DsRNA targeting BdSpt7 gene, biocontrol agent and application thereof

Abstract

The invention discloses dsRNA of a targeted BdSpt7 gene, a biocontrol agent and application of the dsRNA. The sequence of the dsRNA of the targeted BdSpt7 gene is as shown in SEQ ID No.2. The invention further discloses a preparation method of the biocontrol agent of the targeted BdSpt7 gene. The active ingredient of the biocontrol agent is the dsRNA of the target BdSpt7 gene. The invention also provides an application of the botryosphaeria dothidea BdSpt7 gene in prevention and treatment of apple botryosphaeria dothidea, and a nucleotide sequence of the botryosphaeria dothidea BdSpt7 gene is shown as SEQ ID No.1. The invention also provides an African trichoderma harzianum engineering strain for expressing dsRNA, the African trichoderma harzianum engineering strain is integrated with a dsRNA expression box for interfering gene expression of a target gene BdSpt7, the dsRNA expression box is shown as SEQ ID No.1, and the African trichoderma harzianum is an African trichoderma harzianum with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No. 41709. The dsRNA targeting the BdSpt7 gene can significantly reduce the pathogenicity of the physalospora piricola through interference, and the African trichoderma harzianum engineering strain can colonize on the plant surface and continuously express the dsRNA so as to play a role in preventing and treating the physalospora piricola, and is green and environment-friendly.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a dsRNA targeting the BdSpt7 gene, a biocontrol agent, and its application. Background Technology

[0002] Apple ring rot is caused by pathogenic fungi ( Botryosphaeria dothidea Apple ring rot, a fruit tree disease, is one of the most significant diseases affecting fruit production and quality. It is widespread in major apple-producing areas of my country, infecting branches, trunks, and fruits, causing branch cankers and fruit rot, severely hindering normal tree growth, and significantly reducing fruit quality, resulting in substantial economic losses. Therefore, the prevention and control of apple ring rot is a crucial issue facing the fruit tree cultivation industry.

[0003] Currently, the control of apple ring rot still relies mainly on chemical fungicides. However, this traditional method easily leads to two major problems: first, it causes the pathogen to develop resistance, gradually reducing the control effect; second, the widespread use of chemical pesticides pollutes the environment. Therefore, with the promotion of green agriculture, it is necessary to seek more environmentally friendly methods for controlling apple ring rot.

[0004] In recent years, RNA interference (RNAi) technology has shown great application potential in the field of plant disease control due to its high specificity and environmentally friendly characteristics. This technology designs double-stranded RNA (dsRNA) that targets key pathogenic genes in pathogens, which can effectively inhibit the growth and pathogenicity of pathogens.

[0005] However, there is currently limited research on the key pathogenic genes of the fungus causing apple ring rot, and there are no effective dsRNA products that can be applied to the prevention and control of apple ring rot based on RNA interference technology.

[0006] Therefore, existing technologies need further improvement. Summary of the Invention

[0007] To address the above problems, the present invention provides a targeted BdSpt7 The dsRNA of the gene, biocontrol agents containing dsRNA and their application in the control of apple ring rot.

[0008] To address the above problems, this application provides the following technical solution: Firstly, this application provides a *Rhizoctonia solani* pathogen. BdSpt7 Application of genes in the prevention and control of apple ring rot, the ring rot pathogen BdSpt7 The nucleotide sequence of the gene is shown in SEQ ID No. 5.

[0009] This application constructs a fungus for the treatment of ringworm using molecular biology techniques. BdSpt7Gene knockout strains were identified, and the functional morphology of the knockout bacteria was studied. BdSpt7 This gene plays a crucial regulatory role in the nutritional metabolism, host adaptation, and pathogenicity of *Rhizoctonia solani*. Compared to the wild type, the colony expansion diameter is only 30%-50% of that of the wild type. The growth ability of this knockout bacterium is significantly weakened in different culture media (PDA, MM) and host simulation environments (fruit homogenate, bark extract). Furthermore, the pathogenicity of this knockout bacterium on apple fruits and branches is reduced by 70%-80%, and the lesion diameter is reduced from 2.7-3.7 cm in the wild type to 0.6-0.7 cm, confirming that this gene is an essential gene for the pathogenesis of *Rhizoctonia solani*.

