Endophytic sphingomonas SE-S32 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to an endophytic sphingomonas strain SE-S32 and application of the endophytic sphingomonas strain SE-S32. The invention provides endophytic sphingomonas SE-S32, the preservation number of the endophytic sphingomonas SE-S32 is CCTCC NO: M 20241464, when the endophytic sphingomonas SE-S32 is applied to oilseed rape, the disease resistance of the oilseed rape to sclerotiniose can be improved, the germination rate and quality of oilseed rape seeds and the yield of rapeseeds can be improved, the growth and disease resistance of next-generation seedlings can be improved, and the endophytic sphingomonas SE-S32 has good application potential.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to an endophytic Sphingomonas SE-S32 and an application thereof. Background Art

[0002] Sclerotinia sclerotiorum is an important disease with worldwide distribution. This pathogen can infect more than 450 plant species, including some important economic crops such as rapeseed, soybean, lettuce and sunflower. Sclerotinia sclerotiorum can cause disease in all parts of crops and has a wide host range.

[0003] At present, the prevention and control of Sclerotinia sclerotiorum is still mainly chemical, but the long-term and repeated use of chemical agents also threatens crop safety and the ecological environment. Effective biological control can reduce the application of chemical pesticides, reduce environmental pollution, and improve the disease resistance of crops while controlling the growth and reproduction of pathogens. However, there are currently few biocontrol agents for Sclerotinia sclerotiorum, and the existing biocontrol agents are only effective for contemporary plants and cannot guarantee the health of future generations of plants. Therefore, it is the focus of research in this field to explore the antagonistic strains of Sclerotinia sclerotiorum and improve the disease resistance of future generations of plants after applying them to contemporary plants. Summary of the invention

[0004] The invention aims to provide an endophytic Sphingomonas SE-S32 and its application to improve the disease resistance of rapeseed to sclerotinia sclerotiorum, improve the germination rate, quality and yield of rapeseed seeds, and improve the growth and disease resistance of the next generation of seedlings.

[0005] The invention provides an endophytic Sphingomonas endophytica SE-S32, whose preservation number is CCTCC NO: M 20241464.

[0006] The present invention also provides a bacterial agent, which includes the endophytic Sphingomonas SE-S32 described in the above technical solution.

[0007] Preferably, the OD of the bacterial agent is 600 The value is 0.05~0.4.

[0008] Preferably, the bacterial agent further comprises a surfactant; the surfactant comprises Silwet L-77 and / or Tween 20.

[0009] Preferably, the concentration of Silwet L-77 in the bacterial agent is 0.03 to 0.07 v / v%;

[0010] The concentration of Tween 20 in the bacterial agent is 0.8-1.2 v / v‰.

[0011] The present invention also provides the use of the intrathecal aminomonas SE-S32 or the bacterial agent described in the above technical solution in rapeseed cultivation.

[0012] Preferably, the rapeseed planting includes one or more of improving the germination rate of rapeseed seeds, improving the quality of rapeseed seeds, improving the yield of rapeseed, improving the growth ability of the next generation of rapeseed seedlings, and improving the disease resistance of the next generation of rapeseed seedlings;

[0013] The disease resistance includes resistance to rapeseed sclerotinia;

[0014] Improving the growth capacity of the next generation of rapeseed seedlings includes increasing the aboveground root length and / or aboveground fresh weight of the next generation of rapeseed seedlings;

[0015] The improving the quality of rapeseed seeds comprises increasing the glucosinolate content of rapeseed seeds.

[0016] The present invention also provides a method for improving rapeseed seed quality, rapeseed yield, next generation seedling growth ability and disease resistance, comprising the following steps: applying the bacterial agent described in the above technical solution during the rapeseed flowering period.

[0017] Preferably, the bacterial agent is applied twice, and the time interval between two adjacent applications is 7 to 10 days.

