Rhizoctonia solani low-toxicity strain and application thereof in improving heat tolerance of perennial ryegrass

Abstract

The application discloses a low-toxic Rhizoctonia solani strain and application thereof in improving heat resistance of perennial ryegrass, and belongs to the technical field of microorganisms. Rhizoctonia solani The Rhizoctonia solani (Rhizoctonia solani) DF-SX-1 is preserved in the China General Microbiological Culture Collection Center, and the preservation date is April 24, 2026, and the preservation number is CGMCC No. 42766. The application finds that the Rhizoctonia solani DF-SX-1 carries three fungal viruses, has low pathogenicity characteristics, has a good inhibiting effect on the Rhizoctonia solani of sugar beets, has a biocontrol potential for preventing and treating the Rhizoctonia solani of sugar beets, can promote the growth of perennial ryegrass and improve the heat stress tolerance of the perennial ryegrass, and provides a new resource for development of a new pesticide and improvement of the perennial ryegrass.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a low-toxicity strain of Rhizoctonia solani and its application in improving the heat resistance of perennial ryegrass. Background Technology

[0002] Fungal viruses are a class of viruses that use fungi and oomycetes as hosts, obtaining nutrients from the host's tissue cells and replicating within them. Most fungal viruses do not affect the characteristics of the host fungus; however, some fungal viruses not only affect the biological characteristics of the host fungus, including pathogenicity, but can also influence plant growth, immune responses, and tolerance to abiotic stresses through virus-fungus complexes. For example, the virus *Sclerotinia gemycirculvirus* 1 (SsHADV-1), which infects *Sclerotinia gemycirculus*, can reduce the pathogenicity of the host fungus, effectively controlling sclerotinia rot in rapeseed, and can also promote plant growth and increase the yield of crops such as rapeseed. SsHADV-1 can also grow endophytically in cereal crops, enhancing their resistance to pathogens. Fusarium graminearum and Magnaporthe oryzae Resistance. Curvularia thermaltolerance virus (CThTV) infects Crescentella spp. Curvularia protuberata This leads to the upregulation of genes related to melanin, trehalose, and catalase in the host fungus. Furthermore, those carrying CThTV... C. protuberata Inoculation with bluegrass ( Dichanthelium lanuginosum After that, it can improve the heat resistance of bluegrass, which is speculated to be because the virus-carrying strain protects the host plant by clearing the reactive oxygen species produced by the plant under heat stress, thereby resisting abiotic stress.

[0003] Perennial ryegrass ( Lolium perenne (Lolium perfoliatum) is a herbaceous plant belonging to the genus Lolium in the Poaceae family. It possesses advantages such as strong tillering ability, rapid turf establishment, tolerance to mowing and trampling, good palatability, high crude protein content, and tolerance to cutting, making it an important dual-purpose forage and turfgrass. As a cool-season grass, its optimal growth temperature is 10-27℃, and heat stress is the main factor limiting its growth and development. Regions where perennial ryegrass is grown often experience summer temperatures as high as 38℃, far exceeding its optimal growth temperature, disrupting the balance between growth and development and accelerating its metabolic and physiological changes. The challenges posed by global climate change are escalating, with continuously rising environmental temperatures and increasingly frequent and intense extreme weather events, severely limiting the growth, geographical distribution, and yield of perennial ryegrass. However, there are currently no reports of using fungal viruses to regulate the growth and heat tolerance of ryegrass. Summary of the Invention

[0004] The purpose of this invention is to provide a low-virulence strain of *Rhizoctonia solani* and its application in improving the heat resistance of perennial ryegrass, thereby addressing the problems existing in the prior art. This invention discovers a low-virulence strain of *Rhizoctonia solani*, DF-SX-1, which carries three fungal viruses and exhibits low pathogenicity. It shows good inhibitory effects against beet damping-off, demonstrating biocontrol potential for controlling beet damping-off. Simultaneously, it can promote the growth of perennial ryegrass and improve its tolerance to heat stress, providing new resources for the development of novel pesticides and the improvement of ryegrass traits.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a low-virulence strain of Rhizoctonia solani ( ). Rhizoctonia solani DF-SX-1, deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 24, 2026, with accession number CGMCC No. 42766.

