CN118931801A

CN118931801A - Biocontrol strain SZ-T8 and its application in controlling corn root rot

Info

Publication number: CN118931801A
Application number: CN202411331262.6A
Authority: CN
Inventors: 胡伟民; 董文霞; 黄奇; 任禹琪; 冯忠平; 权晓康; 盛呈泽; 曲思宇; 石依萍
Original assignee: Huzhou Agricultural Science Research Institute Huzhou Agricultural Science And Technology Development Center; Zhejiang University ZJU
Current assignee: Huzhou Agricultural Science Research Institute Huzhou Agricultural Science And Technology Development Center; Zhejiang University ZJU
Priority date: 2024-09-24
Filing date: 2024-09-24
Publication date: 2024-11-12

Abstract

The present invention belongs to the field of development and utilization of microbial resources, and specifically relates to a biocontrol strain SZ-T8 and an application thereof in preventing and controlling corn root rot. The classification name of the biocontrol strain is Bacillus velezensis SZ-T8, and the deposit unit is China Center for Type Culture Collection, the deposit number is CCTCC NO: M 20241102, and the deposit date is May 30, 2024. The biocontrol bacteria SZ-T8 provided by the present invention has a significant inhibitory effect on three main pathogens of corn root rot, "Fusarium graminearum", "Fusarium pseudo-verticillium" and "Fusarium spp.", and the average inhibition rate is more than 90%; the use of the live bacterial suspension prepared by the present invention to soak the seeds of the sweet corn inbred line can significantly reduce the incidence of root rot in the seedling stage, and is better than the commonly used agent thiophanate-methyl, and at the same time has a promoting effect on the growth of corn seedlings, which can ensure the safety of seed production and breeding. In addition, SZ‑T8 also has the ability to prevent and treat a variety of plant diseases and has a broad-spectrum biological control effect.

Description

Biocontrol strain SZ-T8 and application thereof in preventing and controlling corn root rot

Technical Field

The invention belongs to the field of development and utilization of microbial resources, and particularly relates to a biocontrol strain SZ-T8 and application thereof in preventing and controlling corn root rot.

Background

Corn (Zea mays l.) is an annual gramineous plant of the genus Zea, which not only plays an important role in grain production, but is also widely used in a variety of fields (Nuss ET.Maize:A paramount staple crop in the context of global nutrition.Compr.Rev.Food Sci.Food Saf.2010,9,417-436.). such as food, feed, energy, industry, etc., however, corn is often subjected to damage from corn root rot (Maize fusarium root rot, MFRR) during growth and development, sweet corn is inhibited by mutated endosperm genes due to starch synthesis in the kernel, resulting in shrinkage and low vigor of seeds, especially sweet corn inbred line seeds that are not subjected to coating treatment are susceptible to attack by pathogenic bacteria of root rot at the early stage of germination due to weak resistance. (Lu Wenjia. Screening of sweet corn seed coating agent and optimization of coating technological parameters [ J ]. Chinese seed, 2014,11,56-58.). Infection with root rot causes not only browning and rot of root system, tissue necrosis, browning and rot of mesocotyl connected to grain, but also yellowing and wilting of plant leaf tips and even death of seedlings (Li Jun. Occurrence and control of root rot in maize seedling stage [ J. Agricultural development and equipment, 2014 (07): 123.). The disease is widely generated in corn production areas on the global scale, the incidence rate of serious plots is even more than 80 percent, and the disease is one of important factors for restricting the high and stable yield of corn (Díaz Arias.Aggressiveness of Fusarium species and impact of root infection on growth and yield of soybeans.Phytopathology2013,103,822–832.).

