Seed endophyte for preventing and controlling wheat fungal diseases and application thereof

Abstract

The present invention discloses a seed endophyte for preventing and treating wheat fungal diseases and its application, belonging to the technical field of crop planting and crop disease biological control. The wheat endophyte (Pseudescherichia vulneris) GSE25 in the present invention was deposited in the China General Microbiological Culture Collection Center (CGMCC) on November 21, 2022, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC NO.26158. The GSE25 strain has the ability to significantly reduce the pathogenicity of gibberellins and stripe rust on wheat plants, and can significantly reduce the mycotoxins produced by pathogens on wheat grains, has a promoting effect on crop growth and development, and has a broad-spectrum effect. The GSE25 strain of the present invention is of great value in the healthy growth, yield increase, control of pathogenic fungi and mycotoxin pollution of crops.

Description

Technical Field

[0001] The invention relates to seed endophytes for preventing and controlling wheat fungal diseases and application thereof, belonging to the technical field of crop planting and crop disease biological control. Background Art

[0002] Diseased grains contain a variety of mycotoxins produced by pathogens. Consumption can cause poisoning in humans and animals, posing a serious threat to food safety. The best way to combat crop diseases is prevention. Traditionally, chemical pesticides have been used, but long-term and frequent use has led to an increasing problem of pesticide resistance. Furthermore, the carcinogenic, teratogenic, and mutagenic effects of chemical pesticides pose a significant threat to human health. Microbial pesticides are an important biocontrol tool, offering advantages such as environmental friendliness, strong selectivity, low residue levels, high efficiency, resistance to pesticide resistance, and the ability to significantly reduce pesticide usage without compromising crop yield and quality. Therefore, under the purview of sustainable and green agriculture, the use of microbial pesticides for crop disease control is a current research hotspot.

[0003] Endophytes are an important source of microbial pesticides. They possess a stable habitat and a reliable source of nutrients within plants, making them more effective. Endophytes can be isolated from plant tissues (roots, stems, leaves, seeds, etc.) that have undergone rigorous surface disinfection. Currently, they are a hot area of microbial research both domestically and internationally, focusing on agricultural disease control, environmental remediation, and growth promotion, though primarily focused on cash crops such as cotton, tomatoes, and tobacco. Studies on the use of endophytic bacteria in wheat for disease control are limited, and research on endophytic bacteria in wheat seeds is even rarer. Endophytic bacteria in wheat seeds act as the last barrier against pathogen infection and toxin production during wheat growth. Since wheat seeds are long consumed by humans, biocontrol bacteria isolated from them are safer and more reliable, with more pronounced antibacterial and toxin control effects. Furthermore, seed endophytes possess a broad spectrum of control capabilities, making them suitable for fungal disease control not only in grain crops like wheat and corn, but also in cash crops like apples, tomatoes, and wolfberries. In the prior art, patent CN109294945B discloses that the wheat endophyte Bacillus CM134L-2 has an inhibitory effect on Fusarium graminearum and Fusarium wilt pathogens. The document Endophytic bacteria from wheat grain as biocontrol agents of Fusarium graminearum and deoxynivalenol production in wheat discloses that Bacillus megaterium BM1 and Bacillus subtilis BS43 isolated from wheat ears have an inhibitory effect on Fusarium graminearum. However, on the one hand, the prior art does not contain any research on the use of endophytic bacteria in wheat seeds to prevent wheat diseases. On the other hand, the above reports do not evaluate the biosafety of endophytes, which still poses a risk of low safety in practical applications. In addition, existing endophytes for preventing and controlling wheat diseases still have problems such as a narrow antibacterial spectrum and poor antibacterial effect. Summary of the Invention

[0004] In order to address the deficiencies in the prior art, the present invention provides a seed endophyte (Pseudescherichia vulneris) GSE25 for preventing and controlling wheat fungal diseases and its application. The strain provided by the present invention can significantly inhibit the pathogenicity of gibberellins and stripe rust, while reducing the mycotoxins produced by gibberellins. It has a significant antibacterial effect, good experimental repeatability, high specificity, simple operation, and good stability and safety.

