Vp-1 and application of fermentation broth thereof

Abstract

The present application belongs to the field of microbial technology, and particularly relates to a strain of controvirus VP-1 and a fermentation liquor and application thereof. The present application provides a strain of controvirus VP-1, which has been biologically preserved with a preservation number of CCTCC NO: M20221633. The controvirus VP-1 of the present application can be used for preventing and treating plant diseases and promoting plant growth. The results of examples show that the controvirus VP-1 obtained by the present application can promote the growth of rape roots and has a good biocontrol effect on sclerotinia stem rot of rape, thereby providing a new and beneficial biocontrol resource for biological control of crop fungal diseases.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a controversial strain of glutathione VP-1, its fermentation broth, and its applications. Background Technology

[0002] Plant diseases are one of the natural disasters that seriously endanger agricultural production. Plant diseases can not only reduce crop yields, but also seriously threaten the quality and safety of agricultural products and their international trade to a certain extent. Rapeseed, as one of the world's four major oil crops, has multiple utilization values ​​such as oil, rapeseed, flowers, fertilizer, honey, and feed. However, in recent years, the global supply and demand gap for rapeseed has been large and the situation is not optimistic. In 2020, the global rapeseed initial inventory was 7.565 million tons, a decrease of 22.9% year-on-year; the rapeseed stock-to-consumption ratio was 6.15%, a decrease of 2.52 percentage points from 2019, hitting a record low since 2016 (He Wei, Li Jun, Wang Xiaomei, Lin Qiao, Yang Xiaowei. The current status of the global rapeseed industry and the problems and countermeasures of my country's rapeseed industry [J]. China Oils and Fats, 2022, 47(02):1-7.DOI:10.19902 / j.cnki.zgyz.1003-7969.210430.). Therefore, my country still needs to work hard to improve its self-sufficiency level in rapeseed.

[0003] Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, poses the greatest threat to rapeseed production. It is prevalent and widespread worldwide, especially in temperate regions. The pathogenic fungus Sclerotinia sclerotiorum can infect more than 700 plant species from over 75 families, including important field crops, fruit trees, flowers, trees, shrubs, and many weeds (National Fungus Collections, ARS, USDA).

[0004] Currently, the control of rapeseed sclerotinia stem rot largely relies on chemical fungicides. However, the emergence of drug-resistant strains and the increasing global demand for environmentally friendly and sustainable production have driven the development of environmentally friendly and safe biological control. The use of beneficial microorganisms for biological control is receiving increasing attention. Previous research on the biocontrol mechanisms of beneficial microorganisms mainly focused on competition between microorganisms and the antagonistic effects of antibiotics, neglecting many microbial resources that promote crop growth and enhance crop resistance to diseases. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of the controversial phlegmon VP-1, which can effectively prevent and control plant diseases and promote plant growth.

[0006] This invention provides a strain of the glutathione VP-1, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO: M 20221633.

[0007] The present invention also provides a fermentation broth of *Vallisneria natans*, which is obtained by fermentation of *Vallisneria natans* VP-1 as described in the above technical solution.

[0008] Preferably, the OD of the fermentation broth of the gluttonous bacteria is... 600 It equals 2.

[0009] This invention also provides a method for preparing the fermentation broth of the controversial gluttonous bacteria described in the above technical solution, comprising the following steps:

[0010] The controversial gluttonous bacterium VP-1 described in the above technical solution was placed in a seed culture medium to obtain a seed culture solution;

[0011] The seed culture was inoculated into a fermentation medium for fermentation culture to obtain the fermentation broth of the controversial gluttonous bacteria.

[0012] Preferably, both the seed culture medium and the fermentation culture medium include LB medium.

[0013] The present invention also provides the application of the above-described glutathione VP-1 or the fermentation broth of the glutathione in the prevention and control of plant diseases and / or the promotion of plant growth.

[0014] Preferably, the plant disease includes sclerotinia rot; and the plant includes rapeseed.

[0015] The present invention also provides a method for preventing and controlling Sclerotinia sclerotiorum, characterized in that it includes:

[0016] The fermentation broth of the controversial gluttonous bacteria described in the above technical solution is mixed with Tween 80 and sprayed on rapeseed leaves.