The main pathogenic bacteria of the corn root rot are fusarium graminearum (Fusarium graminearum), fusarium verticillatum (Fusarium verticillioides) and fusarium graminearum (Fusarium proliferatum), which are used as pathogens or endophytes to be combined with corn seeds and can generate a large amount of toxins, so that the sowing period which damages the growth and development of corn seedlings to (Paola Pereira.Effects of maize inoculation with Fusarium verticillioides and with two bacterial biocontrol agents on seedlings growth and antioxidative enzymatic activities.Soil Ecology,2010,51,52-59.). years is the optimal infection period of the corn root rot pathogens, the pathogens overwintering in soil or disease residues in the form of sclerotium and mycelium, the pathogens start to infect the corn seeds or seedlings once encountering proper disease conditions, and then spread in the corn plants, thereby causing the root rot and stem rot in sequence and spreading to the ear to cause corn ear rot (Bacon CW.Fusarium verticillioides:managing the endophytic association with maize for reduced fumonisins accumulation.Toxin Rev.,2008,27,1-36.)..

Compared with traditional chemical Control, biological Control is a disease and pest Control technology combining a green production concept with agricultural production practice, and can effectively avoid the problems of pesticide residues, disease-resistant variety failure and the like (Dubey M.biological Control of PLANT DISEASES IN CHANGING environmental. Agronomy,2021,11,2000.). Along with the continuous deep research on the ecology and diversity of related microorganisms of corn rhizosphere at home and abroad, the plant-microorganism interaction control technology is considered to have high application value in corn production, and by taking Bacillus bailii BM21 screened by Wang Shuang et al as example (Wang S.Bacillus velezensis BM21,a potential and efficient biocontrol agent in control of corn stalk rot caused by Fusarium graminearum.Egypt J Biol Pest Control,2020,30,9.),, the growth inhibition rate of Fusarium graminearum hyphae causing corn stalk rot is 79.2%, and in a potting test, the incidence rate of corn stalk rot can be reduced by 72.4% -77.4% by root irrigation of a live bacterial extract of BM 21.

In conclusion, the development of biocontrol bacteria with high specificity to corn root rot can reduce the dependence on environment-friendly chemicals, improve crop productivity and inject new vigor for sustainable development of agriculture.

Disclosure of Invention

Aiming at the demand of biocontrol bacteria with high specificity on corn root rot in the prior art, the invention provides biocontrol strain SZ-T8 and application thereof in preventing and treating corn root rot, and the specific technical scheme is as follows:

In a first aspect, the invention provides a biocontrol strain SZ-T8, which is classified and named as Bacillus bailii (Bacillus velezensis) SZ-T8, and has a preservation unit of China Center for Type Culture Collection (CCTCC) NO: m20241102, the preservation date is 2024, 5 and 30.

Further, the 16S rDNA sequence of the biocontrol strain SZ-T8 is shown as SEQ ID NO. 1.

The biocontrol bacterial strain SZ-T8 was isolated in 2023 from the rhizosphere soil of maize Tian Zhizhu in Tokyo village, shangzhi, qinglong Jiangkou, shangzhi, and has remarkable inhibition effect on three pathogenic bacteria of Fusarium graminearum, fusarium verticillium and Fusarium layering, which cause corn root rot.

In a second aspect, the invention provides a biocontrol microbial agent, wherein the active ingredient of the biocontrol microbial agent is the biocontrol strain SZ-T8 of claim 1.

Further, in the biocontrol microbial inoculum, the concentration of the biocontrol strain SZ-T8 is 1 multiplied by 10 ⁷～1×10⁹CFU·mL^-1.

Further, in the biocontrol microbial agent, the concentration of the biocontrol strain SZ-T8 is 1 multiplied by 10 ⁸CFU·mL^-1.

In a third aspect, the invention provides the use of the biocontrol strain SZ-T8 or the biocontrol agent in antagonizing one or more pathogenic bacteria from Fusarium graminearum, fusarium verticillium, fusarium layering.

In a fourth aspect, the invention provides application of the biocontrol strain SZ-T8 or the biocontrol microbial inoculum in preventing and controlling corn seedling root rot.

In a fifth aspect, the invention provides a method for preventing root rot in maize seedling stage, comprising:

Before sowing corn seeds, the biocontrol microbial inoculum is used for seed soaking.

Further, the corn is a sweet corn inbred line.