[0005] The present invention provides a wheat seed endophyte (Pseudescherichia vulneris) GSE25. The (Pseudescherichia vulneris) GSE25 has been deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC NO.26158. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit date is November 21, 2022.

[0006] The wheat seed endophyte (Pseudescherichia vulneris) GSE25 was isolated from wheat seeds. Through whole genome sequencing and phylogenetic evolutionary tree analysis, the strain was identified as a new strain of the genus Pseudescherichia and was named (Pseudescherichia vulneris) GSE25.

[0007] The wheat seed endophyte (Pseudescherichia vulneris) GSE25 is a Gram-negative bacterium; the bacteria are short rod-shaped, the colonies are milky white, the edges are rounded, and the protrusions are shiny.

[0008] The wheat seed endophyte (Pseudescherichia vulneris) GSE25 can significantly reduce the pathogenicity of gibberellosis and stripe rust on wheat plants, and can greatly reduce the fungal toxins produced by the pathogens on wheat grains.

[0009] The wheat seed endophyte (Pseudescherichia vulneris) GSE25 has a promoting effect on the growth and development of crops, wherein the crops include grass plants, or the crops include but are not limited to wheat, corn, apple, tomato or wolfberry.

[0010] The present invention also provides a microbial agent containing the wheat seed endophyte (Pseudescherichia vulneris) GSE25.

[0011] In one embodiment, the microbial agent is a liquid agent or a solid agent containing one or more of living cells of the wheat seed endophyte (Pseudescherichia vulneris) GSE25 strain, freeze-dried wheat seed endophyte (Pseudescherichia vulneris) GSE25 dry bacteria, and immobilized wheat seed endophyte (Pseudescherichia vulneris) GSE25 cells.

[0012] In one embodiment, the content of wheat seed endophyte (Pseudescherichia avulneris) GSE25 in the microbial agent is not less than 10 6 CFU / mL, or not less than 10 8 CFU / mL.

[0013] In one embodiment, the liquid inoculum is prepared by inoculating the activated seed solution into a culture medium at an inoculum size of 5-15%, and culturing at 20-35°C for 16-48 hours. Preferably, the activated seed solution is inoculated into a culture medium at an inoculum size of 10%, and culturing at 25°C for 24-48 hours.

[0014] In one embodiment, the culture medium comprises: 5-15 g / L peptone, 1-10 g / L yeast powder, and 5-15 g / L sodium chloride.

[0015] The present invention also provides the use of the wheat seed endophyte (Pseudescherichia vulneris) GSE25 or the microbial agent in promoting plant growth and development.

[0016] In one embodiment, the plant includes but is not limited to grass, or the plant includes but is not limited to wheat, corn, apple, tomato or wolfberry.

[0017] In one embodiment, the application is to irrigate the plants with a bacterial suspension containing the wheat seed endophyte (Pseudescherichia vulneris) GSE25 or the microbial agent, wherein the content of the wheat seed endophyte (Pseudescherichia vulneris) GSE25 in the bacterial suspension is not less than 1×10 6 CFU / mL.

[0018] The present invention also provides the use of the wheat seed endophyte (Pseudescherichia vulneris) GSE25 or microbial agent in preventing and controlling plant diseases.

[0019] In one embodiment, the plant disease comprises a plant disease caused by at least one of Fusarium graminearum or stripe rust.

[0020] In one embodiment, the plant disease comprises head blight and / or rust.

[0021] In one embodiment, the plant diseases include but are not limited to wheat fusarium head blight, wheat stripe rust, corn ear rot, apple rot, tomato leaf spot or wolfberry black fruit disease.

[0022] In one embodiment, the controlling of plant diseases includes reducing mycotoxin levels.

[0023] In one embodiment, the mycotoxins include but are not limited to deoxynivalenol (DON), zearalenone, fumonisins, or patulin.

[0024] In one embodiment, the application is spraying a liquid bacterial agent containing the wheat seed endophyte (Pseudescherichia avulneris) GSE25 on the ears, fruits or leaves of the plant.