[0017] The present invention also provides a method for promoting rapeseed germination, comprising: soaking rapeseed seeds in a preparation containing the fermentation broth of the *Gnaphalium affine* described in the above technical solution.

[0018] The present invention also provides a method for promoting rapeseed growth, characterized in that it includes: treating rapeseed seedlings with root irrigation using the fermentation broth of the glutathione bacteria described in the above technical solution.

[0019] This invention provides a strain of *Sclerotinia sclerotiorum* VP-1, which has been biopreserved at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20221633. This invention isolated *Sclerotinia sclerotiorum* VP-1 from the soil surrounding the sclerotia of the low-virulence strain SH051. This strain and its liquid fermentation product can promote root growth in rapeseed seedlings. Spraying with *Sclerotinia sclerotiorum* VP-1 can inhibit the occurrence of sclerotinia rot in rapeseed. Field trials show that *Sclerotinia sclerotiorum* VP-1 has good biocontrol effects against sclerotinia rot in rapeseed. The discovery of this strain provides a new and beneficial biocontrol resource for the biological control of fungal diseases in crops.

[0020] Biological Preservation Information

[0021] The controversial glutathione (Variovorax paradoxus) VP-1 was deposited on October 21, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, with accession number CCTCC NO: M20221633. Attached Figure Description

[0022] 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.

[0023] Figure 1 The colony morphology of strain VP-1 on LB medium;

[0024] Figure 2 The morphology of strain VP-1 under transmission electron microscopy;

[0025] Figure 3 The results of agarose gel electrophoresis of the PCR amplification products of strain VP-1;

[0026] Figure 4 To debate the phylogenetic tree of the gluttonous bacterium VP-1;

[0027] Figure 5 To investigate the inhibitory and preventive effects of the glutathione VP-1 on sclerotinia stem rot in potted rapeseed;

[0028] Figure 6 The effects of different concentrations (cfu / ml) of *Vitis vinifera* VP-1 on the promotion of rapeseed root growth were debated.

[0029] Figure 7 To investigate the effect of root irrigation with *Variococcus viridissima* VP-1 bacterial solution on the growth promotion of rapeseed seedlings. Detailed Implementation

[0030] This invention provides a strain of the glutathione VP-1, which has been biodeposited with accession number CCTCC NO: M 20221633.

[0031]

[0032] Based on the characteristics of the valvularia VP-1 described in this invention, the application of valvularia VP-1 in the prevention and control of plant diseases and / or the promotion of plant growth also falls within the scope of protection of this invention. The plant diseases described in this invention preferably include sclerotinia rot; the plants preferably include rapeseed.

[0033] The present invention also provides a fermentation broth of *Vamectinia valerate* VP-1, which is obtained by fermentation of *Vamectinia valerate* VP-1 as described in the above technical solution.

[0034] In this invention, the OD of the fermentation broth of the gluttonous bacteria in question 600 The preferred value is 2.

[0035] In this invention, the method for preparing the fermentation broth of the glutathione preferably includes the following steps:

[0036] The controversial gluttonous bacterium VP-1 described in the above technical solution was placed in a seed culture medium to obtain a seed culture solution;

[0037] The seed culture was inoculated into a fermentation medium for fermentation culture to obtain the fermentation broth of the controversial gluttonous bacteria.

[0038] This invention involves seed culture of *Vallis fulva* VP-1 in a seed culture medium to obtain a seed culture solution. Preferably, the seed culture medium includes LB medium; the seed culture time is preferably 24 hours; and the seed culture temperature is preferably 28°C. The seed culture method is preferably shaker culture, with a shaker rotation speed preferably of 180 rpm. This seed culture method enables the activation of *Vallis fulva* VP-1.