Still further, the maize variety is S622-432 or S745-1211.

Further, the seed soaking time is 4-12 h.

Further, the seed soaking time was 8 hours.

In a sixth aspect, the invention provides the use of the biocontrol strain SZ-T8 or the biocontrol microbial agent in promoting corn seedling survival.

In a seventh aspect, the invention provides the biocontrol strain SZ-T8 or the application of the biocontrol microbial inoculum in promoting the growth of the overground parts of corn seedlings.

Further, the application of the biocontrol microbial inoculum in promoting the survival of corn seedlings and promoting the growth of the overground parts of the corn seedlings is as follows: before sowing corn seeds, the biocontrol microbial inoculum is used for seed soaking.

Further, the seed soaking time is 4-12 h.

Further, the seed soaking time was 8 hours.

The biocontrol microbial inoculum prepared by the biocontrol strain SZ-T8 provided by the invention is used for soaking corn seeds, so that not only can the inhibition effect on Fusarium graminearum, fusarium verticillium and Fusarium layering be achieved, but also the survival rate of seedlings after corn seeds germinate can be remarkably improved. In the aspect of seedling growth, after seed soaking, the fresh weight of the overground part of the seedling can be remarkably improved, which indicates that the seed soaking by using the biocontrol microbial agent or the viable bacteria suspension of SZ-T8 can promote the healthy and strong growth of the corn seedling.

In an eighth aspect, the invention provides application of the biocontrol strain SZ-T8 in preventing and treating cucumber target spot disease, tomato early blight, crocodile leaf spot disease, cotton anthocyanin mildew, rice sheath blight or peanut southern blight.

Further, the pathogenic bacteria of the cucumber target spot disease are corynespora polymorpha Corynespora cassiicola (Berk.& Curt.), the pathogenic bacteria of the tomato early blight are alternaria solani ALTERNARIA SOLANI, the pathogenic bacteria of the crocodile leaf spot disease are corynespora polymorpha Corynespora cassiicola, the pathogenic bacteria of the cotton bud spot disease are penicillium Cotton Penicillium, the pathogenic bacteria of the rice sheath blight are rhizoctonia solani Rhizoctonia solani, and the pathogenic bacteria of the peanut southern blight are sclerotium rolfsii Sclerotium rolfsii.

In the invention, the biocontrol bacteria SZ-T8 are found to have inhibition effects on pathogenic bacteria causing cucumber target spot disease, tomato early blight, alligator leaf spot disease, cotton anthocyanin mildew, rice sheath blight disease and peanut southern blight, wherein the average inhibition effects on two pathogenic bacteria of Alternaria solani causing tomato early blight and sclerotium rolfsii causing peanut southern blight are best, and the average inhibition effects respectively reach 98.34% and 97.47%, so that the biocontrol bacteria SZ-T8 provided by the invention have broad-spectrum control effects on plant diseases.

Compared with the prior art, the invention has the following beneficial effects:

The biocontrol bacteria SZ-T8 provided by the invention has remarkable inhibition effects on 3 main pathogenic bacteria of corn root rot, namely Fusarium graminearum, fusarium verticillium and Fusarium layering, and the average inhibition rate is more than 90%; the viable bacteria suspension prepared by the method can be used for carrying out seed soaking treatment on sweet corn inbred line seeds, so that the incidence rate of root rot in the seedling stage can be remarkably reduced, the viable bacteria suspension is superior to a common medicament of thiophanate methyl, has a promoting effect on the growth of corn seedlings, and can ensure the safety of seed production and propagation. And SZ-T8 also has control capability on various plant diseases, and has broad-spectrum biological control effect.

Drawings

FIG. 1 shows the results of an in vitro plate stand-off experiment of 8 biocontrol strains selected in example 1 and Fusarium graminearum; wherein CK represents a control group using sterile water, and SZ-T8, SZ-S3, etc. are numbers of the strains used.

