Sclerotinia sclerotiorum strain SD4-8V with double deficiency of pathopoiesis and reproduction, fungicide and application of fungicide

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a sclerotinia sclerotiorum strain SD4-8V with double deletion of pathopoiesis and reproduction, a fungicide and application of the sclerotinia sclerotiorum strain SD4-8V. The invention provides a sclerotinia sclerotiorum strain SD4-8V with double deficiency of pathogenicity and reproduction, and the sclerotinia sclerotiorum strain SD4-8V with double deficiency of pathogenicity and reproduction has no pathogenicity and is lack of sclerotium forming ability, so that the sclerotinia sclerotiorum strain SD4-8V can be used as a plant vaccine for preventing sclerotiniose, and can be used as a plant vaccine for preventing sclerotiniose. And the biological safety is high, the incidence rate of sclerotiniose is reduced, and the growth and flowering of crops are promoted. The sclerotinia sclerotiorum strain SD4-8V with double deficiency of pathopoiesis and reproduction is simple and convenient to operate in field application, low in cost, good in effect and easy to popularize and use in production.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a pathogenic and reproductively absent Sclerotinia sclerotiorum strain SD4-8V, its inoculum agent, and its application. Background Technology

[0002] Rapeseed is a major oilseed crop, and its yield is crucial for ensuring food and oil security. However, *Sclerotinia sclerotiorum* (… Sclerotinia sclerotiorum Sclerotinia stem rot, caused by fungal infections, is the primary biological stressor affecting rapeseed yield, with a wide range of incidence and severe impact. This disease not only leads to reduced yield but also lowers rapeseed oil quality, such as altering fatty acid composition, or potentially accumulating mycotoxins, endangering food safety. Furthermore, infected stems are prone to rotting, often causing lodging and hindering mechanized harvesting. Therefore, utilizing beneficial biological resources to promote rapeseed growth and control sclerotinia stem rot is an environmentally friendly and sustainable control strategy.

[0003] Existing technologies disclose that non-pathogenic strains of *Sclerotinia sclerotiorum* can be used as plant vaccines to control sclerotinia rot in rapeseed. For example, the non-pathogenic strain DT-8 of *Sclerotinia sclerotiorum* carrying the fungal virus SsHADV-1 can lead to a decline in the pathogenicity of *Sclerotinia sclerotiorum* populations in the field and enhance the broad-spectrum resistance of rapeseed, showing good application prospects. To protect my country's rapeseed industry, increase rapeseed yield, and further improve the control effect of sclerotinia rot, this is an urgent problem to be solved in this field. Summary of the Invention

[0004] To further improve the control efficacy against sclerotinia stem rot, this invention provides a *Sclerotinia sclerotiorum* strain SD4-8V that lacks both pathogenicity and reproduction, an inoculum agent, and its application, specifically including the following technical solutions: This invention provides a strain of *Sclerotinia sclerotiorum* that is deficient in both pathogenicity and reproduction. Sclerotinia sclerotiorum The strain SD4-8V is deposited at the China Center for Type Culture Collection, with accession number CCTCC M 2026256.

[0005] The present invention also provides a microbial agent comprising the *Sclerotium sclerotiorum* strain SD4-8V, which is doubly deficient in pathogenicity and reproduction as described above.

[0006] This invention also provides a method for preparing a microbial agent, comprising the following steps: The pathogenic and reproductively absent *Sclerotinia sclerotiorum* strain SD4-8V, as described above, was inoculated into a culture medium and cultured. After culture, the OD was adjusted with ddH2O. 600 The concentration was increased to 1.5-2.5 to obtain the Sclerotinia sclerotiorum inoculum.

[0007] The present invention also provides the application of the pathogenic and reproductively deficient Sclerotinia sclerotiorum strain SD4-8V, the inoculum agent or the inoculum agent prepared by the preparation method described above, the application including any one or more of the following: 1) promoting crop growth; 2) promoting crop flowering; 3) controlling crop sclerotinia disease.

[0008] The present invention also provides, as described above, methods for promoting crop growth, including increasing crop plant height and / or fresh weight of above-ground parts.

[0009] Preferably, the pathogen causing the crop sclerotinia stem rot includes Sclerotinia sclerotiorum.

[0010] Preferably, the crop includes rapeseed.

[0011] The present invention also provides a method for preventing and controlling sclerotinia stem rot in crops, comprising: initiating crop seeds with the inoculant or inoculant prepared by the method described above.

[0012] Preferably, the steps of the above method include: adding crop seeds to the inoculant for seed treatment; placing the treated seeds at 20°C for initiation; and drying the initiated seeds until the mycelium on the seed surface is no longer sticky.

[0013] Preferably, the mass-to-volume ratio of the crop seeds and the inoculant is 5-15 g: 1-10 mL.