[0018] The present invention also provides a method for improving the germination rate of rapeseed seeds, which comprises soaking the rapeseed seeds for 4 to 5 hours using the bacterial agent described in the above technical solution.

[0019] Beneficial effects:

[0020] The invention provides an endophytic sphingomonas SE-S32, with a deposit number of CCTCC NO: M20241464. Pot experiments and field experiments have shown that applying the endophytic sphingomonas SE-S32 to rapeseed can improve the disease resistance of rapeseed to sclerotinia sclerotiorum, improve the germination rate, quality and yield of rapeseed seeds, and improve the growth and disease resistance of the next generation of seedlings. The invention has good application potential.

[0021] Biological deposit information

[0022] Endophytic Sphingomonas SE-S32, biologically classified as Sphingomonas endophytica, was deposited in the China Center for Type Culture Collection on July 3, 2024, with the address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M 20241464. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0024] Figure 1 is the colony morphology of SE-S32 strain;

[0025] Figure 2 is the phylogenetic tree of the SE-S32 strain;

[0026] Figure 3 The germination of rapeseed seeds in different treatment groups at different times in Example 2; where * represents p<0.05, ** represents p<0.01, and NS represents no significant difference;

[0027] Figure 4 The disease resistance of potted rapeseed in different treatment groups in Example 3; the red arrows indicate the diseased areas;

[0028] Figure 5 The seed quality of potted rapeseed in different treatment groups in Example 3; where * represents p<0.05, and NS represents no significant difference;

[0029] Figure 6 The phenotypes, aboveground fresh weight and root length of the next generation seedlings of potted rapeseed seeds in different treatment groups in Example 3; where * represents p<0.05, and NS represents no significant difference;

[0030] Figure 7 The phenotype and lesion size of the next generation seedlings of potted rapeseed in different treatment groups in Example 3 inoculated with Sclerotinia sclerotiorum; where * represents p<0.05;

[0031] Figure 8 The disease resistance and rapeseed yield of rapeseed in the field under different treatments in Example 4; where * represents p<0.05. DETAILED DESCRIPTION

[0032] The present invention provides an endophytic sphingomonas SE-S32, with a deposit number of CCTCC NO: M20241464. The present invention isolates a bacterium SE-S32 from rapeseed Huashuang No. 4 seeds, identifies the bacterium as endophytic sphingomonas through morphology and molecular biology, can improve the disease resistance of rapeseed to sclerotinia sclerotiorum, affects the germination rate, quality and yield of rapeseed seeds, improves the growth and disease resistance of the next generation of seedlings, and has good application potential.

[0033] The present invention also provides a bacterial agent, which comprises the endophytic Sphingomonas SE-S32 described in the above technical solution. In the present invention, the OD of the bacterial agent is 600The value is preferably 0.05-0.4, further preferably 0.08-0.15, and more preferably 0.10; the bacterial agent preferably also includes a surfactant; the surfactant preferably includes Silwet L-77 and / or Tween 20; the concentration of Silwet L-77 in the bacterial agent is preferably 0.03-0.07 v / v%, further preferably 0.04-0.06 v / v%, and more preferably 0.05 v / v%; the concentration of Tween 20 in the bacterial agent is preferably 1 v / v‰.

[0034] In the present invention, the preparation method of the bacterial agent preferably includes the following steps: inoculating the endophytic Sphingomonas SE-S32 into R2A liquid culture medium, culturing at 30°C and 150rpm for 16 to 24h, centrifuging at 5000 to 6000rpm for 10 to 12min, discarding the supernatant metabolites, and collecting the bacterial bodies; resuspending the bacterial bodies with 0.01mol / LPBS solution to obtain the bacterial agent. The present invention preferably uses 0.01mol / LPBS solution to resuspend the bacterial bodies and then mixes with a surfactant to obtain a bacterial agent containing a surfactant. The definition of the surfactant in the present invention is preferably consistent with the above, and will not be repeated here. The 0.01mol / LPBS solution of the present invention preferably includes 10mmol / L phosphate and 150mmol / LNaCl, and the pH value is preferably 7.2 to 7.4. The addition of a surfactant in the present invention can reduce the surface tension of water and increase the wetting area of ​​the bacterial solution on rapeseed.

