Compound microbial agent and application thereof in prevention and treatment of plant fungal diseases

Abstract

The invention discloses a compound microbial agent and application thereof in prevention and treatment of plant fungal diseases. The compound microbial agent is prepared from a Burkholderia ambifara strain Aa27 and a Bacillus velezensis strain Gc8, and the plant fungal disease is the sugarcane smut, and the plant fungal disease is the sugarcane smut. Experiments prove that both the Burkholderia ambifida strain Aa27 and the Bacillus velezensis strain Gc8 can inhibit the germination of teliospore and the sexual coordination of basidiospore of the sugarcane smut, and the composite microbial agent composed of the Burkholderia ambifida strain Aa27 and the Bacillus velezensis strain Gc8 not only can effectively prevent and treat the sugarcane smut, but also can be used for promoting the growth of the sugarcane and increasing the yield of the sugarcane. The compound microbial agent provided by the invention has important significance on prevention and treatment of fungal diseases of plants.

Description

Technical Field

[0001] The invention relates to the field of biological control, and in particular to a composite microbial agent and application thereof in preventing and controlling plant fungal diseases. Background Art

[0002] Sugarcane (Saccharum officinarum) is an important cash crop and an important raw material for producing sugar. About 65% of the world's total sugar production comes from sugarcane, and China accounts for more than 90%. Therefore, the level of sugarcane production has a serious impact on national sugar security. Sugarcane smut is a disease caused by infection with Sporisorium scitanminea, and is one of the more common and serious diseases in the process of sugarcane cultivation.

[0003] At present, the measures adopted to prevent and control sugarcane smut include breeding disease-resistant varieties, chemical control, physical control and field management. Winter spores in the soil and infected sugarcane seeds are the initial infection sources of the disease, and it is difficult to remove the pathogens in the soil by common farming methods. After the pathogen invades the sugarcane, the sugarcane will not show symptoms for a long time, which makes it difficult to kill the pathogen in the early stage. Once the diseased ear is found, it is irreversible and the only way is to remove the diseased plants to prevent the black powder from scattering and infecting healthy sugarcane. Therefore, this brings great difficulties to early prevention and control.

[0004] Biological control is gradually gaining attention due to its advantages such as no pesticide residue, environmental protection, and sustainability. It is currently a very promising means of agricultural control. However, the application of single microbial agents in the field faces many challenges, including interference from biological factors and multiple influences from the non-biological environment, so it is difficult to achieve the best effect. In contrast, compound microbial agents can more effectively inhibit the growth of pathogenic microorganisms through the synergistic effect between different microorganisms, enhance the disease resistance and adaptability of plants, and thus more efficiently prevent and control diseases. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a composite microbial agent that can effectively prevent and treat plant fungal diseases, so as to overcome the disadvantages of using a single agent in the field.

[0006] In order to solve the above technical problems, the present invention first provides a composite microbial agent.

[0007] The active ingredients of the composite microbial inoculant provided by the invention are Burkholderia ambifaria and Bacillus velezensis.

[0008] Furthermore, the CFU ratio of Burkholderia ambifaria to Bacillus velezensis is 1:1.

[0009] Furthermore, the Burkholderia ambifaria is the Burkholderia ambifaria strain Aa27.

[0010] The Bacillus velezensis is the Bacillus velezensis strain Gc8.

[0011] Furthermore, in the composite microbial agent, the concentrations of the Burkholderia ambifaria strain Aa27 and the Bacillus velezensis strain Gc8 are both 5×10 8 ~5×10 9 cfu / mL.

[0012] In order to solve the above technical problems, the present invention also provides a method for preparing a composite microbial agent.

[0013] The preparation method of the composite microbial inoculant provided by the invention is to mix a Burkholderia ambifaria inoculant and a Bacillus velezensis inoculant uniformly.

[0014] Furthermore, the concentration of the Burkholderia ambifaria agent is 1×10 9 ~1×10 10 CFU / mL.

[0015] The concentration of the Bacillus velezensis inoculant is 1×10 9 ~1×10 10 CFU / mL.