[0014] The beneficial effects of this invention are as follows: This invention provides a strain of *Sclerotinia sclerotiorum* that is deficient in both pathogenicity and reproduction. Sclerotinia sclerotiorum The *Sclerotinia sclerotiorum* strain SD4-8V, deposited at the China Center for Type Culture Collection (CCTCC) at Wuhan University, with accession number CCTCC M2026256, is a pathogenic and reproductively absent strain of *Sclerotinia sclerotiorum*. This strain SD4-8V exhibits no pathogenicity, lacks sclerotium formation ability, and has high biosafety. The *Sclerotinia sclerotiorum* strain described in this invention shows significant control efficacy against sclerotinia rot. Compared to the conventional non-pathogenic *Sclerotinia sclerotiorum* strain DT-8, the control efficacy of the *Sclerotinia sclerotiorum* strain SD4-8V is increased by approximately 150.48%. In summary, the *Sclerotinia sclerotiorum* strain SD4-8V, which lacks both pathogenicity and reproduction, can be used as a plant vaccine, activating plant resistance to *Sclerotinia sclerotiorum* and thus preventing sclerotinia rot. This strain possesses multiple advantages, including high biosafety, reduced sclerotinia rot incidence, and promotion of crop growth and flowering. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0016] Figure 1Schematic diagram of colony morphology of mutant strains SD4-8 and Sclerotinia sclerotiorum DT-8VF at different culture stages; Figure 2 The growth rates of the mutant strains SD4-8 and Sclerotinia sclerotiorum DT-8VF are statistically analyzed. Figure 3 The pathogenicity of mutant strains SD4-8 and Sclerotinia sclerotiorum DT-8VF on rapeseed leaves; the scale bar is 2 cm. Figure 4 The results of PCR detection of SsHADV-1 carried in the pathogenic and reproductive double-deficient Sclerotinia sclerotiorum strain SD4-8V are shown; where M represents the molecular weight marker, SD4-8V represents the detection result of the pathogenic and reproductive double-deficient Sclerotinia sclerotiorum strain SD4-8V, H2O represents water as a negative control, and DT-8 represents the detection result of Sclerotinia sclerotiorum. Figure 5 Schematic diagrams of colony morphology at different culture stages for Sclerotinia sclerotiorum strains SD4-8V and DT-8VF, which are both lacking in pathogenicity and reproduction. Figure 6 The growth rate statistics of Sclerotinia sclerotiorum strains SD4-8V and DT-8, which are both lacking in pathogenicity and reproduction; Figure 7 The pathogenicity of *Sclerotinia sclerotiorum* strains SD4-8V and DT-8VF (both lacking pathogenicity and reproduction) on rapeseed leaves is statistically shown; hpi (Hours Post-Inoculation) represents the number of hours after inoculation; the scale bar is 2 cm. Figure 8 The growth status of rapeseed seedlings in each treatment on day 21 after sowing; Figure 9 The statistical results of rapeseed seedling height in each treatment are as follows; Figure 10 The aboveground fresh weight of rapeseed seedlings in each treatment is statistically analyzed. Figure 11 The flowering rate of rapeseed in each treatment was statistically analyzed as of March 18, 2025. Figure 12 This shows the incidence of sclerotinia stem rot in rapeseed in the field on March 26, 2025; the red arrows indicate the affected areas. Figure 13 Statistical results of incidence rates under different treatments; The bar chart in the attached figure above, This indicates that there is a significant difference. P <0.05; This indicates a highly significant difference. P <0.01; Indicates highly significant difference P <0.001; Indicates highly significant difference P <0.0001; ns indicates no significant difference P >0.05.

[0017] Biological Preservation Instructions Sclerotium sclerotiorum ( Sclerotinia sclerotiorum The strain SD4-8V was deposited on January 27, 2026, at the China Center for Type Culture Collection (CCTCC) at Wuhan University, China, with accession number CCTCC M 2026256. Detailed Implementation

[0018] This invention provides a pathogenic and reproductive-deficient Sclerotinia sclerotiorum strain SD4-8V, which is deposited at the China Center for Type Culture Collection (CCTCC) at Wuhan University, with accession number CCTCC M 2026256.

