Bacillus velezensis sfj-27682 and applications thereof

Abstract

The present application belongs to the technical field of plant disease control, and particularly relates to Bacillus velezensis and application thereof. The Bacillus velezensis strain of the present application is obtained by screening from the rhizosphere soil of wheat in main wheat areas in Hebei province. The colony growth characteristics and physiological and biochemical characteristics of the strain are identified, and then 16S rDNA sequencing is carried out. Blast homology comparison is carried out in the GenBank database. Through the above analysis, the strain is identified as Bacillus velezensis. The inoculant containing the Bacillus velezensis has good control effect on wheat foot rot through seed coating.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant disease control, and particularly relates to Bacillus velezensis and application thereof. BACKGROUND

[0002] Wheat Fusarium Crown Rot is a soil-borne disease caused by multiple Fusarium, which occurs and causes serious damage in Australia, the United States, Canada and other countries. In normal years, it can cause an average yield loss of about 9.5%, and in epidemic years, it can reach 35%. The main symptom of wheat Fusarium Crown Rot is brown necrosis at the base of the stem, and severe white ear, with a local white ear rate of 20% to 50%. In recent years, the disease has developed rapidly in the main wheat areas of China. According to reports, the wheat Fusarium Crown Rot disease rate in some areas of Henan and Hebei provinces reached 65.1% and 81.4% respectively, and the field white ear rate in serious plots reached 31.5% and 50.0% respectively, which has seriously threatened China's wheat production and has a trend of increasing year by year.

[0003] At present, there is no ideal disease-resistant variety and efficient control strategy for wheat Fusarium Crown Rot, and no fungicide registered for the prevention and control of the disease. Only seed coating agents registered for the prevention and control of other soil-borne diseases are used to prevent and control the disease. However, these agents have high prevention and control effect at the seedling stage, but the prevention and control effect decreases at the grain filling stage, and the use of azoles and other agents in high doses can easily affect the emergence and growth of wheat. The large-scale use of chemical agents also poses a great threat to the ecological environment and food safety. Therefore, it is an urgent problem to find an environmentally friendly and effective control measure for wheat Fusarium Crown Rot.

[0004] Biocontrol microorganisms themselves or their metabolites have various biological activities, can be used for antibacterial, growth promotion and plant resistance improvement, so as to control the occurrence of diseases. Through microbial fermentation, microbial active body pesticides and microbial metabolite pesticides can be prepared, which can realize the green and sustainable development of agriculture. SUMMARY

[0005] The present application aims to provide a Bacillus velezensis SFJ-27682.

[0006] The present application further aims to provide the application of the above-mentioned Bacillus velezensis strain.

[0007] The Bacillus velezensis SFJ-27682 of the present application is preserved in the China General Microbiological Culture Collection Center on September 19, 2024, and the specific preservation address is No. 1, Beichen West Road, Yard 3, Beijing Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences, and the preservation number is CGMCC No: 32000.

[0008] The present application isolates and preserves a Bacillus velezensis from the rhizosphere soil of wheat in the main wheat area of Hebei Province. The strain has a significant inhibitory effect on Fusarium pseudograminearum and a good control effect on wheat foot rot.

[0009] Based on the above effects of the Bacillus velezensis strain, the present application proposes the application of the strain, especially in the prevention and control of wheat foot rot. For example, according to factors such as plant growth state or soil conditions, the strain is uniformly sprayed on the soil surface and covered with a thin layer of soil to prevent wheat foot rot, or the strain is used after being made into a microbial agent.

[0010] The above microbial agent contains the Bacillus velezensis strain, a suspension seed coating agent containing its fermentation product, etc.

[0011] The present application provides a composite biocontrol agent, which includes the fermentation liquid of the above-mentioned Bacillus velezensis strain, a dispersing agent, a synergist, a thickening agent, and an antifoaming agent.

[0012] Preferably, the biocontrol agent includes the fermentation liquid of the Bacillus velezensis strain (the concentration of the Bacillus velezensis strain is 10 11 cfu / mL), a dispersing agent 5-10%, a synergist 1.2-2.5%, a thickening agent 1-3%, and an antifoaming agent 0.5-1.5%.