[0016] Furthermore, the volume ratio of the Burkholderia ambifaria bacterial agent to the Bacillus velezensis bacterial agent is 1:1.

[0017] Furthermore, the Burkholderia ambifaria is the Burkholderia ambifaria strain Aa27.

[0018] The Bacillus velezensis is the Bacillus velezensis strain Gc8.

[0019] In some embodiments, the preparation method of the Burkholderia ambifaria inoculum comprises the following steps: activating the Burkholderia ambifaria strain Aa27 on an LB solid plate and culturing at 28°C for 1 day; picking a single colony and inoculating it into an LB liquid culture medium, shaking and culturing at 28°C and 200rpm / min for 1 day to obtain a seed solution; inoculating the seed solution into an NB culture medium at a ratio of 5%, shaking and culturing at 28°C and 200rpm / min for 4 days to obtain the Burkholderia ambifaria inoculum.

[0020] In some embodiments, the preparation method of the Bacillus velezensis inoculum comprises the following steps: activating the Bacillus velezensis strain Gc8 on an LB solid plate and culturing at 28°C for 1 day; picking a single colony and inoculating it into an LB liquid culture medium, shaking and culturing at 28°C and 200rpm / min for 1 day to obtain a seed solution; inoculating the seed solution into a Landy modified culture medium at a ratio of 5%, shaking and culturing at 28°C and 200rpm / min for 4 days to obtain the Bacillus velezensis inoculum.

[0021] In order to solve the above technical problems, the present invention also provides new uses of the above composite microbial agent or the composite microbial agent prepared by the above method or the above Burkholderia ambifaria agent or the above Bacillus velezensis agent.

[0022] The present invention provides the use of the composite microbial agent or the composite microbial agent prepared by the above method or the Burkholderia ambifaria agent or the Bacillus velezensis agent in any one of the following (1)-(10):

[0023] (1) Inhibit plant pathogenic fungi;

[0024] (2) Preparation of products that inhibit plant pathogenic fungi;

[0025] (3) Prevent and control plant fungal diseases;

[0026] (4) Preparation of products for preventing and controlling plant fungal diseases;

[0027] (5) Promote plant seed germination;

[0028] (6) preparing products for promoting plant seed germination;

[0029] (7) Promote plant growth;

[0030] (8) Preparation of products that promote plant growth;

[0031] (9) Increase plant yield;

[0032] (10) Preparation of products for increasing plant yield.

[0033] In order to solve the above technical problems, the present invention also provides a method for inhibiting plant pathogenic fungi.

[0034] The method for inhibiting plant pathogenic fungi provided by the present invention comprises the step of treating plant pathogenic fungi with the composite microbial agent or the composite microbial agent prepared by the above method or the Burkholderia ambifaria agent or the Bacillus velezensis agent.

[0035] In some embodiments, the treatment method may be a plate confrontation culture method.

[0036] In order to solve the above technical problems, the present invention finally provides a method for preventing and controlling plant fungal diseases or promoting plant seed germination or promoting plant growth or increasing plant yield.

[0037] The method for preventing and controlling plant fungal diseases or promoting plant seed germination or promoting plant growth or increasing plant yield provided by the present invention comprises the step of applying the above-mentioned composite microbial agent or the composite microbial agent prepared by the above-mentioned method or the above-mentioned Burkholderia ambifaria agent or the above-mentioned Bacillus velezensis agent to plants.

[0038] In some embodiments, the frequency and amount of application are as follows: dilute the composite microbial agent 50 times, and soak plant seeds or seed stems (such as dry sugarcane segments) with the diluted composite microbial agent before sowing; spray the plants with the diluted composite microbial agent during the tillering stage.

[0039] In some preferred embodiments, the soaking time is 1 hour.

[0040] In some preferred embodiments, the spraying amount is 50 L / mu.

[0041] Any of the above plants may be dicotyledons or monocotyledons. Further, the monocotyledons are grass plants. Further, the grass plants are Saccharum plants. Further, the Saccharum plants are sugarcane.

[0042] Any of the above-mentioned plant pathogenic fungi is a sugarcane pathogenic fungus.

