Bacillus Velez HMF10, liquid bacterial agent and application thereof

Abstract

The present invention relates to a Bacillus Velezii ( Bacillus from Velez ) HMF10, whose deposit number is CGMCC No. 30476. This strain can effectively antagonize rice stem tip nematode and Pseudomonas syringae pv. syringae, and can effectively prevent and control rice stem tip nematode disease and rice bacterial brown spot disease.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural microorganisms, and in particular to Bacillus Velez subtilis HMF10, a liquid bacterial agent containing the Bacillus Velez subtilis HMF10 and applications thereof. Background Art

[0002] Rice stem tip nematode disease, also known as white tip disease and nematode dieback disease. The pathogen is the rice stem tip nematode ( Aphele- nchoides besseyi ). The rice dry tip nematode is a species of the genus Aphelenchus bessi, and its main host is rice, which can cause rice "dry tip" or "white tip" disease. Rice may be harmed by the rice dry tip nematode throughout its entire growth period, and the symptoms mainly occur in the leaves and panicles. When the leaves are damaged, the seedlings begin to show symptoms when they have 4 to 5 true leaves. The upper leaf tips shrank and died 2 to 4 cm apart, and were yellow-white or yellow-brown, and twisted into a twisted paper shape, the so-called "dry tip", with a clear boundary between the dead part and the green part. The top leaves of the diseased plants at the booting stage, especially the sword leaves, gradually died 1 to 8 cm from the leaf tips, turned yellow-brown and translucent, and then dried and curled into a twisted paper shape, with brown boundaries between the diseased and healthy parts; when the whole plant is damaged, most plants can head normally, and the affected rice plants grow weak, short, short panicles, and many barren grains. Rice with rice dry tip nematode disease generally reduces yield by 10% to 20%, and in severe cases, it can reach more than 30%.

[0003] Rice dry tip nematodes hibernate in the husks and between grains as adults and larvae, and seeds with worms are the main source of initial infection. Nematode adults and larvae have strong survival ability under dry conditions and can survive for about 3 years in dry rice seeds. They are cold-resistant but not heat-resistant. The optimum temperature for activity is 20-26℃, the critical temperature is 13℃ and 42℃, and the lethal temperature is 54℃ for 5min or 44℃ for 4h or 42℃ for 16h. Nematodes need 70% relative humidity for normal development, are very active in water, and can survive for about 30 days.

[0004] The main methods for preventing and controlling rice stem tip nematode disease are: (1) Select disease-free seeds, strengthen quarantine, and strictly prohibit the transportation of seeds from diseased areas. (2) Soak seeds in warm water or with chemicals. At present, rice that develops stem tip nematode disease after transplanting can only be controlled by spraying with chemicals, such as avermectin, methamectin, miltiorrhiza, and chlorpyrifos, but the control effect is poor.

[0005] Rice bacterial brown spot disease is caused by Pseudomonas syringae pv. syringae ( Pseudomonas syringae pv.syringae) is a disease caused by and occurring in rice, mainly affecting leaves, leaf sheaths, stems, nodes, ears and grains. In the seedling stage, it starts from the leaf tips and leaf edges, and gradually spreads to the entire leaf. The lesions are initially small brown water-soaked spots, and then expand into spindle-shaped, oblong or irregular stripes. Finally, the center of the lesion turns gray-brown, and the tissue necrotizes, but there is no perforation. In recent years, bacterial brown spot disease of rice has been common in Hebei Province and the Northeast, especially in plots planted with repeated crops. The disease spreads quickly and infects a wide range. After the disease becomes prevalent, if timely protective measures are not taken, it can easily threaten the healthy growth of rice and affect the yield and quality of rice. Summary of the invention

[0006] The invention aims to provide a Bacillus velez HMF10 which can effectively antagonize rice stem sharp nematodes and Pseudomonas syringae pv. syringae and application thereof.

[0007] The present invention adopts the following technical solution:

[0008] A species of Bacillus velez Bacillus velezensis ) HMF10, with the deposit number CGMCC No.30476, was deposited in the China General Microbiological Culture Collection Center, Beijing, China on April 30, 2024.