[0019] As one implementation method, the *Sclerotinia sclerotiorum* strain SD4-8V, which lacks both pathogenicity and reproduction, is derived from the *Sclerotinia sclerotiorum* strain DT-8VF as a base strain. This strain was then subjected to ultraviolet mutagenesis to obtain the reproduction-deficient mutant strain SD4-8. The virus SsHADV-1 was then horizontally propagated through the reproduction-deficient mutant strain SD4-8 to obtain the *Sclerotinia sclerotiorum* strain SD4-8V, which lacks both pathogenicity and reproduction. The reproduction-deficient mutant strain SD4-8 described in this invention grows slowly, does not form sclerotia, and is non-pathogenic to rapeseed. The *Sclerotinia sclerotiorum* strain SD4-8V, which lacks both pathogenicity and reproduction, is non-pathogenic, lacks sclerotia formation ability, and carries the non-pathogenic DNA virus SsHADV-1. It grows slowly, does not form sclerotia, and is non-pathogenic to rapeseed. The *Sclerotium sclerotiorum* strain SD4-8V carrying SsHADV-1 in this invention can achieve a functional transformation from a necrotrophic pathogen to an endophytic fungus. Its endophytic colonization not only promotes the growth and development of the host plant but also reshapes the crop's immune system, enhancing its broad-spectrum disease resistance. This effectively strengthens the crop's resistance to both biotic stress (such as pathogen infection) and abiotic stress (such as drought stress). Growing within the crop, it can protect crop health throughout its entire growth cycle. Furthermore, the *Sclerotium sclerotiorum* strain SD4-8V, lacking both pathogenicity and reproduction, possesses biosafety and immune-stimulating capabilities. When used as a plant vaccine, it can utilize the fungal virus SsHADV-1 to attack the pathogenic fungus, preventing infection and causing the decline of the pathogenic fungal population in the field. Therefore, the *Sclerotium sclerotiorum* strain SD4-8V, lacking both pathogenicity and reproduction, can be used as a plant vaccine to control sclerotinia disease.

[0020] The present invention also provides a microbial agent comprising the *Sclerotium sclerotiorum* strain SD4-8V, which is doubly deficient in pathogenicity and reproduction as described above.

[0021] As one implementation method, the OD of the bacterial agent 600 The concentration is 1.5~2.5. As one embodiment, the inoculum is in the form of a liquid inoculum. As a preferred embodiment, the inoculum comprises a fermentation broth.

[0022] This invention also provides a method for preparing fermentation broth of a *Sclerotinia sclerotiorum* strain SD4-8V that is deficient in both pathogenicity and reproduction, comprising: The pathogenic and reproductive-deficient *Sclerotium sclerotiorum* strain SD4-8V was inoculated into PDB medium and cultured. After culture, OD was adjusted with ddH2O. 600 The temperature was increased to 1.5-2.5 to obtain the Sclerotinia sclerotiorum fermentation broth.

[0023] As one embodiment, the culture conditions are 20℃ and 150 r / min for 4-5 days. As another embodiment, the OD of the *Sclerotinia sclerotiorum* fermentation broth...600 It can be any one of 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, and 2.5, or the midpoint of any two point values.

[0024] This invention also provides the application of the pathogenic and reproductively absent *Sclerotium sclerotiorum* strain SD4-8V, the bacterial agent, or the bacterial agent prepared by the method described above, wherein the application includes any one or more of the following: 1) Promote crop growth; 2) Promote crop flowering; 3) Prevent crop sclerotinia disease.

[0025] In one embodiment, promoting crop growth includes increasing crop plant height and / or above-ground fresh weight. In one embodiment, the pathogen causing sclerotinia stem rot includes *Sclerotinia sclerotiorum*. In one embodiment, the crop includes rapeseed.

[0026] The present invention also provides a method for preventing and controlling sclerotinia stem rot in crops, comprising: initiating crop seeds with the inoculant described above.

[0027] In one embodiment, the method of using the inoculant includes: mixing crop seeds with the inoculant; placing the mixed seeds at 20°C for initiation; and air-drying the initiated seeds at room temperature until the mycelium on the seed surface is no longer sticky. In one embodiment, the mass-to-volume ratio of crop seeds to inoculant during seed mixing is 10-100 g:5 mL. In another embodiment, the mass-to-volume ratio of crop seeds to inoculant can be any one or any two values ​​from 10 g:5 mL, 20 g:5 mL, 30 g:5 mL, 40 g:5 mL, 50 g:5 mL, 60 g:5 mL, 70 g:5 mL, 80 g:5 mL, 90 g:5 mL, and 100 g:5 mL. As one implementation method, the initiation time is 10-20 hours. Alternatively, the initiation time can be any one of 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, and 20 hours, or the midpoint between any two values. As another implementation method, the initiated seeds are air-dried at room temperature until the mycelium on the seed surface is no longer sticky, and then they can be sown. As yet another implementation method, the initiated seeds are stored at room temperature after air-drying to constant weight.

[0028] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a pathogenic and reproductively absent Sclerotium sclerotiorum strain SD4-8V, its inoculum agent, and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1: Method for Creating Mutagenic Strains SD4-8 This embodiment uses Sclerotium sclerotiorum strain DT-8VF as the basic strain, which is disclosed in the literature "Yu et al. A Geminivirus-related DNA mycovirus that confers hypovirulence to a plant pathogenic fungus. Proc Natl Acad Sci USA 2010, 107: 8387-8392".