[0010] based on BdSpt7 The aforementioned functional role of the gene, the *Rhizoctonia solani* BdSpt7 In the application of genes in the prevention and control of apple ring rot, the method of application is as follows: knocking out the pathogen of apple ring rot... BdSpt7 Genes, or those of the fungus that causes apple ring rot. BdSpt7 Genes undergo RNA interference, thereby inhibiting... BdSpt7 The expression.

[0011] Secondly, this application provides a targeted BdSpt7 The dsRNA of the gene, the sequence of which is shown in SEQ ID No. 2.

[0012] This application is aimed at BdSpt7 The above-mentioned dsRNA was designed. This dsRNA significantly inhibited the pathogenicity of *Rhizoctonia solani* in vitro. After treatment with dsRNA at a concentration of 80-120 ng / μL for 5 days, the lesions of *Rhizoctonia solani* decreased by 0.5-1.0 cm, showing a dose-response effect. The inhibitory effect was more significant at 400 ng / μL.

[0013] Thirdly, this application also provides a biocontrol agent, the active ingredient of which is the aforementioned targeted agent. BdSpt7 dsRNA of genes.

[0014] Fourthly, this application also provides the aforementioned targets BdSpt7 Application of dsRNA of the gene or the above-mentioned biocontrol agents in the preparation of biological agents for the prevention and control of apple ring rot.

[0015] Optionally, in the application, the concentration of dsRNA is 80~400 ng / μL, and the application method is to spray it onto the plant leaves.

[0016] Fifthly, this application also provides an engineered strain of *Trichoderma harzianum* expressing dsRNA, wherein the engineered strain of *Trichoderma harzianum* integrates an interfering target gene. BdSpt7 Trichoderma harzianum (a species of Trichoderma) expressing dsRNA gene cassettes Trichodermaatroviride The dsRNA expression cassette is shown in SEQ ID No. 1; the *Trichoderma harzianum* used is *Trichoderma harzianum* with accession number CGMCC No. 41709 (…). Trichodermaatroviride )T10.

[0017] Because dsRNA products suffer from poor stability and low delivery efficiency in natural environments, they can be transferred into microbial expression systems for stable expression to improve their stability and efficacy.

[0018] Extensive screening revealed that Trichoderma ( Trichodermaspp As a widely used biocontrol bacterium, it has stable colonization ability and efficient dsRNA delivery potential, giving it a great advantage as a biocontrol vector.

[0019] The *Trichoderma afroharzianum* used in this application was deposited on December 11, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41709, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0020] The aforementioned *Trichoderma harzianum* T10 not only exerts a long-term control effect against *Trichoderma harzianum* by stably expressing dsRNA based on the principle of RNA interference, but also has a control effect against a variety of other pathogens. Furthermore, while controlling diseases, it acts as a growth promoter, enhancing the growth of fruit trees. In addition, this fungus has a certain ability to degrade the main components of rot virin, reducing the damage of such toxins produced by pathogens in the soil to fruit trees.

[0021] Sixthly, this application also provides a method for preparing the above-mentioned engineered Trichoderma harzianum strain, which includes the following steps: (1) Construction of dsRNA expression cassette: It consists of arm sequences with identical sequences at both ends but opposite directions and a central sequence that acts as a connector, thus constructing a cassette containing the target gene. BdSpt7 The dsRNA expression cassette, the nucleotide sequence of which is shown in SEQ ID No. 1; (2) Construction of recombinant expression vector: The dsRNA expression cassette was ligated into the Trichoderma expression vector to construct a recombinant expression vector containing the dsRNA expression cassette; (3) Protoplast transformation: The recombinant expression vector containing the dsRNA expression cassette was successfully constructed and introduced into Trichoderma harzianum T10 via PEG-mediated protoplast transformation. Positive transformants were screened to obtain the Trichoderma harzianum engineered strain.

[0022] Preferably, the Trichoderma expression vector is pYF11.

[0023] Seventhly, this application also provides a method for preventing and controlling apple ring rot, the method being: applying the above-mentioned dsRNA or one or two of the above-mentioned engineered strains of Trichoderma harzianum to the fruit trees.

[0024] Optionally, the application method of the aforementioned engineered *Trichoderma harzianum* fungus is as follows: prepare a spore suspension of the aforementioned engineered *Trichoderma harzianum* fungus, and apply the spore suspension to the surface of apple plants or fruits. Preferably, the concentration of the *Trichoderma harzianum* spore suspension is not less than 1 × 10⁻⁶. 6 CFU / mL.

