A strain of Paenibacillus polymyxa and its suspension and application

Abstract

The present invention provides a strain of Paenibacillus polymyxa, which is "Paenibacillus polymyxa Paenibacillus polymyxa Pp‑HK11‑9” was deposited in the “General Microbiology Center of China National Microbiological Culture Collection Committee” on April 1, 2022, with the deposit number of “CGMCC No.24634”. The present invention provides a polymyxa suspension, which comprises a sterile buffer solution and polymyxa bacterial bodies uniformly dispersed in the sterile buffer solution. The present invention also provides an application of the polymyxa suspension. The polymyxa suspension can strongly inhibit the growth of pathogenic hyphae and conidia germination, prevent pathogenic bacteria from invading, improve the immunity of plants, prevent and control cucumber coryspore leaf spot, powdery mildew and downy mildew, promote plant growth, and help improve yield and quality.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a strain of Paenibacillus polymyxa and a suspension thereof and application thereof. Background Art

[0002] There are many beneficial microorganisms in nature that can survive and reproduce by attaching themselves to plants, produce antagonistic active substances, hormone-like substances, siderophores, etc., have functions such as dissolving phosphorus, potassium, and nitrogen fixation, directly or indirectly participate in plant growth and development, promote plant growth, enhance plant resistance, and prevent and control plant diseases.

[0003] Paenibacillus polymyxa is a type of facultative anaerobic Gram-positive bacteria with peritrichous flagella that is widely found in nature. It can produce spores with strong resistance when encountering adverse environments. Among them, beneficial strains with the ability to promote growth and prevent diseases can effectively colonize in plants, and have the effects of promoting growth, preventing diseases, increasing production and improving quality. Research and development and application in agricultural production can reduce the use of pesticides and fertilizers, reduce environmental pollution and pesticide residues, increase production and quality, and provide a favorable path for the sustainable development of green agriculture. The application prospects are very broad.

[0004] The invention patent application with application number 202111390742.6 discloses a method for promoting vegetable growth and disease resistance using Paenibacillus polymyxa AF01, and specifically discloses a Paenibacillus polymyxa AF01 that has an inhibitory effect on tomato black spot pathogen, tomato anthracnose pathogen, cucumber target spot pathogen, and cucumber wilt pathogen, and also has a good preventive effect on powdery mildew of bitter melon. However, the Paenibacillus polymyxa only records the antibacterial effect under the plate experiment, and does not disclose its application and control effect in farmland production. The invention patent application with application number 201710623589.4 discloses a Paenibacillus polymyxa and its application in the prevention and control of cucumber powdery mildew and downy mildew, but does not disclose that the strain can also simultaneously prevent and control Coryspore leaf spot disease. At the same time, there are few reports that existing Paenibacillus polymyxa has the effect of preventing and controlling more than two leaf fungal diseases in the field at the same time. Summary of the invention

[0005] In view of this, the present invention provides a strain of Paenibacillus polymyxa and a suspension and application thereof to solve the above problems.

[0006] The present invention provides a strain of Paenibacillus polymyxa, which is "Paenibacillus polymyxa Pp-HK11-9", which was deposited in the "General Microbiology Center of China Microorganism Culture Collection Committee" on April 1, 2022, and the deposit number is "CGMCC No.24634". The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101. The Paenibacillus polymyxa Pp-HK11-9 strain was obtained by isolation and screening from the rhizosphere of Coreopsis plants collected in Jigong Mountain, Xinyang, Henan on November 11, 2008, and can also be cultured using TSA medium.

[0007] The invention also provides a polymyxa bacillus suspension, which comprises a sterile buffer solution and the polymyxa bacillus cells uniformly dispersed in the buffer solution.

[0008] Based on the above, the formula of the sterile buffer solution is: glycerol 0.01mL~0.1mL, (NH4)2SO4 0.01g~0.1g, K2HPO4 0.07g~0.1g, KH2PO4 0.02g~0.1g, MgSO4 0.001g~0.1g, and distilled water 1000mL.

[0009] Based on the above, in the polymyxa suspension, the concentration of the polymyxa bacillus cells is (4-6)×10 8 CFU / mL.

[0010] The invention also provides an application of the Paenibacillus polymyxa suspension, wherein the Paenibacillus polymyxa suspension is used for preventing and controlling coryspore leaf spot, powdery mildew and downy mildew of cucumber.