FIG. 2 shows the results of in vitro plate-challenge experiments of biocontrol strain SZ-T8 and Fusarium graminearum, fusarium verticillium and Fusarium layering in example 1; wherein CK represents a control group using sterile water, SZ-T8 is the number of the strain used, fg is Fusarium graminearum, fv is Fusarium verticillium, fp is Fusarium layering.

FIG. 3 is a schematic diagram showing growth of biocontrol strain SZ-T8 of the invention on LB plates.

FIG. 4 is a schematic diagram of a phylogenetic tree of biocontrol bacteria SZ-T8 constructed based on a 16S rDNA sequence.

FIG. 5 is a schematic diagram showing the inhibition of biocontrol bacteria SZ-T8 against 6 different pathogenic bacteria; wherein CK represents a control group using sterile water, c.cassiicola represents clavulans causing cucumber target spot disease, a.solani represents alternaria solani, c.cassiicola represents clavulans causing crocodile leaf spot disease, penicillium represents Penicillium, r.solani represents rhizoctonia solani, s.rolfsii represents rhizoctonia solani, in the order of the figure.

Detailed Description

In order to make the present invention more well understood by those skilled in the art, the following description of the present invention will be made with reference to specific embodiments. It should be noted that the following detailed description is exemplary and is merely an example of a portion, but not all, of the present invention.

All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, shall fall within the scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The experimental materials used in the embodiment of the application are all conventional in the field and can be purchased through commercial channels. Experimental methods without specifying detailed conditions were performed according to conventional experimental methods or according to the instructions recommended by the suppliers.

In the following examples, the pathogenic bacteria "Fusarium graminearum" (Fg), "Fusarium verticillium" (Fv) and "Fusarium layering" (Fp) of corn root rot: is provided by the institute of corn and special dry grain of agricultural academy of sciences of Zhejiang province and stored in a refrigerator at 4 ℃ in a laboratory.

Test soil sample: the rhizosphere soil of the maize plants which are respectively picked from Hainan, zhejiang, heilongjiang and other places and are healthily grown is stored in a refrigerator at the temperature of 4 ℃ in a laboratory.

Corn seeds: zheFeng glutinous No. 3, purchased from Zhejiang Don forget agriculture Co., ltd. Sweet corn inbred line S622-432, provided by Zhejiang university, is stored in a low temperature low humidity seed storage cabinet. Sweet corn inbred line S745-1211, offered by university of Zhejiang, is stored in a low temperature and low humidity seed storage cabinet.

Potato Dextrose Agar (PDA) (1000 mL): 6.0g of potato soaked powder, 20.0g of grape and 20.0g of agar, the volume of the potato soaked powder is 1000mL, and the potato soaked powder is sterilized by heat and humidity at 121 ℃ for 20min.

LB medium (1000 mL): 10.0g of tryptone, 5.0g of yeast extract, 10.0g of sodium chloride, 1000mL of constant volume and 20min of heat sterilization at 121 ℃.

Spore-forming medium (1000 mL): 2.0g of sodium dihydrogen phosphate, 2.0g of sodium nitrate, 1.0g of sodium chloride, 1.0g of sucrose, 1.0g of magnesium sulfate, 0.0002g of manganese sulfate, 0.0002g of ferrous sulfate and 0.0002g of zinc sulfate, and sterilizing the mixture to 1000mL of volume by moist heat at 115 ℃ for 20min.

Hoagland nutrient solution (1000 mL): 607mg of potassium sulfate, 115mg of ammonium dihydrogen phosphate, 493mg of magnesium sulfate, 20mg of EDTA iron sodium salt, 15mg of ferrous sulfate, 2.86mg of boric acid, 4.5mg of borax and 3mg of trace elements, and carrying out wet heat sterilization for 20min at 115 ℃ to 1000 mL.

Thiophanate methyl: the main component is thiophanate methyl, the chemical formula is C ₁₂H₁₄N₄O₄S₂, the broad-spectrum systemic low-toxicity bactericide can effectively prevent and treat diseases of various crops, and is produced by Ningbo Sanjiang Yinong chemical Co.