[0006] The present invention also provides a fungal virus RsCV1, which is obtained from the above-mentioned low-virulence strain DF-SX-1 of Rhizoctonia solani.

[0007] The present invention also provides a fungal virus RsCV2, which is obtained from the low-virulence strain DF-SX-1 of Rhizoctonia solani mentioned above.

[0008] The present invention also provides a fungal virus RsMV94-AG4, which is obtained from the above-mentioned low-virulence strain of Rhizoctonia solani DF-SX-1.

[0009] The present invention also provides the application of the above-mentioned fungal virus RsCV1 and / or the above-mentioned fungal virus RsCV2 in reducing the pathogenicity of plant pathogens, including Rhizoctonia solani.

[0010] The present invention also provides the application of the above-mentioned fungal virus RsCV1 and / or the above-mentioned fungal virus RsCV2 in the preparation of products for the prevention and control of plant diseases, wherein the pathogens causing the plant diseases include Rhizoctonia solani.

[0011] The present invention also provides a microbial preparation comprising the above-mentioned low-virulence strain DF-SX-1 of Rhizoctonia solani or microorganisms infected with the above-mentioned fungal viruses RsCV1 and RsCV2.

[0012] The present invention also provides the use of the above-mentioned low-virulence strain of Rhizoctonia solani DF-SX-1, the above-mentioned fungal virus RsCV1, the above-mentioned fungal virus RsCV2, or the above-mentioned microbial preparation in the preparation of products that promote the growth of ryegrass.

[0013] The present invention also provides the use of the above-mentioned low-virulence strain of Rhizoctonia solani DF-SX-1, the above-mentioned fungal virus RsCV1, the above-mentioned fungal virus RsCV2, or the above-mentioned microbial preparation in the preparation of products that improve the heat resistance of ryegrass.

[0014] The present invention also provides a product for promoting the growth of ryegrass, the product comprising the above-mentioned low-virulence strain of Rhizoctonia solani DF-SX-1, the above-mentioned fungal virus RsCV1, the above-mentioned fungal virus RsCV2, or the above-mentioned microbial preparation.