[0011] The invention also provides an application method of the above-mentioned Paenibacillus polymyxa suspension, wherein the Paenibacillus polymyxa suspension is diluted 10 to 50 times and then sprayed on leaves for preventing and controlling cucumber coryspore leaf spot, powdery mildew and downy mildew.

[0012] Among them, the Bacillus polymyxa suspension is diluted 10 to 50 times and then sprayed on the leaves; in the early stage of diseases such as powdery mildew, downy mildew and coryneformis leaf spot of melons, spray once every 7 days, for a total of 3 to 4 times; when the powdery mildew, downy mildew and coryneformis leaf spot of melons are serious, spray once every 4 days, for a total of 4 to 5 times.

[0013] The present invention also provides a biological preparation, comprising the uniformly mixed Paenibacillus polymyxa suspension and a premix, wherein the premix is ​​formed by uniformly mixing lecithin, sunflower oil and distilled water.

[0014] Based on the above, the volume ratio of the Bacillus polymyxa suspension to the premix is ​​1:(5-15); in the premix, the mass ratio of lecithin, sunflower oil and distilled water is (3-10):(10-50):(30-100).

[0015] Specifically, the preparation method of the premix is: weigh soybean lecithin, sunflower oil and distilled water respectively according to the mass ratio of lecithin, sunflower oil and distilled water being (3-10): (10-50): (30-100) and mix them for 3-5 minutes using a wall-breaking machine. The main reason for using the wall-breaking machine for mixing is that the oil particles formed by soybean lecithin and sunflower oil in water are relatively large, and the wall-breaking machine is used to break the oil particles into small particles.

[0016] The invention also provides an application of the biological agent, which is used for preventing and controlling cucumber coryspore leaf spot, powdery mildew and downy mildew.

[0017] Based on the above, the biological agent is diluted 100 to 200 times and used as a foliar spray for cucurbit leaf diseases.

[0018] Among them, the above-mentioned biological preparations are diluted 100 to 200 times and then sprayed on the leaves; in the early stage of diseases such as powdery mildew, downy mildew and coryneformis leaf spot of melons, spray once every 7 days, for a total of 3 to 4 times; when the powdery mildew, downy mildew and coryneformis leaf spot of melons are serious, spray once every 4 days, for a total of 4 to 5 times.

[0019] The polymyxa bacillus Pp-HK11-9 strain provided by the present invention has a relatively wide antibacterial spectrum, and can inhibit a variety of pathogens such as cucumber coryneform leaf spot, tomato leaf mold, gray mold, celery early blight, tomato early blight, wilt, pepper blight, wheat root rot flat navel worm spores, etc. In particular, it has high antibacterial activity against coryneform mold, can strongly inhibit the hyphae growth and spore germination of coryneform leaf spot; and has stable colonization ability in cucumber leaves. At the same time, experiments show that the suspension of the polymyxa bacillus Pp-HK11-9 strain can achieve a control effect of more than 50% on cucumber coryneform leaf spot, powdery mildew and downy mildew, and can be actually applied in agricultural production.

[0020] Therefore, the polymyxa bacillus suspension provided by the present invention has the effect of preventing and treating cucumber coryneform leaf spot, powdery mildew and downy mildew. Further, experiments have shown that the biological preparation mainly composed of the polymyxa bacillus suspension and the premix can significantly improve the effect of preventing and treating cucumber coryneform leaf spot, powdery mildew and downy mildew, and only needs to be sprayed on the leaves during application. The application method is simple and easy to be applied in agricultural production, and can also improve the immunity of cucumber, which is beneficial to improve yield and quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The present invention provides the colony morphology of the Paenibacillus polymyxa Pp-HK11-9.

[0022] Figure 2 This is the phylogenetic tree of Pp-HK11-9 constructed based on the 16Sr DNA sequencing results. The numbers on the branches represent the confidence levels.

[0023] Figure 3 This is a diagram of the biological characteristics of the Paenibacillus polymyxa Pp-HK11-9 provided by the present invention.

[0024] Figure 4 This is the antibacterial spectrum of the polymyxa bacillus Pp-HK11-9 provided by the present invention against plant pathogens.

[0025] Figure 5 The invention discloses an inhibitory effect of the Paenibacillus polymyxa Pp-HK11-9 suspension on the conidia germination of cucumber Corynespora leaf spot pathogen.

[0026] Figure 6 This is a graph showing the control effect of Bacillus polymyxa suspension (Pp-HK1-9) and biological agent (LSO-Pp-HK11-9) on cucumber Coryspore leaf spot under in vitro inoculation conditions.