[0035] The present invention also provides the use of the endophytic Sphingomonas SE-S32 or the bacterial agent described in the above technical solution in rapeseed cultivation. In the present invention, the rapeseed preferably includes but is not limited to Huashuang No. 4, Shishan Caitai and Dadi 199.

[0036] In the present invention, the rapeseed planting preferably includes one or more of improving the germination rate of rapeseed seeds, improving the quality of rapeseed seeds, improving the yield of rapeseed, improving the growth ability of the next generation of rapeseed seedlings, and improving the disease resistance of the next generation of rapeseed seedlings, and further preferably improving the germination rate of rapeseed seeds, improving the quality of rapeseed seeds, improving the yield of rapeseed, improving the growth ability of the next generation of rapeseed seedlings, and improving the disease resistance of the next generation of rapeseed seedlings. The disease resistance of the present invention preferably includes the ability to resist rapeseed sclerotinia; the improvement of the growth ability of the next generation of rapeseed seedlings preferably includes increasing the aboveground root length and / or aboveground fresh weight of the next generation of rapeseed seedlings, and further preferably increasing the aboveground root length and aboveground fresh weight of the next generation of rapeseed seedlings; the improvement of the quality of rapeseed seeds preferably includes increasing the glucosinolate content of rapeseed seeds.

[0037] The present invention also provides a method for improving rapeseed seed quality, rapeseed yield, next generation seedling growth ability and disease resistance, comprising the following steps: applying the bacterial agent described in the above technical solution during the rapeseed flowering period. In the present invention, the bacterial agent is preferably applied twice, and the time interval between two adjacent applications is preferably 7 to 10 days, and more preferably 8 days.

[0038] In the present invention, when the rapeseed is planted in potted plants, the application amount of the microbial agent is preferably 25-30 mL / plant / time, and more preferably 28 mL / plant / time; when the rapeseed is planted in the field, the application amount of the microbial agent is preferably 0.9-1.1 L / 10 m 2 / time, more preferably 1L / 10m 2 / times; the rapeseed sowing density is 340-360 plants / 10m 2 , more preferably 350 plants / 10m 2 Applying rapeseed during its flowering period can improve its disease resistance to sclerotinia, affect the quality of rapeseed seeds and rapeseed yield, and improve the growth and disease resistance of the next generation of seedlings, which has good application potential.

[0039] The present invention also provides a method for improving the germination rate of rapeseed seeds, wherein the rapeseed seeds are soaked for 4 to 5 hours using the bacterial agent described in the above technical solution, which is further optimized to 4 hours. The endophytic Sphingomonas SE-S32 provided by the present invention can promote the germination rate of rapeseed seeds. After the soaking, the effect of promoting germination lasts for 72 hours, making the rapeseed seeds vigorous.

[0040] In order to further illustrate the present invention, an endophytic Sphingomonas SE-S32 and its application provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0041] The materials and culture medium used in the present invention are as follows:

[0042] Rapeseed varieties: Huashuang 4, Shishan Caitai and Dadi 199;

[0043] R2A liquid medium: tryptone 0.25g / L, acid hydrolyzed casein 0.5g / L, yeast extract powder 0.5g / L, soluble starch 0.5g / L, dipotassium hydrogen phosphate 0.3g / L, magnesium sulfate 0.1g / L, sodium pyruvate 0.3g / L, peptone 0.25g / L and glucose 0.5g / L. Use after sterilization at 121℃ for 30min;

[0044] R2A solid medium: R2A liquid medium containing 10 g / L agar;

[0045] Potato Dextrose Agar (PDA): Heat 200 g / L of potatoes to a boil, filter out the solid potatoes, add 10 g / L agar and 20 g / L glucose to the remaining liquid, add distilled water to 1 L, and sterilize at 121°C for 30 min before use;