[0015] The present invention discloses the following technical effects: This invention discovers a low-virulence strain of Rhizoctonia solani, DF-SX-1, which carries three fungal viruses and exhibits low pathogenicity. Co-infection with the viruses RsCV1 and RsCV2 significantly reduces the pathogenicity of the host strain, demonstrating a good inhibitory effect on sugar beet damping-off and showing potential for biocontrol of the disease. Furthermore, the low-virulence Rhizoctonia solani strain DF-SX-1 of this invention can also promote the growth of perennial ryegrass and improve its tolerance to heat stress, providing a new resource for the development of novel pesticides and the improvement of ryegrass traits. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 A schematic diagram of the colony morphology of Rhizoctonia solani strain DF-SX-1 on potato dextrose agar (PDA) medium plates; Figure 2 Image of dsRNA extracted from strain DF-SX-1 on a 1.5% agarose gel (DNase I and S1 nuclease digestion); where M is DL 5000 DNA Marker; Figure 3 The diagram shows the genome structure and phylogenetic tree of RsMV94-AG4; where A is the genome structure diagram and B is the phylogenetic tree, with the red asterisks in the phylogenetic tree indicating the location of RsMV94-AG4. Figure 4 This represents the conservative motif of RsMV94-AG4; where the red asterisk indicates the location of RsMV94-AG4. Figure 5 Genome structure diagrams of RsCV1 and RsCV2; Figure 6 This is the terminal conservation and phylogenetic tree of RsCV1 and RsCV2; where A is the terminal conservation region of RsCV1 and RsCV2; B is the phylogenetic tree of RsCV1 and RsCV2, and the red asterisks in the phylogenetic tree indicate the locations of RsCV1 and RsCV2. Figure 7 Let RsCV1 and RsCV2 be the conserved basis orders; where the red asterisks indicate the positions of RsCV1 and RsCV2. Figure 8 The images show the virus detection and rDNA-ITS gel images of the parental virus-carrying and detoxified strains; where A is the gel image of virus detection of the parental virus-carrying and detoxified strains, and B is the gel image of PCR amplification of the rDNA-ITS region of the parental virus-carrying and detoxified strains. Figure 9 Observation of colony morphology of parental virus-carrying and virus-free strains after 21 days of growth; Figure 10 This study aims to determine the pathogenicity of the parental virus-carrying strain DF-SX-1 and the virus-free strain DF-SX-1-D3 to sugar beets. In this study, A is a bar chart comparing the incidence rate of sugar beet seedlings with DF-SX-1 and DF-SX-1-D3, and B is a bar chart comparing the disease index of sugar beet seedlings with DF-SX-1 and DF-SX-1-D3. The error bars in the bar charts represent standard errors; * indicates p < 0.05, and ** indicates p < 0.01. Figure 11 Bar chart showing the effects of the parental virus-carrying strain DF-SX-1 and the virus-free strain DF-SX-1-D3 on the growth of perennial ryegrass; where A: plant height of perennial ryegrass; B: root length of perennial ryegrass; the letters in the bar chart indicate the significance of different treatments. Figure 12 The growth of perennial ryegrass before and after heat stress is shown in the figure. AC: no inoculation (CK); DF: inoculated strain DF-SX-1; GI: inoculated strain DF-SX-1-D3. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Example 1 Rhizoctonia solani ( Rhizoctonia solani Isolation and identification of DF-SX-1 A bacterial strain was isolated from root samples of sugar beet seedlings infected with damping-off disease in Datong City, Shanxi Province, China. After incubation in the dark at 25°C on PDA medium, pure colonies were obtained using the mycelial tip isolation method and named DF-SX-1. The colonies of this strain are light brown, flexible, with well-developed aerial hyphae, and are nearly circular in shape (see...). Figure 1 ).

[0024] Strain DF-SX-1 was inoculated onto PDA agar plates lined with cellophane and cultured in the dark at 25°C for 5 days. Mycelia were scraped off, and DNA was extracted using the CTAB method. The rDNA-ITS region was amplified by PCR. The obtained rDNA-ITS region sequence was BLASTed in NCBI and then compared with fusion groups of all Rhizoctonia solani in DNAMAN v7.0. Finally, the strain was identified as belonging to *Rhizoctonia solani* (*Rhizoctonia solani*). Rhizoctonia solani ) Fusion group AG-4HGII.

[0025] Rhizoctonia solani ( Rhizoctonia solani DF-SX-1 was deposited on April 24, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 42766.