[0009] Furthermore, the Bacillus Velezii HMF10 can antagonize rice stem nematodes.

[0010] Furthermore, the Bacillus Velezii HMF10 can antagonize Pseudomonas syringae pv. syringae.

[0011] Furthermore, the Bacillus Velezii HMF10 can simultaneously antagonize rice stem nematodes and Pseudomonas syringae pv. syringae.

[0012] A microbial agent comprising the above-mentioned Bacillus Velezii HMF10 bacterial body, spores, fermentation liquid and / or extracellular metabolites.

[0013] In the microbial agent, the viable count of the Bacillus Velezii HMF10 is not less than 3.0×10 7 cfu / mL. Preferably, the viable count of Bacillus Velezii HMF10 is 3.0×10 7 ~6.0×10 7 cfu / mL.

[0014] Microbial agents also include preservatives, UV protectants and dispersants.

[0015] Preferably, the preservatives are ketone and methylparaben, the UV protective agent is sorbitol, and the dispersant is Tween-20.

[0016] Furthermore, the amount of kaison added is 0.1‰~0.3‰ of the mass of the fermentation broth, the amount of methyl paraben added is 0.05%~0.2% of the mass of the fermentation broth, the amount of sorbitol added is 0.5%~2.0% of the mass of the fermentation broth, and the amount of Tween-20 added is 0.05%~0.2% of the mass of the fermentation broth.

[0017] Furthermore, the microbial agent is a liquid agent.

[0018] An application of the above-mentioned Bacillus Velezii HMF10 in preventing and controlling rice stem tip nematode disease.

[0019] An application of the above-mentioned Bacillus Velez subtilis HMF10 in preventing and controlling rice bacterial brown spot disease.

[0020] The beneficial effects of the present invention are as follows: the Bacillus Velez HMF10 strain of the present invention can effectively antagonize rice stem tip nematodes and effectively antagonize Pseudomonas syringae pv. syringae. The liquid microbial agent containing Bacillus Velez HMF10 bacteria, spores and / or extracellular metabolites can effectively prevent and control rice stem tip nematode disease caused by rice stem tip nematodes, prevent and control rice bacterial brown spot disease caused by Pseudomonas syringae pv. syringae, effectively reduce various hazards of rice, and is suitable for large-scale production and application. Compared with the blank control, the Bacillus Velez HMF10 liquid microbial agent of the present invention has a control effect of more than 80.50% on rice stem tip nematode disease, and increases rice yield by more than 5.65% per mu; the control effect of rice bacterial brown spot disease is 72.86%, and increases rice yield by more than 3.62% per mu. Compared with the conventional control, the control effect of Bacillus Velez HMF10 liquid microbial agent on rice stem tip nematode disease increased by more than 24.77%, and the per-acre yield increased by more than 1.78%; the control effect on rice bacterial brown spot disease increased by more than 21.43%, and the per-acre yield increased by more than 0.93%. Therefore, the control effect of Bacillus Velez HMF10 liquid microbial agent is significantly better than that of commercially available pesticide preparations, and it can replace pesticides for promotion, which is of great significance to increasing farmers' income and protecting people's health. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The toxic effect of Bacillus Velez HMF10 suspension on rice stem nematode.

[0022] Figure 2 This is the colony morphology of Bacillus velez HMF10 cultured on solid LB medium for 48 hours.

[0023] Figure 3 The figure shows the morphology of Bacillus velezensis HMF10 under an optical microscope after Gram staining.

[0024] Figure 4Phylogenetic tree constructed based on the 16S rDNA sequence of Bacillus velez HMF10.

[0025] Figure 5 This is a confrontation test between Bacillus Velez HMF10 and Pseudomonas syringae pv. syringae (LB medium, the right picture is the control, and the left picture is the confrontation result). DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with the embodiments and drawings. The protection scope of the present invention is not limited to the embodiments, and any changes made by those skilled in the art within the scope defined by the claims also fall within the protection scope of the present invention.