[0046] Hoagland medium: potassium chloride 152.828 mg / L, potassium hydrogen phosphate trihydrate 456.638 mg / L, magnesium sulfate heptahydrate 246.48 mg / L, EDTA sodium iron 11.17 mg / L, boric acid 1.546 mg / L, manganese sulfate monohydrate 0.338 mg / L, copper sulfate 0.125 mg / L, zinc sulfate monohydrate 0.576 mg / L, sodium molybdate 0.102 mg / L. Add 2 mmol / L calcium nitrate as a nitrogen source and 6 g / L agar, sterilize at 121°C for 20 min before use;

[0047] Nutrient soil: purchased from Jiangsu Zhenjiang Beilei Organic Fertilizer Co., Ltd.;

[0048] Buffer I: 25 mmol / L NaOH and 0.2 mmol / L ETA, pH 12;

[0049] BufferII: 40mmol / LTris-HCl, pH 7.5;

[0050] 0.01 mol / L PBS (Phosphate Buffered Saline) solution: 10 mmol / L phosphate, 150 mmol / L NaCl, pH 7.2-7.4; Sclerotinia sclerotiorum strain Ep-1 PNA367: disclosed in Yu X, Li B, Fu Y, Jiang D, Ghabrial SA, Li G, Peng P, Xie J, Cheng J, Huang J, Yi X. A Geminivirus-related DNA mycovirus that confers hypovirulence to a plantpathogenic fungus. Proc Natl Acad Sci USA 2011, 107: 8387-8392.

[0051] Example 1

[0052] 1. Isolation and pure culture of bacterial strains

[0053] The dilution plate method was used to isolate microorganisms. Specifically, 0.2 g of rapeseed Huashuang No. 4 seeds were weighed and added to 1 mL of 0.01 mol / L PBS solution. The rapeseed seeds were ground into a homogenate using a tissue grinder at 35 Hz for 1 min. The turbid solution was set at 10 0 The solution was diluted by taking 100 μL and adding it to 900 μL of 0.01 mol / L PBS solution, and then diluted 10 -1 and 10 -2 Concentration gradient was used to obtain the diluted plant tissue homogenate. 100 μL of each was applied to R2A solid medium to obtain a single colony. A sterilized toothpick was used to pick a single colony, and it was streaked and purified in a new R2A solid medium to obtain a bacterial strain named SE-S32.

[0054] 2. Identification of strain morphology

[0055] The SE-S32 strain was activated in R2A solid medium for two generations, and a single colony of SE-S32 was cultured in R2A liquid medium for 48 hours. The concentration of the bacterial solution after shaking was OD 600 Reach 0.6-0.8. Dilute the bacterial solution 10 -7 The concentration gradient was applied to R2A solid medium and grown at 25°C for 5 days. Figure 1 Middle left: Observed under a stereomicroscope, strain SE-S32 forms round, yellow colonies with smooth edges ( Figure 1 center right).

[0056] 3. DNA Extraction from Bacterial Strains

[0057] Reference is made to the prior art (Bai Y, Müller DB, Srinivas G, Garrido-Oter R, Potthoff E, Rott M, Dombrowski N, Münch PC, Spaepen S, Remus-Emsermann M, Hüttel B, McHardy AC, Vorholt JA, Schulze-Lefert P. Functional overlap of the Arabidopsis leaf and root microbiota. Nature. 2015, 528: 364-369). Use a sterilized toothpick to pick up a single colony on R2A solid culture medium, and use a toothpick without picking up a colony as a negative control, and shake and culture overnight at 30°C and 150rpm in R2A liquid culture medium. If the negative control is not contaminated, DNA extraction is performed by alkaline lysis. Take 6 μL of turbid bacterial solution, add 10 μL of buffer I, and place it in a PCR instrument at 95°C for 30 minutes for lysis. After the completion, 10 μL of buffer II was added to balance the pH value to complete the extraction of bacterial whole genome DNA. The DNA concentration was measured by Nanodrop2000 (ThermoFisher Scientific), and its range was stable between 30-100 ng / μL. 1 μL was taken as a DNA template to perform PCR amplification of 16S rDNA sequence.