[0026] Example 2: Detection of virus carried by strain DF-SX-1 The dsRNA of the strain was extracted using the cellulose adsorption method, and the specific procedures are as follows: Fresh or cryopreserved Rhizoctonia solani DF-SX-1 hyphae were placed in a mortar, cooled thoroughly with liquid nitrogen, and rapidly ground into a fine powder. The powder was then transferred to sterile 2 mL centrifuge tubes. Extraction buffer was added to each 2 mL tube: 500 μL of 2×GPS, 115 μL of 10% SDS, 500 μL of Tris-saturated phenol, and 500 μL of chloroform (Tris-saturated phenol and chloroform were added in a fume hood). The tubes were vigorously shaken at room temperature for 10 min. After centrifugation at 12000 r / min for 10 min at 16℃, 600 μL of the supernatant was transferred to a freshly sterilized 2 mL centrifuge tube (the tube contained two layers: a lower organic phase and an upper aqueous phase, with nucleic acids in the upper aqueous phase). 114 μL of anhydrous ethanol was added and mixed thoroughly, followed by 0.04 g of C6280 cellulose powder. The mixture was shaken and incubated on ice for 30 min. The tubes were then centrifuged at 12000 r / min for 3 minutes at 4℃. After elution for 3 minutes, discard the supernatant (white cellulose powder at the bottom of the tube), add 600 μL of elution buffer, mix the cellulose powder well, and let stand at room temperature for 3 minutes. Repeat the elution 2-3 times, centrifuge at 12000 r / min for 3 minutes, discard the supernatant, add 600 μL of 1×STE, mix well and vortex for 10 minutes; centrifuge at 12000 r / min for 3 minutes at 4℃, transfer 500 μL of supernatant to a freshly sterilized 1.5 mL centrifuge tube; add an equal volume of isopropanol, precipitate at -20℃ for at least 30 minutes; centrifuge at 12000 r / min for 10 minutes at 4℃, discard the supernatant; rinse the precipitate with 200 μL of 75% ethanol, centrifuge for 3 minutes and discard the residual liquid; repeat the rinsing once, air dry for 10 minutes, dissolve the precipitate with 25 μL of ddH2O, and store at -20℃ for later use. The dsRNA sample extraction results were detected by 1% agarose gel electrophoresis. The extracted dsRNA sample was digested sequentially with S1 nuclease and DNase I to remove single-stranded RNA and single-stranded DNA, respectively. The specific digestion steps were as follows: 20 μL of dsRNA sample was taken, and 2.5 μL of S1 nuclease and 2.5 μL of 10×S1 nuclease buffer were added. After mixing thoroughly, the mixture was reacted at 23℃ for 30 min. Then, 3 μL of DNase I and 3 μL of 10×DNase I nuclease buffer were added to the reaction solution from step one, and the mixture was reacted at 37℃ for 30 min.

[0027] The digested dsRNA samples were detected by 1.5% agarose gel electrophoresis at 80 V, and the target dsRNA was recovered by gel extraction using an agarose gel purification and recovery kit. For specific procedures, please refer to the Adley Recovery Kit instructions.

[0028] The 1.5% agarose gel electrophoresis image of the extracted dsRNA is shown below. Figure 2Lane M represents the DL 5000 DNA Marker; lane DF-SX-1 represents the extracted viral dsRNA. Multiple distinct bands were observed at 2000 bp, each corresponding to a different virus. This result indicates that strain DF-SX-1 carries a fungal virus.

[0029] Example 3: Identification of Fungal Viruses Total RNA was extracted from the strain using the TRIzol method: 0.2 g of mycelium was placed in a 2 mL sterile EP tube, two sterile steel balls were added, and the tube was ground for 60 s at 40 Hz using an automated high-speed grinder. If grinding is incomplete, repeat grinding for 60 seconds until the hyphae are completely powdered; add 1 mL of TRIzol lysis buffer and let stand at room temperature for 10 min; centrifuge at 12000 rpm and 4℃ for 10 min; take the supernatant and add it to a freshly sterilized 2 mL EP tube, add 200 µL of chloroform, shake thoroughly to mix, and incubate on ice for 5-10 min until separation occurs; centrifuge at 12000 rpm and 4℃ for 10 min; take 400 µL of the supernatant and add it to a freshly sterilized 1.5 mL EP tube, add an equal amount of isopropanol, and let stand at -80℃ for 10 min to precipitate; centrifuge at 12000 rpm and 4℃ for 15 min; remove the supernatant, add 200 µL of 75% anhydrous ethanol, and centrifuge at 12000 rpm and 4℃ for 5 min. Repeat this step 1-2 times to wash away the precipitate; remove the supernatant and carefully aspirate excess liquid from the bottom of the tube with a pipette, incubate at 37°C for 5 min until dry; add 25 µL of DEPC-treated water to dissolve the RNA, and use NanoDrop 2000 to detect the concentration and quality of total RNA. Perform reverse transcription on the qualified total RNA sample. The specific reverse transcription steps are as follows: take 1000 ng of total RNA and 0.8 µL of hexabase random primers into a 0.1 mL enzyme-free microcentrifuge tube, and add DEPC-treated water to bring the volume to 12 µL; incubate the above system at 65°C for 5 min, then immediately place on ice for 2 min; add 4 µL of 5× Reverse Transcriptase M-MLV Buffer, 2 µL of dNTP, 0.2 µL of RRI (40 U / µL), and 0.5 µL of Reverse Transcriptase M-MLV (RNase H) sequentially to the system. - 200 U / µL), 1.3 µL of DEPC-treated water, mixed thoroughly; the above system was placed in a 25℃ metal bath for 5 min, reacted at 42℃ for 1 h, and then treated at 65℃ for 5 min to terminate the reaction, thus completing the synthesis of the first strand of cDNA.

