Application of non-pathogenic strain DT-8 of Sclerotinia sclerotiorum in the control of root-knot nematode disease in crops

Abstract

This invention discloses the application of a non-pathogenic strain of *Sclerotinia sclerotiorum*, DT-8, in the control of root-knot nematode disease in crops. The preservation number of the non-pathogenic *Sclerotinia sclerotiorum* strain DT-8 is CCTCC NO: M2019328. This invention is the first to discover that the non-pathogenic *Sclerotinia sclerotiorum* strain DT-8 can be used to control root-knot nematode disease through mechanisms such as inducing resistance, thereby reducing the use of chemical pesticides. This invention provides new ideas and methods for the control of root-knot nematode disease, contributing to the promotion of green and sustainable agricultural development and ensuring the yield and quality safety of agricultural products.

Description

Technical Field

[0001] This invention relates to the field of biological control technology for crop diseases, specifically to a non-pathogenic strain of Sclerotinia sclerotiorum (Sclerotinia sclerotiorum). Sclerotinia sclerotiorum Application of DT-8 in the control of root-knot nematode disease in crops. Background Technology

[0002] Root-knot nematodes are plant parasitic nematodes that infect the roots of host plants, inducing the formation of root knots and hindering healthy plant growth. They are numerous and widely distributed, infecting over 5,500 plant species, including food crops, cash crops, fruit trees, vegetables, and ornamental plants. Root-knot nematodes are a significant global plant pathogen, posing a serious threat to agricultural production.

[0003] *Sclerotinia sclerotiorum* is a necrotrophic pathogenic fungus, and the sclerotinia rot it causes in rapeseed is a significant fungal disease. *Sclerotinia sclerotiorum* DT-8 carries the DNA virus SsHADV-1, which alters the fungus's growth pattern, transforming it from a parasitic pathogen into a beneficial fungus capable of endophytic growth within plants. Field trials have shown that spraying rapeseed with mycelial solution of *Sclerotinia sclerotiorum* DT-8 containing SsHADV-1 significantly promotes rapeseed growth, inhibits sclerotinia rot, and increases rapeseed yield. *Sclerotinia sclerotiorum* DT-8 can also grow endophytically within wheat plants, enhancing plant disease resistance, promoting wheat growth, and increasing yield. Furthermore, *Sclerotinia sclerotiorum* DT-8 can grow endophytically within various gramineous crops such as corn and rice. While previous studies have found that the non-pathogenic strain DT-8 of *Sclerotinia sclerotiorum* has a promoting effect on crop growth and can effectively increase crop yield, there are currently no reports on its efficacy in controlling root-knot nematodes. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a non-pathogenic strain of *Sclerotinia sclerotiorum*. Sclerotinia sclerotiorum Application of DT-8 in the control of root-knot nematode disease in crops. This invention aims to explore the biocontrol potential of the non-pathogenic strain DT-8 of Sclerotinia sclerotiorum against root-knot nematodes, providing new ideas and methods for the control of root-knot nematode disease, and contributing to the promotion of green and sustainable agricultural development, ensuring the yield and quality safety of agricultural products.

[0005] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides a sclerotium ( Sclerotinia sclerotiorumThe application of non-pathogenic strain DT-8 in the control of crop root-knot nematode disease. The preservation number of the non-pathogenic strain DT-8 of *Sclerotinia sclerotiorum* is: CCTCC NO: M2019328. Root-knot nematode disease is a type of plant parasitic nematode disease caused by root-knot nematodes infecting crop roots; the root-knot nematode is *Phytophthora spp.* (…). Meloidogyne graminicola ) or Southern root-knot nematode ( Meloidogyne incognita ).

[0006] The aforementioned non-pathogenic strain of *Sclerotinia sclerotiorum*, DT-8, was deposited on May 5, 2019, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, Hubei Province, China, with accession number CCTCC NO: M2019328. This non-pathogenic strain DT-8 is a weakened strain of *Sclerotinia sclerotiorum*, disclosed in Chinese invention patent publication number CN116918833A, entitled "Application of *Sclerotinia sclerotiorum* strain DT-8 in Increasing Rice Yield."

[0007] Furthermore, the crops infected by the grass-like root-knot nematode are rice or rapeseed, and the crops infected by the southern root-knot nematode are tomatoes.

[0008] This invention also provides a sclerotium ( Sclerotinia sclerotiorum The application of non-pathogenic strain DT-8 in the preparation of pesticides for the control of root-knot nematode disease in crops, wherein the plant is rice, rapeseed or tomato, and the preservation number of the non-pathogenic strain DT-8 of Sclerotinia sclerotiorum is: CCTCC NO: M2019328.