[0025] In one embodiment, the wheat seed endophyte (Pseudescherichia vulneris) GSE25 content is not less than 10 8 CFU / mL.

[0026] In one embodiment, the amount of liquid microbial agent sprayed is 1-10 mL / m 2 , preferably 5mL / m 2 .

[0027] Beneficial effects:

[0028] (1) The present invention provides a new strain of the genus Pseudescherichia (Pseudescherichia vulneris) GSE25, which can simultaneously inhibit crop fungal diseases, reduce fungal toxins and promote crop growth.

[0029] (2) (Pseudescherichia vulneris) GSE25 strain has a promoting effect on plant growth. 6 Watering wheat with a bacterial agent containing CFU / mL can significantly promote the growth of wheat seedlings.

[0030] (3) The biological evaluation of (Pseudescherichia vulneris) GSE25 strain was safe. The hemolytic activity test showed that the (Pseudescherichia vulneris) GSE25 strain had no hemolytic phenomenon; the plant pathogenicity test showed that the (Pseudescherichia vulneris) GSE25 strain had no pathogenicity to wheat plants and tobacco leaves.

[0031] (4) (Pseudescherichia vulneris) GSE25 strain concentration was 10 6 Under the condition of 100 CFU / mL, it can significantly inhibit the growth of Gibberella and Stripe Rust in vitro.

[0032] (5) (Pseudescherichia vulneris) GSE25 strain was isolated at 10 8 After spraying the fungicide at a concentration of 1.5 CFU / mL for 3 days, the incidence of ergot disease can be prevented by 85.48%. At the same time, the fungal toxins produced by ergot can be reduced, and the fungal toxin inhibition rate can be as high as 91.18%. The antibacterial effect is obvious, the test has good repeatability, high specificity, simple operation, and good stability and safety.

[0033] (6) The fungus is prepared into a liquid agent and sprayed on the ears, fruits or leaves of crops, which can effectively prevent the occurrence of fungal diseases and reduce the content of mycotoxins in crops, thereby increasing crop yield and quality, and achieving the purpose of safely and effectively preventing and controlling fungal diseases in humans and animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The colony morphology of Pseudescherichia vulneris GSE25 is shown;

[0035] Figure 2 The maximum likelihood method was used to construct the phylogenetic tree for the genome of Pseudescherichia vulneris GSE25 strain and the distribution of species compared to the genome sequence in the Nr database. Different colors represent different species.

[0036] Figure 3 This is a growth time curve of the (Pseudescherichia vulneris) GSE25 strain;

[0037] Figure 4 For the cytotoxicity assay of (Pseudescherichia vulneris) GSE25 strain;

[0038] Figure 5 For the hemolytic test of Pseudescherichia vulneris GSE25 strain, 0 is the negative control (PBS), 1 is the culture supernatant stock solution, 2 refers to the 2-fold diluted culture supernatant, and the numbers 4, 8, 16, 32, 64, and 128 all represent the dilution multiples of the culture supernatant;

[0039] Figure 6 Blood plate culture results of Pseudescherichia vulneris GSE25 strain;

[0040] Figure 7 Figure 1 is the result of plant pathogenicity experiment of Pseudescherichia vulneris GSE25 strain;

[0041] Figure 8 This is a diagram showing the effect of Pseudescherichia vulneris GSE25 strain on wheat growth promotion;

[0042] Figure 9 Figure 2 is the inhibitory effect of (Pseudescherichia vulneris) GSE25 strain on the growth of Fusarium graminearum hyphae, where A is Fusarium graminearum hyphae (control group, normal morphology), and B is Fusarium graminearum hyphae after (Pseudescherichia vulneris) GSE25 action (treatment group);

[0043] Figure 10 This is a graph showing the inhibitory effect of the (Pseudescherichia vulneris) GSE25 strain on stripe rust;

[0044] Figure 11 This is a diagram showing the inhibitory effect of the (Pseudescherichia vulneris) GSE25 strain on Fusarium graminearum. DETAILED DESCRIPTION

[0045] The culture medium formula involved in the embodiment is:

[0046] Potato dextrose agar (PDA) medium: 46.0 g of PDA powder was added with distilled water to make up to 1 L. After mixing, it was sterilized by high-pressure steam at 115°C for 20 min and kept at room temperature for use.

