Trichoderma koningiopsis dmpk1 and its use in preparing conidiospore-producing fungicide

Abstract

The present application belongs to the field of genetic engineering and microbial technology. The present application provides a technology for promoting the sporulation of Trichoderma koningiopsis in liquid culture medium by editing the pk1 gene, and provides a Trichoderma koningiopsis DMPK1 capable of rapidly producing conidia in liquid culture medium, and the preservation number of the Trichoderma koningiopsis DMPK1 is CGMCC NO.41236. The strain DMPK1 has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on May 21, 2024, and the address of the center is No. 1, Yihuangyuan 3rd, Beichen West Road, Chaoyang District, Beijing. The strain DMPK1 produces a large amount of conidia in liquid culture conditions for 2 days, and the sporulation amount reaches 3.25x10 7 cfu / mL.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and microbial biotechnology. Technical Background

[0002] Trichoderma is a type of filamentous fungus with excellent lignocellulose degradation capabilities and antagonistic effects against various pathogenic microorganisms. Furthermore, some Trichoderma strains possess strong protein secretion abilities, thus exhibiting significant economic value. In agriculture, Trichoderma is widely used for biological control and straw degradation, while industrially it is used to produce enzyme preparations such as cellulase, chitinase, and protease. Due to shelf-life considerations, most Trichoderma inoculants used in agricultural production are conidial preparations. Compared to mycelium, conidia have stronger resistance to adverse conditions, stable biological properties, and are easier to store and transport. Obtaining large quantities of conidia is a prerequisite for the industrialized production of Trichoderma inoculants. Solid-state fermentation is commonly used to prepare Trichoderma conidia. However, solid-state fermentation presents challenges in controlling nutrients, temperature, humidity, aeration, and pH levels. Furthermore, maintaining consistent substrate moisture holding capacity is difficult, leading to unstable conidia production and even fermentation failure during solid-state fermentation. In recent years, researchers have attempted to optimize culture conditions to produce spores from *Trichoderma viride*, *Trichoderma harzianum*, and other fungi through liquid fermentation (de Rezende LC et al., 2020). However, most naturally isolated *Trichoderma* strains exhibit weak or no conidial production under liquid fermentation conditions. Even when some *Trichoderma* strains can produce conidia through liquid fermentation, the fermentation cycle is often lengthy, and the conidial yield is low. Rapid and efficient preparation of *Trichoderma* spores via liquid fermentation remains a key factor hindering the industrialization of *Trichoderma* inoculants.

[0003] Like most filamentous fungi, the life cycle of *Trichoderma* strains includes growth, development, and morphological differentiation. The transition from hyphal growth to conidia is influenced not only by nutrients and environmental factors (such as temperature, humidity, osmotic pressure, aeration, and light), but also by its own regulatory genes. Studies in the model fungus *Aspergillus nidulans* have found that the genes *fluG*, *flbA*, *flbB*, *flbC*, *flbD*, and *flbE* are involved in conidia formation (Adams et al., 1998). The expression of these genes is subject to complex and precise regulation; for example, a cAMP-dependent protein kinase K inhibits conidia production (Shimizu & Keller, 2001). External factors such as nutrients and environmental conditions exert their influence through this complex regulatory network. Although research on the conidia formation system in *Trichoderma* is still lacking, advancements in genome sequencing and bioinformatics have provided the means to achieve mass conidia production in *Trichoderma* under liquid fermentation conditions through targeted genetic modification.

[0004] *Trichoderma koningiopsis* is a strain capable of degrading lignocellulose, obtained through screening using Congo red staining and liquid shake-flask fermentation. On various substrates, this strain exhibits superior cellulase production compared to *Trichoderma reesei* QM9414. After 84 hours of fermentation using microcrystalline cellulose as the sole carbon source, the activities of filter paper cellulase, carboxymethyl cellulase, β-glucosidase, and xylanase in the supernatant of *Trichoderma koningiopsis* were 1.72, 1.70, 6.35, and 1.12 times higher than those in QM9414, respectively. However, *Trichoderma koningiopsis* does not exhibit strong conidial production; conidia are only produced after prolonged incubation in liquid medium.

[0005] References:

[0006] de Rezende LC, de Andrade Carvalho AL, Costa LB, et al. Optimizing massproduction of Trichoderma asperelloides by submerged liquid fermentation and its antagonism against Sclerotinia sclerotiorum. World J MicrobiolBiotechnol. 2020, 36(8): 113.