[0025] The present invention has the following beneficial effects: 1. This invention discloses for the first time BdSpt7 The study explores the core role of genes in the pathogenesis of apple ring rot fungus and provides technical solutions for dsRNA interference and Trichoderma vector delivery, offering a new strategy for the green control of apple ring rot.

[0026] 2. The engineered strain of *Trichoderma harzianum* of this invention can stably express the target pathogen *Trichoderma harzianum*. BdSpt7 The dsRNA expression cassette of the gene plays a role in controlling apple ring rot, with stable effects and high delivery efficiency. This engineered strain can colonize on the plant surface and continue to exert its effects. It can be used to prepare biocontrol agents for the control of apple ring rot, providing a new technical means for plant disease control.

[0027] 3. Compared with traditional chemical control methods, the RNA interference technology and Trichoderma engineered fungus control method adopted in this invention are more environmentally friendly, reducing the environmental pressure caused by pesticide use. It provides a feasible technical solution for the field of fruit tree disease control and has certain practical value and promotion significance. Attached Figure Description

[0028] Figure 1 Apple ring rot fungus BdSpt7 PCR validation results of gene knockout strains; Figure 2 for BdSpt7 Phenotypes of the deletion strains on PDA and MM media; Figure 3 for BdSpt7 Phenotypes of the missing strain on apple twigs and fruit culture media; Figure 4 The effect of BdSpt7 on the pathogenicity of *Rhizoctonia solani*; Figure 5 The interference effect of dsRNA-Spt7 on *Pseudomonas aeruginosa*; Figure 6 A schematic diagram of the Trichoderma-mediated delivery vector for ds-Spt7; Figure 7 PCR identification of DNA delivered by Trichoderma ds-Spt7 strain; Figure 8 RT-PCR identification of RNA from Trichoderma ds-Spt7 delivery strain; Figure 9 The interference effect of Trichoderma ds-Spt7 delivery strain on young apple fruits infected with ring rot fungus; Figure 10 The interference effect of Trichoderma ds-Spt7 delivery strain on the infection of ripe apples by Trichoderma ring rot; Figure 11 The interference effect of Trichoderma ds-Spt7 delivery strain on apple tree branches infected with ring rot fungus; Figure 12 A is the morphology and phylogenetic tree of Trichoderma T10; B is the colony morphology of strain T10 on PDA medium; C is the morphology of conidiophores of strain T10; D is the phylogenetic tree of ITS gene of strain T10. Figure 13 The degradation ability of Trichoderma T10 against three toxic substances in apple tree rot disease; Figure 14 A represents the growth-promoting effect of strain T10 treatment on apple seedlings; B represents the growth-promoting effect of strain T10 treatment on apple seedlings; C represents the effect of strain T10 treatment on the height, fresh weight, dry weight, and chlorophyll content of apple seedlings. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0030] To ensure a complete and unambiguous understanding of the technical solution of this invention, the term "apple" as used in this invention refers to an apple tree (sapling). Malus domestica This refers to tissue culture seedlings obtained through tissue culture, rather than simply edible apple fruit. The "..." described in this invention... BdSpt7 The italicized "BdSpt7" in this invention refers to the gene; the non-italicized "BdSpt7" in this invention refers to the gene "BdSpt7". BdSpt7The protein encoded by "". Of course, those skilled in the art can clearly and completely understand the meaning and description of the relevant genes and their encoded proteins according to the description of the present invention.

[0031] Example 1: Ringworm BdSpt7 Molecular verification of gene knockout strains In yeast, Spt7 is a core protein constituting the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, responsible for maintaining the stability of the SAGA complex structure. It accounts for over 10% of intracellular gene transcription and plays a crucial role in the growth process (DOI: 10.1016 / j.mad.2021.111480), and its sequence is shown in SEQ ID No. 5. This example aims to explore the role of *Rhizoctonia solani*. Spt7 To understand the gene function, a BdSpt7 gene knockout strain of *Rhizoctonia solani* was constructed.

[0032] 1. Experimental Methods Using a three-fragment homologous recombination strategy, a 1.2kb fragment containing DNA amplified from wild-type genomic DNA was constructed. BdSpt7 Upstream and downstream gene sequences, and hygromycin B phosphotransferase ( HPH Knockout cassettes were selected. The knockout cassettes were introduced into wild-type apple ring rot strain LW03 via PEG-mediated protoplast transformation. Resistant transformants were screened on PDA medium containing 50 μg / mL hygromycin. Genomic DNA was extracted from the transformants and amplified by PCR using primers specific to the knockout region.