[0043] In some embodiments, the sugarcane pathogenic fungus is Ustilago canephora.

[0044] In some specific embodiments, the inhibition of Sugarcane Whip Ustilago may be embodied in inhibiting the sexual mating of basidiospores of Sugarcane Whip Ustilago and / or inhibiting the germination of teliospores of Sugarcane Whip Ustilago.

[0045] Any of the above plant fungal diseases is a sugarcane fungal disease.

[0046] In some embodiments, the sugarcane fungal disease is sugarcane smut.

[0047] In some specific embodiments, the controlling of sugarcane smut is manifested as reducing the incidence of sugarcane smut and / or improving the control effect of sugarcane smut.

[0048] Any of the above-mentioned methods for promoting plant seed germination is promoting sugarcane stem germination.

[0049] In some embodiments, the promoting sugarcane stem germination is manifested as increasing the germination rate of the sugarcane stem.

[0050] Any of the above-mentioned promoting plant growth is promoting sugarcane growth.

[0051] In some embodiments, the promoting sugarcane growth is manifested in increasing the height of sugarcane plants and / or increasing the number of sugarcane stalks (effective stalks).

[0052] Any of the above-mentioned methods for increasing plant yield is increasing sugarcane yield.

[0053] In some embodiments, the increasing sugarcane yield is reflected in increasing the yield of sugarcane plots and / or increasing the yield of sugarcane per mu.

[0054] Any of the above-mentioned Burkholderia ambifaria strains Aa27 was deposited in the China General Microbiological Culture Collection Center on November 14, 2023, with the deposit number CGMCC No.28992.

[0055] Any of the above-mentioned Bacillus velezensis strains Gc8 was deposited in the China General Microbiological Culture Collection Center on November 14, 2023, with the deposit number CGMCC No.28991.

[0056] The present invention provides a composite microbial agent, the active ingredients of which are Burkholderia ambifaria strain Aa27 and Bacillus velezensis strain Gc8. Experiments have shown that both Burkholderia ambifaria strain Aa27 and Bacillus velezensis strain Gc8 can inhibit the germination of winter spores of sugarcane whip smut fungus and the mating between "+" and "-" basidiospores, and the composite microbial agent composed of the two can not only effectively prevent and control sugarcane smut, but also can be used to promote sugarcane growth and increase sugarcane yield. The composite microbial agent provided by the present invention is of great significance for the prevention and control of plant fungal diseases.

[0057] Collection Instructions

[0058] Bacteria species name: Burkholderia bifida

[0059] Latin name: Burkholderia ambifaria

[0060] Strain ID: Aa27

[0061] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0062] Abbreviation of depository institution: CGMCC

[0063] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0064] Deposit date: November 14, 2023

[0065] CGMCC Registration Number: CGMCC No.28992

[0066] Bacteria name: Bacillus Velez

[0067] Latin name: Bacillus velezensis

[0068] Strain ID: Gc8

[0069] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0070] Abbreviation of depository institution: CGMCC

[0071] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0072] Deposit date: November 14, 2023

[0073] CGMCC Registration Number: CGMCC No.28991 BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 This is the phylogenetic tree of strain Aa27 based on 16S rRNA.

[0075] Figure 2 Phylogenetic tree of strain Gc8 based on 16S rRNA.

[0076] Figure 3 Plates are facing each other; A: blank control; B: bispecific Burkholderia Aa27; C: Bacillus Velezii Gc8.

[0077] Figure 4 Scanning electron microscopy of teliospores under different treatments; A: blank control; B: Burkholderia bifida Aa27; C: Bacillus velezensis Gc8. DETAILED DESCRIPTION

[0078] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0079] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0080] The culture medium and its formulation involved in the following embodiments are as follows:

[0081] The solvent of LB liquid culture medium is water, and the solutes and their concentrations are as follows: sodium chloride 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, and the pH value is 7.2.

[0082] The solvent of LB solid medium is water, and the solutes and their concentrations are as follows: sodium chloride 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, agar 15 g / L, and the pH value is 7.2.