[0027] Figure 7 This is a graph showing the control effect of Bacillus polymyxa suspension (Pp-HK1-9) and biological agent (LSO-Pp-HK11-9) on cucumber Coryspore leaf spot under live inoculation conditions.

[0028] Figure 8 The colonization density of Paenibacillus polymyxa Pp-HK11-9 strain on cucumber leaves treated with Paenibacillus polymyxa suspension (Pp-HK1-9) and biological agent (LSO-Pp-HK11-9). DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described in detail below through specific implementation methods.

[0030] Example 1

[0031] The present invention provides a strain of Paenibacillus polymyxa, which is "Paenibacillus polymyxa Pp-HK11-9", which was deposited in the "General Microbiological Center of China Microbiological Culture Collection Committee" on April 1, 2022, with a deposit number of "CGMCC No.24634". The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101. The Paenibacillus polymyxa Pp-HK11-9 strain was isolated and screened from the rhizosphere of Coreopsis plants collected in Jigong Mountain, Xinyang, Henan on November 11, 2008.

[0032] Strain screening: 1-2 g of the roots of healthy Coreopsis tinctoria with soil were collected from Jigong Mountain in Xinyang, Henan Province, crushed with a mortar, mixed and cultured in 100 mL of sterile water for 30 minutes, and then treated in a constant temperature water bath at 80°C for 20 minutes, diluted with sterile water to 1000-10000 times, and the diluted mixture was evenly spread on a 1 / 10 TSA medium plate, inverted and placed in a constant temperature incubator at 30°C for 48-72 hours. After the strain grows, bacterial colonies of different sizes, colors and shapes are picked up with a sterile toothpick, and separated and purified by streaking on a TSA medium plate. The purified strains were cultured against the leaf spot pathogen of Coryneformis, and the polymyxa bacillus Pp-HK11-9 with antagonistic activity against the leaf spot pathogen of Coryneformis was screened out, and the polymyxa bacillus Pp-HK11-9 strain was obtained by gradually increasing the induction concentration to obtain stable resistance to rifampicin (100 μL·mL -1 ) strains and stored them in a -80℃ ultra-low temperature freezer.

[0033] Identification of strains: The Pp-HK11-9 strain was cultured in TSA medium at 28-32°C. The colonies on the TSA medium were milky white with jagged edges and were opaque. After 3-5 days of culture, the colonies formed Figure 1 At the same time, the morphological characteristics and physiological and biochemical properties of Paenibacillus polymyxa Pp-HK11-9 were identified, as shown in Table 1.

[0034] Table 1 Morphological characteristics and physiological and biochemical properties of Paenibacillus polymyxa Pp-HK11-9

[0035]

[0036]

[0037] As can be seen from Table 1: Pp-HK11-9 bacteria are straight rod-shaped, Gram staining reaction is positive, spores are produced, and they are motile; the growth temperature range is 4-50°C, the optimum growth temperature is 30°C, the optimum pH is 6.2-7.5, the highest salt tolerance is 4%, sucrose, glucose, glycerol, galactose, fructose, L-arabinose and sorbitol can be utilized, nitrite reduction, nitrate reaction, starch hydrolysis and catalase reaction are positive, no hydrogen sulfide is produced, and gelatin liquefaction is negative. Glucose oxidation fermentation is a fermentation type.

[0038] like Figure 2 As shown, the full length of the 16S rDNA sequence of the bacterium is 1449 bp, and the similarity with the 16S rDNA sequence of Paenibacillus polymyxa is 99.72%, so the strain is identified as Paenibacillus polymyxa Pp-HK11-9.

[0039] The biological function of the strain: The plate confrontation method was used to study the antibacterial effect of Paenibacillus polymyxa Pp-HK11-9. The results are shown in Table 2 and Figure 4 shown.

[0040] Table 2 Antibacterial ability of Paenibacillus polymyxa Pp-HK11-9 against plant pathogens

[0041]

[0042]

[0043] From Table 2 and Figure 4 It can be seen that the polymyxa bacillus Pp-HK11-9 provided by the present invention has a relatively wide antibacterial spectrum, and can inhibit a variety of pathogens such as cucumber corynespora leaf spot pathogen (Corynespora cassiicola), tomato leaf mold pathogen (Fulvia fulva), tomato gray mold pathogen (Botrytis cineria), celery early blight pathogen (Cercospora apci), tomato early blight pathogen (Alternaria solani), wilt pathogen (Fusarium oxysporum), pepper blight pathogen (Phytophthora capsici), and wheat root rot flat navel worm spore (Bipolaris sorokiniana). In particular, the antibacterial activity against corynespora (cucumber corynespora leaf spot pathogen) is high.