[0033] 2. Experimental Results and Analysis Agarose gel electrophoresis results are as follows Figure 1 As shown, wild-type LW03 amplified a 2.1kb specific band, while the knockout strain... ΔSpt7 The gene deletion did not produce this band. This validation result indicates that: BdSpt7 The gene has been successfully knocked out, and a BdSpt7 knockout transformant of *Rhizoctonia solani* has been obtained.

[0034] Example 2: Effect of BdSpt7 deletion on the growth of *Rhizoctonia solani* in nutrient medium. 1. Experimental Methods To explore BdSpt7 The biological function of the gene will activate the wild-type LW03 and the knockout strain. ΔSpt7 Use a punch to create 5mm-sized mycelial cakes, which are then inoculated into PDA medium and MM basal medium, respectively. Under constant temperature incubation at 25℃, the colony expansion diameter is observed periodically.

[0035] 2. Experimental Results The results are as follows Figure 2As shown, in PDA and MM media, after 2.5 days, the wild-type strain LW03 of *Rhizoctonia solani*, the causal agent of apple ring rot, had grown to the edge of the culture medium, while... ΔSpt7 The mutant colonies were less than 1 cm in diameter; after further culture for 14 days, the wild-type strain had already shown signs of aging. ΔSpt7 The mutant colonies were only 2 cm in diameter. This experimental data indicates that... BdSpt7 The deletion of the gene significantly affected the growth ability of *Rhizoctonia solani* under nutritional conditions, suggesting that the gene is involved in regulating the nutritional metabolic pathways of the pathogen.

[0036] Example 3 BdSpt7 Effect of deletion on the growth of *Rhizoctonia solani* in apple fruit and bark culture media 1. Experimental Methods To simulate the colonization environment of the pathogen in apple fruit and bark, fruit homogenate medium (Fruit) and bark extract medium (Bark) were prepared for growth assays. Each strain was inoculated according to the method in Example 2 and cultured at 25°C.

[0037] Fruit homogenate medium: Boil 200g of fruit for 30min, bring the volume to 1L, add 15g of agar, and sterilize at 121℃ for 20min.

[0038] Bark extract culture medium (Bark): Boil 100g of bark for 30 minutes, bring the volume to 1L, add 15g of agar, and sterilize at 121℃ for 20 minutes.

[0039] 2. Experimental Results and Analysis like Figure 3 The results showed that after 7 days of culture on fruit homogenate medium and bark extract medium, the wild-type strain LW03 of *Aureobasidium pinnatifida*, the causal agent of apple ring rot, had grown to the edge of the medium and had abundant aerial hyphae. ΔSpt7 The mutant colonies are less than 1 cm in diameter.

[0040] The experimental results show that BdSpt7 Gene deletion not only affects the growth of *Rhizoctonia solani* in artificial culture media, but also significantly impacts its ability to spread within the host tissue-associated nutrient environment, suggesting... BdSpt7 Genes play a crucial role in the adaptation of *Rhizoctonia solani* to the host microenvironment.

[0041] Example 4 BdSpt7 Effect of deletion on the pathogenicity of *Rhizoctonia solani* 1. Experimental Methods The pathogenicity of BdSpt7 was verified through inoculation experiments on apple fruits and branches. Activated wild-type LW03 and knockout plants were used. ΔSpt7Use a punch to create 5mm-sized fungal cakes, and inoculate mature fruits, young fruits, and one-year-old branches using the puncture method. After 5-7 days of moist cultivation, measure the expansion of lesions.

[0042] 2. Experimental Results and Analysis like Figure 4 As shown, the average diameter of lesions formed by wild-type apple ring rot fungus LW03 on mature apples is 2.7 cm, while... ΔSpt7 The resulting lesions were only 0.6 cm in diameter, and their infectivity in the fruit was significantly reduced; on the apple branches, ΔSpt7 The resulting lesions were only 0.7 cm in diameter, significantly smaller than those formed by the wild-type apple ring rot fungus LW03 (3.7 cm in diameter), indicating a significant decrease in its infectivity on branches. ΔSpt7-C was a complement strain of the Spt7 knockout mutant, while Mock was the blank control.

[0043] This experiment confirms that, BdSpt7 The gene is essential for the infection of apple host by *Rhizoctonia solani*. The deletion of this gene leads to a significant decrease in the pathogenicity of *Rhizoctonia solani* in different tissues of the host.

