and place at -20 °C overnight. Centrifuge at 10000 rmp for 10 min, discard the supernatant. Wash the precipitate with 75% ethanol aqueous solution by volume percentage to remove salt, wash with absolute ethanol to dehydrate, and dry in an incubator at 37 °C for 10 min. Add 50 μL of ddH2O solution to dissolve DNA.

[0072] The ITS sequence is a small gene fragment located between the IDNA coding genes 18S, 5.8S, and 28S. The ITS sequence in eukaryotic cells is conserved and not affected by changes in the environmental conditions. Therefore, by using universal primers to analyze the homology of the ITS base sequences between eukaryotic cells and those of other microbial species, the distance of their genetic relationships and phylogenetic status can be analyzed.

[0073] The ITS universal primers are as follows:

[0074] ITS1: 5'GGAAGTAAAAGTCGTAACAAGG-3, as shown in SEQ ID NO.2;

[0075] ITS4: 5'TCCTCC GCTTATTGATATGC-3', as shown in SEQ ID NO.3.

[0076] The PCR amplification program was as follows: After pre-denaturation at 94°C for 5 min, cycling started with denaturation at 94°C for 30 s, annealing at 52°C for 60 s, and extension at 72°C for 2 min, for a total of 30 cycles, followed by extension at 72°C for 10 min. 2 μL of the reaction solution was taken for agarose gel electrophoresis detection with a mass percentage of 1%. The PCR amplification product was sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd. for sequencing, and the sequence was input into GenBank for sequence homology comparison and analysis.

[0077] 4) Construction of the ITS sequence phylogenetic tree

[0078] The ITS sequences of strains with a sequence similarity greater than 97% to the sequence of the degrading strain ZC3 were downloaded from the Genbank gene database. After clustering analysis by ClustalX, a phylogenetic evolution tree was generated using the MEGA4.1 software. The results were as Figure 3 shown.

[0079] Example 3

[0080] Determination of the degradation ability of strain ZC3 on Zanthoxylum bungeanum twig powder

[0081] Liquid medium: 20 g of glucose, 5 g of yeast extract powder, 1 g of KH2PO4, 0.5 g of MgSO4·7H2O, 50 mg of ZnSO4·7H2O, made up to 1 L with water, natural pH, dispensed into 250 mL conical flasks, 100 mL of liquid medium was poured into each flask, sterilized at 121°C for 30 min, and then 40 μL of vitamin B1 was added.

[0082] Solid medium of Zanthoxylum bungeanum twigs: The Zanthoxylum bungeanum twigs were dried to constant weight at 60°C and then crushed, passed through a 40-mesh sieve, and added to the petri dish according to the ratio of Zanthoxylum bungeanum twigs: water = 1:2.5 (mass ratio), and sterilized at 121°C for 30 min.

[0083] The seed fermentation broth was inoculated into the solid medium of Zanthoxylum bungeanum twigs at a ratio of seed fermentation broth volume: Zanthoxylum bungeanum twig mass = 1 ml:1 g, cultured at 28°C for 40 d, sampled every 10 d (plus sampling on the 5th d), and after 40 d, the samples were taken out, dried to constant weight at 60°C, and put into self-sealing bags for standby. The degradation of the samples was observed by scanning electron microscopy, as Figure 4 shown, where A was the CK untreated with bacteria, and B was after treatment with strain ZC3.

[0084] The lignin, cellulose, and hemicellulose contents of the Zanthoxylum bungeanum twigs in the solid medium after 40 d of fermentation were measured, as shown in Table 1-3. (Note: CK1 was the white rot fungus NDM3-2 of the second generation, purchased from the Beijing Bioresource Collection Center; CK2 was the compound straw decomposing agent, purchased from Zhongxi Biotechnology Co., Ltd., the same below)

[0085] Table 1 Lignin degradation rate of Zanthoxylum bungeanum branches treated with Trichoderma asperellum ZC3 for 40 days

[0086]

[0087] Table 2 Cellulose degradation rate of Zanthoxylum bungeanum branches treated with Trichoderma asperellum ZC3 for 40 days

[0088]

[0089] Table 3 Hemicellulose degradation rate of Zanthoxylum bungeanum branches treated with Trichoderma asperellum ZC3 for 40 days

[0090]

[0091]

[0092] It can be seen from Tables 1 - 3 that the Trichoderma asperellum ZC3 strain of the present invention has obvious advantages in the degradation of lignin, cellulose, and hemicellulose of Zanthoxylum bungeanum branches.

[0093] Finally, it should be noted that the above - preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail through the above - preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. Trichoderma asperellum strain ZC3, characterized in that, Its classification name is Trichoderma asperellum ZC3, which has been deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The deposit number is CGMCC No. 40236, the deposit date is July 1, 2022, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The Trichoderma asperellum ZC3 strain according to claim 1, characterized in that, The ITS sequence of Trichoderma asperellum ZC3 strain is shown in SEQ ID NO.

1.

3. Application of the Trichoderma asperellum ZC3 strain according to claim 1 or 2 in the degradation of lignin, cellulose or hemicellulose.

4. Application of the Trichoderma asperellum ZC3 strain according to claim 1 or 2 in the degradation of Zanthoxylum bungeanum branches.

5. A microbial inoculant, characterized in that, Its active ingredient is the Trichoderma asperellum ZC3 strain according to claim 1 or 2, and its mass content is 5-10%.

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