[0013] Preferably, according to the mass ratio of the fermentation liquid of the Bacillus velezensis strain, the biocontrol agent contains a dispersing agent 5%, a synergist 1.5%, a thickening agent 2%, and an antifoaming agent 1%.

[0014] Preferably, the dispersing agent is a mixture of sodium lignosulfonate, phenylethyl phenol polyoxyethylene ether, and Tween 80. More preferably, the mass ratio of sodium lignosulfonate, phenylethyl phenol polyoxyethylene ether, and Tween 80 in the dispersing agent is 1:1-3:2-5, and the mass ratio of sodium lignosulfonate, phenylethyl phenol polyoxyethylene ether, and Tween 80 can be selected as 1:1:3.

[0015] More preferably, the synergist is a nitrogen synergist, the thickening agent is xanthan gum, and the antifoaming agent is an organic silicon antifoaming agent.

[0016] Preferably, the application also provides a composite biocontrol agent, which also has the above-mentioned bacterial agent and 6% oligosaccharide · chain protein.

[0017] The 6% oligosaccharide · chain protein wettable powder is a product of Ate Ling, which is an antiviral protein pesticide with a wide range of applications, including corn, rice, wheat, vegetables, fruits, tobacco, fruit trees, flowers, medicinal materials and other crops, and can be used to prevent various viral diseases. The application method is to mix 6% oligosaccharide · chain protein wettable powder with water (usually 15-20 g of Ate Ling is mixed with 15 kg of water), and to spray the leaves 2-3 times.

[0018] Preferably, the mass ratio of the bacterial agent to 6% oligosaccharide · chain protein is 1-10:1-10, and more preferably, the mass ratio of the bacterial agent to 6% oligosaccharide · chain protein is 1:1.

[0019] The application also creatively finds that the above-mentioned Bacillus velezensis bacterial agent can be used in combination with 6% oligosaccharide · chain protein for preventing and treating wheat basal stem rot, and has a more significant and excellent effect than single Bacillus velezensis or 6% oligosaccharide · chain protein wettable powder, which indicates that the Bacillus velezensis bacterial agent and 6% oligosaccharide · chain protein have a synergistic effect on the above-mentioned effects, and in particular, the mass ratio of the Bacillus velezensis bacterial agent to 6% oligosaccharide · chain protein is 1:1, which has the best effect.

[0020] The application also provides a culture medium of the above-mentioned Bacillus velezensis, which comprises 1-5% sucrose, 1-5% soybean powder, 0.5-2% NaCl, 0.1-0.5% CaCO3, 0.01-0.05% KH2PO4 and 0.01-0.05% MgSO4·7H2O.

[0021] Preferably, the culture medium comprises 3% sucrose, 1% soybean powder, 1% NaCl, 0.2% CaCO3, 0.02% KH2PO4 and 0.03% MgSO4·7H2O.

[0022] The application has the following beneficial effects:

[0023] The Bacillus velezensis strain of the application is isolated and screened from the rhizosphere soil of wheat in the main wheat area of Hebei Province. The colony growth characteristics and physiological and biochemical characteristics of the strain are identified, and then 16S rDNA sequencing is performed. After Blast homology comparison in the GenBank database, the strain is identified as Bacillus velezensis through the above analysis.

[0024] The Bacillus velezensis strain SFJ-27682 of the present application has obvious inhibitory effect on Fusarium pseudograminearum, and the prevention effect on wheat foot rot can reach 41.47%.

[0025] The fermentation broth of Bacillus velezensis SFJ-27682 is mixed with dispersants, thickeners, synergists and other ingredients in a specific ratio to prepare a microbial agent, and the wheat is treated by seed dressing, which can form a uniform coating on the seed and play a long-lasting protective role, and the prevention effect on wheat foot rot can be improved to 64.43%.