[0030] The method for preparing potato glucose culture medium (PDA) used in this embodiment is as follows: boil 200 g of peeled potatoes for 20 min, filter and keep the juice, add 20 g of glucose, add distilled water to 1 L, add 15 g of agar, and autoclave at 121℃ for 20 min.

[0031] The preparation method of PDB medium is as follows: boil 200 g of peeled potatoes for 20 min, filter and keep the juice, add 20 g of glucose, add distilled water to 1 L, and autoclave at 121℃ for 20 min.

[0032] The RM medium was prepared as follows: 0.7 M Sucrose, 1 g yeast extract, water added to 1 L, 15 g agar added, and autoclaved at 121℃ for 20 min.

[0033] The raw materials for the STC solution are: 1 M sorbitol, 50 mM Tris·HCl (pH 8.0) and 50 mM CaCl2·2H2O.

[0034] The cell wall enzymatic hydrolysate described in this embodiment was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0035] 1. Preparation of Sclerotinia sclerotiorum protoplasts Sclerotium sclerotiorum strain DT-8VF was inoculated onto PDA medium and cultured at 20°C for 24–36 h. Young and active mycelial blocks of DT-8VF were picked and inoculated onto fresh PDA medium lined with cellophane. The medium was then cultured at 20°C in the dark. After about 24–36 h, young DT-8VF mycelia were scraped off and inoculated into 100 mL of PDB medium. The medium was then shaken at 150 r / min and 20°C for about 24–36 h. The medium was removed by filtration with a layer of sterile lens paper, and the mycelia were washed 1–2 times with 0.7 M NaCl solution and collected.

[0036] The concentration of the cell wall enzymatic hydrolysate was diluted to 0.01 g / mL using 0.7 M NaCl as the solvent. The hydrolysate was centrifuged at 10,000 r / min and 4℃ for 10 min. The supernatant was collected and filtered twice through a bacterial filter to obtain the enzyme solution. This sterilization step was to ensure the purity of the enzyme solution and avoid contamination by exogenous microorganisms.

[0037] Mix the mycelia and enzyme solution evenly. For every 0.25-0.35 g of mycelia, mix 1 mL of enzyme solution to obtain a mixture. Incubate the mixture at 28℃ and 80-100 r / min for 2 h with shaking to obtain a culture mixture. Filter the culture mixture through a funnel lined with 3-4 layers of sterile lens paper to remove unlysaturated mycelia. Collect the filtrate and centrifuge it at 6000-7000 r / min and 4℃ for 10 min. Collect the precipitate to obtain protoplasts.

[0038] After dissolving the protoplasts in 500 µL of STC, the protoplasts were then diluted with STC to a concentration of 5 × 10⁻⁶. 5 Protoplast fluid was obtained by collecting samples per mL.

[0039] 2. Ultraviolet mutagenesis of Sclerotinia sclerotiorum Take 200 µL of protoplast fluid, spread it on RM medium, and place it in a clean bench to dry.

[0040] Preheat the UV lamp for 20 min on a clean bench. The UV lamp has a power of 12 W and an effective wavelength of 254 nm. The distance between the UV lamp and the petri dish is 60 cm. Under dark conditions, open the lid of the RM medium and irradiate it with UV light for 50 s. Then, place the RM medium in a 20°C incubator and incubate in the dark for 36 h. After incubation, pick the formed single colonies and inoculate them onto fresh PDA medium and incubate them in the dark at 20°C.

[0041] During 3-4 days of incubation in the dark, the *Sclerotium sclerotiorum* strain DT-8VF was found to form sclerotia primordia on fresh PDA medium. By day 7, approximately 50% of the 486 protoplast regenerated progeny strains had formed sclerotia; by day 14, approximately 90% of the protoplasts had formed sclerotia.

[0042] One of the mutant strains that could not form sclerotia was selected and named mutant strain SD4. Further purification was performed using protoplast regeneration: protoplasts of mutant strain SD4 were prepared using the above method, spread on RM medium, and cultured in a 20℃ incubator in the dark for 14 days to obtain mutant strain SD4-8 with sclerotia formation defects.

[0043] Example 2: Biological characteristics of the mutant strain SD4-8 The sclerotium-forming defective mutant strain SD4-8 and the *Sclerotinia sclerotiorum* strain DT-8VF were inoculated onto PDA medium and cultured at 20°C for 36 h to activate the strains. Mycelial blocks were obtained by punching holes at the vigorous growth edges of the colonies using a sterile 5 mm diameter punch. These mycelial blocks were then transferred to the center of 90 mm diameter PDA medium and cultured at 20°C in the dark for 30 days. During the culture period, the biological characteristics of the mutant strains SD4-8 and DT-8VF were observed and recorded. The results are as follows: Figures 1-2 As shown.