Seed germination bag (30 cm. Times.25.5 cm): purchased from phytoTC company.

In the following examples, the preparation steps of the biocontrol bacterium live bacterium suspension are as follows: inoculating biocontrol bacteria on an LB agar medium, culturing for 24 hours in a biochemical incubator at 28 ℃ for activation, taking activated bacterial cakes by a bacterial colony puncher with the diameter of 5mm, placing the bacterial cakes into a shaking tube filled with the LB medium, shaking and culturing for 24 hours on a shaking table at 200 r.min ^-1 and 30 ℃, and then sucking 5mL of bacterial liquid into a conical flask containing 250mL of LB medium for shaking for 48 hours to prepare bacterial suspension. Transferring the bacterial suspension into a 50mL centrifuge tube, centrifuging for 10min at 8000 r.min ^-1 and 28 ℃, pouring out the supernatant, and regulating the supernatant into bacterial suspension with the concentration of 1X 10 ⁸CFU·mL^-1 by using sterile water for later use.

In the following examples, the preparation steps of the pathogenic bacteria suspension are as follows: inoculating pathogenic bacteria to PDA agar medium, culturing at 28deg.C for 7d for activation, taking activated bacterial cake with colony puncher with diameter of 5mm, placing into 10mL shaking tube, adding 5mL spore-producing medium, placing into 180 r.min ^-1, shake culturing at 28deg.C for 3d on shaking table, sucking 5mL bacterial liquid, adding into 500mL conical flask, adding 250mL spore-producing medium, shaking for 7d to obtain bacterial suspension. Transferring the bacterial suspension into a centrifuge tube with a capacity of 50mL, centrifuging for 5min at the temperature of 4500 r.min ^-1 and the temperature of 25 ℃, pouring out the supernatant, diluting live bacteria at the bottom of the centrifuge tube by using sterile water, and regulating the bacterial suspension into bacterial suspension with the concentration of 1 multiplied by 10 ⁷CFU·mL^-1 for later use.

In the following examples, the analysis of the significance of differences between the different processed data (P < 0.05) was performed using the Waller-Duncan method using the SPSS27.0 software system.

In the following examples, the base sequence of the 16S rDNA gene sequence of Bacillus bailii SZ-T8 is shown in SEQ ID NO. 1.

SEQ ID NO.1：

GGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTG TACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAAACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATCTGTCCGCTCGACTA.

Example 1 isolation, screening and identification of biocontrol bacteria for corn seedling stage root rot

(1) Pathogenic bacteria pathogenicity determination

Firstly, carrying out high-pressure moist heat sterilization on a soil matrix suitable for seed growth at 121 ℃ for 20min for later use. Fully mixing pure water with a soil matrix, and filling the mixture into a culture box; the weight ratio of the pure water to the soil matrix is 0.7:1. Each treatment of 90 corn seeds (30 seeds per cassette, 3 cassettes combined into one repetition) was repeated three times. The culture was performed alternately at 25℃for 14 days under 12h of light (20000 lux) and 12h of darkness. The corn seed is ZheFeng glutinous No. 3.

Inoculating pathogenic bacteria to PDA agar medium, culturing at 28deg.C for 7d for activation, taking activated bacterial cake with colony puncher with diameter of 5mm, placing into 10mL shaking tube, adding 5mL spore-producing medium, placing into 180 r.min ^-1, shake culturing at 28deg.C for 3d on shaking table, sucking 5mL bacterial liquid, adding into 500mL conical flask, adding 250mL spore-producing medium, shaking for 7d to obtain bacterial suspension. The bacterial suspension is transferred into a centrifuge tube with the capacity of 50mL, the centrifugation is carried out for 5min at the temperature of 4500 r.min ^-1 and the temperature of 25 ℃, the living bacteria at the bottom of the centrifuge tube are diluted by sterile water after the supernatant is poured out, and the bacterial suspension with the concentration of 1 multiplied by 10 ⁷CFU·mL^-1 is prepared.