[0026] The Bacillus velezensis SFJ-27682 microbial agent is further compounded with 6% oligosaccharide · chain protein to form a composite microbial agent, and when the combination is 1:1, obvious synergistic effect is obtained. The above-mentioned composite microbial agent is used for seed dressing treatment of wheat, and the prevention effect on wheat foot rot is further improved to 83.54%, the disease index and white head rate prevention effect of field plant stage reaches 62.25% and 65.32%, and at the same time, the composite microbial agent can improve the emergence rate of wheat, promote the growth of wheat, and significantly improve the yield of wheat. DETAILED DESCRIPTION

[0027] To make the purpose, technical scheme and advantages of the present application clearer, the technical scheme of the present application will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope protected by the present application.

[0028] The Bacillus velezensis SFJ-27682 of the present application was preserved in the China General Microbiological Culture Collection Center on September 19, 2024, and the specific preservation address is: No. 3, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the preservation number is CGMCC No: 32000.

[0029] Example 1 Screening and identification of Bacillus velezensis SFJ-27682

[0030] 1.1 Screening of Bacillus velezensis SFJ-27682

[0031] The rhizosphere soil of wheat in main wheat areas in Hebei Province was taken, and then sterile physiological saline was added for grinding, gradient dilution (diluted to 10 1 ~ 10 5The bacteria were inoculated on beef extract peptone medium (NA) and cultured in an inverted incubator at 37°C for 2-3 days, with the last rinse water as a control. Different single colonies were selected according to the morphology, color and size of the colonies on the plate, and then purified by streaking until single colonies were obtained. The slants were stored at 4°C for standby use.

[0032] Each strain was screened by confrontation culture. The prepared 5-mm-diameter F. pseudograminearum cake was placed in the center of a PDA plate, and the bacteria to be tested were inoculated in the center of the plate using a bacterial loop, and then the isolated bacteria were inoculated at equal distances at the cross intersection 2.5 cm from the center of the plate. The control (CK) was not inoculated with the isolated bacteria. Each treatment was repeated three times. The culture was carried out at a temperature of 25°C. When the control (CK) was fully grown, the size of the inhibition zone was recorded. The five strains with the largest inhibition zone (SFJ-27634, SFJ-27682, SFJ-27684, SFJ-27698 and SFJ-28725) were selected for greenhouse pot bioassay. Each strain was cultured in an LB medium in a shaking incubator at 37°C and 180 r / min for 2 days. The bacterial liquid was concentrated by centrifugation to a viable bacterial count of 10 10 CFU / mL of bacterial liquid. The bacterial liquid and wheat seeds (variety Shixin 828) were mixed at 300 g / 100 kg of seeds at room temperature. After mixing, the wheat seeds were taken out and dried at 35°C for 24 h to obtain coated wheat seeds. A blank control group (without seed dressing treatment) was also set up. The coated seeds were planted in nutrient pots (15 cm in diameter x 15 cm in height), and the soil was induced to be infected with F. pseudograminearum at a concentration of 1 x 10 5 The disease level and disease index of the wheat were investigated after the culture in a 22°C, 12 h light / dark alternating greenhouse for 30 days, and the greenhouse control effect was calculated.

[0033] The greenhouse pot culture was investigated according to the modified Bovill grading method: the stem base rot of the wheat was graded according to the length of the browning of the stem below the first leaf sheath.

[0034] 0 grade: no browning symptoms;

[0035] 1 grade: the browning length below the first leaf sheath accounts for less than 25% of the stem;

[0036] 2 grade: the browning length below the first leaf sheath accounts for 25-50% of the stem;

[0037] 3 grade: the browning length below the first leaf sheath accounts for 50-75% of the stem, and the browning is deepened;

[0038] 4 grade: the browning length below the first leaf sheath accounts for more than 75% of the stem, or the whole plant is browning and dead.

[0039] Disease index = ∑(number of diseased plants x each level representative value) / (total number of plants surveyed x highest level representative value) x 100;

[0040] Control effect (%) = (control disease index - treatment disease index) / control disease index x 100.

[0041] Table 1: Inhibition zone and greenhouse potting control effect of biocontrol bacteria on wheat foot rot

[0042]

[0043] As shown in Table 1, the strain SFJ-27682 has the best control effect, and thus, the SFJ-27682 with the highest control effect is selected for identification and further testing.