[0044] Example 5: In vitro interference of dsRNA-Spt7 against *Rhizoctonia solani* 1. Experimental Methods Using the primer design website (http: / / primerexplorer.jp / e / index.html) for... BdSpt7 A 442bp dsRNA fragment was designed outside the non-conserved region of the gene coding region, as shown in SEQ ID No. 2. It was synthesized and purified by the Thermo Fisher in vitro dsRNA synthesis and kit MEGAscript™ RNAi (catalog number AM1626) through an in vitro transcription system, and then the interference efficiency was screened.

[0045] Different concentrations (80, 100, 120 ng / μL) of the above dsRNA were mixed with 1*10 6 / mL of ring spot bacteria ( Botryosphaeria dothidea The spore suspensions were mixed and incubated, and the disease development (lesion size) was observed after 1, 3, and 5 days, with the untreated group (CK) as the control.

[0046] 2. Experimental Results and Analysis Experimental results are as follows Figure 5In the initial treatment period (1 dpi), there was no significant difference in lesion size between the concentration groups and the control group. As the treatment time was extended to 3 dpi and 5 dpi, the lesions showed a clear concentration-dependent decreasing trend. The lesions in the 100 ng / μL and 120 ng / μL treatment groups were 0.5-1.0 cm smaller than those in the control group. This result indicates that dsRNA-Spt7 can effectively inhibit the pathogenicity of the bacteria.

[0047] Further supplementary experiments confirmed that the inhibitory effect was more significant when the concentration of the *Rhizoctonia solani* spore suspension was increased to 200 ng / μL and 400 ng / μL.

[0048] These results demonstrate that dsRNA-Spt7 can significantly reduce the pathogenicity of *Rhizoctonia solani* through interference, and the inhibitory effect is time- and dose-dependent, making it applicable to the biocontrol of *Rhizoctonia solani*.

[0049] Example 6: Construction and Molecular Verification of Recombinant Trichoderma strains 1. Experimental Methods (1) To achieve in vivo delivery of dsRNA via homologous recombination, the expression cassette of the highly efficient interfering sequence dsRNA-Spt7 was inserted into the Trichoderma expression vector pYF11 (purchased from BioVector NTCC Plasmid Vector Culture Collection Center (BioVector NTCC Inc.)).

[0050] The dsRNA expression cassette consists of arm sequences with identical sequences at both ends but opposite orientations and a central sequence serving as a connector. The arm sequences at both ends are partial coding sequences of the BdSpt7 gene, as shown in SEQ ID No. 2. The central sequence is derived from an endogenous gene from *Rhizoctonia solani*. Bdβ-TubulinA The first intron, sequenced as shown in SEQ ID No. 3, was ligated into the pYF11 vector using homologous recombination (Takara). The sense arm of the Bdspt7 sequence and the first intron of the β-tublin gene were then ligated together. Furthermore, the antisense arm of the Bdspt7 sequence was ligated to the other end of the intron using T7 ligase (Takara), forming... Figure 6 The sequence structure was used to construct an expression cassette for the target gene dsRNA. The sequence of the expression cassette is shown in SEQ ID No. 1, and the specific structure is as follows. Figure 6 As shown.

[0051] Express box BdSpt7 The dsRNA was ligated to the multiple cloning site of the pYF11 vector, which contains the constitutive promoter RP27 suitable for Trichoderma. The multiple cloning site sequence of the pYF11 vector is shown in SEQ ID No. 4. The pYF11 vector contains an antibiotic resistance gene (G418), which can be used as a selection marker to screen transformants.

[0052] Plasmids were extracted from positive transformants, and the target sequence was recovered by enzyme digestion. After confirming the successful construction of the vector by sequencing the target sequence, it was introduced into Trichoderma harzianum T10 via PEG-mediated protoplast transformation. Positive transformants were selected by G418 resistance and named Mu10-ds.

[0053] (2) DNA of the transformants was extracted and positive transformants were detected using SPT7 primers. RNA was then extracted and reverse transcribed to obtain cdna of the transformants, which was then detected by RT-PCR.

[0054] 2. Experimental Results and Analysis PCR amplification results as follows Figure 7 As shown, the recombinant strain *Trichoderma harzianum* 10-ds amplified a ds-Spt7-specific band of approximately 440 bp, while the wild-type *Trichoderma* did not exhibit this band. This result indicates that the ds-Spt7 expression cassette has been integrated into the *Trichoderma harzianum* genome. The RT-PCR results are shown below. Figure 8 As shown, the results indicate that the ds-Spt7 sequence in 10-ds was successfully transcribed, and the cDNA amplification product was consistent with the expected size.