[0030] Based on the contig sequence information obtained from metatranscriptomics, nested primers were designed for RT-PCR to verify the sequence accuracy. The bases at each position were determined by at least three bidirectional cloning fragments obtained from sequencing. Specific primers were designed based on the verified viral sequence (see Table 1), and the complete viral genome sequence was amplified using RACE technology. The specific steps of the RLM-RACE method are as follows: 1) Adding adapters to the ends: Take 14 μL of enzymatically purified dsRNA, add 1 μL of PC3-T7 loop (5´-p-GGATCCCGGGAATTCGGTAATACGACTCACTATATTTTTATA GTGAGTCGTATTA-OH-3´, SEQ ID NO.1), mix well, treat in a 65℃ metal bath for 2 min, then immediately in an ice bath for 3 min. Add 5 μL of 10× T4 RNA ligase buffer, 25 μL of 50% PEG 6000, 3 μL of 0.1% BSA, 1 μL of RRI, and 1 μL of T4 RNA ligase in sequence, mix well, and ligate in a 16℃ metal bath for 24 h; 2) Precipitating and dissolving the adapter-added dsRNA: Add 550 μL of sterile enzyme-free water and 600 μL of chloroform to the ligation system, centrifuge at 12000 rpm for 10 min at 4℃, and take 500 μL of the supernatant and 5 After mixing μL of nucleic acid precipitation aid, 50 μL of NaAC (3 mol / L), and 555 μL of isopropanol, the mixture was allowed to stand at -20℃ for 30 min to precipitate. The mixture was then centrifuged at 12000 rpm for 10 min at 4℃, the supernatant was discarded, and the precipitate was washed twice with 200 μL of 75% ethanol. After the last wash, the precipitate was briefly centrifuged, and residual liquid was removed with a pipette. The precipitate was dried in a clean bench for 5 min, and dsRNA was dissolved in 14.5 μL of sterile, enzyme-free water. 3) dsRNA reverse transcription: 2 μL of DMSO was added to the 14.5 μL of dissolved dsRNA from the previous step. The mixture was treated in a 65℃ metal bath for 5 min, then immediately placed on ice for 3 min. 5 μL of 5× M-MLV buffer, 0.5 μL of RRI, 2 μL of dNTP, and 1 μL of reverse transcriptase M-MLV were added and mixed. The mixture was then reacted in a 42℃ metal bath for 1 minute. h; 4) Specific primer PCR: Design specific primers based on the validated viral sequence and form a primer pair with PC2 (designed based on the PC3-T7 loop sequence, 5´-CCGAATTCCCGGGATCC-3´, SEQ ID NO.2) to amplify the ends. Use at least 2 pairs of specific primers for PCR at each end.

[0031] Table 1 Virus-specific terminal primers designed based on the known viral genome. The terminal sequences were amplified using the TaKaRa SMAERTer RACE 5' / 3' kit, following the instructions. Sequencing was performed by Beijing Nuosai Genome Research Center Co., Ltd., ensuring that each position was obtained from sequencing at least three bidirectional clones.

[0032] The sequence fragments obtained from sequencing were assembled and aligned using DNAMAN 7.0 in conjunction with metatranscriptome sequencing data to obtain partial viral cDNA sequences. The terminal cDNA sequences obtained from the validated partial cDNA sequences were assembled into the complete viral genome sequence. The obtained complete genome sequence was then used to perform a BLASTx search on the NCBI NR database to obtain information on the most similar viruses. The viral nucleic acid sequences were used to predict viral genome ORFs using NCBI's ORF Finder, and the amino acid sequences of the ORFs were obtained. Based on the amino acid sequences, a BLASTp search was performed on the NCBI NR database to obtain information on each ORF. Furthermore, CDD search was used to predict conserved regions and related information of the ORFs based on the amino acid sequences. Based on the information obtained above, a viral genome structure simulation was constructed. CLUSTAL_X software was used to predict and analyze conserved viral motifs, and some closely related viruses were analyzed simultaneously with the target virus. Based on the predicted amino acid sequence of RdRp from the virus, the optimal phylogenetic tree model was found using the ModelFinder model of IQ-TREE 2.2.2.6, the tree was constructed using the maximum likelihood method, and the phylogenetic tree was edited using FigTree 1.4.4.