[0044] 1.2 Identification of Bacillus velezensis SFJ-27682

[0045] 1. Morphological identification

[0046] The colony morphology of the antagonistic strain is inoculated on a beef extract peptone medium (NA) by streaking method, and cultured at 37°C for 1-2 days, and the colony morphological characteristics are observed. The cell morphology is observed by optical microscope after staining by single staining method and Gram staining method.

[0047] The results show that the colony formed by the strain SFJ-27682 on the beef extract peptone medium is thin, oval or nearly round, milky white, wrinkled on the surface, raised in the middle, with neat edges, and opaque. The cells are rod-shaped, the strain is Gram-positive, and the strain produces spore bacteria.

[0048] 2. Physiological and biochemical identification

[0049] The physiological and biochemical tests of the endophytic antagonistic bacteria are carried out according to the "Berger Bacterium Identification Manual" and "Common Bacterium System Identification Manual".

[0050] The results show that the contact enzyme reaction is positive, the starch hydrolysis reaction is positive, the glucose acid production test is negative, the glucose gas production test is negative, the methyl red determination is negative, the acetyl methyl methanol test (V.P.) is positive, the gelatin liquefaction reaction is positive, the nitrate reduction reaction is positive, the citrate utilization determination is positive, the indole test is negative, the phenylalanine dehydrogenase test is negative, and the urea hydrolysis test is positive.

[0051] The physiological and biochemical reaction determination results of the strain SFJ-27682 are compared with the Bacillus velezensis described in the literature, and it is found that the physiological and biochemical test results of the strain are basically consistent with those of the Bacillus velezensis.

[0052] 3. 16S rDNA sequence analysis

[0053] Genomic DNA was extracted using Shanghai Shengong SK1201-UNIQ-10 column bacterial genomic DNA extraction kit, and the 16S rDNA fragments in the total DNA of the strains were amplified using 16S rDNA universal primers 7F (5'-CAGAGTTTGATCCTGGCT-3') and 1540R (5'-AGGAG GTGATCCAGCCGCA-3'), and the reaction conditions were as follows:

[0054] Pre-denaturation 98℃, 5min,

[0055] Cycling 95℃ 35S, 55℃ 35S, 72℃ 90s, 35 cycles,

[0056] Extension 8min.

[0057] PCR system establishment (50μL):

[0058]

[0059]

[0060] After the amplification product was recovered by UNIQ-10 column DNA gel recovery kit (Shanghai Shengong), sequencing was performed by Shanghai Shengong Bioengineering Company, the sequenced sequence was submitted to Genbank, and through Blast comparison, it was found that the sequence had 100% similarity with multiple strains of Bacillus velezensis strains reported, and according to the morphological characteristics and physiological and biochemical reaction determination results, it could be determined that the strain SFJ-27682 was Bacillus velezensis.

[0061] The strain was preserved in the China General Microbiological Culture Collection Center on September 19, 2024, and the specific preservation address was No. 3, Beichen West Road, Haidian District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, and the preservation number was CGMCC No: 32000.

[0062] Example 2 Optimization of fermentation medium of Bacillus velezensis SFJ-27682

[0063] The highest field control effect of the screened biocontrol strain SFJ-27682 was optimized. Corn flour, sucrose, glucose and soluble starch were used as carbon sources, respectively, soybean powder, sodium nitrate, ammonium nitrate and ammonium sulfate were used as nitrogen sources, respectively, and a certain amount of trace elements, NaCl, KH2PO4, MgSO4·7H2O and CaCO3 were added. Through single factor determination test, the effects of four kinds of carbon sources and four kinds of nitrogen sources on the growth of strain SFJ-27682 were determined.

[0064] The results show that among the four carbon sources, the best carbon source is sucrose, and the highest concentration is 1.47 x 10 10 cfu / ml after 30 hours of fermentation, followed by glucose, soluble starch, and corn flour. Among the four nitrogen sources, the best nitrogen source is soybean meal, and the highest concentration is 1.22 x 10 10 cfu / mL after 30 hours of fermentation, followed by ammonium sulfate, sodium nitrate, and ammonium nitrate.