[0047] Sodium carboxymethyl cellulose (CMC) liquid culture medium: CMC 15.0 g, yeast extract 1.0 g, KH2PO4 1.0 g, MgSO4·7H2O 0.5 g, NH4NO3 1.0 g, distilled water to 1 L, mix well, and sterilize by high-pressure steam at 121°C for 20 min. Keep at room temperature for use.

[0048] Yeast extract peptone glucose (YEPD) liquid medium: glucose (Dextrose) 20.0g, Yeast Extract 3.0g, peptone (Peptone) 10.0g, add distilled water to 1L, mix well, and sterilize with high pressure steam at 121℃ for 20min, and set aside at room temperature.

[0049] The Fusarium graminearum PH-1 strain and the Puccinia striiformis CYR32 strain were deposited by the School of Food Science and Engineering, Dalian Polytechnic University.

[0050] Example 1 Screening, Identification, and Physiological and Biochemical Characterization of Pseudescherichia vulneris GSE25 Strain

[0051] (1) Screening of (Pseudescherichia vulneris) GSE25 strain

[0052] Wheat seeds (Triticum aestivum L.) used for the isolation of endophytic bacteria were obtained from healthy wheat plants (variety Fielder) at Caoxinzhuang Wheat Research Farm, Yangling, Shaanxi, China (108°10'28", 34°30'63") in 2021. Healthy wheat seeds were first disinfected with 75% alcohol for 30 seconds, then with 1% sodium hypochlorite for 2 minutes, and then rinsed eight times with sterile water under aseptic conditions. For isolation, 10 g of sterile tissue was ground in 50 mL of 0.85% saline, and the suspension was diluted to 10 -3 . Take 100 μL of the suspension and spread it on an LB agar plate and culture it at 25°C for 1-2 days. Each dilution was set to three replicates. Transfer the single colony to a new LB agar plate to purify the colony. After purification, interact with the PH-1 strain of Fusarium graminearum to verify the inhibitory effect of different endophytes on Fusarium graminearum and stripe rust, and finally obtain a strain that can inhibit wheat fungal diseases and mycotoxins.

[0053] (2) Identification of (Pseudescherichia vulneris) GSE25 strain

[0054] 1) Genome sequencing and phylogenetic tree analysis and identification

[0055] The strain obtained in step (1) was subjected to whole genome sequencing, and a phylogenetic tree was constructed using the genome sequence. The phylogenetic tree is shown in FIG. Figure 2 As shown in A, it can be seen that although the strain is closely related to Pseudescherichia vulneris, there is still a significant difference of 42.57%. In addition, by comparing the strain genome sequence with the species distribution in the Nr database ( Figure 2 B) revealed significant differences between the strain genome and existing species, with the highest similarity being that of Pseudescherichia vulneris, but with only 68.29% homology. In summary, the strain obtained in step (1) was a Pseudescherichia strain, significantly different from Pseudescherichia vulneris, and was named (Pseudescherichia vulneris) GSE25.

[0056] 2) Physiological and biochemical characterization of Pseudescherichia vulneris GSE25 strain

[0057] Gram and morphological identification

[0058] The obtained single bacterial strain was identified by Gram staining under an optical microscope and was found to be a Gram-negative bacterium, e.g. Figure 1 As shown, the bacteria are short rod-shaped, the colonies are milky white, with rounded edges and shiny protrusions.

[0059] (Pseudescherichia vulneris) GSE25 bacterial growth curve assay

[0060] Pseudescherichia vulneris GSE25 bacteria and LB medium were added to the flask at a ratio of 1:100. Four replicates were set up and placed in an incubator. The culture conditions were set at 25°C and 170 rpm for 30 hours. During this time, the instrument collected the OD value every two minutes. Finally, the bacterial growth curve was drawn with time as the horizontal axis and OD value as the vertical axis, as shown in the figure. Figure 3 shown.