[0007] Adams TH, Wieser JK, Yu JH. Asexual sporulation in Aspergillusnidulans. Microbiol Mol Biol Rev. 1998, 62(1): 35-54.

[0008] Shimizu K,Keller NP.Genetic involvement of a cAMP-dependent proteinkinase in a G protein signaling pathway regulating morphological and chemical transitions in Aspergillus nidulans.Genetics.2001,157(2):591-600. Summary of the Invention

[0009] For the reasons stated above, the purpose of this invention is to provide a strain of *Trichodermakoningiopsis* DMPK1, with accession number CGMCC NO.41236. This strain DMPK1 was deposited on May 21, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Compared with the wild-type strain of *Trichodermakoningiopsis*, strain DMPK1 has the pk1 gene sequence knocked out; the pk1 gene sequence is shown in SEQ ID NO.1 of the sequence listing.

[0010] This invention also provides an application of Trichoderma koningiopsis DMPK1 for the rapid production of conidia under liquid fermentation conditions.

[0011] The present invention also provides a method for constructing Trichoderma koningiopsis DMPK1, comprising the following steps:

[0012] Based on CRISPR / Cas9 technology, the plasmid pDpk1 for pk1 gene knockout was constructed.

[0013] Plasmid pDpk1 was introduced into Trichoderma synergae cells via PEG-mediated genetic transformation, successfully obtaining strain DMPK1 with the pk1 gene knocked out.

[0014] In a specific embodiment of the present invention, the method for constructing the plasmid pDpk1 includes: using plasmid pUC19-AMA1-Hyg-Cas9 as the starting plasmid, adding a gRNA expression cassette and homologous arms; using the *Trichoderma reesei* 5S rRNA promoter and the *Trichoderma konjac* glycine tRNA promoter ligated together as a dual promoter to drive gRNA expression, and using 6 Ts to terminate gRNA expression; and using CRISPR Guide RNA Design Tools (www.benchling.com / crispr) to select a 20 bp sequence for targeting the pk1 gene.

[0015] The *Trichoderma koningiopsis* DMPK1 gene knockout strain provided by this invention can produce a large number of conidia under liquid culture conditions, with a conidial yield of 3.25 × 10⁻⁶. 7 cfu / mL. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be described in detail below.

[0017] Figure 1 This is a schematic diagram illustrating the construction strategy of the pDpk1 plasmid for pk1 gene knockout.

[0018] Figure 2 This image shows the pDpk1 plasmid map and enzyme digestion verification diagram used for pk1 gene knockout.

[0019] Figure 3 This diagram shows the construction and validation of the pk1 gene deletion mutant strain DMPK1.

[0020] In this study, (A) amplification primers were internal primers for the pk1 gene; and (B) amplification primers were external primers for the pk1 gene. M: DNA marker; 1-3: pk1 gene deletion mutant DMPK1; P: plasmid pDpk1; WT: wild-type Trichoderma synergae; H2O: negative control.

[0021] Figure 4 Growth of wild-type strain (WT) and pk1-deficient strain DMPK1.

[0022] (A) Growth on media containing different carbon sources; (B) Comparison of colony diameters on media containing different carbon sources. Data are expressed as Mean±SD (n=3), *p<0.05, **p<0.01, ***p<0.001.

[0023] Figure 5 Sporulation of wild-type strain and pk1 gene-deleted strain DMPK1 in solid culture medium.

[0024] Among them, (A) wild-type strain WT and (B) DMPK1.

[0025] Figure 6 Sporulation of strain DMPK1 and wild-type strain in liquid culture medium.

[0026] Among them, (A) the color of the culture after 48 hours. The left is the wild-type strain (WT), and the right is the pk1 deletion mutant strain DMPK1; (B) microscopic observation of the wild-type strain growing in liquid culture medium; (C) microscopic observation of the pk1 deletion strain DMPK1 growing in liquid culture medium.

[0027] Figure 7 Microscopic images of DMPK1 cultured in liquid culture medium for different times.

[0028] Among them, (A) the culture time is 10h; (B) the culture time is 28h; and (C) the culture time is 30h. Detailed Implementation

[0029] This invention provides a technical method for promoting sporulation of Trichoderma synergae in liquid culture medium through gene editing. The technical method includes constructing an engineered strain DMPK1 with the pk1 gene deleted based on CRISPR / Cas9 technology.

[0030] The present invention also provides the application of the engineered strain DMPK1 in sporulation in liquid culture medium.