[0009] This invention also provides a microbial preparation for controlling root-knot nematode disease in crops, wherein the microbial preparation contains Sclerotinia sclerotiorum (Sclerotinia sclerotiorum). Sclerotinia sclerotiorum The non-pathogenic strain DT-8, wherein the plant is rice, rapeseed or tomato, and the preservation number of the non-pathogenic strain DT-8 of Sclerotium sclerotiorum is: CCTCC NO: M2019328.

[0010] Furthermore, the microbial preparation is any one of the following: Sclerotinia sclerotiorum DT-8 mycelial suspension, Sclerotinia sclerotiorum DT-8 mycelia, Sclerotinia sclerotiorum DT-8 fermentation broth, and Sclerotinia sclerotiorum DT-8 fermentation filtrate.

[0011] Furthermore, the method for preparing the *Sclerotinia sclerotiorum* DT-8 mycelial suspension is as follows: Sclerotinia sclerotiorum DT-8 was activated by slicing strain DT-8 into SPDB / PDB medium, placing it on a shaker, weighing the mycelium, adding sterile water to make up to a final volume, and obtaining a mycelial suspension. The content of Sclerotinia sclerotiorum DT-8 in the mycelial suspension was 0.07~0.28 g / mL. The preparation method of the Sclerotinia sclerotiorum DT-8 fermentation broth is as follows: Sclerotinia sclerotiorum DT-8 was activated by scrambling strain DT-8 onto SPDB medium and placing it on a shaker to obtain Sclerotinia sclerotiorum DT-8 fermentation broth, wherein the content of Sclerotinia sclerotiorum DT-8 in the fermentation broth was 0.14~0.30 g / mL; The preparation method of the Sclerotinia sclerotiorum DT-8 fermentation filtrate is as follows: To activate Sclerotinia sclerotiorum DT-8, strain DT-8 was sliced ​​into SPDB medium, placed on a shaker, centrifuged to remove mycelia, and the supernatant was taken as the fermentation filtrate of Sclerotinia sclerotiorum DT-8.

[0012] Furthermore, the content of Sclerotinia sclerotiorum DT-8 in the fermentation broth is 0.14 g / mL.

[0013] The present invention also provides a method for controlling rice root-knot nematode disease, the method comprising any one of the following application methods: (1) Coat rice seeds with the above-mentioned Sclerotium sclerotiorum DT-8 mycelial suspension; (2) Apply the above-mentioned Sclerotium sclerotiorum DT-8 fermentation broth, Sclerotium sclerotiorum DT-8 fermentation filtrate, or Sclerotium sclerotiorum DT-8 mycelial suspension to the roots of rice. (3) Inoculate the above-mentioned Sclerotium DT-8 mycelium into the roots of rice.

[0014] Furthermore, when the seed coating is performed using the method described above, the content of Sclerotinia sclerotiorum DT-8 mycelial suspension is 0.07 g / mL, the coating ratio is 50 mL mycelial suspension / 500 g seeds, and the treatment time is 12 h.

[0015] Furthermore, when the root irrigation is performed using the method described above, the content of Sclerotinia sclerotiorum DT-8 in the fermentation broth is 0.14 g / mL; The concentration of Sclerotinia sclerotiorum DT-8 mycelial suspension was 0.28 g / mL.

[0016] This invention also provides a method for controlling root-knot nematode disease in rapeseed, wherein the method involves coating rapeseed seeds with a suspension of *Sclerotinia sclerotiorum* DT-8 mycelium, and the root-knot nematode is a *Phraceae* root-knot nematode (…). Meloidogyne graminicola The preservation number of the *Sclerotium sclerotiorum* DT-8 is: CCTCC NO: M2019328.

[0017] Furthermore, the content of Sclerotinia sclerotiorum DT-8 mycelial suspension in the suspension was 0.20 g / mL, the coating ratio was 1 mL mycelial suspension / 2 g seeds, and the coating time was 18 h.

[0018] This invention also provides a method for controlling tomato root-knot nematode disease, wherein the method involves drenching the roots of tomatoes with the fermentation broth of *Sclerotinia sclerotiorum* DT-8, and the root-knot nematode is *Symplocos septemlobus* (Southern root-knot nematode). Meloidogyne incognita The preservation number of the *Sclerotium sclerotiorum* DT-8 is: CCTCC NO: M2019328.