On day 6 of corn seed germination, bacterial suspensions of three pathogenic bacteria such as fusarium graminearum (Fg), fusarium graminearum (Fv) and fusarium graminearum (Fp) are inoculated to corn seedlings for root rot pathogenicity measurement, and the results are shown in table 1, and compared with unvaccinated seedlings (CK), the incidence of the seedlings treated by the three pathogenic bacteria is obviously increased, wherein the incidence and the disease index of the fusarium graminearum treated are the highest, and reach 73.33% and 29.33, which indicates that the fusarium graminearum is the most dominant pathogenic bacteria.

TABLE 1 disease conditions after 7 days of inoculation of different pathogenic bacteria onto maize seedlings

The different lower case letters after the same column of data in the table represent the significance of the difference in pathogenicity of different pathogens (P <0.05, duncan).

(2) Isolation and purification of rhizosphere soil bacteria

Healthy and growing corn plant rhizosphere soil samples are collected from phoenix and white chicken villages (south numerous base of Zhejiang university), si An Zhenxian mountain villages (agricultural test station of Zhejiang university) of Zhejiang province and Tokyo village of Harbin Shangzhi of Heilongjiang province respectively, and a dilution flat plate coating method is adopted to separate corn plant rhizosphere soil bacterial groups. Weighing 5g of soil sample, placing into a conical flask, adding 5 steel balls and 100mL of sterile water, placing into a constant temperature shaking table at 37 ℃ and 180 r.min ^-1, sufficiently shaking for 1h, taking out and standing for 15min, taking 5mL of supernatant under sterile conditions, carrying out gradient dilution to obtain 1X 10 ^-1、1×10^-2、1×10^-3、1×10^-4、1×10^-5 of dilution, respectively sucking 100 mu L of dilution, coating on an LB plate, culturing for 72h, after single colony grows out, selecting single colony with different morphological characteristics, inoculating into a new LB culture medium, separating and purifying, mixing the bacterial liquid with 50% glycerol 1:1 after pure culture bacteria are obtained, and placing into a refrigerator at-80 ℃ for standby. And (3) co-separating and purifying from the rhizosphere soil of the corn plants to obtain 47 strains of bacteria.

(3) Screening of biocontrol bacteria for corn root rot

The bacterial strain with antagonism to pathogenic bacteria is initially screened by adopting a plate counter method. Firstly, carrying out opposite culture on separated and purified bacteria and main pathogenic bacteria of corn root rot, namely fusarium graminearum (Fg), taking pathogenic bacteria cakes with the diameter of 5mm by a colony puncher in a super clean workbench, inoculating four bacterial cakes with the diameter of 5mm at four symmetrical points which are 2.5cm away from the center of a flat plate by a crisscross method, inoculating the bacterial cakes with four strains to be screened with the diameter of 5mm by taking the flat plate which is only connected with the pathogenic bacteria as a reference, reversely buckling the flat plate which is placed in a biochemical incubator at 28 ℃ for culturing for 7d, uploading the opposite pictures to an imageJ image processing system, measuring the growth area of pathogenic bacteria hypha, calculating the antibacterial rate, screening out the strains with obvious biocontrol activity on the pathogenic bacteria, and then carrying out co-culture on the screened biocontrol strains with fusarium graminearum (Fv) and fusarium graminearum (Fp) respectively so as to verify the inhibition effect of the strains on other pathogenic bacteria causing corn root rot.

Antibacterial ratio (%) = [ (control group pathogenic bacteria area-treatment group pathogenic bacteria area)/control group pathogenic bacteria area ] ×100%.

As shown in fig. 1 and table 2, 8 strains of 47 strains of bacteria had a better antagonism against the hyphal growth of fusarium graminearum; as shown in figure 2, the strain SZ-T8 can obviously inhibit the growth of fusarium graminearum, fusarium verticillium and fusarium venenatum hypha, and the bacteriostasis rates respectively reach 96.36%, 93.76% and 93.39%, which shows that the SZ-T8 has obvious inhibition effect on corn root rot pathogens. SZ-T8 was a bacterium isolated from the rhizosphere soil of maize Tian Zhizhu in Tokyo, amaranthaceae, shangzhi, shangzhi, of Harbin, junoron, 2023, and grown on LB plates as shown in FIG. 3.