[0055] This experiment confirms that the ds-Spt7 expression cassette has been stably integrated into the Trichoderma harzianum genome and transcribed, laying the foundation for the construction of an RNAi delivery system.

[0056] Example 7: Inhibitory effect of recombinant Trichoderma strains on the pathogenicity of *Trichoderma repens*. 1. Experimental Methods (1) Fruit treatment: Prepare a suspension of wood 10-ds spores (concentration 1×10⁻⁶). 6 Inoculation with CFU / mL of the fungus was performed on mature and young apple fruits, using wild-type Trichoderma T10 and sterile water as controls. After 48 h of moistening treatment, activated apple ring rot pathogens (5 mm fungal cakes) were collected and applied to areas treated with Trichoderma spore suspension. Moistening treatment was continued for 3-5 days, and the diameter of the lesions was measured and photographed to evaluate the dsRNA delivery effect. The experiment compared the effects of different treatment groups (CK: blank control; T10: wild-type Trichoderma; T10-ds: Trichoderma strain expressing ds-Spt7) on the pathogenicity of the ring rot pathogen.

[0057] (2) Treatment of apple branches and trunks: Cut sections of Fuji apple branches with a diameter of 1-1.5 cm. Disinfect the branches with 75% alcohol and allow them to dry. Then, make wounds on the branches using a 5 mm diameter punch. Apply 20 μL of Trichoderma suspension (1 × 10⁶ CFU / mL) to the wounds. Two days later, inoculate with 5 mm pieces of apple ring rot fungus. Measure the diameter of the lesions 5 days after inoculation.

[0058] 2. Experimental Results and Analysis (1) The results are as follows Figure 9 and 10 As shown, on young and mature apple fruits, the diameter of lesions in the Trichoderma 10-ds treatment group (M. 10-ds) was significantly smaller than that in the wild-type Trichoderma 10 treatment group and the blank control group (CK). This indicates that the Trichoderma 10-ds expressing strain can effectively inhibit the pathogenicity of Trichoderma ringworm through its produced double-stranded RNA, providing a new and effective strategy for the biological control of plant diseases. Specific implementation conditions, including the culture method of Trichoderma and the concentration of spore treatment, can be adjusted according to actual application needs.

[0059] (2) The results are as follows Figure 11 As shown, the diameter of lesions in the Trichoderma strain expressing ds-Spt7 on apple branches (Mu10-ds) was significantly smaller than that in the wild-type Trichoderma strain (Mu10) and the blank control group (CK).

[0060] Table 1. Results of the inhibition of pathogenicity of Trichoderma recombinant strains against *Trichoderma variegata*.

[0061] Example 8 Isolation and strain identification of Trichoderma harzianum T10 1. Isolation and screening of strains Apple branches infected with apple tree rot were collected from apple orchards. After surface disinfection, the branch tissue was ground and sterile water was added. The tissue was then isolated using a conventional gradient dilution spreader method and cultured on Trichoderma selective medium at 25°C. Colonies with significant morphological differences were selected, purified, and preserved on PDA medium. The fungal strain was then screened for antagonistic bacteria against the apple tree rot pathogen, and the strain was eventually identified as T10.

[0062] 2. Identification of strain T10 (1) Morphological identification A. Experimental Methods: The isolated strain was inoculated onto a PDA plate and cultured at 25°C. The morphology and color of the colonies formed were observed, as well as the morphology of the conidia and sporogenous cells produced by the strain.

[0063] B. Results and Analysis According to observations, such as Figure 12 As shown, the isolated strain forms white, circular colonies on PDA plates. With prolonged incubation, the colonies gradually turn green, indicating the formation of conidia (such as...). Figure 12 A), the sporogenous cells are flask-shaped (e.g., Figure 12 B), conidia are round (e.g. Figure 12 C); the morphological observation results indicate that this strain is a Trichoderma strain.

[0064] (2) Gene identification A. Experimental Methods: Genomic DNA was extracted from the hyphae and used as a template to amplify the strain's ITS using universal primers ITS1 and ITS4. The amplified fragments were sequenced and BLAST aligned, followed by MEGA(X) phylogenetic analysis.