[0033] Three viruses were ultimately identified and named RsMV94-AG4, RsCV1, and RsCV2. Among them, RsMV94-AG4 belongs to... Mitoviridae The genome consists of a single fragment plus ssRNA. Its genome structure, conserved motifs, and phylogenetic tree are shown below. Figure 3 and Figure 4 RsCV1 and RsCV2 are Curvulaviridae The genome consists of two segments (dsRNA1 and dsRNA2). Its genome structure, terminal conservation, conserved motifs, and phylogenetic tree are shown in [link to phylogenetic tree]. Figures 5-7 .

[0034] Example 4: Obtaining and confirming the virus-free strain (DF-SX-1-D3) Two detoxification methods were used to detoxify strain DF-SX-1, and the specific methods are as follows: (1) Detoxification of mycelial tips, the specific method is as follows: 1) Incubate in the dark at 25℃ for 2-3 days. Take 0.2 g of mycelium from the edge of the colony and put it into a 2 mL sterile centrifuge tube. Add two sterile steel balls and grind twice with an automatic rapid grinder at 40 Hz for 60 s. 2) Place the above-ground mycelium into a 150 mL Erlenmeyer flask containing 50 mL PDB (liquid PDA medium) and incubate with shaking at 180 rpm for 24 h; 3) Take the above mycelial culture medium into a 2 mL sterile centrifuge tube, add two sterile steel balls, and grind twice with an automatic rapid grinder at 40 Hz for 60 s; 4) Spread the above mycelial suspension evenly on PDA medium and incubate in the dark at 25°C for 24 h; 5) Pick the tips of the above hyphae and place them on PDA medium, and incubate in the dark at 25°C for 5 days.

[0035] (2) Protoplast regeneration and detoxification, the specific method is as follows: 1) Activate the strain for 3-4 days, culture in the dark at 25℃, pick the cultured hyphae, add PDB and grind in a 20 Hz grinder for 2 min, place in an Erlenmeyer flask, add PDB and culture, shake at 25℃ and 180 rpm for 36 h; 2) Collect the shake culture mycelium, filter it with sterile gauze, and rinse twice with 0.7 M NaCl; 3) After rinsing, add the mycelium to the enzymatic hydrolysate and enzymatically hydrolyze it in a metal bath at 32°C for 4 hours, shaking it up and down every half hour to ensure more complete enzymatic hydrolysis. 4) After the enzymatic hydrolysis is complete, fold the lens paper into a funnel shape, filter the enzymatic hydrolysis mixture through the funnel end with three layers of cleaning paper to remove mycelial fragments, and finally rinse once with a small amount of STC solution and collect the filtrate. 5) Centrifuge the filtrate at 7000 rpm and 4℃ for 10 min, and discard the supernatant; 6) Gently suspend the protoplast precipitate in 100 μL of STC solution. Observe the number of cells in the suspension using a microscope on a hemocytometer. Adjust the concentration of the protoplast suspension to 1 × 10⁻⁶. 4 cells / mL; 7) Mix 100 μL of suspension into 15 mL of RM medium (heat and then cool until completely warm to the touch), incubate at 25℃ for 3-4 days, and observe every day after 2 days.

[0036] RT-PCR was used to verify the presence of the corresponding specific primers in the detoxified strain. Three generations of detoxified strains were verified, and if no bands were observed in any of them, the detoxification was considered successful.