[0027] The experimental methods in the following examples are conventional methods unless otherwise specified. The reagents used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0028] Example 1 Isolation, purification and screening of strains

[0029] Six soil samples were collected from the rice fields of Qilizhuang Village, Ruhe Township, Changli County, Hebei Province, with 100 g of each sample. The six soil samples were evenly mixed.

[0030] Prepare LB solid medium: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g NaCl, 18 g agar, 1000 mL distilled water, adjust pH to 7.2-7.4, sterilize at 121 °C for 20 min before use. (Do not add agar when preparing LB liquid medium).

[0031] Take 10g of the mixed soil sample and add it to 100mL of distilled water and stir evenly. After standing for 1min, take 1mL of the supernatant and add it to a test tube containing 9mL of sterile water to obtain 1×10 -1 Diluent, gradient dilution, dilute to 1×10 -4 , 1×10 -5 , 1×10 -6 Concentration, from 1×10 -4 , 1×10 -5 , 1×10 -6 Take 0.1 μL of the diluted solution of the concentration and spread it on the LB plate, spread it evenly, and place it in a 30℃ constant temperature incubator for 2-4 days, and observe the growth of the colonies regularly. After the colonies grow, 3 purifications are performed on LB solid medium, and finally 5 strains are selected and numbered as 1, 2, 3, 4, and 5, respectively, and stored in a 4℃ refrigerator for future use.

[0032] The toxicity test of different strains was carried out with the larvae of rice stem nematode as the target, and the strain with the highest corrected mortality rate was screened out.

[0033] The specific method is: first prepare different strains into effective viable bacteria with an effective count of 1.0×10 7 cfu / mL of bacterial suspension, then take out the rice dry sharp nematode larvae suspension with a content of 200 cfu / mL prepared in advance, and finally mix 0.5mL of bacterial suspension and 0.1mL of larvae suspension in a 24-well plate, place it in a constant temperature incubator at 28℃, and count the number of dead rice dry sharp nematode larvae after 24 hours (such as Figure 1 The dead nematodes were stiff, and the surviving nematodes were bent). An equal amount of sterile water was added instead of the bacterial suspension as a blank control. The experiment was repeated three times, and the nematode mortality rate and the adjusted mortality rate were calculated. The adjusted mortality rate results were averaged.

[0034] The formula for calculating nematode mortality and corrected mortality is:

[0035] Nematode mortality (%) = Ca÷Ta×100.

[0036] Among them, Ca represents the number of nematodes that died in the treatment, and Ta represents the total number of nematodes in the treatment.

[0037] Corrected nematode mortality (%) = (treated nematode mortality - control nematode mortality) / (1 - control nematode mortality) × 100.

[0038] Table 1 shows the results of the mortality test of the larvae of rice stem-tipped nematodes killed by suspensions of 5 different strains. As shown in Table 2, the corrected mortality of the suspension of strain No. 4 to kill rice stem-tipped nematodes was the highest, at 95.15%, and was named HMF10.

[0039] Table 1 Results of the mortality test of different strains of suspensions against rice stem nematode larvae

[0040] .

[0041] Table 2 Calculation results of corrected mortality rates of different strains of suspensions killing rice stem nematode larvae

[0042] .

[0043] Example 2 Determination of the genetic stability of antagonistic strains

[0044] The strain HMF10 was subcultured on LB medium by streaking method. After 15 generations, the toxicity of the subcultured HMF10 strain to rice stem nematode was determined by toxicity correction mortality method. The results showed that the original HMF10 strain had the same toxicity as the original strain after 15 generations of culture, indicating that the strain had good genetic stability. The bacterial beads were cultured in LB liquid medium, and the bacterial solution was aspirated and stored in glycerol at -80℃.

[0045] Example 3 Identification of strain HMF10

[0046] (1) Microbiological characteristics

[0047] The colony morphology of strain HMF10 after constant temperature culture on LB plate at 30℃ for 3 days is as follows Figure 2 As shown in the figure, the single colony of this strain is milky white, with a rough surface and irregular edges. Under an optical microscope, the bacteria are rod-shaped, with a size of 0.5μm × (1.5-3.5μm), and the spores are oval, mid-spore, and the end is swollen into a sporangium; Gram staining is positive, such as Figure 3 According to the Bergey's Manual of Bacterial Identification, the strain was morphologically identified and it was preliminarily determined that strain HMF10 belonged to Bacillus.