[0031] In this invention, the strain used for pk1 gene knockout is Trichoderma koningiopsis 8985 (Li Peng et al., 2021).

[0032] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions or conditions recommended in the manufacturer's instructions.

[0033] The main experimental materials involved in the following examples are:

[0034] Table 1 Primer sequences used in this invention

[0035]

[0036] Table 2 Primer sequences used in this invention

[0037]

[0038] Table 3 Primer sequences used in this invention

[0039]

[0040]

[0041] The culture medium involved in the embodiments includes the following components:

[0042] PDA medium (g / L): 200g potato, 20g glucose. Cut 200g peeled potatoes into small pieces, boil in boiling water for 30 minutes, filter through eight layers of gauze, add glucose to the filtrate, and bring the volume to 1L. PDA solid medium requires the addition of 1.5% agar powder; without agar powder, it becomes PDB medium. The pH should be natural. Autoclave at 115℃ for 30 minutes.

[0043] LMM medium (g / L): glucose 10g, yeast extract 1g, 20× Nitrate salts 50mL, pH natural. Autoclave at 115℃ for 30min. Add 1mL of 1000× Trace elements (filter sterilized) before use. 20× Nitrate Salts (g / L): NaNO3 120g, KCl 10.4g, MgSO4·7H2O 10.4g, KH2PO4 30.4g; 1000× Trace Elements (g / L): ZnSO4·7H2O 22g, H3BO3 11g, MnCl2·4H2O 5g, FeSO4·7H2O 1.6g, CoCl2·6H2O 1.6g, CuSO4·5H2O 1.6g, (NH4)6Mo7O 24 • 4H2O 1.1g, Na4EDTA 50g.

[0044] Protoplast transformation supernatant (g / L): Potato broth 39g, sorbitol 218.6g, agar powder 0.5%, pH natural. Autoclave at 115℃ for 30min.

[0045] Protoplast transformation sub-medium medium (g / L): 39g potato broth medium, 218.6g sorbitol, 1.5% agar powder, pH natural. Autoclave at 115℃ for 30min.

[0046] Basic culture medium (g / L): KH₂PO₄ 15g, (NH₄)₂SO₄ 5g, MgSO₄·7H₂O 0.6g, CaCl₂ 0.8g, urea 0.3g, pH 4.8. Autoclave at 115℃ for 30min. Before use, add trace element solution (MnSO₄·H₂O 0.0016g, FeSO₄·7H₂O 0.0005g, ZnSO₄·7H₂O 0.0014g, CoCl₂ 0.0002g, filter sterilize).

[0047] Single carbon source medium (g / L): Add 20g glucose / 20g glycerol / 20g lactose to the basic medium.

[0048] The buffer solution involved in the embodiments includes the following components:

[0049] Osmotic Medium:

[0050] MgSO4·7H2O 1.2M

[0051] Phosphate buffer (pH 6.5) 0.01M

[0052] Adjust the pH to 5.8 using 1M Na2HPO4.

[0053] Trapping buffer:

[0054] Sorbitol 0.6M

[0055] Tris-HCl (pH 7.5) 0.1M

[0056] STC buffer:

[0057] Sorbitol 1.2M

[0058] CaCl2 0.01M

[0059] Tris-HCl (pH 7.5) 0.01M

[0060] PEG buffer:

[0061] PEG4000 60% (w / v)

[0062] CaCl2 0.05M

[0063] Tris-HCl (pH 7.5) 0.05M

[0064] The present invention will be further illustrated below with reference to the embodiments.

[0065] Example 1

[0066] The pk1 gene-deleted engineered strain DMPK1 was constructed using CRISPR / Cas9 technology.

[0067] (1) Construction of pDpk1 plasmid for pk1 gene knockout

[0068] See the schematic diagram of the plasmid construction strategy. Figure 1 .

[0069] The pk1 gene (GenBank record PP505844) in *Trichoderma koningiopsis* was knocked out using CRISPR / Cas9-based gene editing technology. The amino acid sequence of the product encoded by the pk1 gene is shown in SEQ ID NO.1 of the sequence listing.

[0070] The plasmid pUC19-AMA1-Hyg-Cas9 contains the gene encoding Cas9 (4,266 bp in length, including sequences encoding the nuclear localization signal NLS and the 3xFlag tag), and utilizes the promoter PgpdA from the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase gene to drive the expression of Cas9 (GenBank record WYB68443) (Chen et al., 2020). The nucleotide sequence of the promoter PgpdA is shown in SEQ ID NO.2 of the sequence listing.