TABLE 2 inhibition of Fusarium graminearum growth by different biocontrol strains

The different lower case letters after the same column of data in the table represent the significance of the differences in inhibition of fusarium graminearum by different strains (P <0.05, duncan).

(4) Identification of biocontrol strain

The screened antagonistic strain DNA was extracted using OMEGA bacterial DNA extraction kit. The 16S rDNA gene fragment of the biocontrol bacterium is amplified by adopting a PCR amplification kit, and the extracted genome DNA product is taken as a template. The forward primer sequence adopted by the amplified gene is 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), the reverse primer sequence is 1492R (5'-TACGGCTACCTTGTTACGACTT-3'), the product is subjected to agarose gel electrophoresis, the obtained product is sent to Hangzhou Shangya bioengineering company for DNA sequencing, the 16S rDNA gene sequence with the length of 1398 is obtained, the nucleotide sequence is shown as SEQ ID NO.1, the sequencing result is subjected to BLAST sequence comparison on NCBI, mega 11 software is utilized for sequence analysis, a Neighbor-joining tree is adopted for constructing an evolutionary tree, the evolutionary tree diagram is shown as fig. 4, and the strains SZ-T8 and Bacillus veelezensis can be seen to be on the same branch, and are identified as Bacillus bailii.

The biocontrol strain SZ-T8 is classified and named as bacillus belicus (Bacillus velezensis) and is preserved in China center for type culture collection (in eight-path 299-th university of Wuhan in Wuhan, hubei province), and the strain preservation number is CCTCC NO: m20241102, the preservation date is 2024, 5 and 30.

Example 2 biocontrol bacterium SZ-T8 test for controlling root rot of sweet corn inbred line S622-432 at seedling stage

The growth environment of the plots with serious corn root rot disease is simulated by adopting a method that fusarium graminearum suspension and Hoagland nutrient solution are mixed and added into a germination bag according to the proportion of 1:3. Soaking seeds of the surface-sterilized sweet corn inbred line S622-432 in a biocontrol bacterium suspension of 1X 10 ⁸CFU·mL^-1 for 8 hours, pouring out the bacterium suspension, and airing the seeds at room temperature; sterile water soaked seeds are used as CK-1, and 1500 times liquid methyl thiophanate soaked seeds are used as CK-2. The treated seeds were transferred into germination bags, and 100mL of a culture solution prepared by mixing a Fusarium graminearum suspension with Hoagland nutrient solution in a ratio of 1:3 was added to each germination bag. The germination bags were transferred to an incubator and incubated for 7d at 25℃under 10000lux illumination for 12h and alternate darkness for 12h. Counting germination vigor of the seeds subjected to different treatments on the 4 th day; and (7) counting the rooting rate, the survival seedling rate, the seedling morbidity, the disease index and the prevention and treatment effect of the seeds, randomly selecting 10 survival seedlings each time, measuring the height and the root length of the seedlings, peeling off the seed residues on the seedlings, and measuring the fresh weight of the overground part and the fresh weight of the underground part respectively. Each 100 seeds were replicated 3 times per treatment.

Referring to Liu Zhigang (Liu Zhigang. Screening of trichoderma biocontrol of root rot in maize seedling stage [ J ]. Guizhou agricultural science, 2010,38 (9): 114-115) for disease classification: level 0: root health, no root rot disease symptoms are seen; stage 1: the root has sporadic brown spots; 2 stages: the ratio of the brown spot or rotten part of the root to the length of the whole root system is less than 1/4;3 stages: the ratio of the root rotting part to the whole root system length is 1/4-1/2; 4 stages: the ratio of the root rotting part to the whole root system length is 1/2-3/4; 5 stages: the diseased part of the root is more than 3/4, and even the whole root system is rotten and browned.