[0044] The mutant strains SD4-8 and DT-8VF of *Sclerotinia sclerotiorum* were activated and subcultured separately in PDA medium. When the mycelia reached 2 / 3 of the volume of the culture dish (DT-8VF typically reaches this size after 36-48 hours of culture, while SD4-8 typically reaches it after 48-60 hours), uniformly sized mycelial blocks were obtained using a 5 mm diameter punch. Rapeseed leaves of uniform age were selected, and before inoculation, the leaf surfaces were rinsed with distilled water. The rapeseed leaves were then placed on trays lined with moistened absorbent paper. Mycelial blocks of DT-8VF and SD4-8 were inoculated onto fresh, healthy detached rapeseed leaves, with DT-8VF inoculated on the left side and SD4-8 on the right side. The inoculated rapeseed leaves were kept moist with plastic wrap and incubated at 20°C. The size of the lesions was measured at 36 h, 48 h, and 72 h post-inoculation, and the disease progression was recorded by photograph. The results are as follows: Figure 3 As shown.

[0045] Depend on Figure 1 As can be seen, the *Sclerotinia sclerotiorum* strain DT-8VF spreads rapidly on PDA medium, forming dense colonies covered with white aerial hyphae. White sclerotia primordia are visible after 72 hours, and black sclerotia are visible after 7 days. Strain SD4-8 grows more slowly on PDA medium, forming gray colonies, and fails to form sclerotia primordia or sclerotia after 30 days of culture.

[0046] Depend on Figure 2 It can be seen that the average growth rate of Sclerotium sclerotiorum strain DT-8VF is 19.8±1.2 mm / d, while the average growth rate of strain SD4-8 is 13.15±0.78 mm / d. The growth rate of strain SD4-8 is significantly lower than that of Sclerotium sclerotiorum strain DT-8VF.

[0047] Depend on Figure 3 It can be seen that lesions appeared on the leaves inoculated with Sclerotinia sclerotiorum strain DT-8VF 24 h after inoculation with mycelial blocks, while no obvious lesions were observed on the leaves inoculated with strain SD4-8 until 72 h. It is evident that strain SD4-8 lost its pathogenicity to rapeseed.

[0048] Example 3: Biological characteristics of the *Sclerotium sclerotiorum* strain SD4-8V, which lacks both pathogenicity and reproduction. The *Sclerotium sclerotiorum* DT-8 carrying the SsHADV-1 virus used in this embodiment is disclosed in the literature “Yu et al. AGeminivirus-related DNA mycovirus that confers hypovirulence to a plantpathogenic fungus. Proc Natl Acad Sci USA 2010, 107: 8387-8392”. The sequence of the SsHADV-1 is accessed in GenBank under accession number GQ365709.

[0049] The 2×Hieff PCR Master Mix kit used in this example was purchased from Shanghai Yisheng Biotechnology Co., Ltd.

[0050] 1. Creation of a *Sclerotinia sclerotiorum* strain SD4-8V that lacks both pathogenicity and reproduction. This study employed the confrontation culture method, the operation of which was referenced from "Yu Xiao. Research on the characteristics and application potential of non-pathogenic DNA virus 1 in Sclerotinia sclerotiorum [D]. Wuhan: Huazhong Agricultural University, 2013." The virus SsHADV-1 in Sclerotinia sclerotiorum DT-8 was transferred to the mutant strain SD4-8. The specific steps are as follows: Following the method described in Example 1, *Sclerotinia sclerotiorum* strain DT-8 was activated. Then, using a sterile punch, 5 mm diameter mycelial blocks were inoculated onto fresh PDA medium, approximately 1 cm from the edge of the medium. The culture dishes were incubated at 20°C for 3 days. When *Sclerotinia sclerotiorum* strain DT-8 grew to a diameter of 1.5–2 cm, 5 mm diameter mycelial blocks of the mutant strain SD4-8 were inoculated 1 cm from the edge of the *Sclerotinia sclerotiorum* DT-8 colony. The culture dishes were then incubated at 20°C for 2–3 days. Mycelial blocks located near the mutant strain SD4-8 and away from the region of *Sclerotinia sclerotiorum* DT-8 were transferred to fresh PDA medium and cultured to obtain *Sclerotinia sclerotiorum* strain SD4-8V, which lacks both pathogenicity and reproduction.