[0058] 4. Sequencing and identification of bacterial 16S rDNA sequences

[0059] Figure 2 As shown. Figure 2 It can be seen that the 16S rDNA sequence bootstrap support rate of the SE-S32 strain and the Sphingomonas endophytica Y72 strain is 98, and the sequence identity is 97.61%. Therefore, the SE-S32 strain was determined to be Sphingomonas endophytica, named Sphingomonas endophytica SE-S32, and was deposited in the China Center for Type Culture Collection on July 3, 2024, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a deposit number of CCTCC NO: M 20241464.

[0060] Example 2

[0061] Effects of strain SE-S32 on the germination rate of rapeseed seeds

[0062] (1) Streak the SE-S32 strain on R2A solid medium for two generations. Rinse the plate directly with 0.01 mol / L PBS solution to obtain a bacterial suspension. Dilute the SE-S32 strain to an OD of 600 =0.4, used for inoculation of rapeseed seeds.

[0063] (2) The rapeseed seeds (variety: Huashuang No. 4) were surface disinfected by soaking in 75% (v / v) alcohol for 1 min, 3.5% (v / v) sodium hypochlorite for 3 min, and 75% (v / v) alcohol for 1 min. Finally, they were rinsed with sterile water 3 times, each time for 1 min, and the disinfection effect was tested.

[0064] (3) The disinfected rapeseed seeds were randomly divided into an experimental group (Sph.) and a control group (PBS). The experimental group soaked the rapeseed seeds in the bacterial solution of step (1); the control group soaked the rapeseed seeds in 0.01 mol / L PBS solution; after soaking for 4 hours, the experimental group and the control group were respectively soaked in a 10 cm × 10 cm square culture dish, lined with a layer of filter paper, and added with 5 mL of sterilized distilled water. 100 treated rapeseed seeds were added to each dish, and the seed germination rates were measured after 24 hours, 48 ​​hours, 72 hours and 96 hours (n = 3), and the seeds were grown in a plant greenhouse at 20°C with 12 hours of alternating light and dark. The differences between the groups were statistically analyzed by one-way analysis of variance using Excel, and the results are shown in Tables 1 and Figure 3 shown.

[0065] Table 1 Germination rate of rapeseed seeds in different treatment groups at different times (%)

[0066]

[0067] Note: Different lowercase letters in the table indicate significant differences at the p<0.05 level.

[0068] According to Table 1 and Figure 3 It can be seen that the SE-S32 strain inoculated in the experimental group significantly improved the seed germination rate (p<0.05). At 24h, the germination rate of the control group was 14.33±4.93%, and the germination rate of the experimental group was 32.33±3.51%; at 48h, the germination rate of the control group was 38.00±7.21%, and the germination rate of the experimental group was 59.67±3.21%; at 72h, the germination rate of the control group was 55.00±7.00%, and the germination rate of the experimental group was 74.67±3.05%; at 96h, the germination rate of the control group was 79.33±2.52%, and the germination rate of the experimental group was 84.33±2.52%. That is, the inoculation of the seeds with the SE-S32 strain significantly promoted the germination rate of rapeseed seeds, and the effect lasted until 72h. Until 96h, all seeds with vitality completed germination, and there was no longer any biological difference between the groups ( Figure 3 ).

[0069] Example 3

[0070] Pot experiment

[0071] (1) The strain SE-S32 was cultured in a flask containing 200 mL of R2A liquid medium at 30°C and 150 rpm overnight, the cells were collected by centrifugation, the supernatant metabolites were discarded, and the cells were resuspended in 0.01 mol / L PBS solution. The bacterial solution was diluted to OD 600 =0.1, add surfactant, 0.05% (v / v) Silwet L-77 as a bacterial agent for subsequent treatment.