[0039] After obtaining the seed culture solution, the present invention inoculates the seed culture solution into a fermentation medium for fermentation culture to obtain the fermentation broth of the *Gnaphalium affine*. In the present invention, the fermentation medium is preferably LB medium; the fermentation temperature is preferably 28°C. The fermentation culture method is preferably shaker culture, and the shaker rotation speed is preferably 180 rpm. In an embodiment of the present invention, it is preferred that 100 μL of the seed culture solution is inoculated into 100 mL of the fermentation medium. The OD of the fermentation medium of the present invention... 600 Preferably, it is 2. This invention does not have strict requirements on the fermentation time, allowing the final obtained *Gnaphalium affine* fermentation broth to have an OD value of... 600 The answer is 2.

[0040] This invention also provides the application of the fermentation broth of the aforementioned *Sclerotinia sclerotiorum* in the prevention and control of plant diseases and / or the promotion of plant growth. In this invention, the plant disease preferably includes sclerotinia rot, more preferably sclerotinia rot caused by *Sclerotinia sclerotiorum*; the plant preferably includes rapeseed. The fermentation broth of the aforementioned *Sclerotinia sclerotiorum* has a particularly good preventive and control effect on rapeseed sclerotinia rot caused by *Sclerotinia sclerotiorum*.

[0041] The present invention also provides a method for preventing and controlling Sclerotinia sclerotiorum, characterized in that it includes:

[0042] The fermentation broth of the controversial gluttonous bacteria described in the above technical solution is mixed with Tween 80 and sprayed on rapeseed leaves.

[0043] This invention mixes the *Bufo valerate* fermentation broth described in the above technical solution with Tween 80 to obtain a reagent. Preferably, this invention mixes the *Bufo valerate* fermentation broth with a 0.5% (w / w) Tween 80 solution to achieve a Tween 80 (w / w) concentration of 0.05% in the *Bufo valerate* fermentation broth, thereby obtaining the reagent. This invention, by mixing the *Bufo valerate* fermentation broth with Tween 80, can improve the adsorption effect of the *Bufo valerate* fermentation broth.

[0044] This invention involves spraying the pesticide onto rapeseed leaves. Preferably, the rapeseed plant height is 150 cm. Preferably, the pesticide is sprayed onto each rapeseed leaf. There are no strict requirements on the amount of pesticide applied; it is sufficient that the pesticide adheres to the surface of each rapeseed leaf in the form of water droplets. The method described in this invention can effectively control sclerotinia stem rot in rapeseed.

[0045] The present invention also provides a method for promoting rapeseed germination, comprising: soaking rapeseed seeds in a preparation containing the fermentation broth of the *Gnaphalium affine* described in the above technical solution.

[0046] In this invention, the effective viable count of *Variococcus valerate* VP-1 in the formulation is preferably 10. 5 ~10 9 cfu / mL, more preferably 10 6 ~10 8 cfu / mL. The soaking time described in this invention is preferably 12 hours. In specific implementation of this invention, rapeseed seeds are preferably submerged in a preparation containing the fermentation broth of the aforementioned *Gnaphalium affine*.

[0047] This invention also provides a method for promoting rapeseed growth, comprising: irrigating rapeseed seedlings with the fermentation broth of *Gnaphalium affine* as described in the above technical solution. In the root irrigation treatment of this invention, the preferred dosage of the *Gnaphalium affine* fermentation broth is 5 mL / seedling. This invention utilizes the *Gnaphalium affine* fermentation broth for root irrigation to promote root length growth in rapeseed seedlings and increase the biological yield (dry weight) of rapeseed.

[0048] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a controversial glutathione strain VP-1, its fermentation broth, and its applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0049] Unless otherwise specified, the culture media and analytical methods used in the specific implementation of this invention are all conventional methods. The *Sclerotinia sclerotiorum* strain used in the examples is EP-1PNA367, hereinafter referred to as *Sclerotinia sclerotiorum* A367 (Yu Xiao. Research on the characteristics and application potential of *Sclerotinia sclerotiorum* attenuated viral DNA virus 1 [D]. Huazhong Agricultural University, 2013).

[0050] Example 1

[0051] 1.1 Isolation and Identification of Strains

[0052] (1) A soil sample was collected from the sclerotium surrounding the sclerotium of the low-toxicity Sclerotium sclerotiorum strain SH051, and a strain was isolated and named VP-1.