[0061] Example 2 (Pseudescherichia vulneris) GSE25 strain biosafety verification

[0062] 1. Cytotoxicity assay of (Pseudescherichia vulneris) GSE25 culture supernatant

[0063] Pseudescherichia vulneris GSE25 was cultured overnight in a shaker at 25°C and 170 rpm. 175 μL was added to a 96-well plate and the OD was measured. 600 =0.81. The bacterial suspension was collected and centrifuged at 8000 rpm for 10 min. The supernatant was collected and filtered through a 0.2 μM filter membrane before cytotoxicity testing. The supernatant was serially diluted 2, 4, 8, 16, 32, 64, 128, and 256 times.

[0064] The specific operation process is as follows:

[0065] (1) Bacterial culture. Add 100 μL of bacterial solution to 500 μL of pre-prepared LB medium, set the incubator conditions to 25°C, 170 rpm, and culture overnight. Pipette 175 μL of the shaken bacterial solution into a 96-well plate and measure the OD 600 =0.8.

[0066] (2) Preparation of bacterial suspension. Collect the shaken bacterial suspension into a centrifuge bottle and centrifuge at 8000 rpm for 10 min. Collect the supernatant and filter through a 0.2 μm filter membrane for later use.

[0067] (3) Dilution of bacterial supernatant: The filtered bacterial solution was diluted with DMEM complete medium in a 2-fold gradient to 8 concentrations, ensuring that the supernatant after dilution was at least 650 μl.

[0068] (4) Cell plating. Dilute MDCK cells to 1×10 one day in advance. 5 Cells / ml were evenly added into 96-well plates, 100 μl per well, and cultured in a cell culture incubator for 24 h until the cell density in each well was about 80% before subsequent experiments were performed.

[0069] (5) Cytotoxicity assay. Remove the 96-well plate pre-plated with MDCK cells, discard the culture supernatant, and wash 2-3 times with sterile PBS. Add the diluted bacterial solution from left to right, 100 μl per well, and set up 3 replicates for each concentration. The wells with only culture medium added serve as blank controls, and the wells with 100 ng / ml of Clostridium perfringens epsilon toxin (ETX) added serve as positive controls.

[0070] (6) After culturing for 3 hours, discard the supernatant and wash with PBS 2-3 times. Add 100 μl DMEM complete medium and 20 μl MTS to each well and incubate in a cell culture incubator in the dark for 3 hours. Detect OD 492 Cell survival rate = OD of experimental group 492 / blank control group OD 492 × 100%. Add different concentrations of bacterial supernatant to another group and culture for 24 hours, then repeat step 6.

[0071] The results showed that ( Figure 4 ), the culture supernatant of the bacteria had no toxicity to canine kidney epithelial cells (MDCK cells).

[0072] 2. Identification of hemolytic activity of Pseudescherichia vulneris GSE25 strain

[0073] 5% red blood cells of human, mouse, rat, rabbit and sheep were selected for hemolysis experiment. The specific operation process is as follows:

[0074] (1) Preparation of red blood cell suspension: Take 5 ml of whole blood from humans, mice, rats, rabbits and sheep respectively, and transfer the blood samples to several 1.5 ml centrifuge tubes, 1 ml per tube. Centrifuge at 1000 × g for 10 min at room temperature. After centrifugation, use a pipette to remove as much plasma as possible in the upper layer of the centrifuged product and the white blood cell layer covering the surface of the red blood cell layer to avoid contamination of the red blood cells. Subsequently, resuspend the red blood cells in phosphate buffered saline (PBS) and wash them. Repeat this process several times until the upper layer of the centrifuged product is colorless and transparent, and there is no obvious white blood cell layer. This indicates that most of the plasma and other cells have been removed. Finally, aspirate the remaining red blood cells and resuspend them in PBS to make a red blood cell suspension with a hematocrit of about 5%. Store the red blood cell suspension in a refrigerator at 4°C (can be stored for about 5 days).