[0083] The solvent of NB medium is water, and the solutes and their concentrations are as follows: beef extract 3 g / L, peptone 10 g / L, and sodium chloride 5 g / L.

[0084] The solvent of Landy's modified medium is water, and the solutes and their concentrations are as follows: sodium L-glutamate 5.0 g / L, glucose 10.0 g / L, magnesium sulfate heptahydrate 0.5 g / L, potassium chloride 0.78 g / L, potassium dihydrogen phosphate 1.0 g / L, ferrous sulfate 0.05 mg / L, manganese sulfate 5.0 mg / L, copper sulfate 0.16 mg / L, pH 7.2.

[0085] The solvent of the PDA liquid culture medium is water, and the solutes and their concentrations are as follows: peeled potatoes 200 g / L, glucose 20 g / L.

[0086] The solvent of PDA solid culture medium is water, and the solutes and their concentrations are as follows: peeled potato 200 g / L, glucose 20 g / L, agar powder 15 g / L.

[0087] The sugarcane variety Liucheng 05-136 in the following examples is recorded in the document “Zhao Jun, Wu Caiwen, He Mingde, Zan Fenggang, Xia Hongming, Zhong Yongchun, Luo Zhiqiang, Analysis on the high yield and stable yield of Yunzhe 05-51 and Liucheng 05-136 in Gengma sugarcane area, Yunnan Province, Subtropical Research, 2016, 12(03): 151-155.”

[0088] The positive and negative basidiospores JG35 and JG36 of sugarcane smut fungus in the following examples are both recorded in the document “Hao-yang Z, Yan-fang Y, Feng G, et al. SsRSS1 mediates salicylic acid tolerance and contributes to virulence in sugarcane smut fungus. Journal of Integrative Agriculture, 2023, 22(07): 2126-2137.”

[0089] The 25% pyraclostrobin in the following examples is a product of BASF Crop Protection GmbH.

[0090] Example 1: Isolation, identification and preservation of biocontrol strains Aa27 and Gc8

[0091] I. Isolation and Screening of Biocontrol Strains

[0092] 1. Isolation and Screening of Strain Aa27

[0093] Weigh 5 g of rhizosphere soil (rhizosphere soil from tobacco in Sanming City, Fujian Province), put it into 100 mL of sterilized water, shake it on a shaker for 30 min, and dilute it serially to 10 -4 、10 -5 . Respectively, pipette 100 μL of the diluted solution and spread it on an LB plate, incubate it at 28 °C, pick colonies with different morphologies and colors for streak purification, and finally store them in a glycerol solution with a final concentration of 25% and store them in a -80 °C refrigerator. Through plate confrontation screening, strains with inhibitory effects on Sporisorium scitamineum were obtained. Finally, 1 strain with relatively good inhibitory effects on Sporisorium scitamineum was obtained and named Strain Aa27.

[0094] 2. Isolation and Screening of Strain Gc8

[0095] Weigh 5 g of rhizosphere soil (rhizosphere soil from tobacco in Enshi City, Hubei Province), put it into 100 mL of sterilized water, shake it on a shaker for 30 min, and dilute it serially to 10 -4 、10 -5 . Respectively, pipette 100 μL of the diluted solution and spread it on an LB plate, incubate it at 28 °C, pick colonies with different morphologies and colors for streak purification, and finally store them in a glycerol solution with a final concentration of 25% and store them in a -80 °C refrigerator. Through plate confrontation screening, strains with inhibitory effects on Sporisorium scitamineum were obtained. Finally, 1 strain with relatively good inhibitory effects on Sporisorium scitamineum was obtained and named Strain Gc8.

[0096] II. Molecular Identification of Biocontrol Strains

[0097] Use the Bacterial Genomic DNA Extraction Kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract the genomic DNA of strains Aa27 and Gc8, perform PCR amplification with universal primers 27F and 1492R, and sequence the amplified products by Beijing Tianyihuiyuan Biotechnology Co., Ltd. to obtain the 16S rRNA sequences of strain Aa27 and strain Gc8 respectively. Among them, the 16S rRNA sequence of strain Aa27 is shown as SEQ ID NO.1, and the 16S rRNA sequence of strain Gc8 is shown as SEQ ID NO.2.