[0044] In addition, from Figure 3It can be seen that the Bacillus polymyxa Pp-HK11-9 strain has the functions of decomposing starch, cellulose, protein, and producing siderophore, which is beneficial to its disease prevention and growth promotion role.

[0045] Example 2

[0046] This embodiment provides a polymyxa suspension, which includes a sterile buffer solution and the polymyxa cells of the polymyxa in Example 1 uniformly dispersed in the sterile buffer solution. In this embodiment, the concentration of the polymyxa is 6×10 8 CFU / mL.

[0047] The formula of the sterile buffer solution is: 0.01 mL of glycerol, 0.01 g of (NH4)2SO4, 0.07 g of K2HPO4, 0.02 g of KH2PO4, 0.001 g of MgSO4, and 1000 mL of distilled water.

[0048] Specifically, the preparation method of the polymyxa bacillus suspension comprises the following steps:

[0049] The polymyxa bacillus strain provided in Example 1 was cultured in TSA medium to obtain the polymyxa bacillus Pp-HK11-9 strain; the polymyxa bacillus Pp-HK11-9 strain was inoculated into a fermentation medium, and fermented at 32° C. for 48 hours to obtain the polymyxa bacillus fermentation liquid;

[0050] The formula of the fermentation medium includes 20 g of peptone, 5 g of yeast extract, 0.5 g of NaCl, 10 mL of 0.25 mol / L KCl, 5 mL of 2 mol / L MgCl2, 20 mL of 1 mol / L glucose and 1000 mL of distilled water, and the pH value of the fermentation medium is 7.0;

[0051] The polymyxa Paenibacillus fermentation broth is centrifuged to obtain the polymyxa Paenibacillus Pp-HK11-9 bacterial cells; the polymyxa Paenibacillus Pp-HK11-9 bacterial cells are diluted with a sterile buffer solution to obtain the polymyxa Paenibacillus suspension.

[0052] The experiment showed that the polymyxa suspension had an inhibitory effect on the germination of conidia of cucumber coryneformis. The specific test method is as follows:

[0053] The hanging drop method was used to test the conidia germination of cucumber coryneformis in "sterile water" and "Pp-HK11-9 suspension". Equal amounts of cucumber coryneformis conidia suspension and Pp-HK11-9 suspension were mixed evenly, dropped onto a concave glass slide and placed in a 28°C constant temperature incubator for moisturizing culture. The spore germination was observed under a microscope every 2 hours, and the spore germination rate was calculated. A total of 3 fields of view were observed each time, with approximately 100 conidia in each field of view. The results are as follows: Figure 5 As shown in the figure, "sterile water" in the figure is a mixture of the conidia suspension of cucumber coryneformis and sterile water, and "Pp-HK11-9" is a mixture of the conidia suspension of cucumber coryneformis and Pp-HK11-9 suspension. Among them, spore germination rate (%) = number of germinated spores / total number of investigated spores × 100.

[0054] from Figure 5 It can be seen that the conidia of cucumber coryneformis leaf spot pathogen began to germinate after 2 hours of culture in sterile water, and the spore germination rate reached 100% at the 6th hour of culture, while there was no germination at all after 8 hours of culture in Pp-HK11-9 suspension. Therefore, Pp-HK11-9 suspension can strongly inhibit the spore germination of coryneformis leaf spot pathogen.

[0055] This embodiment also provides the above-mentioned Paenibacillus polymyxa suspension for preventing and controlling cucumber corysporium leaf spot, powdery mildew and downy mildew. Specifically, the Paenibacillus polymyxa suspension is diluted 10 to 50 times for foliar spraying; in the early stage of diseases such as powdery mildew, downy mildew and corysporium leaf spot of melons, spray once every 7 days, for a total of 3 to 4 times; when the diseases such as powdery mildew, downy mildew and corysporium leaf spot of melons are seriously ill, spray once every 4 days, for a total of 4 to 5 times.

[0056] This embodiment also provides a biological preparation using the above-mentioned Paenibacillus polymyxa suspension. The biological preparation is prepared by uniformly mixing the above-mentioned Paenibacillus polymyxa suspension with a premix, wherein the premix is ​​composed of a uniform mixture of lecithin, sunflower oil, and distilled water.

[0057] In this embodiment, the volume ratio of the premix to the Paenibacillus polymyxa suspension is 5:1; in the premix, the mass ratio of lecithin, sunflower oil and distilled water is 3:10:30.