[0071] In addition, the plasmid pUC19-AMA1-Hyg-Cas contains the autonomously replicating sequence AMA1 (Aleksenko & Clutterbuck, 1997), so it can be introduced into Trichoderma koningiopsis in a non-integrated genome manner via PEG-mediated transformation.

[0072] Under resistance pressure, the plasmid can exist stably in the cell outside the genome; if the transformant is passaged under resistance-free conditions, the autonomously replicating plasmid will be lost due to the lack of selection pressure, thus avoiding the plasmid from integrating into the genome and causing adverse effects on the strain.

[0073] The plasmid pUC19-AMA1-Hyg-Cas contains the hygromycin B phosphotransferase gene hyg. Antibiotic susceptibility testing of *Trichoderma koningiopsis* showed that *Trichoderma koningiopsis* is sensitive to hygromycin B and can be used for screening *Trichoderma koningiopsis* transformants.

[0074] Using plasmid pUC19-AMA1-Hyg-Cas9 as the starting plasmid, after adding a gRNA expression cassette and homologous arms, the plasmid pDpk1 for pk1 gene knockout was constructed. The sequence of the gRNA expression cassette is shown in SEQ ID NO.3 of the sequence listing. The homologous arms include the upstream homologous arm pk1-L and the downstream homologous arm pk1-R of pk1. The sequence of the upstream homologous arm pk1-L is shown in SEQ ID NO.4 of the sequence listing, and the sequence of the downstream homologous arm pk1-R is shown in SEQ ID NO.5 of the sequence listing.

[0075] A dual promoter was constructed by tandemly using the *Trichoderma reesei* 5S rRNA promoter and the *Trichoderma koningiopsis* glycine tRNA promoter to drive gRNA expression, with 6 Ts used to terminate gRNA expression. A 20 bp sequence targeting the pk1 gene, 5'-CAAGTTAAAATAAGGCTAGT-3', was designed using CRISPR Guide Design Tools. The *Trichoderma reesei* 5S rRNA promoter sequence is shown in SEQ ID NO. 6 of the sequence listing, and the *Trichoderma koningiopsis* glycine tRNA promoter sequence is shown in SEQ ID NO. 7 of the sequence listing.

[0076] Using *Trichoderma reesei* genomic DNA as a template, primers 5S rRNA-F / R were designed to amplify the *Trichoderma reesei* 5S rRNA promoter sequence. Using *Trichoderma koningiopsis* genomic DNA as a template, primers tRNA-F / R were designed to amplify the *Trichoderma koningiopsis* glycine tRNA promoter sequence. The two promoter sequences were then ligated into a single fragment by PCR amplification using primer gRNA-1-F / R to obtain the promoter sequence driving gRNA expression.

[0077] Primers gRNA-2-F / R were designed to amplify the gRNA expression cassette via PCR. This cassette contained a 20 bp crRNA guide sequence targeting the knockout gene and a tracrRNA sequence for binding to the Cas9 protein. After recovering the fragment, primer gRNA-3-F / R was used to amplify the fragment via PCR and ligate it to the promoter sequence that drives gRNA expression, ultimately obtaining a DNA fragment (full length 642 bp) containing the gRNA expression cassette coding sequence.

[0078] Using Trichoderma koningiopsis genomic DNA as a template, primers Pk1-1-F / R were designed to amplify the DNA fragment containing the upstream homologous arm of the pk1 gene (full length 1040 bp) by PCR. Primers Pk1-2-F / R were designed to amplify the DNA fragment containing the downstream homologous arm of the pk1 gene (full length 1030 bp) by PCR.

[0079] The plasmid pUC19-AMA1-Hyg-Cas9 was digested with SmaI to obtain a linearized plasmid. The above fragment was ligated to the linearized plasmid pUC19-AMA1-Hyg-Cas9 using the Gibson assembly method (Gibson et al., 2009) to obtain the plasmid pDpk1 for pk1 knockout. Figure 1 ).

[0080] pDpk1 was digested with HindIII, yielding three fragments consistent with the theoretical size, confirming the correct construction of the plasmid. Figure 2 ).

[0081] (2) Construction of the pk1 gene-deleted strain DMPK1

[0082] The plasmid pDpk1 was introduced into *Trichoderma koningiopsis* cells via PEG-mediated protoplast transformation. et al., 1987.