[0048] (2) Physiological and biochemical characteristics

[0049] The physiological and biochemical characteristics of strain HMF10 were identified, and the results are shown in Table 3.

[0050] Table 3 Physiological and biochemical characteristics of strain HMF10

[0051] .

[0052] (3) Molecular biological characteristics

[0053] The genomic DNA of strain HMF10 was extracted and used as a template to perform PCR amplification using universal primers for bacterial 16S rDNA. The amplified product was recovered and sequenced to obtain a DNA sequence containing 767 bp (as shown in SEQ ID No. 1). The sequencing results were entered into the GeneBank database for BLAST comparison analysis. The 16S rDNA sequence was compared with the NCBI database, and it was found that the consistency between HMF10 and Bacillus Velezii reached 99%. The results of constructing the phylogenetic tree are shown in the following figure. Figure 4 According to the strain morphology, sequencing analysis and physiological and biochemical test results, HMF10 was identified as Bacillus velezinis ( Bacillus velezensis ).

[0054] Bacillus velez HMF10 was deposited in the China General Microbiological Culture Collection Center on April 30, 2024, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the culture collection number CGMCC No.30476.

[0055] Example 4 Confrontation test between Bacillus velez HMF10 and Pseudomonas syringae pv. syringae

[0056] Preparation of PDA solid culture medium: 200 g fresh potatoes (peeled), boil for 20 min, filter, add 20 g glucose, 20 g agar powder, add distilled water to make up to 1000 mL, sterilize at 121 °C for 30 min, and set aside.

[0057] Preparation of LB solid culture medium: Same as Example 1.

[0058] Take out the stored rice bacterial brown spot pathogen (Pseudomonas syringae pv. syringae) slant from the refrigerator, pick up 1 loop with an inoculation loop, streak it on the LB medium plate, and activate and culture it in an incubator at 37°C for 3 days.

[0059] Confrontation test between Bacillus Velez and Pseudomonas syringae pv. syringae: Pseudomonas syringae pv. syringae bacteria beads were mixed in the dissolved LB solid medium and poured into a plate, then a 5mm Bacillus Velez bacterial cake was inoculated in the middle of the medium, and the LB solid plate inoculated with only Pseudomonas syringae pv. syringae was used as a control, and the plate was placed in a 37℃ incubator for 72h to observe the antagonistic effect of Bacillus Velez strains on Pseudomonas syringae pv. syringae and detect the diameter of the inhibition zone (mm). The test was repeated three times and the results were averaged.

[0060] Judgment criteria: The diameter of the inhibition zone is greater than or equal to 10 mm, indicating a strong antagonistic effect.

[0061] Depend on Figure 5 As shown in Table 4, the diameter of the inhibition zone of strain HMF10 against Pseudomonas syringae pv. syringae is greater than 11.5 mm, indicating that strain HMF10 has a strong inhibitory effect on Pseudomonas syringae pv. syringae.

[0062] Table 4 Results of confrontation test between strain HMF10 and Pseudomonas syringae pv. syringae

[0063] .

[0064] Example 5 Preparation of liquid bacterial agent of Bacillus velez HMF10

[0065] The specific steps are:

[0066] (1) Preparation of LB liquid culture medium: (same as Example 1).

[0067] (2) Activation of strains: Pick 2 loops of HMF10 colonies from the preserved test tube, inoculate them into a test tube containing 20 mL of LB liquid culture medium, and culture them at a constant temperature of 180 rpm and 37°C for 12 h for activation.

[0068] (3) Preparation of seed solution: 7.5 mL of the activated bacterial solution was inoculated into a 1000 mL Erlenmeyer flask containing 250 mL of LB liquid culture medium. The culture was shaken at 180 rpm and 37°C for 48 h to obtain the seed solution.

[0069] (4) Preparation of fermentation broth: The prepared seed liquid was inoculated into a 6-L small fermentation tank containing 4.2 L of LB liquid culture medium, and cultured at a constant temperature of 180 rpm and 37°C for 48 h to obtain HMF10 fermentation broth.