[0065] Table 2 Single-factor determination of fermentation carbon and nitrogen sources of biocontrol bacteria

[0066]

[0067]

[0068] Based on the single-factor experiment, under the condition that the two trace elements KH2PO4 and MgSO4·7H2O remain unchanged, an L9(34) orthogonal experiment is conducted to screen the concentrations of the four factors, i.e., carbon source sucrose (A), nitrogen source soybean meal (B), and the two inorganic salts NaCl (C) and CaCO3 (D) with higher usage, so as to determine the optimal fermentation medium for the strain.

[0069] Table 3 Screening of the usage of carbon and nitrogen sources and inorganic salts by orthogonal experiment

[0070]

[0071] The orthogonal experiment results show that the preferred medium formula of the optimal selection of each factor is A3B1C3D2, with a concentration of 6.43 x 10 11 cfu / mL, which is more than 30 times higher than the lowest fermentation medium concentration. That is, the optimal fermentation medium for SFJ-27682 is 3% sucrose, 1% soybean meal, 1% NaCl, 0.2% CaCO3, 0.02% KH2PO4, and 0.03% MgSO4·7H2O.

[0072] Using the above medium, Bacillus velezensis SFJ-27682 is cultured in a shaking incubator at 37°C and 180 r / min for 30 days to obtain the fermentation broth of Bacillus velezensis SFJ-27682.

[0073] Example 3 Preparation of biocontrol agent

[0074] Example 3: The fermentation broth of SFJ-27682 is obtained according to the method of Example 2, and is diluted with sterile water to a concentration of 10 11Example 3: The fermentation broth of SFJ-27682 was obtained according to the method of Example 2, and diluted with sterile water to a concentration of 10

[0075] Comparative Example 1: The fermentation broth of SFJ-27682 was obtained according to the method of Example 2, and diluted with sterile water to a concentration of 10 11 cfu / mL. According to the mass ratio with the fermentation broth, dispersant 5%, nitrogen synergist 1.5%, xanthan gum (thickening agent) 2.0%, silicone defoaming agent 1.0% were added; the dispersant was mixed by lignin sulfonate sodium, phenylethyl phenol polyoxyethylene ether, Tween 80 in a mass ratio of 1:1:3.

[0076] Comparative Example 2: The fermentation broth of SFJ-27682 was obtained according to the method of Example 2, and diluted with sterile water to a concentration of 10 11 cfu / mL. According to the mass ratio with the fermentation broth, dispersant 8%, nitrogen synergist 1.5%, xanthan gum (thickening agent) 2.0%, silicone defoaming agent 1.0% were added; the dispersant was mixed by lignin sulfonate sodium, phenylethyl phenol polyoxyethylene ether, Tween 80 in a mass ratio of 1:1:1.

[0077] Comparative Example 3: The fermentation broth of SFJ-27682 was obtained according to the method of Example 2, and diluted with sterile water to a concentration of 10 11 cfu / mL. According to the mass ratio with the fermentation broth, dispersant 8%, nitrogen synergist 1.5%, xanthan gum (thickening agent) 1.0%, silicone defoaming agent 1.0% were added; the dispersant was mixed by lignin sulfonate sodium, phenylethyl phenol polyoxyethylene ether, Tween 80 in a mass ratio of 1:1:3.

[0078] The control effects of the biocontrol agents prepared in Example 3 and Comparative Examples 1, 2, and 3 were determined according to the greenhouse pot bioassay method in Example 1.

[0079] Table 4: Control effect of biocontrol agent on wheat foot rot in greenhouse pot

[0080]

[0081] The results are shown in Table 4. The decrease in the content of dispersant in Comparative Example 1 and the change in the formulation of dispersant in Comparative Example 2 resulted in the uniformity of seed coating being affected, the protective film of the seedling being incomplete, and the control effect being reduced; the decrease in the content of thickening agent in Comparative Example 3 affected the sustained-release property of the agent, the persistence of the agent was reduced, and the control effect was reduced. The selection of the appropriate component ratio in Example 3 made the seed coating uniform, formed a complete protective film for the seedling, and could play a protective role for a relatively long time, so the control effect was obviously higher than that of the comparative examples, and therefore the formulation of Example 3 was selected for further test and study.