[0019] Furthermore, the content of Sclerotinia sclerotiorum DT-8 in the fermentation broth is 0.14~0.30 g / mL; the amount used for root irrigation per tomato plant is 15~30 mL.

[0020] This invention also provides a non-pathogenic strain of Sclerotinia sclerotiorum (Sclerotinia sclerotiorum). Sclerotinia sclerotiorum The application of DT-8 or the above-mentioned microbial preparations in promoting rice root growth, wherein the preservation number of the non-pathogenic strain of Sclerotinia sclerotiorum DT-8 is: CCTCC NO: M2019328.

[0021] The principle of this invention: *Sclerotinia sclerotiorum* is a plant pathogenic fungus with a wide host range and serious damage, causing sclerotinia rot which has severely impacted oilseed crop production in my country. The non-pathogenic strain DT-8 of *Sclerotinia sclerotiorum* possesses a DNA virus (*Sclerotinias clerotiorum* hypovirulence associated DNA virus 1, SsHADV-1), enabling endophytic growth within gramineous crops such as wheat, barley, corn, and rice. This not only offers advantages in controlling the pathogen but also enhances crop growth and disease resistance, increasing yield. These characteristics make the non-pathogenic strain DT-8 an important target for research on the biological control of root-knot nematodes and other plant diseases.

[0022] The beneficial effects of this invention are: This invention marks the first discovery that the non-pathogenic strain DT-8 of *Sclerotinia sclerotiorum* can be used to control root-knot nematode disease, reducing the use of chemical pesticides. This invention provides new ideas and methods for the control of root-knot nematode disease, contributing to the promotion of green and sustainable agricultural development and ensuring the yield and quality safety of agricultural products. Attached Figure Description

[0023] Figure 1 Root irrigation with Sclerotinia sclerotiorum DT-8 fermentation broth induced systemic resistance in rice to control root-knot nematode disease; In the diagram, A is a schematic diagram of rice root division; B is a diagram of rice roots (scale bar = 2 cm), with yellow arrows indicating root knots; C is a diagram of rice plant height; D is a diagram of the number of rice root knots; E is a diagram of the number of rice nematodes; the method used to test rice plant height, number of root knots, and number of root nematodes in C and D is the t-test. represent P <0.001, represent P <0.01, represent P <0.05.

[0024] Figure 2 A schematic diagram illustrating the nematicidal activity of Sclerotinia sclerotiorum DT-8 fermentation filtrate against root-knot nematodes of the Poaceae family; In the figure, A is a schematic diagram of the J2 mortality rate (scale bar = 250 µm); B is a graph of the J2 mortality rate, and the test method is ttest. represent P <0.0001.

[0025] Figure 3 The effect of biological initiation and mycelial inoculation of Sclerotinia sclerotiorum DT-8 mycelial suspension on the control of rice root-knot nematodes of the Poaceae family. In the figure, A is a diagram of rice roots (scale bar = 1 cm); B is a diagram of rice root knots after acid fuchsin staining (scale bar = 1 mm); C is a diagram of the number of nematodes in the roots of a single rice plant. The test method was one-way ANOVA. Different letters above the bars represent... P A value <0.05 indicates a significant difference. MS represents DT-8 bacterial suspension; Myc represents DT-8 hyphae; yellow arrows indicate root knots (A) and root nematodes (B).

[0026] Figure 4 The effect of root irrigation with Sclerotinia sclerotiorum DT-8 fermentation broth on the control of rice root-knot nematode disease; In the figure, A is a diagram of rice roots (scale bar = 2 cm); B is a diagram of rice roots after acid fuchsin staining (scale bar = 10 mm); C is a diagram of rice root knots after acid fuchsin staining (scale bar = 2 mm); D is a diagram of the fresh weight of a single rice root. The test method is one-way ANOVA. Different letters above the bars represent... P A value <0.05 indicates a significant difference; E represents the number of nematodes in the roots of a single rice plant, and the test method is t-test. represent P <0.05, yellow arrows indicate root knots (A, B) and root nematodes (C); Mg DT-8+ was used to inoculate against root-knot nematodes of the Poaceae family. Mg To control root-knot nematode disease in rice caused by sclerotiorum fermentation broth.