[0072] (2) On December 10, 2023, during the flowering period of rapeseed in Shishan, the plants were randomly divided into an experimental group and a control group. Each rapeseed in the experimental group was treated with 25 mL of the bacterial agent in step (1), and each rapeseed in the control group was treated with 25 mL of a 0.01 mol / L PBS solution containing 0.05% (v / v) Silwet L-77. The application was repeated once in the experimental group and the control group, respectively.

[0073] (3) Rapeseed plants were naturally diseased. On February 18, 2024, with reference to the grading standards of the prior art (Li G, Huang H, Miao H, Erickson RS, Jiang D, Xiao Y. 2006. Biological control of Sclerotinia diseases of rapeseed by aerial applications of the mycoparasite Coniothyrium minitans. European Journal of Plant Pathology 111: 345-355), the severity of Sclerotinia disease in the experimental group and the control group was statistically analyzed, and the incidence rate of Sclerotinia disease in rapeseed and the average disease index per rapeseed plant were statistically analyzed.

[0074] The disease index statistical formula is divided into 0-4 levels according to the disease severity of rapeseed, level 0: no lesions; level 1: less than 1 / 3 of the branches have lesions, or the size of the main stem lesions is less than 3 cm; level 2: 1 / 3 to 2 / 3 of the branches have lesions, or less than 1 / 3 of the branches are diseased, but the main stem lesions are larger than 3 cm; level 3: 2 / 3 of the branches are diseased, or less than 2 / 3 of the branches are diseased, but the main stem lesions are larger than 3 cm; level 4: more than 2 / 3 of the branches are diseased, or less than 2 / 3 of the branches are diseased but the main stem lesions are larger than 10 cm.

[0075] The disease index is calculated as follows: 100×(P0×0+P1×1+P2×2+P3×3+P4×4) / 4×(P0+P1+P2+P3+P4). P0 to P4 are the number of rapeseeds with disease at each level.

[0076] The results are shown in Table 2 and Figure 4 shown.

[0077] Table 2 The incidence of sclerotinia sclerotiorum in different treatment groups

[0078] Experimental group (Sph.Spraying) disease level Control group (Control) disease level Repeat 1 1 1 Repeat 2 0 4 Repeat 3 1 0 Repeat 4 0 4 Repeat 5 3 4 Repeat 6 0 0 Repeat 7 0 1 Repeat 8 1 4 Repeat 9 / 1 Repeat 10 / 1 Repeat 11 / 0 Repeat 12 / 3 Disease index 18.75 48.00

[0079] According to Table 2 and Figure 4 It can be seen that compared with the control group, the experimental group sprayed the strain SE-S32 on the rapeseed plants, which significantly reduced the incidence of rapeseed and the average disease index per rapeseed. The incidence of rapeseed was statistically analyzed, and the red arrows in the figure are the diseased areas. The experimental group applied the strain SE-S32, and 4 plants were infected, with an incidence of 50%; the control group had 9 plants infected, with an incidence of 75%. The average disease index per rapeseed was statistically analyzed. The experimental group applied the strain SE-S32, and the disease index was 18.75, and the control group was 48.00. For potted experiments, the application of strain SE-S32 improved the disease resistance of rapeseed to sclerotinia, and it has a high application potential.

[0080] (4) The rapeseed seeds of the experimental group and the control group were harvested and their oil content, glucosinolates, oleic acid, linoleic acid and α-linolenic acid contents were measured by near infrared analyzer (NIRSYSTEM 3750, Bio-Rad) at the Rapeseed Engineering Center of Huazhong Agricultural University. The differences among the groups were statistically analyzed by one-way ANOVA using Excel. The results are shown in Table 3 and Figure 5 shown.