[0098] The sequence obtained was uploaded to the EzBioCloud database for strain identification, and the sequence with the highest similarity was selected using MEGA 7.0 software to construct a phylogenetic tree using the neighbor-joining method. The results showed that the similarity between strain Gc8 and Bacillus velezensis CR-502 was 99.86%, and strain Gc8 was between Bacillus velezensis CR-502 and Bacillus siamensis KCTC 13613 on the evolutionary tree, so strain Gc8 was identified as Bacillus velezensis CR-502 ( Figure 1 The similarity between strain Aa27 and Burkholderia ambifaria AMMD was 99.79%, and strain Aa27 was divided into a branch with Burkholderia ambifaria AMMD on the evolutionary tree, so strain Aa27 was identified as Burkholderia ambifaria ( Figure 2 ).

[0099] 3. Preservation of biocontrol strains

[0100] According to the molecular identification results in step 2, strain Aa27 was determined to be Burkholderia ambifaria and strain Gc8 was determined to be Bacillus velezensis.

[0101] The strain Aa27 is named Burkholderia ambifaria and has been deposited in the China General Microbiological Culture Collection on November 14, 2023, with the deposit number CGMCC No. 28992. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0102] The strain Gc8 is named Bacillus velezensis and has been deposited in the China General Microbiological Culture Collection Center on November 14, 2023, with the deposit number CGMCC No. 28991. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0103] Example 2: Inhibitory activity of biocontrol strains Aa27 and Gc8 against sugarcane whip smut fungus

[0104] The life cycle of sugarcane whip smut goes through three stages: winter spore, basidiospore, and binucleate mycelium. Under suitable conditions, winter spores germinate to produce positive and negative mating basidiospores. The colony morphology of basidiospores of the same mating type on the culture medium is yeast-like. Basidiospores of different mating types form binucleate mycelium through sexual mating. Only binucleate mycelium has the ability to infect sugarcane. After infecting sugarcane, winter spores can be produced, thus completing the entire life cycle. Therefore, reducing the germination rate of winter spores and interfering with the formation of binucleate mycelium are crucial for the prevention and control of sugarcane whip smut.

[0105] 1. Biocontrol strains Aa27 and Gc8 inhibit sexual maturation of basidiospores of Ustilago officinalis

[0106] 1. Take out the strains Aa27 and Gc8 stored in the -80℃ refrigerator, streak them on LB solid culture medium, pick single colonies and inoculate them on LB liquid culture medium, shake and culture them on a shaker at 28℃ for 2 days to obtain Aa27 biocontrol bacteria liquid and Gc8 biocontrol bacteria liquid.

[0107] 2. Take out the positive and negative basidiospores of sugarcane whip smut fungus JG35 and JG36 from the -80℃ refrigerator, spread them on PDA plates, and culture them at 28℃ for 2 days. Scrape an appropriate amount of colonies into a sterile centrifuge tube, fully suspend them with sterile water, and adjust the OD 600 is 1.0, and JG35 suspension and JG36 suspension are obtained respectively.

[0108] 3. Mix equal volumes of JG35 suspension and JG36 suspension of equal concentration to obtain a mixed suspension; take 10 μL of the mixed suspension and spot-spread it in the center of the PDA plate, and spot-spread 5 μL of biocontrol bacteria solution (Aa27 biocontrol bacteria solution or Gc8 biocontrol bacteria solution) at a position of about 1 cm around it. At the same time, use a plate without biocontrol bacteria inoculation as a control (CK), and observe the results after culturing at 28°C for 2 days.

[0109] The results are as follows Figure 3 As shown, the results showed that on the control plate, the sugarcane whip smut fungus was white binucleate mycelium ( Figure 3 A), but the colonies treated with Burkholderia bifida Aa27 and Bacillus velezensis Gc8 did not produce binucleate hyphae and remained yeast-like ( Figure 3 B and Figure 3 C), indicating that the biocontrol bacteria Aa27 and Gc8 can inhibit the sexual maturation of basidiospores of Ustilago officinalis.