[0037] RsCV1 and RsCV2 were removed from strain DF-SX-1 using a high-precision detoxification method, yielding detoxified strains DF-SX-1-D1 and DF-SX-1-D2. RsCV2 was removed from detoxified strain DF-SX-1-D1 using protoplast regeneration detoxification, resulting in detoxified strain DF-SX-1-D3, which simultaneously eliminated both viruses (RsCV1 and RsCV2). RT-PCR amplification and sequencing using specific primers confirmed the removal of dsRNA1 (RdRp) and dsRNA2 (hypothetical protein, HP) from both RsCV1 and RsCV2. Furthermore, the rDNA-ITS region of the detoxified strains was amplified by PCR and sequenced. Results are as follows: Figure 8 As shown, detoxified strains DF-SX-1-D1, DF-SX-1-D2 and DF-SX-1-D3 with the same genetic background were successfully obtained.

[0038] Example 5: Comparison of biological characteristics and pathogenicity determination of virus-carrying and virus-free strains 1. Comparison of biological characteristics The parental virus-carrying strain (DF-SX-1) and virus-free strains (DF-SX-1-D1, DF-SX-1-D2, and DF-SX-1-D3) were cultured in PDA medium at 25°C in the dark for 21 days, during which colony morphology was observed. At least three replicates were performed for each strain. Results are as follows: Figure 9 As shown, after 21 days of growth, some differences in colony morphology emerged. All four strains exhibited sparse aerial hyphae attached to the culture medium surface. Compared to the light-white hyphae of the original virus-carrying strain DF-SX-1, the hyphae of the other three virus-free strains had a darker pigment (yellowish-brown) and denser aerial hyphae. Sclerotium morphology and distribution are also important indicators for evaluating Rhizoctonia solani colony morphology. The sclerotia of strain DF-SX-1 were mostly distributed near the center of the colony, forming a ring shape with a high density; the sclerotia of strains DF-SX-1-D1 and DF-SX-1-D2 were mostly distributed at the colony edge; while the sclerotia of strain DF-SX-1-D3 were relatively evenly distributed across the entire culture medium surface.

[0039] 2. Pathogenicity determination Pathogenicity tests on sugar beet seedlings were conducted using the wheat grain inoculation method. The specific method is as follows: 1) After activating the parental virus-carrying strain DF-SX-1 and the virus-free strain DF-SX-1-D3, they were transferred to PDA plates. A layer of wheat grains was placed on top of the colonies that had grown for about 3 days, and the plates were incubated in the dark at 25°C for 3 days before use. 2) After surface sterilization, beet seeds are sown in 50-cell trays containing sterilized soil, with two seeds per cell at a depth of 5 mm. Observe every 3-4 days and water according to soil moisture. Wait until two true leaves have grown before use. 3) Near the roots of beet seedlings, wheat grains inoculated with the fungus were placed in the soil to a depth of about 10 mm and covered with sterile soil. The control group was inoculated with wheat grains that had been cultured in the dark at 25°C for 3-4 days on sterile PDA plates. Ten seedlings were used for each treatment, with three replicates per treatment, in a randomized plot experiment. Seven days after inoculation, beet seedlings were harvested, and the morbidity and disease index were measured; the formulas are as follows: Incidence rate (%) = (Number of infected plants / Total number of plants) × 100%; Disease index = 100 × [∑ (number of diseased plants at each level × representative value at each level) / (total number of plants surveyed × highest representative value)]; 4) Grading standards: 1-7 grade grading standards, 1=root epidermal damage; 2-6 are root surface lesion areas of 5, 25, 50, 75 and 100% respectively; 7=100% damage to the root and epidermis.

[0040] Seven days after inoculation (dpi), beet seedlings were removed from the soil. Disease incidence was recorded in beet seedlings inoculated with both the parental virus-carrying strain DF-SX-1 and the virus-free strain DF-SX-1-D3, and the disease incidence and disease index were calculated. Results are as follows: Figure 10 As shown, the parental virus-carrying strain DF-SX-1 exhibited significantly lower morbidity and disease index in sugar beet seedlings compared to the virus-free strain DF-SX-1-D3. The results indicate that co-infection with viruses RsCV1 and RsCV2 can reduce the virulence of the host in sugar beet seedlings, demonstrating a weakened virulence characteristic.