[0058] Specifically, the biological agent is used to prevent and control cucumber coryspore leaf spot, powdery mildew and downy mildew. When used in agricultural production, the biological agent is diluted 100 to 200 times for foliar spraying; in the early stage of melon powdery mildew, downy mildew and coryspore leaf spot, spray once every 7 days, for a total of 3 to 4 times; when melon powdery mildew, downy mildew and coryspore leaf spot are serious, spray once every 4 days, for a total of 4 to 5 times.

[0059] Example 3

[0060] This embodiment provides a polymyxa suspension, which includes a sterile buffer solution and the polymyxa cells of Example 1 uniformly dispersed in the sterile buffer solution. In this embodiment, the concentration of the polymyxa suspension is 4×10 8 CFU / mL.

[0061] The formula of the sterile buffer solution is: 0.1 mL of glycerol, 0.1 g of (NH4)2SO4, 0.1 g of K2HPO4, 0.1 g of KH2PO4, 0.1 g of MgSO4, and 1000 mL of distilled water.

[0062] Specifically, the preparation method of the polymyxa bacillus suspension comprises the following steps:

[0063] The polymyxa bacillus strain provided in Example 1 was cultured in TSA medium to obtain the polymyxa bacillus Pp-HK11-9 strain; the polymyxa bacillus Pp-HK11-9 strain was inoculated into a fermentation medium, and fermented at 30° C. for 56 hours to obtain a polymyxa bacillus fermentation liquid;

[0064] The formula of the fermentation medium includes 20 g of peptone, 5 g of yeast extract, 0.5 g of NaCl, 10 mL of 0.25 mol / L KCl, 5 mL of 2 mol / L MgCl2, 20 mL of 1 mol / L glucose and 1000 mL of distilled water, and the pH value of the fermentation medium is 7.0;

[0065] The polymyxa Paenibacillus fermentation broth is centrifuged to obtain the polymyxa Paenibacillus Pp-HK11-9 bacterial cells; the polymyxa Paenibacillus Pp-HK11-9 bacterial cells are diluted with a sterile buffer solution to obtain the polymyxa Paenibacillus suspension.

[0066] This embodiment also provides the above-mentioned Paenibacillus polymyxa suspension for preventing and controlling cucumber coryneformis leaf spot, powdery mildew and downy mildew.

[0067] The present embodiment also provides a biological preparation using the above-mentioned Paenibacillus polymyxa suspension, which is prepared by uniformly mixing the above-mentioned Paenibacillus polymyxa suspension with a premix, wherein the premix is ​​composed of a uniform mixture of lecithin, sunflower oil, and distilled water. The biological preparation is used to prevent and treat cucumber coryneformis leaf spot, powdery mildew, and downy mildew. In the present embodiment, the volume ratio of the premix to the Paenibacillus polymyxa suspension is 15:1; in the premix, the mass ratio of lecithin, sunflower oil, and distilled water is 1:5:10.

[0068] Example 4

[0069] This embodiment provides a polymyxa suspension, which includes a sterile buffer solution and the polymyxa cells of Example 1 uniformly dispersed in the sterile buffer solution. In this embodiment, the concentration of the polymyxa suspension is 5×10 8 CFU / mL.

[0070] The formula of the sterile buffer solution is: 0.05 mL of glycerol, 0.05 g of (NH4)2SO4, 0.09 g of K2HPO4, 0.08 g of KH2PO4, 0.01 g of MgSO4, and 1000 mL of distilled water.

[0071] This embodiment also provides a method for preparing a suspension of Paenibacillus polymyxa, which comprises the following steps:

[0072] The polymyxa bacillus strain provided in Example 1 was cultured in TSA medium to obtain the polymyxa bacillus Pp-HK11-9 strain; the polymyxa bacillus Pp-HK11-9 strain was inoculated into a fermentation medium, and fermented at 30° C. for 56 hours to obtain a polymyxa bacillus fermentation liquid;

[0073] The formula of the fermentation medium includes 20 g of peptone, 5 g of yeast extract, 0.5 g of NaCl, 10 mL of 0.25 mol / L KCl, 5 mL of 2 mol / L MgCl2, 20 mL of 1 mol / L glucose and 1000 mL of distilled water, and the pH value of the fermentation medium is 7.0;

[0074] The polymyxa Paenibacillus fermentation broth is centrifuged to obtain the polymyxa Paenibacillus Pp-HK11-9 bacterial cells; the polymyxa Paenibacillus Pp-HK11-9 bacterial cells are diluted with a sterile buffer solution to obtain the polymyxa Paenibacillus suspension.