[0082] Example 4. Screening and control effect determination of Bacillus velezensis SFJ-27682 agent combined with 6% oligosaccharide · chain protein

[0083] 4.1 Greenhouse synergistic ratio screening

[0084] The Bacillus velezensis SFJ-27682 agent prepared according to Example 3 was screened for synergistic combination with 6% oligosaccharide · chain protein according to the greenhouse potting bioassay method in Example 1. The seed dressing amount of the SFJ-27682 agent and 6% oligosaccharide · chain protein wettable powder was set as 900 g / 100 kg seeds, 600 g / 100 kg seeds, 300 g / 100 kg seeds, 100 g / 100 kg seeds, 50 g / 100 kg seeds, 25 g / 100 kg seeds, and 12.5 g / 100 kg seeds, respectively. Then, the seed dressing amount of each was prepared into a combination with a mass ratio of 1:1, 1:3, 1:5, 1:7, 1:9, 3:1, 5:1, 7:1, and 9:1, respectively. Two single agents were used as controls. The control effects of each treatment were determined. The logarithmic values of each seed dressing concentration and the probability values of the control effects were used to calculate the linear regression equation. The theoretical and actual values of EC 50 were calculated, and the joint virulence was evaluated by Wadley method.

[0085] EC 50 (th) = (a + b) / [a / EC 50 (A) + b / EC 50 (B)].

[0086] Synergistic coefficient (SR) = EC 50 (th) / EC 50 (ob).

[0087] In the formula, A and B represent two agents respectively, a and b represent the ratio of the two agents respectively, ob is the actual observation value, and th is the theoretical value.

[0088] Table 5. Screening of Bacillus velezensis SFJ-27682 and 6% oligosaccharide · chain protein wettable powder for greenhouse synergistic combination

[0089]

[0090] The results are shown in Table 5 that Bacillus velezensis SFJ-27682 microbial agent combined with 6% oligosaccharide · chain protein wettable powder at 1:1, 1:3, 1:5, 1:9, 3:1 has certain synergistic effect on the prevention and control of wheat foot rot in greenhouse seedling stage, among which the most obvious synergistic effect is the 1:1 combination, and the synergistic coefficient can reach 2.59, therefore, the synergistic combination is selected for greenhouse and field control effect determination.

[0091] 4.2 Synergistic combination greenhouse bioassay

[0092] According to the mass ratio of 1:1, Bacillus velezensis SFJ-27682 microbial agent prepared in Example 3 is combined with 6% oligosaccharide · chain protein (Atiling) wettable powder, and 300g / 100kg seeds are coated, with 6% chemical fungicide tebuconazole seed coating 66.7mL / 100kg seeds as control, and blank control group CK (i.e. without seed coating treatment) is added, and the method is carried out according to the greenhouse bioassay method in Example 1, and the plant height, root length, fresh weight per plant of each treatment are determined, and the disease index and greenhouse control effect are calculated.

[0093] Table 6 Bacillus velezensis SFJ-27682 microbial agent combined with 6% oligosaccharide · chain protein wettable powder greenhouse bioassay

[0094]

[0095] The results show that the combination of Bacillus velezensis SFJ-27682 microbial agent and 6% oligosaccharide · chain protein (Atiling) wettable powder at 1:1 can improve the emergence rate of wheat, promote the growth of wheat, and the control effect is obviously better than that of chemical fungicide 6% tebuconazole seed coating, and does not affect the emergence of wheat.

[0096] 3.3 Synergistic combination field efficacy test

[0097] Bacillus velezensis SFJ-27682 microbial agent is combined with 6% oligosaccharide · chain protein (Atiling) wettable powder at a mass ratio of 1:1, and 300g / 100kg seeds are coated, and the variety is selected as Shixin 828, and a 6% tebuconazole seed coating control group is set.

[0098] The wheat seeds of each treatment group and the control group are planted in the field of the Experimental Farm of the Institute of Plant Protection of Hebei Academy of Agriculture and Forestry from October 2020 to June 2021. Investigation is carried out during the grain filling period, 20-30 wheat plants are taken from each sample point, and the disease index is calculated according to the grading standard modified by Yang Yun et al., and the control effect is obtained according to the formula.