[0041] Example 6: Effects of parental virus-carrying strain DF-SX-1 and virus-free strain DF-SX-1-D3 on the growth and heat tolerance of perennial ryegrass. A bacterial suspension of DF-SX-1 and DF-SX-1-D3 mycelia with a concentration of 100 CFU was prepared. 10 mL of the bacterial suspension was applied as a root drench to the roots of perennial ryegrass seedlings 14 days after emergence. A blank control group (CK) without inoculation was also included. The perennial ryegrass was then placed in a day / night (25℃ for 10 h / 16℃ for 14 h) environment for growth. Each treatment contained 60 ryegrass plants, and plant height and root length were measured 7 days after inoculation. Results are as follows: Figure 11 As shown, inoculation with strain DF-SX-1 can promote the growth of perennial ryegrass, with root length and plant height significantly higher than those of uninoculated ryegrass and ryegrass inoculated with DF-SX-1-D3.

[0042] Perennial ryegrass was inoculated with DF-SX-1 and DF-SX-1-D3 using the same treatment method. Seven days after inoculation, it was subjected to heat stress at 42°C for 48 hours, and then placed under normal conditions (25°C for 10 hours / 16°C for 14 hours) for 7 days to recover. The growth of the ryegrass before and after heat stress treatment was observed. Figure 12 As shown, after 48 h of heat stress at 42℃, compared with DF-SX-1-D3, the yellowing rate of ryegrass inoculated with DF-SX-1 was significantly reduced, and the survival rate was significantly improved after 7 days of recovery.

[0043] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A low-virulence strain of Rhizoctonia solani ( Rhizoctonia solani DF-SX-1, characterized in that, It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 24, 2026, with accession number CGMCC No. 42766.

2. A fungal virus RsCV1, characterized in that, The fungal virus RsCV1 was isolated from the low-virulence strain DF-SX-1 of Rhizoctonia solani described in claim 1.

3. A fungal virus RsCV2, characterized in that, The fungal virus RsCV2 was isolated from the low-virulence strain DF-SX-1 of Rhizoctonia solani described in claim 1.

4. A fungal virus RsMV94-AG4, characterized in that, The fungal virus RsMV94-AG4 was isolated from the low-virulence strain DF-SX-1 of Rhizoctonia solani described in claim 1.

5. The application of the fungal virus RsCV1 of claim 2 and / or the fungal virus RsCV2 of claim 3 in reducing the pathogenicity of plant pathogens, characterized in that, The plant pathogens include Rhizoctonia solani.

6. The use of the fungal virus RsCV1 of claim 2 and / or the fungal virus RsCV2 of claim 3 in the preparation of products for the prevention and control of plant diseases, characterized in that, The pathogens causing the plant diseases include Rhizoctonia solani.

7. A microbial preparation, characterized in that, The microbial preparation contains the low-virulence strain DF-SX-1 of Rhizoctonia solani as described in claim 1 or a microorganism that infects the fungal virus RsCV1 as described in claim 2 and the fungal virus RsCV2 as described in claim 3.

8. The use of the low-virulence strain of Rhizoctonia solani DF-SX-1 as described in claim 1, the fungal virus RsCV1 as described in claim 2, the fungal virus RsCV2 as described in claim 3, or the microbial preparation as described in claim 7 in the preparation of products that promote the growth of ryegrass.

9. The use of the low-virulence strain of Rhizoctonia solani DF-SX-1 as described in claim 1, the fungal virus RsCV1 as described in claim 2, the fungal virus RsCV2 as described in claim 3, or the microbial preparation as described in claim 7 in the preparation of products that improve the heat resistance of ryegrass.

10. A product for promoting the growth of ryegrass, characterized in that, The product contains the low-virulence strain of Rhizoctonia solani DF-SX-1 as described in claim 1, the fungal virus RsCV1 as described in claim 2, the fungal virus RsCV2 as described in claim 3, or the microbial preparation as described in claim 7.

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