[0053] (2) Morphological identification

[0054] Strain VP-1 was inoculated onto LB medium and cultured. Colony morphology was observed and analyzed using transmission electron microscopy. The results are as follows: Figures 1-2 As shown.

[0055] according to Figures 1-2 It can be seen that the colonies of strain VP-1 are round, light yellow, smooth, and moist; the bacterial cells are rod-shaped and have flagella, so it is preliminarily identified as bacteria.

[0056] (3) Molecular biological identification

[0057] A single colony of strain VP-1 was inoculated into 2 mL of liquid LB medium and incubated at 28 °C and 180 rpm for 24 h. Using this bacterial culture as template DNA, PCR amplification was performed on the bacterial culture of the unknown bacterium VP-1 using universal bacterial primers 27-f (SEQ ID NO. 2: 5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492-r (SEQ ID NO. 3: 5′-GGTTACCTTGTTACGACTT-3′). The reaction volume was 50 μL. PCR Master Mix (YEASEN) 25 μL, 27-f 1 μL, 1492-r 1 μL, bacterial culture (template) 1 μL, ddH2O 22 μL; Amplification conditions: 94℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 90 s, repeated 35 times; 72℃ extension for 5 min; 16℃ incubation for 10 min;

[0058] 5 μL of PCR amplification product was mixed with loading buffer and spotted into the wells of a 1% agarose gel. 2 μL of DL2000 marker was used as a reference. The 1% agarose gel was then placed in a 120V electrophoresis apparatus for electrophoresis. After electrophoresis, the 1% agarose gel was stained with EB and then observed under a gel imaging system (Gel Doc™ Imager, Bio-RAD) to detect the target bands. Figure 3 ).

[0059] The target band was recovered according to the instructions of the Axygen nucleic acid gel recovery kit. The specific steps were as follows: Cut the target band gel and place it in a 2mL centrifuge tube. Weigh the gel and calculate the volume (100μL = 0.1g). Add three times the volume of DE-A to the tube and heat in a 75°C metal bath to dissolve. Then add half the volume of DE-A of DE-B. Add the mixture to a DNA preparation tube and centrifuge at 12000 rpm for 1 min, discarding the filtrate. Add 500μL of Buffer W1 and centrifuge at 12000 rpm for 1 min, discarding the filtrate. Add 700μL of Buffer W2 and centrifuge at 12000 rpm for 30 s, discarding the filtrate. Repeat the above steps once. Place the empty tube in a centrifuge and centrifuge at 12000 rpm for 1 min. Replace the empty tube with a 1.5mL EP tube and add 30μL of preheated 75°C deionized water to dissolve. Let stand for 1 minute, then centrifuge at 12000 r / min for 1 minute, collect the centrifuged liquid, and obtain the PCR recovered product.

[0060] The purified target fragment was ligated into T-Vector (pMD18-T, Takara) and transformed into E. coli competent cells Top10. The method was as follows: 5 μL of 2×Solution I, 0.5 μL of PMD18-T rector, and 4.5 μL of PCR recovered product were mixed in a centrifuge tube and incubated at 4-16℃ for 12-16 h. Then, 10 μL of the ligation product was mixed with 50 μL of E. coli competent cells Top10, and the mixture was incubated on ice for 30 min, followed by a 1 min incubation at 42℃, and then immediately incubated on ice for 2 min. The mixture was added to 500 μL of 37℃ SOC medium, and the EP tubes were placed in a shaker at 37℃ and 180 rpm for 1 h. 100 μL of the culture medium was spread onto 10 μL / 100 mL LB (Amp) medium. + After drying in a clean bench on the culture medium, place it in a 37℃ incubator for 10-14 hours.