[0051] 2. Genomic DNA was extracted from the pathogenic and reproductive-deficient strain SD4-8V of Sclerotinia sclerotiorum. Genomic DNA samples of *Sclerotinia sclerotiorum* strains SD4-8V and DT-8, which have both pathogenicity and reproductive defects, were extracted using the CTAB method. The extraction method is as follows: Collect 1-2 g of bacterial mycelium as a sample, add the sample to a 2 mL centrifuge tube, and rapidly grind it into powder using liquid nitrogen. Add 800 μL of preheated 2% CTAB extraction buffer, and heat the centrifuge tube in a water bath at 65℃ for 30 min, inverting and mixing once every 5 min during this period. After heating, add 400 μL of chloroform and 400 μL of phenol to the centrifuge tube, gently invert and mix, and centrifuge at 12000 r / min at room temperature for 15 min. Take 650 μL of supernatant, place it in a new centrifuge tube, add 650 μL of chloroform for extraction, invert and mix, and centrifuge at 12000 r / min for 10 min. Take 450 μL of supernatant, place it in a new centrifuge tube, add an equal volume of pre-cooled isopropanol, and perform nucleic acid precipitation at -20℃ for 30 min. After precipitation, centrifuge at 12000 r / min at room temperature for 15 min. Discard the supernatant and add 1 Wash the precipitate with 75% ethanol for more than 2 times, discard the residual liquid, and dry it in a 37℃ oven for 7 min; add 40 μL ddH2O (containing 25 μg / mL RNase A) to dissolve it for 5~10 min. Genomic DNA samples can be stored at 4℃ for short-term storage and at -20℃ for long-term storage.

[0052] 3. PCR detection of SsHADV-1 in the pathogenic and reproductively absent *Sclerotium sclerotiorum* strain SD4-8V: Using the genomic DNA extracted from the non-pathogenic strain SD4-8V of Sclerotinia sclerotiorum as a template and the genomic DNA sample of strain DT-8 as a positive control, PCR detection of SsHADV-1 carried in the strain was performed using the SsHADV-1 specific primers Rep 5 and Rep 6.

[0053] Rep 5 (SEQ ID NO: 1): 5'-TCACATGACTTTCGACTTCCACGC-3'; Rep 6 (SEQ ID NO:2): 5'-CCGGCGCAGCCGATATGGATAAT-3'.

[0054] PCR was performed using the 2×Hieff PCR Master Mix kit.

[0055] The PCR system is as follows: PCR Master Mix 10 µL; 10 µM upstream and downstream specific primers 0.5 µL each; genomic DNA template 1.0 µL; ddH2O to bring the volume to 20 µL.

[0056] PCR program settings: annealing at 95℃ for 5 min; denaturation at 94℃ for 30 s; annealing at 60℃ for 30 s; extension at 72℃ for 20 s; 32 cycles; extension at 72℃ for 5 min; incubation at 16℃ for 2 min.

[0057] After the reaction was complete, the amplification products were subjected to 1% agarose gel electrophoresis, stained with ethidium bromide (0.5 μg / mL), and imaged using a gel imaging system. The results are as follows: Figure 4 As shown. By Figure 4 It is evident that a band of the same size as strain DT-8 was detected in the Sclerotium sclerotiorum strain SD4-8V, which lacks both pathogenicity and reproduction, indicating that the Sclerotium sclerotiorum strain SD4-8V carries SsHADV-1.

[0058] 4. Verification of the physiological characteristics of the *Sclerotium sclerotiorum* strain SD4-8V, which lacks both pathogenicity and reproduction. Sclerotinia sclerotiorum strains SD4-8V and DT-8, both lacking pathogenicity and reproduction, were inoculated onto PDA medium and incubated at 20°C for 24-48 hours to ensure the strains remained active. Mycelial blocks were then obtained by punching holes at the vigorous growth edges of the colonies using a 5 mm diameter sterile punch. These mycelial blocks were transferred to the center of 90 mm diameter petri dishes and incubated at 20°C in the dark for 24-30 days. The growth of the pathogenic and reproductive Sclerotinia sclerotiorum strains SD4-8V and DT-8 was observed, and the results are as follows: Figures 5-6 As shown.

[0059] Depend on Figure 5 It is evident that the growth rates of *Sclerotinia sclerotiorum* strain DT-8 and the pathogenic and reproductively absent strain SD4-8V are comparable. Strain DT-8 colonies exhibit fan-shaped colonies; white sclerotia primordia appear after 7 days of culture in the DT-8 medium, and black sclerotia appear after 20 days. In contrast, the colonies of strain SD4-8V, which lacks both pathogenicity and reproduction, are gray and do not form sclerotia primordia or sclerotia.

[0060] Depend on Figure 6 It can be seen that, after being cultured on a PDA at 20℃ in the dark for 24 h to 48 h, the growth rate of the *Sclerotinia sclerotiorum* strain SD4-8V, which lacks both pathogenicity and reproduction, was not significantly different from that of the *Sclerotinia sclerotiorum* strain DT-8. P >0.05).

[0061] Mycelial blocks of strain SD4-8V were inoculated onto detached rapeseed leaves and cultured at 20℃ under moist conditions. Disease incidence on the detached rapeseed leaves was recorded, and the results are as follows: Figure 7 As shown.