[0075] (2) Preparation of bacterial suspension: Collect the shaken bacterial suspension into a centrifuge bottle, centrifuge at 8000 rpm for 10 min, collect the supernatant, filter through a 0.2 μm filter membrane and set aside.

[0076] (3) Dilution of bacterial supernatant: The filtered bacterial solution was diluted with PBS in a 2-fold gradient to 8 concentrations, ensuring that the supernatant after dilution was at least 1.1 ml.

[0077] (4) Take 20 1.5 ml EP tubes and add 100 μl of human red blood cell suspension to each tube. Then, add 100 μl of bacterial culture supernatant of different concentrations to each tube, with two replicates for each concentration. The group with only 100 μl of PBS as the blank control group and the group with 100 μl of 0.5% Triton X-100 as the positive control group were incubated in a 37°C water bath for 1 h.

[0078] After incubation, remove the EP tube and centrifuge at 1000×g for 10 min at room temperature. Then, take 175 μL of the supernatant into a 96-well plate. Then, use a multifunctional microplate reader to measure the absorbance of the supernatant at 540 nm. Hemolysis rate = (OD 540 -Blank control group OD 540 ) / positive control group OD 540 × 100%. Draw the hemolysis curve.

[0079] The results showed that ( Figure 5 ), the culture supernatant of this bacterium had no hemolytic effect on the above five types of red blood cells.

[0080] 3. (Pseudescherichia vulneris) GSE25 blood plate culture experiment

[0081] LB blood plate medium was prepared by taking blood from human, mouse, rat, rabbit and sheep, respectively. A single GSE25 colony was incubated in LB liquid medium at 25°C for 16 h, and then centrifuged at 12000 rpm for 10 min at 4°C. The bacterial cells were harvested, washed twice with sterile phosphate-buffered saline (PBS, 0.2 M, pH 7.2), and resuspended in PBS buffer to adjust the concentration to 10 8 CFU / mL. Take 20 μL of cell suspension and spread it onto a blood agar plate using the three-zone streak method. Incubate in a 25°C incubator. Observe bacterial growth and hemolysis every 12 hours and take photos. Use Vibrio vulnificus as a positive control and Escherichia coli as a negative control.

[0082] The results can be seen ( Figure 6 ), Vibrio vulnificus formed an obvious hemolytic ring on the blood agar plate, while Escherichia coli and wheat endophytes showed no hemolytic phenomenon.

[0083] 4. Plant pathogenicity test of GSE25 strain: GSE bacterial collection is the same as 3; supernatant preparation: bacterial culture steps are the same as 3, and the supernatant obtained by centrifugation is collected. To ensure that there are no bacterial cells in the supernatant, the cell-free supernatant of GSE25 culture is filtered three times with a 0.22 μm filter.

[0084] 2 μL of bacterial suspension and supernatant were inoculated into the wound of fresh wheat coleoptile and wheat ear at flowering stage. The wheat coleoptile was incubated at 25℃ for 6 days and the disease symptoms were observed ( Figure 7 A), wheat ears were incubated in a greenhouse at 25℃ for 2 weeks and the disease index was observed ( Figure 7 B). At the same time, 1 mL of bacterial suspension and supernatant were slowly injected into 5-week-old Nicotiana benthamiana leaves and cultured in a greenhouse at 25°C and 16 / 8 photoperiod. The degree of damage to the leaves was observed after 3 days. Figure 7 C) The results are as follows Figure 7 As shown in AC (GSE25: bacterial suspension, CFS: supernatant), it can be seen that the (Pseudescherichia vulneris) GSE25 strain has no pathogenicity to wheat plants and tobacco leaves.

[0085] Example 3 Growth-promoting effect of strain GSE25 (Pseudescherichia vulneris)

[0086] 200g sterile saline soil was added to a 10cm×10cm plastic pot, and 9 wheat seeds were planted with a spacing of 2.5cm. The seeds were covered with soil about 1cm. The bacterial suspension (the preparation method of the bacterial suspension was the same as that of Example 2, except that the bacterial concentration was adjusted to 10 6CFU / mL) was used to irrigate the plants, 50 mL per pot, every 5 days, and sterile water was used as a control. The growth of the plants was observed every week. After 3 weeks of cultivation, the plants were removed from the soil and the growth of the seedlings and roots was observed. Figure 8 (GSE25: bacterial suspension, CK: sterile water) As shown, it can be seen that the (Pseudescherichia vulneris) GSE25 strain has a growth-promoting effect on wheat seedlings but has no promoting effect on the root system.