[0061] Pick LB (Amp) + A single colony of Escherichia coli in the culture medium was incubated in 500 μL of LB (Amp) solution. + In liquid culture medium, the bacteria were incubated at 37°C and 200 rpm for 8 hours. After successful transformation confirmed by PCR, the bacterial culture was sent to Quintiles for sequencing. The sequencing results (as shown in SEQ ID NO.1) were compared using BLAST in NCBI. Nucleic acid sequences of strains with high similarity were selected, and phylogenetic analysis was performed using the NJ method with MEGA11. The phylogenetic tree was constructed by obtaining the propagation value (%) of each branch with 1000 replicates. The branch length corresponds to the genetic distance, and the scale bar in the lower left corner corresponds to the genetic distance. Figure 4 Strain VP-1 converged with *Variovorax paradoxus* (accession number: MF101056.1) in one strain, showing 100% similarity. Therefore, strain VP-1 was identified as belonging to *Variovorax paradoxus*, named *Variovorax paradoxus* VP-1, and deposited on October 21, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, with accession number CCTCCNO: M 20221633.

[0062] Example 2

[0063] Pick 2 mL of the controversial glutathione VP-1 culture medium and incubate it in a shaker at 28°C and 180 rpm for 24 h. Then, take 100 μL of the bacterial culture and inoculate it into 100 mL of liquid LB medium. Incubate the fermentation broth until the OD reaches 100°C. 600 The concentration of 2 was used to obtain the fermentation broth of the controversial gluttonous bacteria.

[0064] Example 3

[0065] Pot experiment

[0066] (1) First, activate the Sclerotinia sclerotiorum A367 (EP-1PNA367) strain on a PDA plate lined with cellophane at 20°C for 3 days. Then, scrape the mycelium off the cellophane and inoculate it into an Erlenmeyer flask containing 100 mL of PDB. Incubate at 20°C and 150 r / min for 3 days with shaking. Then, homogenize the mixture to obtain a suspension of Sclerotinia sclerotiorum A367 mycelium.

[0067] (2) Add a 0.5% Tween 80 solution to the fermentation broth of the glutathione obtained in Example 2, so that the final concentration of Tween 80 in the fermentation broth of the glutathione is 0.05%, and obtain the agent.

[0068] (3) The potted rapeseed was divided into control group 1, control group 2 and VP-1 treatment group, with 5 pots in each group and 55 rapeseed plants in each pot. The growth of the live rapeseed plants in control group 1, control group 2 and VP-1 treatment group was similar. The rapeseed leaves of the VP-1 fermentation liquid treatment group were sprayed with the agent in step (2), and the rapeseed leaves of control group 1 and control group 2 were sprayed with the same amount of water.

[0069] (4) After the rapeseed leaves in each treatment group in step (3) have dried, spray the A367 mycelial suspension prepared in step (1) onto the leaves of live rapeseed plants in control group 1 and VP-1 treatment group. Control group 2 is sprayed with an equal amount of water. Each group is repeated 4 times, that is, a total of 5 parallel experiments are carried out. The plants are kept moist at 20℃. After 5 days, the disease situation is observed and recorded, and the morphology of the lesions is recorded. The disease index is calculated (disease index = ∑(number of diseased leaves at each level × representative value of each level) / (total number of leaves investigated × highest level representative value) × %). The disease level is divided into 5 levels according to the diameter of the lesions: level 0, no disease; level 1, leaves are intact, but only small, black dot-like lesions exist; level 2, less than 1 / 3 of the leaves are diseased and obvious lesions appear; level 3, 1 / 3-2 / 3 of the leaves are diseased; level 4, more than 2 / 3 of the leaves are diseased and obvious lesions appear, but the plant has not died; level 5, the whole plant is withered and necrotic. The results are shown in Table 1 and Figure 5 .

[0070] Table 1. Debate on the potted control efficacy of *Gnaphalium affine* against sclerotinia stem rot in rapeseed.

[0071]

[0072] Note: The data in the table are the average values ​​of 5 parallel experiments.

[0073] According to Table 1 and Figure 5It can be seen that the incidence of sclerotinia stem rot in rapeseed was significantly reduced by spraying VP-1 fermentation liquid. Through the investigation of lesions on diseased leaves and the calculation of disease index, it was found that the disease index of control group 1 (A367 treatment group) was 79.27%, while the disease index of VP-1 treatment group was 33.68%. Therefore, the relative control effect of VP-1 fermentation liquid on sclerotinia stem rot in rapeseed in the field was 58.46%.