[0065] The amplification primer sequences for ITS are as follows: ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' ITS4: 5'-TCCTCCGCTTATTGATATGC-3' B. Results and Analysis: Sequencing results showed that the ITS extended fragment was 490 bp in length, and its sequence is shown in the sequence listing SEQ ID NO: . The obtained sequence was compared with nucleic acid sequences in GenBank using BLAST software in the NCBI database (http: / / www.ncbi.nlm.nih.gov) and compared with Trichoderma harzianum ( Trichoderma afroharzianum The ITS sequences of isolate XD19 and isolate ZJ11 (Accession: ON045490.1 and ON649772.1) showed 99.9% homology. Phylogenetic analysis showed that this strain is related to... Trichoderma afroharzianum Clustered on the same branch ( Figure 12 D).

[0066] Based on the aforementioned morphological characteristics, physiological and biochemical characteristics, and sequence analysis results, strain T10 was identified as *Trichoderma harzianum* (African Trichoderma). Trichoderma afroharzianum ).

[0067] Example 9: Degradation ability of strain T10 against putrefactive virus. A. Experimental Methods: Preparation of the toxin solution: Accurately weigh 0.1 g (accurate to 0.0001 g) of the target toxin standard using an analytical balance of 0.0001 g. After ultrasonic-assisted dissolution in ultrapure water, transfer the solution quantitatively into a 100 mL Grade A volumetric flask and dilute to the mark to prepare a 1000 mg / L standard stock solution. Dilute the stock solution to 100 mg / L using a serial dilution method. After drawing the solution with a 2 mL sterile syringe, filter it through a 0.45 μm microporous membrane and collect the filtrate into a 1.5 mL sample vial as the test sample. The target toxins used in this example are p-hydroxybenzoic acid, trihydroxybenzoic acid, and protocatechuic acid.

[0068] Transfer 50 mL of the target toxin into 150 mL Erlenmeyer flasks, and inoculate each flask with 200 μL of a 1×10⁻⁶ toxin concentration. 6 A toxin-cell co-culture system was constructed using a CFU / mL Trichoderma spore suspension. The control group used an equal volume of sterile distilled water instead of the bacterial suspension. All treatment groups were simultaneously cultured in a 25℃ constant-temperature shaker (180 rpm), with samples taken at regular intervals on days 3 and 7. One mL of culture solution was filtered through a 0.45 μm microporous filter and collected in a 2 mL chromatographic vial for toxin quantification using high-performance liquid chromatography (HPLC). The degradation rate was calculated using the following formula: Degradation rate = (Concentration of Vm toxin culture medium in control group - Concentration of Vm toxin culture medium in degradation system) × 100% / Concentration of Vm toxin culture medium in control group B. Results and Analysis: Figure 11 The results showed that after 3 days of degradation of Vm toxin by *Trichoderma harzianum* T10, the content of p-hydroxybenzoic acid was 90.3 μg / mL, with a degradation rate of 82%; the content of trihydroxybenzoic acid was 72.3 μg / mL, with a degradation rate of 75.6%; and the content of protocatechuic acid was 73.08 μg / mL, with a degradation rate of 79.3%. After 7 days of degradation of Vm toxin by *Trichoderma harzianum* T10, the content of p-hydroxybenzoic acid was 3.6 μg / mL, with a degradation rate of 96.6%; the content of trihydroxybenzoic acid was 10.1 μg / mL, with a degradation rate of 90.3%; and the content of protocatechuic acid was 14.4 μg / mL, with a degradation rate of 86.3%. Therefore, *Trichoderma harzianum* T10 has a good degradation effect on the main toxins in rot virins, alleviating the toxicity of these toxins to fruit trees.

[0069] Example 10: Detection of the inhibitory rate of Trichoderma harzianum T10 fermentation broth against multiple plant pathogens. A. Experimental Methods: Fermentation broth of Trichoderma harzianum T10 was prepared, and PDA medium with a concentration of 20% was prepared. PDA without fermentation broth was used as a control. Then, activated and cultured pathogens were inoculated. The specific pathogens used in this example are shown in Table 1.

[0070] B. Results and Analysis: As shown in Table 1, the fermentation broth of Trichoderma harzianum T10 has a significant inhibitory effect on major plant pathogens such as apple tree rot fungus, apple ring rot fungus, pear tree rot, pear ring rot, apple bagged fruit spot disease, apple core rot, peach brown rot, and apple anthracnose leaf blight fungus, exhibiting good broad-spectrum antibacterial properties. Its inhibition rate is shown in Table 1.