[0081] Table 3 Rapeseed quality in different treatment groups

[0082]

[0083] According to Table 3 and Figure 5 It can be seen that the experimental group sprayed with strain SE-S32 significantly increased the glucosinolate content in rapeseed seeds ( Figure 5 b, p<0.05), it is inferred that the inoculation leads to an increase in glucosinolate content, which is one of the main reasons affecting plant disease resistance. It has no adverse effects on other qualities of rapeseed seeds, such as oil content, oleic acid, linoleic acid and α-linolenic acid.

[0084] (5) Surface disinfection of the harvested rapeseed seeds was performed in the manner described in step (2). The surface-disinfected seeds were transferred to Hoagland solid culture medium for seedling cultivation. After 2 days, the rapeseed seeds germinated. The seedlings with uniform germination were selected and transplanted to nutrient soil. They were grown in a plant greenhouse at 20°C with 12 h light-dark alternation. After 3 weeks of growth and development of the rapeseed seeds, the aboveground parts were taken to determine their fresh weight. At the same time, the root length of the seedlings was determined. The biological replicates were 6 to 8 plants. One-way ANOVA was performed using Excel to statistically analyze the differences between the groups. The results are shown in Tables 4 and Figure 6 shown.

[0085] Table 4 Aboveground fresh weight, root length and resistance to sclerotinia in the next generation of rapeseed seedlings in different treatment groups

[0086]

[0087]

[0088] According to Table 4 and Figure 6 It can be seen that when the strain SE-S32 was applied to the previous generation of plants during the flowering period of rapeseed, the aboveground fresh weight of the next generation of seedlings reached 1.496±0.300g, which was significantly increased compared with the control group sample 1.148±0.198g (p<0.05). As for the root length of rapeseed seedlings, the experimental group was 155.34±16.59mm, and the control group was 149.02±18.96mm, with no adverse effects.

[0089] (6) Cultivate 3 generations of Sclerotinia sclerotiorum Ep-1PNA367 in 15 mL PDA medium at 20°C continuously to ensure the activity of the strain. Inoculate the rapeseed leaves in step (5) with Ep-1PNA367 mycelium blocks with a diameter of 2 mm, which are taken from the edge of the colony. Cultivate the whole rapeseed in a greenhouse at 20°C with moisturizing, and alternate light and dark for 12 hours. After 24 hours, the size of the lesions was counted, and biological replicates were performed for more than 6 plants. One-way analysis of variance was performed using Excel to statistically analyze the differences between the groups, and the results are shown in Tables 4 and Figure 7 shown.

[0090] According to Table 4 and Figure 7 It can be seen that the Ep-1PNA367 strain of Sclerotinia sclerotiorum was inoculated on the leaves, and the strain SE-S32 in the experimental group significantly improved the disease resistance of rapeseed seedlings, and the lesion size was 38.60±13.16mm 2 , which is significantly lower than the lesion size of the control group samples (67.36±28.27mm). 2 (p<0.05). Spraying strain SE-S32 during the flowering period significantly improved the disease resistance of the plants and also provided good protection for the next generation of seedlings.

[0091] Example 4

[0092] Field experiments

[0093] (1) The SE-S32 strain was cultured in a flask containing 200 mL of R2A liquid medium at 30°C and 150 rpm overnight, the cells were collected by centrifugation, the supernatant metabolites were discarded, and the cells were resuspended in 0.01 mol / L PBS solution. The bacterial solution was diluted to OD 600 =0.05, and a surfactant Tween 20 at a concentration of 1‰ (v / v) was added as a bacterial agent for subsequent treatment.

[0094] (2) On March 21, 2024, at the 199 flowering period of rapeseed in Huanggang Experimental Field, the subjects were randomly divided into an experimental group and a control group. The experimental group received a 10m 2 Apply 1L of bacterial solution to rapeseed plants, and the rapeseed planting area is 350 plants / 10m 2 , control group every 10m 2 Rapeseed plants were applied with a surfactant Tween 20 at a concentration of 1‰ (v / v). The application was repeated once in the experimental group and the control group on March 28, 2024.