[0074] Example 4

[0075] Daejeon Experiment

[0076] (1) Using the rapeseed variety Huayouza 62 as the test variety and the highly pathogenic strain A367 as the test pathogen, the rapeseed experimental field of Huazhong Agricultural University was used as the test site. The subjects were randomly divided into a negative control group, a positive control group, and a fermentation broth group, and were treated as follows:

[0077] Negative control group: Water was sprayed onto rapeseed plants about 150cm tall in the field, with each leaf sprayed 3 times until the water formed droplets and adhered to the leaf surface; rapeseed was treated in plots of 3×8m, with 4 replicates for each treatment;

[0078] Positive control group: Ioniocin (Jiangxi Heyi Chemical Co., Ltd.) was sprayed on rapeseed plants with a height of about 150cm in the field. Each leaf was sprayed 3 times until the water was applied to the leaf surface in the form of water droplets. Rapeseed was treated in plots of 3×8m, and each treatment was replicated 4 times.

[0079] Fermentation liquid group: The agent obtained in step (2) of Example 3 was sprayed on rapeseed plants with a height of about 150cm in the field. Each leaf was sprayed 3 times until the water was attached to the leaf surface in the form of water droplets. Rapeseed was treated in plots of 3×8m, and each treatment was repeated 4 times.

[0080] (2) After 30 days of treatment, the negative control group, positive control group and fermentation liquid group were treated and the disease incidence was statistically analyzed. The disease index was calculated (disease index = ∑(number of diseased leaves at each level * representative value of each level) / (total number of leaves investigated * highest representative value of the highest level) * %). The disease incidence was divided into 5 levels according to the diameter of the lesions: Level 0, no disease; Level 1, less than 1 / 3 of the branches are diseased but the main stem is not diseased; Level 2, 1 / 3-2 / 3 of the branches are diseased or less than 1 / 3 of the branches are diseased but the stem lesions are less than 3cm; Level 3, 2 / 3 of the branches are diseased or less than 2 / 3 of the branches are diseased but the lesions on the middle and lower parts of the main stem are more than 3cm; Level 4, more than 2 / 3 of the branches are diseased or less than 2 / 3 of the branches are diseased but the lesions on the middle and lower parts of the main stem are more than 10cm. The results are shown in Table 2.

[0081] Table 2. Field control efficacy of different treatment groups against sclerotinia stem rot in rapeseed.

[0082]

[0083] As shown in Table 2, the disease index of the water control group was 44.30%, while that of the fermentation broth treatment group was 17.35%. Therefore, the relative control effect of the fermentation broth of strain VP-1 on sclerotinia rot in rapeseed in the field was 37.92%.

[0084] Example 5

[0085] The impact of the glutathione VP-1 on rapeseed growth is under debate.

[0086] (1) Take the agent obtained in step (2) of Example 3 and dilute it with sterilized ddH2O to obtain an effective viable count of 10 *Pseudomonas virosa* VP-1. 5 cfu / mL (fermentation agent 1), 10 6 cfu / mL (fermentation agent 2), 10 7 cfu / mL (fermentation agent 3), 10 8 CFU / mL (fermentation agent 4) and 10 9 Five concentration gradients of CFU / mL (fermentation agent 5) are prepared for future use.

[0087] (2) Treat rapeseed with 75% alcohol for 30 seconds, wash with sterile water three times for 30 seconds each time, place on a 90 mm plate, add the same volume (5 mL) of fermentation agents 1-5 from step (1) and water to the plate until the seeds are submerged, soak for 12 hours, then evenly spread the rapeseed seeds on a new 90 mm plate lined with absorbent paper, and then cultivate the rapeseed in a 20℃ light incubator for 9 days. Root length, stem length, and dry weight are recorded. The results are shown in Table 3 and... Figure 6 .

[0088] Table 3. Effects of different concentrations of the controversial bacteria VP-1 on rapeseed growth.