[0071] Table 1. Antagonistic effect of Trichoderma harzianum T10 fermentation broth on pathogens causing fruit tree diseases.

[0072] Example 11 Effect of strain T10 treatment on apple growth promotion A. Experimental Methods: Apple seeds were sterilized and germinated, then potted in sterile soil. Experiments were conducted once seedlings emerged. The T10 strain was inoculated onto PDA medium and cultured at 25°C for 8 days. The colonies were then rinsed with purified water, conidia were collected, and the culture was adjusted to a concentration of 10... 7 After achieving a concentration of [number] spores / mL, the treatment group was irrigated with 50 mL of the conidial solution every week, while the control group was irrigated with an equal volume of purified water. After 4 weeks, the plant height, fresh weight, dry weight, chlorophyll content, and other indicators of the apple seedlings were measured.

[0073] Improvement rate (%) = (Treatment group colony diameter - Control group colony diameter) / Control group colony diameter × 100% B. Results and Analysis: The results are as follows Figure 12 As shown in Table 2, compared with the control group, the experimental group treated with T10 conidial solution showed improvements in plant height, fresh weight, dry weight, and chlorophyll content. For example, plant height increased by 46.14%, and the above-ground dry weight increased by 59.16%. These results demonstrate that strain T10 has a good growth-promoting effect on apple seedlings.

[0074] Table 2. Growth-promoting effects of Trichoderma harzianum T10 spores on apple seedlings.

[0075] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A type of ring spot pathogen BdSpt7 The application of genes in the prevention and control of apple ring rot is characterized by, The ring spot bacterium BdSpt7 The nucleotide sequence of the gene is shown in SEQ ID No.

5.

2. The ringworm pathogen according to claim 1 BdSpt7 The application of genes in the prevention and control of apple ring rot is characterized by, The application method is as follows: knocking out the BdSpt7 gene in the pathogen of apple ring rot, or performing RNA interference on the BdSpt7 gene in the pathogen of apple ring rot.

3. A targeted BdSpt7 The dsRNA of a gene is characterized by, The sequence of the dsRNA is shown in SEQ ID No.

2.

4. A biocontrol agent, characterized in that, The active ingredient is the targeted ingredient as described in claim 3. BdSpt7 dsRNA of genes.

5. The target as described in claim 3 BdSpt7 The use of the dsRNA of the gene or the biocontrol agent as described in claim 4 in the preparation of biological agents for the prevention and control of apple ring rot.

6. The application according to claim 5, characterized in that, The concentration of dsRNA used is 80~400 ng / μL, and the application method is to spray it on the plant leaves.

7. An engineered strain of *Trichoderma harzianum* expressing dsRNA, characterized in that, The engineered strain of *Trichoderma harzianum* from Africa is integrated with interfering target genes. BdSpt7 Trichoderma harzianum (a species of Trichoderma) expressing dsRNA gene cassettes Trichoderma afroharzianum The dsRNA expression cassette is shown in SEQ ID No. 1; the *Trichoderma harzianum* used is *Trichoderma harzianum* with accession number CGMCC No. 41709 (…). Trichoderma afroharzianum )T10.

8. A method for preparing an engineered strain of *Trichoderma harzianum* as described in claim 7, characterized in that, The preparation method includes the following steps: (1) Construction of dsRNA expression cassette: It consists of arm sequences with identical sequences at both ends but opposite directions and a central sequence that serves as a connector, thus constructing a cassette containing the target gene. BdSpt7 The dsRNA expression cassette, the nucleotide sequence of which is shown in SEQ ID No. 1; (2) Construction of recombinant expression vector: The dsRNA expression cassette was ligated into the Trichoderma expression vector to construct a recombinant expression vector containing the dsRNA expression cassette; (3) Protoplast transformation: The recombinant expression vector containing the dsRNA expression cassette was successfully constructed and introduced into Trichoderma harzianum T10 via PEG-mediated protoplast transformation. Positive transformants were screened to obtain the Trichoderma harzianum engineered strain.

9. The application of the engineered strain of Trichoderma harzianum as described in claim 7 in the prevention and control of ring spot disease.

10. A method for preventing and controlling apple ring rot, characterized in that, Apply dsRNA as described in claim 3 or one or both of the engineered Trichoderma harzianum strains as described in claim 7 to fruit trees.

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