[0027] Figure 5 The control effect of Sclerotinia sclerotiorum DT-8 mycelial suspension on root-knot nematode disease in rapeseed is shown in the figure. In the figure, A is a diagram of rapeseed roots (scale bar = 2 cm); B is a diagram of root knots after acid fuchsin staining (scale bar = 1 mm); C is a diagram of the aboveground fresh weight of a single rapeseed plant. The test method was one-way ANOVA. Different letters above the bars represent... P A difference of <0.05 is statistically significant; D shows the number of root knots in a single rapeseed plant; E shows the number of nematodes in the roots of a single rapeseed plant. DE: The test method is t-test. represent P <0.0001, represent P <0.01. The yellow arrows indicate root knots (A) and root nematodes (B).

[0028] Mg For inoculation with root-knot nematodes of the Poaceae family; DT-8 is rapeseed root after bio-initiation with a suspension of Sclerotinia sclerotiorum mycelium; DT-8+ Mg A bio-initiation method using *Sclerotinia sclerotiorum* DT-8 mycelial suspension to control root-knot nematode disease in rapeseed (Poaceae family).

[0029] Figure 6 The effect of Sclerotinia sclerotiorum DT-8 fermentation broth on root-knot nematode control in tomatoes. In the figure, A is a diagram of a tomato plant; B is a diagram of tomato roots (scale bar = 1 cm), with yellow arrows indicating root knots; C is a diagram of the fresh weight of a single tomato root. The test method was one-way ANOVA, and the same letters above the bars represent... P When the value is >0.05, there is no significant difference; D is the number of root knots per tomato plant; E is the root knot index per tomato plant; F is the number of egg masses per tomato plant; G is the total number of eggs between two tomato plants. The DG test method is t test. represent P <0.0001, represent P <0.001, represent P <0.05.

[0030] Mi To inoculate only with southern root-knot nematodes, DT-8 was used for root irrigation treatment of tomatoes with DT-8 fermentation broth. Mi DT-8 fermentation broth was used for root irrigation to control tomato root-knot nematode disease.

[0031] Figure 7 Colonization of Sclerotinia sclerotiorum DT-8 on tomato roots; Control refers to tomato roots that were not inoculated with DT-8, serving as a control; DT-8 indicates that DT-8 mycelia stained with WGA fluorescein emitted green fluorescence 5 days after inoculation of tomato roots with Sclerotium sclerotiorum DT-8 mycelium blocks; PI-stained tomato root tissue emitted red fluorescence; the scale bar is 50 μm. Detailed Implementation

[0032] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0033] Example 1: Isolation of Sclerotinia sclerotiorum DT-8, preparation of culture medium, and preparation and inoculation of nematode suspension. 1. Isolation of Sclerotinia sclerotiorum DT-8 The sclerotia, isolated from the stems of rapeseed infected with Sclerotinia sclerotiorum, were found to carry the DNA virus SsHADV-1. This strain of Sclerotinia sclerotiorum (…) Sclerotinia sclerotiorum DT-8 was deposited on May 5, 2019, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, Hubei Province, China, with accession number CCTCC NO: M2019328. The DT-8 strain of Sclerotinia sclerotiorum was disclosed in Chinese invention patent CN116918833A, entitled "Application of Attenuated Virus Sclerotinia sclerotiorum Strains DT-8 in Increasing Rice Yield".

[0034] 2. Preparation of culture medium SPDB medium: Weigh 35 g of potato glucose broth medium, add it to 1000 mL of distilled water, heat to boiling, dispense into containers, and autoclave at 121℃ for 20 min. Potato glucose broth medium components: 5 g potato extract, 10 g peptone, 15 g glucose, 5 g sodium chloride.

[0035] PDB: Weigh 200 g of peeled potatoes, cut them into small pieces, add an appropriate amount of water and boil for 5-10 minutes. Filter the solution through four layers of gauze to obtain the filtrate. Add 20 g of glucose to the filtrate, shake well and make up to 1 L with distilled water. Sterilize at 121℃ for 30 minutes.

[0036] 3. Preparation and inoculation of root-knot nematode suspension a. Barnyard grass root treatment for propagation of grass-like root-knot nematodes and preparation and inoculation of nematode suspensions: First, the barnyard grass is washed to remove surface impurities. After washing, the barnyard grass root tissue is cut into small pieces and homogenized in a homogenizer for 1 minute. The homogenized mixture is then filtered through a 200-mesh sieve to remove excess root tissue that has not been fully homogenized. Next, the egg suspension filtered through the 200-mesh sieve is further filtered through a 350-mesh sieve. During this process, the nematode eggs will remain on the surface of the 350-mesh sieve, while the filtrate is discarded. Subsequently, sterilized tap water cooled to room temperature is added to the sieve containing the nematode eggs, and the entire sieve is placed in a constant temperature incubator set at 28°C for incubation. After an incubation period of 1-2 days, the second instar larvae (J2) of the root-knot nematode (Poaceae) are collected from the filtrate below the 350-mesh sieve.