[0110] 2. Biocontrol strains Aa27 and Gc8 inhibited the germination of teliospores of sugarcane whip smut fungus

[0111] 1. Take out the strains Aa27 and Gc8 stored in the -80℃ refrigerator, streak them on LB solid medium, pick single colonies and inoculate them on LB liquid medium, shake and culture them at 28℃ for 1 day to obtain Aa27 seed solution and Gc8 seed solution respectively.

[0112] 2. Inoculate Aa27 seed solution and Gc8 seed solution into PDB liquid culture medium at a volume ratio of 5%, culture at 28°C, 200 rpm / min for 4 days, centrifuge at 12000 rpm for 2 min, and filter with a 0.22 μm filter membrane to obtain Aa27 sterile fermentation supernatant and Gc8 sterile fermentation supernatant, respectively.

[0113] 3. Use PDB liquid medium to dilute the winter spores of sugarcane whip smut fungus to 1×10 6 100 μL of winter spores and 100 μL of sterile fermentation supernatant (Aa27 sterile fermentation supernatant or Gc8 sterile fermentation supernatant) were mixed and inoculated into a sterile 50 mL inverted centrifuge tube cap to cover the inside of the centrifuge tube cap. An equal amount of PDB liquid medium was used as a control (CK). After the centrifuge tube body was screwed on, the tube was inverted and cultured for more than 8 hours at 28°C incubator. The winter spore germination was examined by scanning electron microscopy (SEM). The sample preparation and electron microscopy were completed by the electron microscopy platform of the Institute of Microbiology, Chinese Academy of Sciences, and the spore germination rate of each treatment was calculated (the production of clear germ tubes was counted as germination). Spore germination rate = (germination spore number / investigation total) × 100%.

[0114] The inhibitory effects of biocontrol bacteria on the germination of teliospores of sugarcane whip smut fungus are shown in Table 1 and Figure 4 As shown, the results showed that the number of germination of winter spores in the strain Aa27 and Gc8 treatment groups was very small, almost not visible, while the PDB control group was full of many germinated spores. In addition, spore perforation and rupture were observed in both strain Aa27 and strain Gc8 treatments, indicating that the fermentation broth of strains Aa27 and Gc8 can not only inhibit the germination of winter spores, but also destroy the shell of winter spores to kill spores.

[0115] Table 1. Germination of teliospores of sugarcane whip smut fungus in the treatment group and the control group

[0116] deal with Teliospore germination rate (%) Aa27 (1.8±0.46)b Gc8 (1.13±0.4)b Control group (92.5±0.8)a

[0117] Note: Value = mean ± standard error, different lowercase letters indicate significant difference (P < 0.05).

[0118] Example 3. Preparation of single bacterial agent and composite microbial agent

[0119] 1. Take out the strains Aa27 and Gc8 stored in the -80℃ refrigerator, streak them on LB solid medium, and invert them at 28℃ for 1 day. Pick a single colony and inoculate it into LB liquid medium, shake and culture it at 28℃ for 1 day to obtain Aa27 seed liquid and Gc8 seed liquid respectively.

[0120] 2. Inoculate the Aa27 seed solution into NB medium at a volume ratio of 5%, and culture at 28°C and 200 rpm / min for 4 days to obtain Aa27 single bacterial agent. The number of viable bacteria in Aa27 single bacterial agent is 1×10 9 ~1×10 10 CFU / mL.

[0121] The Gc8 seed solution was inoculated into Landy modified medium at a volume ratio of 5%, and cultured at 28°C and 200 rpm / min for 4 days to obtain a single bacterial agent of Gc8. The number of viable bacteria in the single bacterial agent of Gc8 was 1×10 9 ~1×10 10 CFU / mL.

[0122] 3. Mix equal volumes of Aa27 single bacterial agent and Gc8 single bacterial agent to obtain a composite microbial agent.

[0123] Example 4: Application of a single bacterial agent in improving the germination rate of sugarcane stems

[0124] The effects of the single bacterial agent Aa27 and the single bacterial agent Gc8 prepared in Example 3 on sugarcane germination were verified in a greenhouse. The specific steps are as follows:

[0125] 1. Select sugarcane material Liucheng 05-136, wash the sugarcane stems with clean water, and make double bud segments.