[0075] This embodiment also provides the above-mentioned Paenibacillus polymyxa suspension for preventing and controlling cucumber coryneformis leaf spot, powdery mildew and downy mildew.

[0076] The present embodiment also provides a biological preparation using the above-mentioned Paenibacillus polymyxa suspension, which is prepared by uniformly mixing the above-mentioned Paenibacillus polymyxa suspension with a premix, wherein the premix is ​​composed of a uniform mixture of lecithin, sunflower oil, and distilled water. The biological preparation is used to prevent and treat cucumber coryneformis leaf spot, powdery mildew, and downy mildew. In the present embodiment, the volume ratio of the premix to the Paenibacillus polymyxa suspension is 10:1; in the premix, the mass ratio of lecithin, sunflower oil, and distilled water is 5:20:73.

[0077] Effect test:

[0078] Taking the Bacillus polymyxa suspension provided in Example 2 as Pp-HK11-9 and the biological agent as LSO-Pp-HK1-9 as an example, the control effect on cucumber coryspore leaf spot was verified by in vitro inoculation method, live inoculation method and field trial; the control effect on powdery mildew and downy mildew was verified by potted artificial live inoculation method and field trial.

[0079] Test sample grouping:

[0080] Test sample group 1 Pp-HK11-9: the suspension of Paenibacillus polymyxa provided in Example 2.

[0081] Test sample group 2 LSO-Pp-HK11-9: the biological preparation provided in Example 2 consisting of the Paenibacillus polymyxa suspension and the premix.

[0082] Chemical agent of test sample group three: 3000 times diluted 25% tebuconazole emulsion in water.

[0083] Test sample group 4 control: clean water.

[0084] 1. In vitro inoculation test of Corynespora leaf spot

[0085] In vitro inoculation of cucumber leaf spot pathogen:

[0086] When the cucumber had two true leaves, the leaves were sprayed with the above four experimental treatments. The front and back sides of the cucumber leaves were sprayed evenly. After drying, the leaves were cut and placed in a moisturizing tray. Then, the leaves were inoculated with a conidia suspension of Corynespora leaf spot pathogen (10 8 spores / mL), 5 μL spore suspension was added to each well, and then the tray was sealed with plastic wrap and placed in a 25°C incubator for moisturizing culture. After 3 to 4 days of culture, the incidence of cucumber corydalis leaf spot was investigated. The results are shown in Table 3 and Figure 6 shown.

[0087] Among them, the disease investigation and prevention effect calculation methods are as follows:

[0088] The disease survey of Coryneformis leaf spot under in vitro and live inoculation conditions is based on the lesion diameter, and its classification standard is:

[0089] Level 0: lesion diameter 0 cm;

[0090] Level 1: lesion diameter ≤ 0.2 cm;

[0091] Level 2: lesion diameter > 0.2 cm, ≤ 0.4 cm;

[0092] Level 3: lesion diameter > 0.4 cm, ≤ 0.6 cm;

[0093] Level 4: Lesion diameter > 0.6 cm.

[0094] The classification standards for powdery mildew and downy mildew under live inoculation conditions are as follows:

[0095] Level 0: no lesions;

[0096] Level 1: The lesions occupy ≤5% of the entire leaf area;

[0097] Level 3: The lesions occupy >5% and ≤10% of the entire leaf area;

[0098] Level 5: The lesions occupy >10% and ≤25% of the entire leaf area;

[0099] Level 7: The lesions occupy >25% and ≤50% of the entire leaf area;

[0100] Level 9: The lesions occupy more than 50% of the entire leaf area.

[0101] Disease index and prevention and treatment effect calculation formula:

[0102] Disease index = [Σ(number of diseased leaves at each level × representative value at each level) / (total number of surveys × highest representative value)] × 100;

[0103] Control effect (%) = (control disease index - treatment disease index) / control disease index × 100.

[0104] Table 3 Control effect on cucumber coryneformis leaf spot under in vitro inoculation conditions

[0105] Test samples Disease index Control effect (%) Pp-HK11-9 25.03±2.57b 57.09 LSO-Pp-HK11-9 19.75±1.89a 66.14 Chemicals 26.21±3.12b 55.07 Comparison 58.33±3.51c -

[0106] From Table 3 and Figure 6 It can be seen that the cucumber coryneformis leaf spot disease treated with the polymyxa suspension (Pp-HK11-9) and the biological agent (LSO-Pp-HK11-9) provided by the embodiments of the present invention is less severe, and the disease index is lower than that of the chemical agent and control treatments.