[0087] Example 4 Preparation of (Pseudescherichia vulneris) GSE25 Liquid Inoculant

[0088] (1) The Pseudescherichia vulneris GSE25 strain obtained in Example 1 was cultured in an LB liquid shake flask at 25°C overnight, and then streaked on the slant surface of an LB test tube to obtain a test tube strain;

[0089] (2) Use a loop to pick the bacteria from the slant of the test tube and inoculate it into a 250 mL LB liquid shake flask. Cultivate at 25°C and 175 rpm with shaking until the logarithmic growth phase.

[0090] (3) Add 400 L of LB liquid medium to a 500 L seed tank, 121 ° C, 1.1 kg / cm 2 Sterilize with high pressure moist heat for 20 minutes, cool naturally to 20°C, inoculate the strain obtained in step (2) at 10% of the volume of the LB culture medium, and culture at 25°C with stirring until the logarithmic growth phase. The culture time is about 48 hours, and the stirring rate is 150-200 rpm. Sterile air is introduced during the culture period at a ratio of 1:0.4-0.6 to obtain a seed solution;

[0091] (4) Add 4 t of fermentation medium to a 5 t fermenter and then heat at 121°C and 1.1 kg / cm 2 Sterilize with high pressure moist heat for 20 minutes, cool naturally to below 22°C, add 10% of the volume of seed liquid, and culture at 25°C with stirring for about 48 hours at a stirring rate of 150-200 rpm. During the culture period, introduce sterile air at a ratio of 1:0.4-0.6 to obtain (Pseudescherichiavulneris) GSE25 liquid inoculum, whose bacterial content is about 10 9 pieces / mL.

[0092] Example 5 In vitro inhibition experiment of Pseudescherichia vulneris GSE25 on hyphal growth of Fusarium graminearum PH-1

[0093] The PH-1 strain of Fusarium graminearum was cultured on a PDA agar plate at 25°C for 5 days. The PH-1 hyphae were collected from the surface and cultured in CMC medium at 25°C in the dark at 175 rpm for 3 days to induce conidia. After counting with a hemocytometer, the spore concentration was adjusted to 10 with sterile water. 6 spores / mL. 1 mL of spore suspension was inoculated into 50 mL of (Pseudescherichia vulneris) GSE25 liquid inoculum (prepared in the same manner as in Example 4, except that it was diluted with sterile water to a bacterial concentration of 10 6 CFU / mL) at 25℃ for 24h, and observe the morphology of hyphae and spores under a microscope. Figure 9 As shown, it can be seen that the (Pseudescherichia vulneris) GSE25 liquid inoculant has a destructive effect on the hyphae of Fusarium graminearum PH-1, and the hyphae are abnormal in morphology, with swollen tips, nearly spherical expansion, damaged cells, and no spore production.

[0094] Example 6 In vitro inhibition experiment of Pseudescherichia vulneris GSE25 against wheat stripe rust CYR32

[0095] 5 mL of (Pseudescherichia vulneris) GSE25 liquid bacterial agent (preparation method is the same as Example 4, the difference is that it is diluted with sterile water to a bacterial concentration of 10 6 CFU / mL) was sprayed onto 9 wheat leaves at the second leaf stage infected with wheat stripe rust CYR32, and the leaves were cultured at 16°C for 12 days to observe the incidence of stripe rust on the leaves. Figure 10 (GSE25: bacterial suspension, CK: sterile water) As shown, it can be seen that (Pseudescherichia vulneris) GSE25 liquid bacterial agent has an inhibitory effect on the rust spore production of stripe rust fungus CYR32.