[0126] 2. Treat the double bud segments as follows:

[0127] Aa27 single bacterial agent group: The Aa27 single bacterial agent prepared in Example 3 was diluted 10 times, and then the sugarcane double-bud segments were soaked in the diluted Aa27 single bacterial agent for 1 hour.

[0128] Gc8 single bacterial agent group: The Gc8 single bacterial agent prepared in Example 3 was diluted 10 times, and then the sugarcane double-bud segments were soaked in the diluted Gc8 single bacterial agent for 1 hour.

[0129] Clear water control group: the bacterial agent in the single bacterial agent group was replaced with an equal amount of clear water.

[0130] 3. Allow to dry naturally after soaking, then plant in a matrix of vermiculite and nutrient soil, culture at 28°C, 16 hours light / 8 hours dark, and count the germination rate after 20 days of culture.

[0131] The statistical results of the germination rates of each treatment group are shown in Table 2. As shown in Table 2, the single bacterial agent Aa27 or the single bacterial agent Gc8 of the present invention can effectively improve the germination rate of sugarcane.

[0132] Table 2. Statistical results of germination rate in treatment group and control group

[0133] deal with Number of buds (46 buds in total) Germination rate Control group 31 67.39％ Aa27 34 73.91％ Gc8 37 80.43％

[0134] Example 5: Application of composite microbial agent in controlling sugarcane smut

[0135] The control effect of the composite microbial agent prepared in Example 3 on sugarcane smut was verified in a field test, and the specific steps were as follows:

[0136] Sugarcane is planted in the sugarcane fields in Fenghuang Town, Xingbin District, Laibin City. The sugarcane variety is Liucheng 05-136. Operations such as watering and fertilizing are consistent with normal field management measures.

[0137] The experiment set up the following three treatment groups: composite bacterial agent group, pyraclostrobin group and clear water control group. The treatment method of each group is as follows:

[0138] Composite microbial agent group: The composite microbial agent in Example 3 was diluted 50 times to obtain the composite microbial agent used in the field test. Before sowing, the composite microbial agent was used to soak the sugarcane segments for 1 hour for the first treatment, and a sprayer was used for the second treatment during the tillering stage, with a spraying rate of 50 L per mu.

[0139] Pyraclostrobin group: Before sowing, 25% pyraclostrobin was diluted 2000 times and soaked in sugarcane seeds for 1 hour before sowing.

[0140] Clean water control group: The composite microbial agent in the composite agent group was replaced with an equal amount of clean water.

[0141] At maturity, the plant height, stem diameter, plot yield, number of effective stems, per-acre yield, smut incidence rate and biocontrol effect of each treatment group were counted. Biocontrol effect (control effect) = {(smut incidence rate of control group - smut incidence rate of treatment group) / smut incidence rate of control group} × 100%.

[0142] The yield index test results of each treatment group are shown in Table 3. As shown in Table 3, the composite microbial agent of the present invention can effectively promote sugarcane growth and increase sugarcane yield. Compared with the clear water control group, the sugarcane yield increased by 0.83 tons per 667 square meters (mu); compared with the pyraclostrobin group, the sugarcane yield increased by 0.22 tons per 667 square meters (mu).

[0143] Table 3. Yield index determination results of treatment group and control group

[0144] Group Compound bacterial agent group Pyraclostrobin group Control group Plant height (cm) 254.57 182 243.37 Stem diameter(mm) 29.13 32.07 29.5 <![CDATA[Yield of the plot (kg / 60m 2 )]]> 624.59 604.86 549.51 <![CDATA[Effective number of stems (stems / 60 m 2 )]]> 450 440 410 Yield per mu (tons) 6.94 6.72 6.11

[0145] The statistical results of the incidence of sugarcane smut in each treatment group are shown in Table 4. It can be seen from the results in Table 4 that the compound microbial inoculant of the present invention can effectively control sugarcane smut, with a control efficacy of 96.36%, exceeding the control efficacy of the pesticide pyraclostrobin, indicating that the compound microbial inoculant has good biocontrol effects when applied in the field.