[0095] (3) Rapeseed plants are naturally diseased. On April 29, 2024, with reference to the grading standards of the prior art (Li G, Huang H, Miao H, Erickson RS, Jiang D, Xiao Y. 2006. Biological control of Sclerotinia diseases of rapeseed by aerial applications of the mycoparasite Coniothyrium minitans. European Journal of Plant Pathology 111: 345-355), the severity of Sclerotinia disease of 100 rapeseed plants in each plot was statistically analyzed; every 10m 2 The rapeseed was dried and stored in the sun. The rapeseed yield of each plot was counted. The differences between the groups were analyzed by one-way analysis of variance using Excel. The results are shown in Table 5 and Figure 8 shown.

[0096] Table 5 Disease index and yield of rapeseed in different treatment groups

[0097]

[0098] According to Table 5 and Figure 8 It can be seen that the disease index of the control group reached 57.13±7.51, and the incidence of sclerotinia was serious. The experimental group applied strain SE-S32, and the disease index was 43.13±4.38, which was 24.5% lower than that of the control group, and had good biocontrol potential. The rapeseed seeds per unit area were dried and collected, and the rapeseed yield per unit area in the control group was 0.78±0.087kg / 10m 2 The experimental group applied strain SE-S32 to reach 0.91±0.027kg / 10m 2 After treatment, rapeseed yield increased by 16.67%.

[0099] Based on the above content, it can be seen that the strain SE-S32 provided by the present invention can improve the disease resistance of rapeseed to sclerotinia, improve the germination rate, quality and rapeseed yield of rapeseed seeds, and improve the growth and disease resistance of the next generation of seedlings, and has good application potential.

[0100] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Sphingomonas endophytica SE-S32, with a deposit number of CCTCCNO: M 20241464.

2. A bacterial agent, characterized in that The bacterial agent includes the endophytic Sphingomonas SE-S32 described in claim 1.

3. The bacterial agent according to claim 2, characterized in that The OD of the inoculum 600 The value is 0.05~0.

4.

4. The bacterial agent according to claim 2 or 3, characterized in that: The bacterial agent also includes a surfactant; the surfactant includes Silwet L-77 and / or Tween 20.

5. The bacterial agent according to claim 4, characterized in that The concentration of Silwet L-77 in the bacterial agent is 0.03-0.07 v / v%; The concentration of Tween 20 in the bacterial agent is 0.8-1.2 v / v‰.

6. Use of the endophytic Sphingomonas SE-S32 according to claim 1 or the bacterial agent according to any one of claims 2 to 5 in rapeseed cultivation.

7. The use according to claim 6, characterized in that: The rapeseed planting includes one or more of improving the germination rate of rapeseed seeds, improving the quality of rapeseed seeds, improving the yield of rapeseed, improving the growth ability of the next generation of rapeseed seedlings, and improving the disease resistance of the next generation of rapeseed seedlings; The disease resistance includes resistance to rapeseed sclerotinia; Improving the growth capacity of the next generation of rapeseed seedlings includes increasing the aboveground root length and / or aboveground fresh weight of the next generation of rapeseed seedlings; The improving the quality of rapeseed seeds comprises increasing the glucosinolate content of rapeseed seeds.

8. A method for improving rapeseed seed quality, rapeseed yield, next generation seedling growth ability and disease resistance, characterized in that: The method comprises the following steps: applying the bacterial agent according to any one of claims 2 to 5 during the rapeseed flowering period.

9. The method according to claim 8, characterized in that The bacterial agent is applied twice, and the time interval between two adjacent applications is 7 to 10 days.

10. A method for improving the germination rate of rapeseed seeds, characterized in that: Soak rapeseed in the bacterial agent described in any one of claims 2 to 5 for 4 to 5 hours.

Images