[0083] This includes the preparation of Trichoderma synergae protoplasts and PEG-mediated transformation of Trichoderma synergae protoplasts.

[0084] Preparation of Trichoderma kangaroo protoplasts:

[0085] ① Spread the glycerol preservation solution of Trichoderma koningiopsis onto PDA medium and incubate upside down in a 28℃ incubator for 5-7 days to produce sporulation;

[0086] ② Use a sterile cotton swab to scrape off the spores from the plate culture and inoculate them into 50 mL of LMM mycelial germination medium. Incubate at 28℃ and 220 rpm for 10–12 h. Observe under a microscope that the length of the mycelium is more than 5 times the diameter of the spores.

[0087] ③ Use a sterile 40μm filter to filter and collect the mycelium, then wash it 2-3 times with sterile water;

[0088] ④ Weigh 0.64g of the cell wall hydrolysis enzyme Vinotaste into a 50ml centrifuge tube, add 10mL of Osmotic Medium, dissolve thoroughly, and then filter to sterilize.

[0089] ⑤ Add 10 mL of filtered enzyme hydrolysate to a sterilized 100 mL Erlenmeyer flask, resuspend the mycelium in the enzyme hydrolysate (mix thoroughly by pipetting), and incubate at 28 °C and 100 rpm for 8–10 h. Stop the reaction and start collecting when a large number of protoplasts are produced in the microscope field of view.

[0090] ⑥ After protoplast formation, pour the enzyme digest into a 50mL centrifuge tube, and slowly add 10mL of Trapping buffer along the wall with a pipette to ensure that the layers are separated.

[0091] ⑦ Centrifuge at 4000 rpm for 18 min at 4℃, and use a 1 mL pipette tip with the tip removed to transfer the white protoplasts (intermediate layer) into a 50 mL centrifuge tube. Add an equal volume of STC buffer, centrifuge at 4000 rpm for 10 min at 4℃, discard the supernatant, and obtain the protoplast precipitate.

[0092] ⑧ Resuspend the precipitate in 5 mL of STC buffer and centrifuge at 4000 rpm for 5 min;

[0093] ⑨ Repeat step ⑧;

[0094] ⑩ Discard the supernatant, resuspend the protoplasm in an appropriate amount of STC buffer, and store at 4°C for later use. et al., 1987.

[0095] PEG-mediated protoplast transformation of Trichoderma kangaroo:

[0096] ① Add about 5 μg of plasmid pDpk1 to 100 μL of Trichoderma synergae protoplast resuspension, mix gently, and incubate on ice for 50 min;

[0097] ② Slowly add 1.25 mL of PEG buffer, mix gently, and let stand at room temperature for 20 min;

[0098] ③ Add 5 mL of STC buffer and mix well;

[0099] ④ Mix 1 mL with 5 mL of Trichoderma synergae protoplast transformation upper medium (temperature below 50℃) and immediately spread it on the protoplast lower medium (the medium contains 200 μg / mL hygromycin B).

[0100] ⑤ Incubate at 28℃ for 4–7 days until transformants grow, then transfer the transformants to screening plates for further incubation.

[0101] Transformants were selected, and their genomic DNA was extracted as templates for subsequent PCR verification. Internal primers for the pk1 gene (Pk1-iF / R, which, when used for PCR verification, should amplify a 992 bp fragment for wild-type strains and no amplification band for pk1 knockout strains) and external primers (Pk1-eF / R, which, when used for PCR verification, should amplify a 2677 bp fragment for wild-type strains and a 1063 bp fragment for pk1 knockout strains) were designed.

[0102] The results showed that the band sizes of the three transformants were as expected, indicating that the pk1 gene knockout strain was successfully obtained. Figure 3The strain was named *Trichoderma koningiopsis* DMPK1. DMPK1 was deposited on May 21, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 41236. The center is abbreviated as CGMCC and is located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0103] Example 2

[0104] Sporulation of the engineered strain *Trichoderma koningiopsis* DMPK1 in liquid culture medium.

[0105] (1) Growth and sporulation of the engineered strain Trichoderma koningiopsis DMPK1 in solid culture medium

[0106] Wild-type Trichoderma koningiopsis strain WT and engineered strain Trichoderma koningiopsis DMPK1 were inoculated into solid media supplemented with different carbon sources, including PDA composite medium and basal media supplemented with glucose / lactose / glycerol respectively. The media were incubated at 28°C inverted positions, and mycelial growth was compared. Figure 4 ).