[0062] Depend on Figure 7As can be seen, lesions began to appear on the edge of the *Sclerotinia sclerotiorum* strain DT-8VF inoculated on the left side of the leaf 24 hours after inoculation, and these lesions expanded rapidly with the extension of the culture time. Later, the mycelium spread to the leaf edge, almost infecting the left half of the rapeseed leaf, resulting in obvious water-soaked lesions, with dense aerial mycelium forming at the edge of the lesions. The *Sclerotinia sclerotiorum* strain SD4-8V, which lacks both pathogenicity and reproduction, did not form visible lesions on rapeseed leaves even after 72 hours of inoculation, indicating that it had lost its pathogenicity to rapeseed leaves and was a non-pathogenic viral strain.

[0063] Example 4: Verification of the effect of the pathogenicity- and reproduction-deficient *Sclerotium sclerotiorum* strain SD4-8V on rapeseed. The Huayouza 62 mentioned in this embodiment was purchased from Hubei Guoke High-tech Co., Ltd.

[0064] 1. Culture of *Sclerotinia sclerotiorum* strain SD4-8V, which lacks both pathogenicity and reproduction. The pathogenic and reproductive-deficient *Sclerotinia sclerotiorum* strain SD4-8V was inoculated into PDB medium and cultured at 20°C and 150 rpm for 4–5 days. The resulting *Sclerotinia sclerotiorum* fermentation broth was adjusted to OD using ddH₂O. 600 =2.0, to obtain the Sclerotinia sclerotiorum fermentation broth.

[0065] 2. Rapeseed seeds caused 10 g of Huayouza 62 rapeseed seeds were mixed with 5 mL of the *Sclerotinia sclerotiorum* fermentation broth described above for seed treatment. The treated rapeseed seeds were placed at 20℃ for 18 h to initiate the fermentation process, then air-dried at room temperature until the mycelium on the seed surface was no longer sticky before sowing. The remaining treated seeds could be air-dried to constant weight and stored at room temperature. Seeds treated with the *Sclerotinia sclerotiorum* fermentation broth served as the experimental group.

[0066] The seeds of Huayouza 62 rapeseed treated with an equal amount of DT-8 strain fermentation broth cultured under the same conditions were used as a positive control group, while the seeds treated with an equal amount of sterile water were used as a negative control group.

[0067] 3. Verification of the seedling growth effects of different rapeseed seed priming groups Rapeseed seeds from the experimental group (SD4-8V), positive control group (DT-8), and negative control group (H2O) were simultaneously sown on October 19, 2024, in the rapeseed experimental field located on the campus of Huazhong Agricultural University in Wuhan, Hubei Province, at a planting density of approximately 18,000 plants / mu. During the seedling stage (3 weeks after sowing), 16 seedlings were randomly collected from each group to record plant height and fresh weight. A schematic diagram of the rapeseed plant height during the seedling stage is shown below. Figure 8 As shown, the plant height data for different groups are as follows: Figure 9 As shown. Fresh weight data for different groups are as follows. Figure 10 As shown.

[0068] Depend on Figure 8 It is evident that the plant height of rapeseed induced in the experimental group was significantly higher than that in the negative control group.

[0069] Depend on Figure 9 As can be seen, in the negative control group, the average height of rapeseed plants induced by water was 16.47±1.51 cm, in the positive control group, the average height of rapeseed plants induced by seeds of strain DT-8 was 20.64±1.43 cm, and in the experimental group, the average height of rapeseed plants induced by seeds of strain SD4-8V was 21.28±1.32 cm. Compared with the water control, strains SD4-8V and DT-8 significantly increased the height of rapeseed plants after seed induced priming. P <0.001); there was no significant difference between strains SD4-8V and DT-8. This indicates that seed priming by both SD4-8V and DT-8 can increase the plant height of rapeseed.

[0070] Depend on Figure 10 It was observed that the average fresh weight of rapeseed induced by water in the negative control group was 3.34±1.08 g, the average fresh weight of the aboveground parts of rapeseed induced by seeds of strain DT-8 in the positive control group was 6.75±1.35 g, and the average fresh weight of the aboveground parts of rapeseed plants induced by seeds of strain SD4-8V in the experimental group was 6.12±1.31 g. Compared with the negative control, both the positive control group and the experimental group showed a significant increase in the fresh weight of the aboveground parts after induced rapeseed seed treatment. P <0.001); there was no significant difference in fresh weight between the positive control group and the experimental group. This indicates that seed priming by strains SD4-8V and DT-8 can significantly increase the aboveground fresh weight of rapeseed plants.

[0071] 4. Verification of flowering period of rapeseed from different seed-induced groups The number of rapeseed plants that flowered at the initial flowering stage on March 2, 2025, was investigated in the experimental group, positive control group, and negative control group sown in step 3. Seven rows of rapeseed were randomly surveyed for each treatment, and the flowering rate was counted. The results are as follows: Figure 11 As shown.