[0107] 2. Live inoculation test of common cucumber leaf diseases

[0108] Artificial inoculation of Coryneformis leaf spot pathogen in potted plants:

[0109] When the cucumber had two true leaves, the leaves were sprayed with the above four experimental treatments respectively. The front and back sides of the cucumber leaves were sprayed evenly. After drying, the leaves were inoculated with the conidia suspension of Corynespora leaf spot pathogen (10 8 spores / mL), 5 μL of spore suspension was added to each well, the leaves were inoculated and bagged to keep them moist, and then cultured in a 25°C greenhouse. The bags were removed after two days. The disease was investigated at 5 to 7 days of culture. The results are shown in Table 4 and Figure 7 shown.

[0110] Artificial inoculation of powdery mildew in potted plants:

[0111] When the cucumber has 2 to 3 true leaves, the leaves are sprayed with the above four test treatments. Spray the cucumber leaves evenly on both sides. After drying, spray the leaves with cucumber powdery mildew spore suspension (10 5-6 Spores / mL), bagged to keep moisture after inoculation, cultured in a greenhouse at 25-28℃, removed the bag after two days and continued to culture. The disease was investigated after 5-7 days of culture, and the results are shown in Table 4.

[0112] Artificial inoculation of downy mildew in potted plants:

[0113] When the cucumber had 2 to 3 true leaves, the leaves were sprayed with the above four experimental treatments. The front and back sides of the cucumber leaves were sprayed evenly. After drying, the leaves were sprayed with the suspension of zoosporangium of cucumber downy mildew (10 4-5 After inoculation, the samples were bagged to keep moisture and cultured in a greenhouse at 25-28°C. After two days, the bags were removed and culture was continued. The disease was investigated after 4-5 days of culture. The results are shown in Table 4.

[0114] Table 4 Control effect on common cucumber leaf diseases under live inoculation conditions

[0115]

[0116] from Figure 7 It can be seen that the cucumber coryneformis leaf spot disease treated with the polymyxa suspension (Pp-HK11-9) and biological agent (LSO-Pp-HK11-9) provided by the embodiments of the present invention is less severe, and the disease index is significantly lower than that of the chemical agent and control treatments.

[0117] As can be seen from Table 4, the effect of the Paenibacillus polymyxa suspension (Pp-HK11-9) in preventing and controlling cucumber coryspore leaf spot, powdery mildew and downy mildew can reach more than 50%, and can be actually applied in agricultural production. Therefore, it can be proved that the Paenibacillus polymyxa suspension provided in this case can simultaneously prevent and control cucumber coryspore leaf spot, powdery mildew and downy mildew.

[0118] At the same time, it can be seen from Table 4 that the application of biological agents can significantly improve the effect of preventing and controlling cucumber coryspore leaf spot, powdery mildew and downy mildew.

[0119] 3. Field cultivation test under common melon disease conditions

[0120] Cucumber field cultivation experiment:

[0121] Cucumber seedlings were transplanted and planted, and then water and fertilizer management was carried out according to the conventional method. During this period, the leaves of the four test sample groups were sprayed every 7 days from the beginning of the cucumber fruiting period, and the front and back of the cucumber leaves were sprayed evenly. During this period, the growth status, disease incidence, fruit yield and quality of cucumbers, and the colonization density of Pp-HK11-9 on the cucumber leaves were investigated. The investigation and test results are shown in Figure 8 , as shown in Tables 5 and 6.

[0122] Specifically, the growth status of cucumbers was determined by measuring plant height, stem diameter and number of leaves during the flowering period of cucumber flowers; the yield was determined by using the fixed-point cumulative yield measurement method to estimate the yield per mu.

[0123] Quality determination of protein, soluble sugar and vitamin C content in cucumber fruit. Protein content was determined by Coomassie Brilliant Blue G-250 method, soluble sugar content was determined by anthrone colorimetry, and vitamin C content was determined by 2,6-dichlorophenol indophenol titration method.

[0124] The colonization density was determined by the dilution plate method. The main veins of cucumber leaves were removed and ground in a sterile mortar. Then, the dilutions were diluted with sterile water to form dilutions of different concentrations. The dilutions were evenly spread on the plate containing rifampicin (50 μg ml -1 ) on TSA medium and cultured in a constant temperature incubator at 28°C for 48 to 72 hours, then the colony count was investigated and the colonization density was estimated.