[0107] In addition, wild-type strain WT and engineered strain Trichoderma koningiopsis DMPK1 were inoculated onto PDA plates and cultured at 28°C for 7 days to observe the sporulation of the strains.

[0108] Compared to the wild-type strain WT, the engineered strain *Trichoderma koningiopsis* DMPK1 showed a slower growth rate and produced almost no sporulation when cultured on PDA composite medium plates. Figure 5 ).

[0109] (2) Sporulation of the engineered strain Trichoderma koningiopsis DMPK1 in liquid culture medium

[0110] When the wild-type strain WT and the engineered strain *Trichoderma koningiopsis* DMPK1, which has a gene mutation of pk1, were inoculated into PDB medium, in stark contrast to the lack of sporulation on PDA solid plates, the engineered strain *Trichoderma koningiopsis* DMPK1 produced a large number of spores in the liquid environment after being cultured at 28℃ and 220rpm for 48 hours, causing the medium to turn dark green. When the bacterial suspensions of wild-type WT and engineered strain *Trichoderma koningiopsis* DMPK1 were observed under a microscope, the engineered strain *Trichoderma koningiopsis* DMPK1 showed a large number of elliptical spores under the microscope. Figure 6 ).

[0111] An experiment was designed to observe the spore production process of the engineered strain *Trichoderma koningiopsis* DMPK1 in a liquid environment. Spores (1.8 × 10⁻⁶) obtained under liquid culture were... 8 500 μL of (cfu / mL) solution was inoculated into 50 mL of LPDB medium and cultured at 28 °C and 220 rpm. Samples were taken every 2 hours starting from 10 h for microscopic observation. Figure 7 As shown, most engineered Trichoderma koningiopsis DMPK1 spores begin to absorb water and swell at 10 hours, germinating and producing germ tubes, which then spread into hyphae, forming mycelium. By 28 hours, spores begin to appear at the tips of the hyphae. By 30 hours, a large number of spores have been produced. At this point, the number of spores was counted, approximately 3.25 × 10⁻⁶. 7 cfu / mL.

[0112] In summary, this invention obtained the engineered strain *Trichoderma koningiopsis* DMPK1 by knocking out the pk1 gene in *Trichoderma koningiopsis*. The engineered strain *Trichoderma koningiopsis* DMPK1 was able to produce spores under liquid culture conditions, with a spore count reaching 3.25 × 10⁻⁶. 7 cfu / mL.

[0113] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made based on it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the main content of the present invention fall within the scope of protection claimed by the present invention.

[0114] References

[0115] rook J,Fritsch EF,Maniatis T.Molecular Cloning:A Laboratory Manual,Second Edition.Cold Spring Harbor Laboratory Press,New York,1989.

[0116] Hao Z,Su X.Fast gene disruption in Trichoderma reesei using in vitroassembled Cas9 / gRNA complex.BMC Biotechnol,2019,19:2.

[0117] Aleksenko A,Clutterbuck AJ.Autonomous plasmid replication inAspergillus nidulans:AMA1 and MATE elements.Fungal Genet Biol,1997,21:373-387.

[0118] Chen C,Liu J,Duan C,Pan Y,Liu G.Improvement of the CRISPR-Cas9mediated gene disruption and large DNA fragment deletion based on a chimericpromoter in Acremonium chrysogenum.Fungal Genet Biol.2020,134:103279.

[0119] Gibson DG,Young L,Chuang R,et al.Enzymatic assembly of DNA moleculesup to several hundred kilobases.Nature Methods,2009,6:343-345.

[0120] M,Nevalainen HK, M,et al.A versatile transformation systemfor the cellulolytic filamentous fungus Trichoderma reesei.Gene,1987,61(2):155-164.

Claims

1. Trichoderma simonii ( Trichoderma koningiopsis DMPK1, with accession number CGMCC NO.41236; the described Trichoderma DMPK1 was knocked out pk1 Genes; the stated pk1 The amino acid sequence of the gene sequence encoding product is shown in SEQ ID NO. 1 of the sequence listing.

2. The *Trichoderma simonii* as described in claim 1 (… Trichoderma koningiopsis The application of DMPK1 in the preparation of conidial-producing agents, wherein the culture medium for producing conidia is PDB liquid culture medium.

3. The *Trichoderma simonii* according to claim 2 ( Trichoderma koningiopsis The application of DMPK1 in the preparation of conidial-producing fungal agents is characterized by, The amount of conidia reached 3.25 × 10⁻⁶. 7 cfu / mL.

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