[0037] The collected second-instar larvae (J2) suspension was mixed with tap water in a specific ratio to prepare a nematode suspension with a density of 150 nematodes / mL. One mL of the nematode suspension was then pipetted into the rice roots, with an inoculation amount of 150 nematodes / mL.

[0038] b. Tomato root tissue treatment for propagation of southern root-knot nematodes and preparation and inoculation of nematode suspension: Southern root-knot nematodes were cultured using tomatoes as the host plant. At least two months after inoculation, nematode eggs were extracted and hatched from the tomato roots. The specific steps were as follows: First, the nutrient soil adhering to the tomato roots was gently rinsed clean in water. Then, 0.8% NaClO was added and shaken for 5 minutes until the egg masses ruptured. The crude extract of eggs was collected by passing the extract through 200-mesh, 350-mesh, and 500-mesh sieves. Subsequently, the eggs were counted under a microscope to prepare a nematode suspension with an egg density of 1500 eggs / plant. 1 mL of the egg suspension was pipetted and inserted around the tomato roots. The egg suspension was then displaced to complete the inoculation. The inoculation amount was 1500 eggs / plant.

[0039] Example 2: Induction of Systemic Resistance in Rice by Sclerotinia sclerotiorum DT-8 Fermentation Broth 1. Preparation of Sclerotinia sclerotiorum DT-8 fermentation broth After activating Sclerotinia sclerotiorum DT-8 for 2-3 days, it was inoculated into 150 mL of SPDB medium and cultured with shaking at 20℃ and 120 r / min for 4 days to obtain fermentation broth with mycelial content of approximately 0.22 g / mL.

[0040] 2. DT-8-induced systemic resistance in Sclerotinia sclerotiorum To verify whether Sclerotinia sclerotiorum DT-8 can induce systemic resistance in rice, a root-separation experiment was conducted. DT-8 fermentation broth was applied to one side of the roots of rice plants cultured for 21 days, with water as a control. The other side of the roots was inoculated with 150 second-instar larvae of the grass-like root-knot nematode per plant. After 7 days, the number of root knots and the number of nematodes in the roots were investigated. Each treatment was replicated 11 times.

[0041] like Figure 1 As shown, DT-8 treatment significantly increased rice plant height, while significantly reducing the number of root knots and nematode numbers in the untreated lateral roots. This indicates that DT-8 treatment not only promotes rice growth but may also inhibit nematode infection of the untreated root zone by inducing a host systemic defense response. These results demonstrate that inducing host systemic resistance is one of the important mechanisms by which DT-8 controls rice root-knot nematode disease.

[0042] Example 3: Nematicidal activity of Sclerotinia sclerotiorum DT-8 fermentation filtrate against root-knot nematodes of the Poaceae family. 1. Preparation of Sclerotinia sclerotiorum DT-8 fermentation filtrate Sclerotinia sclerotiorum DT-8 was activated on a 60 mm diameter PDA plate for 2 days. Mycelial blocks were inoculated into 150 mL of SPDB medium, lysed for 5-10 seconds, and cultured in a shaker at 20℃ and 120 r / min for 9 days. The supernatant was collected by centrifugation and passed through a 0.22 mm bacterial filter to obtain the fermentation filtrate.

[0043] 2. Nematicidal activity of Sclerotinia sclerotiorum DT-8 fermentation filtrate Take out the hatched second-instar nematode larvae and adjust them at a ratio of 100 larvae / 100 μL. Add 100 μL of second-instar nematode larvae to 100 μL of fermentation filtrate and place them in a 96-well plate. Use ddH2O as a control. After incubation at 28℃ for 72 h, count the mortality rate of the second-instar nematode larvae. Each treatment has 4 replicates.

[0044] like Figure 2 As shown, the fermentation filtrate of strain DT-8 exhibits nematicidal activity against root-knot nematodes of the Poaceae family, with a nematicidal activity of 82.79±9.06% after 72 h. This indicates that the fermentation filtrate of strain DT-8 has good application potential in nematicidal activity and can be used as a potential biological control method for controlling root-knot nematodes of the Poaceae family.