[0072] Depend on Figure 11 It was found that the flowering rate of rapeseed induced by water in the negative control group was 2.4±0%, the average flowering rate of rapeseed induced by DT-8 strain seeds in the positive control group was 15.1±4.89%, and the average flowering rate of rapeseed induced by SD4-8V strain seeds in the experimental group was 13.49±1.12%. Compared with the negative control, the flowering rate of both the positive control and the experimental group was significantly increased, indicating that both the positive control and the experimental group could promote earlier flowering of rapeseed. P <0.001); there was no significant difference in flowering rate between the experimental group and the positive control group. This indicates that SD4-8 and DT-8 seed priming can significantly promote earlier flowering in rapeseed.

[0073] 5. Verification of Sclerotinia stem rot incidence in rapeseed from different seed groups Rapeseed is susceptible to Sclerotinia sclerotiorum infection throughout its entire growth cycle, with the flowering period being a critical stage. Infection leads to stem rot and systemic infection, ultimately causing the entire plant to wilt and die. Therefore, the incidence of Sclerotinia sclerotiorum in rapeseed was observed and recorded in the experimental group, positive control group, and negative control group sown in step 3. On March 18, 2025, Sclerotinia sclerotiorum was found in the rapeseed field, and photographs were taken and recorded. The results are as follows: Figure 12 As shown. On March 26, 2025, the incidence of sclerotinia stem rot in rapeseed was investigated in each group. Seven rows were randomly selected from each group to statistically analyze the incidence of sclerotinia stem rot. The results are as follows. Figure 13 As shown.

[0074] Depend on Figure 12 It can be seen that obvious diseased rapeseed plants were found in the negative control group, with stem rot and white mycelium growing on the surface. Black sclerotia were formed on the outside and inside of the stem. However, no obvious diseased plants were found in the experimental group treated with strain SD4-8V and the positive control group treated with DT-8.

[0075] Depend on Figure 13 As can be seen, the average incidence rate in the negative control group was 12.9±6.60%, the average incidence rate in the positive control group was 8.84±5.93%, and the average incidence rate in the experimental group was 2.72±3.47%. Therefore, the incidence rate in the experimental group showed a highly significant difference compared to the negative and positive control groups. P <0.01). The efficacy of the experimental group and the positive control group was 78.9% and 31.5%, respectively. Compared with the positive control group, the efficacy of the experimental group was increased by 150.48%.

[0076] In summary, this invention provides a *Sclerotinia sclerotiorum* strain SD4-8V that is deficient in both pathogenicity and reproduction. This strain can promote rapeseed seed growth, increase rapeseed plant height and fresh weight, promote early flowering of rapeseed, and has a significant disease prevention effect, thus achieving efficient and safe control of crop sclerotinia sclerotiorum disease.

[0077] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments without creative effort, as shown in these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of *Sclerotinia sclerotiorum* lacking both pathogenicity and reproduction ( Sclerotinia sclerotiorum strain SD4-8V, characterized in that, It is deposited at the China Center for Type Culture Collection, with accession number CCTCC M 2026256.

2. A microbial agent, characterized in that, The bacterial agent comprises the *Sclerotium sclerotiorum* strain SD4-8V, which is deficient in both pathogenicity and reproduction as described in claim 1.

3. A method for preparing a microbial agent, characterized in that the steps include... include: The pathogenic and reproductively absent *Sclerotium sclerotiorum* strain SD4-8V, as described in claim 1, was inoculated into a culture medium and cultured. After culture, the OD was adjusted with ddH2O. 600 The concentration was increased to 1.5-2.5 to obtain the Sclerotinia sclerotiorum inoculum.

4. The application of the *Sclerotinia sclerotiorum* strain SD4-8V, which is doubly lacking in pathogenicity and reproduction, as described in claim 1, the bacterial agent described in claim 2, or the bacterial agent prepared by the method described in claim 3, characterized in that... The application includes any one or more of the following: 1) Promotes crop growth; 2) Promotes crop flowering; 3) Control crop sclerotinia disease.

5. The application as described in claim 4, characterized in that, Promoting crop growth includes increasing crop height and / or the fresh weight of the above-ground parts.

6. The application as described in claim 4, characterized in that, The pathogen causing sclerotinia disease in crops includes Sclerotinia sclerotiorum.

7. The application as described in claim 4, characterized in that, The crop mentioned includes rapeseed.

8. A method for controlling crop sclerotinia stem rot, characterized in that, include: The inoculant prepared by the inoculant of claim 2 or the preparation method of claim 3 is used to initiate crop seeds.

9. The method as described in claim 8, characterized in that step include: Use the aforementioned microbial agent to treat crop seeds; The treated seeds were placed at 20℃ to induce germination. After initiation, the seeds are dried until the mycelium on the seed surface is no longer sticky.

10. The method as described in claim 9, characterized in that, The mass-to-volume ratio of the crop seeds and the inoculant is 5-15 g: 1-10 mL.

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