[0070] (5) Add 0.2‰ of the fermentation broth mass of kaison, 0.1% of methyl paraben, 1.0% of sorbitol and 0.1% of Tween-20 to the HMF10 fermentation broth in turn, and stir for 10 minutes to mix evenly to obtain a liquid inoculum of Bacillus velezensis. The effective viable count was 3.0×10 9 cfu / mL.

[0071] Examples 6-9 Field test of the effectiveness of liquid bacterial agents against rice stem nematode disease

[0072] From May to October 2023, a test on the efficacy of liquid microbial agents on rice stem tip nematode disease was conducted in Qilizhuang Village, Changli County, Hebei Province. The rice variety was "Liaoyou 16". The test was set up with 6 treatments. Examples 6 to 9 were samples after dilution of the liquid microbial agents, and their final concentrations were: 3.0×10 7 cfu / mL, 4.0×10 7 cfu / mL, 5.0×10 7 cfu / mL, 6.0×10 7 cfu / mL, the rice seeds were soaked for 24 hours in each treatment, spread out and dried, and then germinated and sown; CK1 was the conventional control, the seeds were soaked for 15 minutes with a conventional dose of nematode, spread out and dried, and then germinated and sown; CK0 was the blank control, the seeds were soaked in water for 24 hours, spread out and dried, and then germinated and sown. After seedlings were raised, they were planted in rice fields, and each treatment was repeated 3 times. The incidence and control effects of rice dry tip nematode disease in different treatments were investigated at the booting stage, and the rice yield was investigated at the harvest stage.

[0073] Prevention effect investigation method: The diagonal five-point sampling method was used at the booting stage, with 20 plants sampled at each point and a total of 100 plants sampled for each treatment. If the rice plants showed symptoms of "dry tip nematode disease", they were considered to be diseased. The total number of investigated plants and the total number of diseased plants were recorded, and the incidence rate and prevention effect were calculated.

[0074] Calculation method of protection effect:

[0075] Disease rate (%) = number of diseased plants / total number of investigated plants × 100;

[0076] Control effect (%) = [(the diseased plant rate of the clean water control - the diseased plant rate of the pesticide treatment) / the diseased plant rate of the clean water control] × 100.

[0077] Yield survey method: Each treatment adopts 5-point sampling method, and each point is surveyed for 1m 2 The yield is calculated per mu (667m 2 ) output and calculate the yield increase rate.

[0078] Yield increase rate (%) = (yield in the control area - yield in the control area) / yield in the control area × 100.

[0079] The control effect is shown in Table 5. The liquid microbial agent has a good control effect on rice dry tip nematode disease. The incidence of dry tip nematode disease in rice soaked with liquid microbial agent is significantly reduced. Compared with the blank control, the disease control effect of different embodiments is more than 80.50%, and the rice yield per mu is increased by more than 5.65%; compared with the conventional control, the disease control effect of different embodiments is increased by more than 24.77%, and the rice yield per mu is increased by more than 1.78%. When the final concentration of the agent is 6.0×10 7 cfu / mL, the disease prevention effect increased slowly, and the optimal application concentration was determined to be 3.0×10 7 ~6.0×10 7 cfu / mL.

[0080] Table 5 Effects of different treatments on the control of rice stem nematode disease and yield

[0081] .

[0082] Examples 10-13 Test on the efficacy of liquid bacterial agent on rice bacterial brown spot disease

[0083] From May to October 2023, a field test on rice bacterial brown spot was conducted in Dapuhe Village, Changli County, Hebei Province. The rice variety was "Golden Xiang No. 3". The test set up 6 treatments. Examples 10 to 13 were samples after dilution of liquid bacterial agents, and their final concentrations were: 3.0×10 7 cfu / mL, 4.0×10 7 cfu / mL, 5.0×10 7 cfu / mL, 6.0×10 7 cfu / mL, the four treatments were sprayed 10 days after rice planting, at the end of jointing and at the end of booting, with the leaves moist but not dripping; the conventional control (CK1) used 25% chlorothalonil wettable powder (100 g / mu) as the control drug; the blank control (CK0) did not apply the control drug but applied the same weight of water as the example group; each treatment was repeated 3 times, the incidence and control effect of rice bacterial brown spot disease in each treatment were investigated, and the rice yield was investigated during the harvest period.