[0096] Example 7 Field Trial on the Prevention of Wheat Scab and Control of DON Toxin by GSE25 (Pseudescherichia vulneris)

[0097] Trial location: Caoxinzhuang Wheat Research Farm, Yangling, Shaanxi, China

[0098] Trial period: April to June 2022 and 2023

[0099] During the heading and flowering period of wheat, the experimental group of wheat ears (variety Fielder) were sprayed with (Pseudescherichia viruliferis) GSE25 liquid bacterial agent (preparation method is the same as Example 4, the difference is that it is diluted with sterile water to a bacterial concentration of 10 8CFU / mL), the control group was sprayed with tap water, and the spraying rate of both groups was 5mL / m 2 Two spraying methods were adopted. Treatment group I: 1 hour after spraying the liquid microbial agent, the spore solution of Fusarium graminearum PH-1 (10 5 / mL, spraying volume is 5mL / m 2 ); Treatment group II was sprayed with liquid microbial agent 3 days later, and then sprayed with PH-1 spore liquid of Fusarium graminearum (10 5 / mL, spraying volume is 5mL / m 2 ), after 14-18 days, the incidence of wheat scab was counted, and the content of DON and 15ADON in wheat grains of each treatment group was determined. The test results are shown in Table 1 and Figure 11 As shown in the results (GSE25: bacterial suspension, PH-1: spore liquid of Fusarium graminearum PH-1), the liquid bacterial agent (Pseudescherichia graminearum) GSE25 has a significant inhibitory effect on Fusarium graminearum PH-1.

[0100] Table 1 Field application effects of (Pseudescherichia vulneris) GSE25 on wheat scab and DON toxin control

[0101]

[0102] As can be seen from Table 1, the liquid inoculant prepared by (Pseudescherichia vulneris) GSE25 can significantly reduce the incidence of wheat fusarium head blight and has a high preventive and therapeutic effect on the occurrence of wheat fusarium head blight. Three days after spraying the inoculant, the prevention efficiency can reach 85.48%; at the same time, the deoxynivalenol (DON) toxin in the harvested wheat was reduced by 91.18%, and the 15ADON toxin content was reduced by 95.99%. It can be seen that the liquid inoculant can effectively reduce the content of deoxynivalenol in wheat.

[0103] The above embodiments are not specific limitations of the present invention. As long as the description of the claims is followed and combined with basic common sense in the field, the (Pseudescherichia vulneris) GSE25 strain is used to prevent and control the occurrence of wheat diseases, reduce the production of fungal toxins, and reduce the content of fumonisins in corn, while promoting the growth and development of crops and having a broad-spectrum effect, in addition to wheat and corn food crops, it can also be used for economic crops such as tomatoes, apples, wolfberries, and other possible unknown plants, all of which should fall within the scope of protection of the present invention.

Claims

1. A strain of Pseudescherichia vulneris GSE25, deposited in the China General Microbiological Culture Collection Center, with the deposit number CGMCC NO.26158 and the deposit date of November 21, 2022.

2. A microbial agent containing the Pseudescherichia vulneris GSE25 of claim 1.

3. The microbial agent according to claim 2, wherein The microbial preparation is a liquid or solid microbial preparation containing one or more of the following: living cells of Pseudomonas aeruginosa GSE25 according to claim 1, freeze-dried dry bacteria of Pseudomonas aeruginosa GSE25, and immobilized Pseudomonas aeruginosa GSE25 cells.

4. The microbial agent according to claim 2 or 3, characterized in that The content of Pseudescherichia vulneris GSE25 in wounds is not less than 1×10 6 CFU / mL.

5. Use of the Pseudescherichia vulneris GSE25 according to claim 1 or the microbial agent according to any one of claims 2 to 4 in promoting the growth and development of the aerial parts of wheat seedlings.

6. Use of Pseudescherichia vulneris GSE25 according to claim 1 or the microbial agent according to any one of claims 2 to 4 for controlling wheat diseases; the diseases include any one of (a) to (b): (a) plant diseases caused by Fusarium graminearum and / or stripe rust; (b) Scab and / or rust.

Images