[0125] (1) Strain colonization density:

[0126] from Figure 8 It can be seen from the colonization density curve of cucumber leaves shown in FIG that when treated with the polymyxa suspension (Pp-HK11-9) and biological agent (LSO-Pp-HK11-9) provided in the embodiment of the present invention, the Pp-HK11-9 strain remained stably on the cucumber leaves for 10 4 A colonization density above CFU / g provides the prerequisite for it to play its disease prevention role.

[0127] (2) Cucumber yield and quality:

[0128] Table 5 Cucumber yield

[0129] deal with Yield (kg / mu) Pp-HK11-9 2894.7±59.7a LSO-Pp-HK11-9 2921.5±71.6a Chemicals 2733.7±43.6b Comparison 2697.3±32.8b

[0130] Table 6 Cucumber fruit quality related indicators

[0131]

[0132] From Table 5, Table 6 and Figure 8It can be seen that the treatments of biological agent (LSO-Pp-HK11-9) and Paenibacillus polymyxa suspension (Pp-HK1-9) significantly increased the yield, protein, soluble sugar and vitamin C content of cucumber fruit compared with chemical agents and control treatments. There was no significant difference in cucumber yield and protein content between the two treatments of biological agent (LSO-Pp-HK11-9) and Paenibacillus polymyxa suspension (Pp-HK1-9). Figure 8 It can be seen that when the biological agent (LSO-Pp-HK11-9) was treated, the colonization density of the Paenibacillus polymyxa Pp-HK11-9 strain on the cucumber leaves was significantly higher than that of the Paenibacillus polymyxa suspension (Pp-HK1-9), which is beneficial to its role in preventing and controlling diseases.

[0133] Therefore, the Bacillus polymyxa suspension and biological preparation provided by the embodiments of the present invention can improve the immunity of cucumbers, inhibit the growth and germination of pathogens and prevent the invasion of pathogens, and can prevent and control cucumber coryspore leaf spot, powdery mildew and downy mildew, which is beneficial to improving yield and quality.

[0134] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or some technical features can be replaced by equivalents without departing from the spirit of the technical solution of the present invention, which should be included in the scope of the technical solution for protection of the present invention.

Claims

1. A strain of Paenibacillus polymyxa ( Paenibacillus polymyxa ), characterized in that, It is "Paenibacillus polymyxa Pp-HK11-9" and its deposit number is "CGMCC No.24634".

2. A polymyxa suspension, characterized in that: The invention is prepared by dispersing the polymyxa bacillus cells described in claim 1 in a sterile buffer solution.

3. The polymyxa bacillus suspension according to claim 2, characterized in that The formula of the sterile buffer solution is: 0.01 mL to 0.1 mL of glycerol, 0.01 g to 0.1 g of (NH4)2SO4, 0.07 g to 0.1 g of K2HPO4, 0.02 g to 0.1 g of KH2PO4, 0.001 g to 0.1 g of MgSO4, and 1000 mL of distilled water.

4. The polymyxa suspension according to claim 3, characterized in that The concentration of the polymyxa bacillus is (4-6)×10 8 CFU / mL.

5. Use of the polymyxa suspension according to claim 2, 3 or 4, characterized in that: The polymyxa bacillus suspension is used for preventing and controlling cucumber corynespora leaf spot, cucumber powdery mildew and cucumber downy mildew.

6. A method for applying the Paenibacillus polymyxa suspension according to claim 2, 3 or 4, characterized in that: The polymyxa bacillus suspension is diluted 10 to 50 times and then sprayed on the leaves.

7. A biological agent, characterized in that: The invention comprises the uniformly mixed Paenibacillus polymyxa suspension according to claim 2, 3 or 4 and a premix, wherein the premix is ​​formed by uniformly mixing lecithin, sunflower oil and distilled water.

8. The biological agent according to claim 7, characterized in that: The volume ratio of the Bacillus polymyxa suspension to the premix is ​​1:(5-15); in the premix, the mass ratio of lecithin, sunflower oil and distilled water is (3-10):(10-50):(30-100).

9. Use of the biological agent according to claim 7 or 8, characterized in that: The biological preparation is used for preventing and controlling cucumber corynespora leaf spot, cucumber powdery mildew and cucumber downy mildew.

10. The use of the biological agent according to claim 9, characterized in that: The biological preparation is diluted 100 to 200 times and used as a foliar spray for cucumber leaf diseases.

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