[0084] Prevention effectiveness investigation method: During the filling period, the diagonal five-point sampling method was used, with 5 plants selected at each point and a total of 75 plants sampled for each treatment. The total number of investigated plants and the total number of diseased plants were recorded, and the incidence rate, disease index and prevention and control effect were calculated.

[0085] Grading standard for bacterial brown spot disease of rice: based on intact leaves, grade 0 means no lesions; grade 1 means the lesion area is less than 10% of the leaf area; grade 3 means the lesion area is 11% to 25% of the leaf area; grade 5 means the lesion area is 26% to 45% of the leaf area; grade 7 means the lesion area is 46% to 59% of the leaf area; grade 9 means the lesion area is more than 60% of the leaf area.

[0086] Calculation method of protection effect:

[0087] Incidence rate (%) = total number of diseased plants / total number of investigated plants × 100;

[0088] Disease index (%) = ∑ (number of diseased plants × the diseased plant level value) / (total number of investigated plants × the highest level value) × 100;

[0089] Control effect (%) = (disease index of control area - disease index of treated area) / disease index of control area × 100.

[0090] Rice yield survey method: same as in Examples 6-9.

[0091] The control effect is shown in Table 6. The liquid microbial agents of different concentrations in the embodiments have good control effects on rice bacterial brown spot disease, and the incidence of rice bacterial brown spot disease plants is controlled below 7.7%. Compared with the blank control, the disease control effects of the different embodiments are all above 72.86%, and the rice yield per mu is increased by more than 3.62%; compared with the conventional control, the disease control effects of the different embodiments are increased by more than 21.43%, and the rice yield per mu is increased by more than 0.93%. When the final concentration of the microbial agent is 6.0×10 7 cfu / mL, the disease prevention effect increased slowly, and the optimal application concentration was determined to be 3.0×10 7 ~6.0×10 7 cfu / mL.

[0092] Table 6 Effects of different treatments on control of rice bacterial brown spot and yield

[0093] .

[0094] The Bacillus Velez HMF10 strain of the present invention has a strong antagonistic effect on rice stem tip nematodes and Pseudomonas syringae pv. syringae. The liquid bacterial agent of Bacillus Velez HMF10 has a good control effect on rice stem tip nematode disease caused by rice stem tip nematodes and rice bacterial brown spot disease caused by Pseudomonas syringae pv. syringae, and both are better than the agent control.

[0095] The above are only preferred embodiments of the present invention, rather than limitations on its protection scope. Any improvements made to the present invention by those skilled in the art without creative work should be considered to be within the protection scope of the present invention.

Claims

1. A Bacillus Velezii ( Bacillus velezensis ) HMF10, characterized in that The deposit number is CGMCC No.30476.

2. The Bacillus Velezii HMF10 according to claim 1, characterized in that It can antagonize rice stem-tipped nematodes.

3. The Bacillus Velezii HMF10 according to claim 1, characterized in that It can antagonize Pseudomonas syringae pv. syringae.

4. The Bacillus Velezii HMF10 according to claim 1, characterized in that It can simultaneously antagonize rice stem-tipped nematodes and Pseudomonas syringae pv. syringae.

5. A microbial agent, characterized in that: It comprises the bacterial body, spore or fermentation liquid of Bacillus Velez subtilis HMF10 according to claim 1.

6. The microbial agent according to claim 5, characterized in that: The viable count of the Bacillus Velez HMF10 is not less than 3.0×10 7 cfu / mL.

7. The microbial agent according to claim 5, characterized in that: It also includes preservatives, UV protectants and dispersants.

8. The microbial agent according to claim 5, characterized in that: It is a liquid bacterial agent.

9. Use of the Bacillus Velezii HMF10 according to claim 1 in preventing and controlling rice stem tip nematode disease and / or rice bacterial brown spot disease.

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