[0045] Example 4: Control effect of Sclerotinia sclerotiorum DT-8 mycelial suspension bio-initiation and mycelial inoculation on rice root-knot nematode disease (Poaceae). 1. Sclerotinia sclerotiorum DT-8 mycelial suspension First, activate the *Sclerotinia sclerotiorum* DT-8 strain on PDA plates at 20℃ for 2-3 days. Then, scrape the mycelium into 150 mL of SPDB medium and incubate it in a shaker at 20℃ and 120 rpm for 3-4 days. Weigh 10 g of mycelium and add it to sterile water to make up to 150 mL. This yields the *Sclerotinia sclerotiorum* DT-8 mycelial suspension, in which the *Sclerotinia sclerotiorum* DT-8 mycelial suspension contains 0.07 g / mL of *Sclerotinia sclerotiorum* DT-8.

[0046] 2. Sclerotinia sclerotiorum DT-8 mycelium First, activate the DT-8 strain of Sclerotinia sclerotiorum on a PDA plate at 20°C for 2-3 days, then cut out mycelial blocks, which are the mycelia of DT-8 Sclerotinia sclerotiorum.

[0047] 3. Seed coating of Sclerotinia sclerotiorum DT-8 mycelial suspension and inoculation of Sclerotinia sclerotiorum DT-8 mycelials a. Treat the seeds with the above mycelial suspension at a ratio of 50 mL / 500 g seeds for 12 h. After 12 h, dry the seeds and proceed with germination and sowing. b. The above mycelial blocks were inoculated into the roots of rice for 24 hours.

[0048] Rice plants were inoculated with 150 nematodes / mL when they were one to two weeks old, with eight replicates per treatment. Seven days later, the control effect of strain DT-8 on rice root nematode disease was evaluated by measuring the fresh weight of rice roots and the number of nematodes.

[0049] like Figure 3 The results showed that seed coating with mycelial suspension, through both biological initiation and mycelial inoculation, effectively inhibited rice root-knot nematode disease caused by grass-like root-knot nematodes, and significantly reduced the number of nematodes in rice roots.

[0050] Example 5: Effect of Sclerotinia sclerotiorum DT-8 fermentation broth on root-knot nematode disease in rice (Poaceae family). 1. Preparation of Sclerotinia sclerotiorum DT-8 fermentation broth Sclerotinia sclerotiorum DT-8 was activated for 2-3 days. Strain DT-8 was then sliced ​​into 150 mL of SPDB medium and cultured in a shaker at 20℃ and 120 r / min for 3-4 days to obtain the fermentation broth of Sclerotinia sclerotiorum DT-8. The fermentation broth contained 0.14 g / mL of Sclerotinia sclerotiorum DT-8.

[0051] 2. Root irrigation treatment with Sclerotinia sclerotiorum DT-8 fermentation broth Rice plants were irrigated with 7.5 mL of fermentation broth per plant during their one to two-week growth period. Two to three days later, the plants were inoculated with 150 nematodes per mL. Each treatment was replicated nine times. Seven days later, the fresh weight of rice roots and the number of nematodes were measured, with nine replicates per treatment, to evaluate the control effect of strain DT-8 on rice root nematode disease.

[0052] 3. Conclusion like Figure 4 As shown, after root irrigation with DT-8 fermentation broth, the fresh weight of rice roots was significantly higher than that of healthy plants and plants inoculated only with nematodes, indicating that this treatment has a certain promoting effect on rice root growth. Compared with plants inoculated only with nematodes, the number of nematodes in the roots decreased significantly after root irrigation with DT-8 fermentation broth, indicating that this treatment can effectively inhibit root-knot nematodes from invading rice roots. Overall, root irrigation with DT-8 fermentation broth has certain control potential against rice root-knot nematode disease (Poaceae) and also promotes root growth.

[0053] Example 6: Control effect of Sclerotinia sclerotiorum DT-8 mycelial suspension on root-knot nematode disease in rapeseed. 1. Preparation of Sclerotinia sclerotiorum DT-8 mycelial suspension Sclerotinia sclerotiorum DT-8 was activated for 2-3 days. Strain DT-8 was then sliced ​​into 100 mL of PDB medium and cultured in a shaker at 20℃ and 120 r / min for 4-5 days. 10 g of mycelium was weighed and added to sterile water to make up to 50 mL. The mycelium was then broken up for 5-10 s to obtain a suspension of Sclerotinia sclerotiorum DT-8 mycelium. The concentration of Sclerotinia sclerotiorum DT-8 in the suspension was 0.20 g / mL.