[0099] 0 level: no browning symptoms;

[0100] Grade 1: the first stem node browning area is less than 25%;

[0101] Grade 2: the first stem node browning area is 25% to 50%;

[0102] Grade 3: the first stem node browning area is 50% to 75%;

[0103] Grade 4: the first stem node is more than 75% or browning to the second and third stem nodes does not cause white smut;

[0104] Grade 5: the wheat plant white smut;

[0105] Grade 6: the plant dwarfing, death, and no heading; record the number of white smut and disease grade of each plant, calculate the disease index, white smut rate, and control effect.

[0106] Disease index = ∑(number of diseased plants x representative value of each grade) / (total number of plants surveyed x representative value of the highest grade) x 100;

[0107] White smut rate (%) = number of white smut / total number of plants surveyed x 100;

[0108] Disease control effect (%) = (disease index of the control-disease index of the treatment) / disease index of the control x 100;

[0109] White smut control effect (%) = (white smut rate of the control-white smut rate of the treatment) / white smut rate of the control x 100.

[0110] At the mature stage, 5 points are sampled, and 0.5 m 2 The number of spikes, 20-30 plants of wheat are investigated, and invalid spikelets with less than 5 grains are removed to investigate the average spike grain number, weigh the thousand-grain weight, and calculate the wheat yield. The calculation formula is as follows:

[0111] Theoretical yield = (number of effective spikes per unit area x number of grains per spike x thousand-grain weight) / 10 x 0.85. 6

[0112] Table 7 Field control effect of Bacillus velezensis SFJ-27682 microbial agent combined with 6% oligosaccharide·chain protein wettable powder

[0113]

[0114] Table 8 Field yield-increasing effect of Bacillus velezensis SFJ-27682 microbial agent combined with 6% oligosaccharide·chain protein wettable powder

[0115]

[0116] ​The field test results show that the SFJ-27682 fungicide combined with 6% oligosaccharide · chain protein wettable powder for seed dressing has a better control effect on wheat stem base rot, the disease index and white head rate control effect at the adult stage is 62.25% and 65.32%, and the control effect is obviously higher than that of the chemical fungicide 6% tebuconazole seed coating agent; and can significantly improve the wheat yield, and the yield is increased by 11.42% compared with the blank control.

[0117] Therefore, the biocontrol fungicide and the plant immune elicitor composition of the present application have higher bacteriostatic activity on wheat stem base rot fungus, have a better control effect on wheat stem base rot in the field, have obvious synergistic effect compared with two components, and are obviously better than the chemical fungicide 6% tebuconazole seed coating agent; at the same time, the fungicidal composition of the present application is safe to wheat, does not affect the emergence of wheat, promotes growth and improves yield. The biocontrol fungicide and the plant immune elicitor composition of the present application have a long efficacy period, have a higher control effect on the occurrence of wheat stem base rot at the adult stage, and are beneficial to reduce the generation of pathogen resistance risk and the influence on the environment.

[0118] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited to this, any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A complex biocontrol agent, characterized by, The complex biocontrol agent comprises a bacterial agent and 6% oligosaccharide · chain protein, the bacterial agent comprises fermentation liquor of bacillus velezensis (SFJ-27682) Bacillus velezensis ), dispersant 5-10%, synergist 1.2-2.5%, thickening agent 1-3%, antifoaming agent 0.5-1.5%, and the preservation number of bacillus velezensis is CGMCC NO.32000.

2. The complex biocontrol agent according to claim 1, characterized in that, The mass ratio of the bacterial agent to 6% oligosaccharide · chain protein is 1-10: 1-10.

3. The biocontrol agent according to claim 1, characterized in that, The dispersant is mixed by sodium lignosulfonate, phenylethyl phenol polyoxyethylene ether and Tween 80.

4. The biocontrol agent according to claim 3, characterized in that, The mass ratio of sodium lignosulfonate, phenylethyl phenol polyoxyethylene ether and Tween 80 in the dispersant is 1:1-3:2-5. The mass ratio of sodium lignosulfonate, phenylethyl phenol polyoxyethylene ether and Tween 80 in the dispersant is 1:1-3:2-5.

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