[0089]

[0090] According to Table 3 and Figure 6 It can be seen that 10 6 ~10 8 The fermentation broth of *Vitis vinifera* VP-1, used to treat rapeseed seedlings, showed that after 9 days, the average root length was significantly higher than the control, with average root lengths of 64.11 cm, 68.23 cm, and 59.47 cm, and dry weights of 0.22 g / seedling, 0.22 g / seedling, and 0.18 g / seedling, respectively. In contrast, the control group (water soaking) had an average root length of only 40.26 cm and a dry weight of only 0.14 g / seedling. This invention demonstrates that the fermentation broth of *Vitis vinifera* VP-1 can promote root length growth and increase biological yield (dry weight) in rapeseed seedlings.

[0091] Example 6

[0092] Pot experiment

[0093] Potted rapeseed seeds were divided into a control group and a VP-1 treatment group, with 5 pots in each group and 1 rapeseed plant in each pot. The growth of the rapeseed plants in the control group and the VP-1 treatment group was similar. The VP-1 treatment group was watered with 5 mL of the agent in step (2) of Example 2 at the base of the rapeseed seedlings, while the control group was watered with the same amount of water at the base of the rapeseed seedlings. The growth of the rapeseed seedlings in the control group and the VP-1 treatment group was observed after 35 days of watering. The root length, stem length and dry weight were recorded. The results are shown in Table 4 and 5. Figure 7 As shown.

[0094] Table 4. Rapeseed growth in different treatment groups

[0095]

[0096] According to Table 4 and Figure 7 It can be seen that after 35 days of root irrigation treatment, the average root length of potted rapeseed was 21.78 cm, and the dry weight reached 1.19 g / plant; while the average root length of the control group was only 15 cm, and the dry weight was only 0.96 g / plant. This invention argues that root irrigation treatment with the fermentation liquid of *Vitis vinifera* VP-1 can promote the growth of rapeseed seedling roots. If applied in the field, it should be applied in the later stage of plant germination or seed soaking treatment.

[0097] As can be seen from the above, this invention argues that the virucidal bacterium VP-1 can prevent and control sclerotinia stem rot in rapeseed and promote rapeseed growth.

[0098] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and 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 controversial strain of gluttonous bacteria ( Variovorax paradoxus VP-1, characterized in that, The controversial gluttonous bacterium VP-1 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: 20221633.

2. A fermentation broth containing a controversial gluttonous bacterium, characterized in that, The controversial gluttonous bacteria fermentation broth is obtained by fermentation of the controversial gluttonous bacteria VP-1 as described in claim 1.

3. The fermentation broth of the gluttonous bacteria according to claim 2, characterized in that, The debate regarding the OD of the gluttonous bacteria fermentation broth 600 It equals 2.

4. The method for preparing the fermentation broth of the controversial *Gnaphalium affine* as described in claim 2 or 3, characterized in that, Includes the following steps: The controversial gluttonous bacterium VP-1 described in claim 1 was placed in a seed culture medium for seed culture to obtain a seed culture solution; The seed culture was inoculated into a fermentation medium for fermentation culture to obtain the fermentation broth of the controversial gluttonous bacteria.

5. The preparation method according to claim 4, characterized in that, Both the seed culture medium and the fermentation culture medium include LB medium.

6. The application of the controversial valerate VP-1 as described in claim 1 or the fermentation broth of the controversial valerate as described in claim 2 or 3 in the prevention and control of plant diseases and / or the promotion of plant growth; The plant disease control mentioned is the control of sclerotinia stem rot in rapeseed. The promotion of plant growth refers to promoting the growth of rapeseed.

7. A method for controlling Sclerotinia sclerotiorum in rapeseed, characterized in that, include: Mix the fermentation broth of the controversial gluttonous bacteria as described in claim 2 or 3 with Tween 80 and spray it on rapeseed leaves.

8. A method for promoting rapeseed germination, characterized in that, include: Rapeseeds were soaked in a preparation containing the fermentation broth of the controversial *Gnaphalium affine* as described in claim 2 or 3.

9. A method for promoting rapeseed growth, characterized in that, include: The fermentation broth of the controversial gluttonous bacteria described in claim 2 or 3 was used to treat the roots of rapeseed seedlings by irrigation.

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