[0054] 2. Seed coating of Sclerotinia sclerotiorum DT-8 mycelial suspension Seeds were coated at a ratio of 2 g seeds / 1 mL mycelial suspension, dried after 18 h, and then germinated with sterile water. Once the rapeseed radicle emerged, the seedlings were transplanted. Two to three days later, the seedlings were inoculated with 500 nematodes / mL, with 10 replicates per treatment. After 20 days, the above-ground fresh weight of the rapeseed, the number of root knots, and the number of nematodes were measured to evaluate the control effect of strain DT-8 on root nematode disease in rapeseed.

[0055] 3. Conclusion like Figure 5 DT-8 effectively inhibited the infection of rapeseed by the root-knot nematode of the Poaceae family, while promoting plant growth. Increased aboveground fresh weight indicates that this treatment is beneficial for biomass accumulation in rapeseed under nematode stress; the significant decrease in root knot number and nematode population confirms the inhibitory effect of this strain on the root-knot nematode. Therefore, DT-8 bacterial suspension can effectively control nematode diseases and promote rapeseed growth, possessing significant agricultural application value.

[0056] Example 7: Effect of Sclerotinia sclerotiorum DT-8 fermentation broth on the control of southern root-knot nematode disease in tomatoes. 1. Preparation of Sclerotinia sclerotiorum DT-8 fermentation broth Sclerotinia sclerotiorum DT-8 was activated for 2-3 days. Strain DT-8 was then sliced ​​into 150 mL of SPDB medium and cultured in a shaker at 20℃ and 120 r / min for 3-4 days. 15 g and 30 g of mycelium were weighed and added to the fermentation filtrate to make up to 100 mL, which yielded the Sclerotinia sclerotiorum DT-8 fermentation broth. The Sclerotinia sclerotiorum DT-8 fermentation broth contained 0.15 g / mL and 0.30 g / mL of Sclerotinia sclerotiorum DT-8.

[0057] 2. Root irrigation treatment with Sclerotinia sclerotiorum DT-8 fermentation broth Five to seven weeks after tomato growth, the plants were inoculated with 1500 eggs / mL of southern root-knot nematodes. Two to three days later, the roots were drenched with 15 mL of fermentation liquid per plant, with a DT-8 content of 0.15 g / mL. Seven days later, the roots were drenched with 30 mL of fermentation liquid per plant, with a DT-8 content of 0.30 g / mL. Each treatment was replicated eight times. Forty-five days after nematode inoculation, the fresh weight of tomato roots, number of root knots, root knot index, number of egg masses, and number of eggs were measured to evaluate the control effect of strain DT-8 on tomato root nematode disease.

[0058] 3. Sclerotinia sclerotiorum DT-8 colonizes the roots of tomatoes. The activated DT-8 mycelial blocks were inoculated into the roots of tomatoes. Five days later, the roots were stained with WGA and PI, and the colonization of DT-8 mycelia was observed using a confocal fluorescence microscope.

[0059] 4. Conclusion like Figure 6 and Figure 7 As shown, root irrigation with the fermentation broth of strain DT-8 effectively alleviated the stress of southern root-knot nematodes on tomatoes, reducing the number of root knots, root knot index, number of egg masses, and number of eggs. This indicates that the DT-8 fermentation broth has good biocontrol effects, providing a new strategy and candidate resource for the green control of tomato nematode diseases. Furthermore, green fluorescence emitted by DT-8 mycelia was observed on both the surface and inside the tomato roots, indicating that DT-8 can not only attach to the surface of tomato roots but also penetrate into the root tissue to exert its biocontrol effect.

[0060] In summary, the *Sclerotinia sclerotiorum* DT-8 can induce systemic resistance in plants by bio-initiation after seed coating with mycelium or by endogenous growth after mycelium colonization in plants, thereby controlling root-knot nematode diseases. Furthermore, the metabolites in the fermentation filtrate of strain DT-8 are toxic to nematodes and can also be used to control root-knot nematode diseases. Strain DT-8 exhibits uniform control effects against root-knot nematode diseases in monocotyledonous rice and dicotyledonous rapeseed and tomato, demonstrating broad-spectrum control against both diseases caused by grass-like root-knot nematodes and those caused by southern root-knot nematodes.

[0061] All other parts not described in detail are existing technologies. 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 based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A non-pathogenic Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum The application of DT-8 in the control of root-knot nematode disease in crops is characterized by: The non-pathogenic Sclerotium sclerotiorum DT-8 has the accession number: CCTCC NO: M2019328. Root-knot nematode disease is caused by root-knot nematode infection, and the root-knot nematode is either the grass-like root-knot nematode or the southern root-knot nematode. The crops infested by the root-knot nematode of the Poaceae family are rice or rapeseed, while the crops infested by the southern root-knot nematode are tomatoes.