[0146] Table 4 Incidence of sugarcane smut in the treatment group and the control group

[0147] Treatment Group Smut incidence (%) Prevention effect (%) Compound bacterial agent group 0.96 96.36 Pyraclostrobin group 2.83 89.27 Control group 26.37 -

[0148] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application is intended to cover any changes, uses or improvements to the present invention, including those that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some basic features can be applied within the scope of the following appended claims.

Claims

1. A composite microbial agent, the active ingredients of which are Burkholderia ambifaria and Bacillus velezensis.

2. The composite microbial agent according to claim 1, characterized in that: The cfu ratio of Burkholderia ambifaria and Bacillus velezensis is 1:

1.

3. The composite microbial agent according to claim 1 or 2, characterized in that: The Burkholderia ambifaria is a Burkholderia ambifaria strain Aa27; the deposit number of the Burkholderia ambifaria strain Aa27 in the China General Microbiological Culture Collection Center is CGMCC No.28992; The Bacillus velezensis is the Bacillus velezensis strain Gc8; the deposit number of the Bacillus velezensis strain Gc8 in the China General Microbiological Culture Collection Center is CGMCC No.28991.

4. A method for preparing a composite microbial agent, which is obtained by mixing a Burkholderia ambifaria agent and a Bacillus velezensis agent.

5. The method according to claim 4, characterized in that: The concentration of the Burkholderia ambifaria inoculant is 1×10 9 ~1×10 10 CFU / mL; The concentration of the Bacillus velezensis inoculant is 1×10 9 ~1×10 10 CFU / mL; Alternatively, the volume ratio of the Burkholderia ambifaria bacterial agent to the Bacillus velezensis bacterial agent is 1:

1.

6. The method according to claim 4 or 5, characterized in that: The Burkholderia ambifaria is a Burkholderia ambifaria strain Aa27; the deposit number of the Burkholderia ambifaria strain Aa27 in the China General Microbiological Culture Collection Center is CGMCC No.28992; The Bacillus velezensis is the Bacillus velezensis strain Gc8; the deposit number of the Bacillus velezensis strain Gc8 in the China General Microbiological Culture Collection Center is CGMCC No.28991.

7. Use of the composite microbial agent according to any one of claims 1 to 3, or the composite microbial agent prepared by the method according to any one of claims 4 to 6, or the Burkholderia ambifaria agent according to claims 4 to 6, or the Bacillus velezensis agent according to claims 4 to 6 in any one of the following (1) to (10): (1) Inhibit plant pathogenic fungi; (2) Preparation of products that inhibit plant pathogenic fungi; (3) Prevent and control plant fungal diseases; (4) Preparation of products for preventing and controlling plant fungal diseases; (5) Promote plant seed germination; (6) preparing products for promoting plant seed germination; (7) Promote plant growth; (8) Preparation of products that promote plant growth; (9) Increase plant yield; (10) Preparation of products for increasing plant yield.

8. A method for inhibiting plant pathogenic fungi, comprising the step of treating plant pathogenic fungi with the composite microbial agent described in any one of claims 1 to 3, or the composite microbial agent prepared by the method described in any one of claims 4 to 6, or the Burkholderia ambifaria agent described in any one of claims 4 to 6, or the Bacillus velezensis agent described in any one of claims 4 to 6.

9. A method for preventing and controlling plant fungal diseases or promoting plant seed germination or promoting plant growth or increasing plant yield, comprising the step of applying the composite microbial agent described in any one of claims 1 to 3 or the composite microbial agent prepared by the method described in any one of claims 4 to 6 or the Burkholderia ambifaria agent described in any one of claims 4 to 6 or the Bacillus velezensis agent described in any one of claims 4 to 6 to plants.

10. The use according to claim 7 or the method according to claim 8 or 9, characterized in that: The plant is sugar cane; Or, the plant pathogenic fungus is Ustilago canefolia; Or, the plant fungal disease is sugarcane smut.

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