2. A non-pathogenic Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum The application of DT-8 in the preparation of pesticides for controlling crop root-knot nematode disease is characterized by: Root-knot nematode disease is caused by infection with root-knot nematodes, which are either root-knot nematodes of the Poaceae family or southern root-knot nematodes. The crops infected by the root-knot nematode of the Poaceae family are rice or rapeseed, while the crops infected by the southern root-knot nematode are tomatoes; the preservation number of the non-pathogenic Sclerotinia sclerotiorum DT-8 is: CCTCC NO: M2019328.

3. A method for controlling rice root-knot nematode disease, characterized in that: Rice root-knot nematode disease is caused by infection with root-knot nematodes belonging to the Poaceae family. The method includes any of the following application methods: (1) Coating rice seeds with a suspension of Sclerotinia sclerotiorum DT-8 mycelium; (2) Apply the fermentation broth of Sclerotinia sclerotiorum DT-8, the fermentation filtrate of Sclerotinia sclerotiorum DT-8, or the mycelial suspension of Sclerotinia sclerotiorum DT-8 to the roots of rice. (3) Inoculate the roots of rice with Sclerotium sclerotiorum DT-8 mycelium; The preservation number of Sclerotium sclerotiorum DT-8 is: CCTCC NO: M2019328.

4. The method according to claim 3, characterized in that: The method for preparing the *Sclerotinia sclerotiorum* DT-8 mycelial suspension is as follows: Sclerotinia sclerotiorum DT-8 was activated by placing strain DT-8 in a culture medium and culturing it on a shaker. The mycelia were weighed and added to sterile water to make up the volume, thus obtaining a suspension. The content of Sclerotinia sclerotiorum DT-8 in the suspension was 0.07~0.28 g / mL. The preparation method of the Sclerotinia sclerotiorum DT-8 fermentation broth is as follows: Sclerotinia sclerotiorum DT-8 was activated by placing strain DT-8 in a culture medium and culturing it on a shaker to obtain the fermentation broth of Sclerotinia sclerotiorum DT-8; wherein the content of Sclerotinia sclerotiorum DT-8 in the fermentation broth was 0.14~0.30 g / mL; The preparation method of the Sclerotinia sclerotiorum DT-8 fermentation filtrate is as follows: To activate Sclerotinia sclerotiorum DT-8, strain DT-8 was sliced ​​into SPDB medium, placed on a shaker, centrifuged to remove mycelia, and the supernatant was taken as the fermentation filtrate of Sclerotinia sclerotiorum DT-8.

5. A method for controlling root-knot nematode disease in rapeseed, characterized in that: Rapeseed root-knot nematode disease is caused by infection with root-knot nematodes of the Poaceae family. The method involves coating rapeseed seeds with a suspension of *Sclerotinia sclerotiorum* DT-8 mycelium, wherein the root-knot nematode is a root-knot nematode of the Poaceae family. Meloidogyne graminicola The preservation number of Sclerotium DT-8 is: CCTCC NO: M2019328.

6. The method according to claim 5, characterized in that: The method for preparing the *Sclerotinia sclerotiorum* DT-8 mycelial suspension is as follows: Sclerotinia sclerotiorum DT-8 was activated by placing strain DT-8 in a culture medium and culturing it on a shaker. The mycelium was weighed and added to sterile water to make up the volume, thus obtaining a suspension. The content of Sclerotinia sclerotiorum DT-8 in the suspension was 0.07~0.28 g / mL.

7. A method for controlling tomato root-knot nematode disease, characterized in that, Tomato root-knot nematode disease is caused by infection with the southern root-knot nematode. The method involves drenching the roots of tomatoes with a fermentation broth of Sclerotinia sclerotiorum DT-8. The root-knot nematode is the southern root-knot nematode. Meloidogyne incognita The content of Sclerotinia sclerotiorum DT-8 in the fermentation broth is 0.14~0.30 g / mL; the amount of Sclerotinia sclerotiorum DT-8 used for root irrigation is 15~30 mL per tomato plant; the preservation number of Sclerotinia sclerotiorum DT-8 is: CCTCC NO: M2019328.

8. The method according to claim 7, characterized in that: The preparation method of the Sclerotinia sclerotiorum DT-8 fermentation broth is as follows: Sclerotinia sclerotiorum DT-8 was activated by placing strain DT-8 in a culture medium and culturing it on a shaker to obtain the fermentation broth of Sclerotinia sclerotiorum DT-8.

Images