Disease index = Σ (number of patients at each stage x number of patients)/(total number of investigation x number of patients at highest) x 100.

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

As shown in Table 3, after 7d of culture of the inoculated Fusarium graminearum suspension, the corn root system of the blank control group (CK-1) turns brown and decays, the disease of the hypocotyl of the seed is particularly obvious, and only slight disease signs appear after the SZ-T8 suspension is treated, compared with the CK-1, the disease degree is obviously reduced, the disease rate and the disease index are respectively reduced by 38.67 percent and 58.92 percent, and the control effect reaches 59.06 percent. Compared with the common chemical agent of thiophanate methyl (CK-2), the incidence rate of the SZ-T8 bacterial suspension after treatment is higher, but the disease index and the prevention and treatment effect are obviously better than those of the CK-2, the disease index is reduced by 47.02%, and the prevention and treatment effect is improved by 36.69 percent.

TABLE 3 control of root rot of inbred S622-432 by SZ-T8 bacterial suspension treatment

The different lower case letters after the same column of data in the table represent the significance of differences between the different treatments (P <0.05, duncan), as follows.

As shown in Table 4, the germination vigor and rooting rate of the seeds treated by the SZ-T8 bacterial suspension are not obviously different from those of the seeds treated by the CK-1, and the rate of the live seedlings is obviously improved, which proves that the seed soaking of the SZ-T8 live bacterial suspension has no adverse effect on the viability and the vitality of the seeds and can promote the healthy growth of the seedlings.

TABLE 4 influence of SZ-T8 bacterial suspension treatment on seed germination and seedling formation of the inbred line S622-432

As shown in Table 5, the root length, seedling height and fresh weight of the lower part of the treated SZ-T8 bacterial suspension are not significantly different from those of CK-1, but the fresh weight of the upper part of the ground is significantly improved, which indicates that the seed soaking of the SZ-T8 viable bacterial suspension can prevent corn root rot and promote the growth of the upper part of corn seedlings.

TABLE 5 influence of SZ-T8 bacterial suspension treatment on the growth of seedlings of the inbred line S622-432

EXAMPLE 3 biocontrol bacterium SZ-T8 control of sweet corn inbred line S745-1211 seedling stage root rot

The growth environment of the plots with serious corn root rot disease is simulated by adopting a method that fusarium graminearum suspension and Hoagland nutrient solution are mixed and added into a germination bag according to the proportion of 1:3. Soaking seeds of the surface-sterilized sweet corn inbred line S745-1211 in 1× ⁸CFU·mL^-1 biocontrol bacteria suspension for 8h, pouring out the bacteria suspension, and airing the seeds at room temperature; sterile water soaked seeds are used as CK-1, and 1500 times liquid methyl thiophanate soaked seeds are used as CK-2. The treated seeds were transferred into germination bags, and 100mL of a culture solution prepared by mixing a Fusarium graminearum suspension with Hoagland nutrient solution in a ratio of 1:3 was added to each germination bag. The germination bags were transferred to an incubator and incubated for 7d at 25℃under 10000lux illumination for 12h and alternate darkness for 12 h. Counting germination vigor of the seeds subjected to different treatments on the 4 th day; and (7) counting the rooting rate, the survival seedling rate, the seedling morbidity, the disease index and the control effect of the seeds, randomly selecting 10 survival seedlings each time, measuring the seedling height and the root length of the 10 survival seedlings, peeling off the seed residues on the seedlings, and measuring the fresh weight of the overground part and the fresh weight of the underground part respectively. Each 100 seeds were replicated 3 times per treatment.

Disease index = Σ (number of disease stages x number of disease stages)/(total number of investigation x number of highest disease stages).

As shown in Table 6, after being inoculated with the fusarium graminearum suspension for 7 days, compared with CK-1, SZ-T8 bacterial suspension, the disease degree is obviously reduced after treatment, wherein the disease index is obviously reduced by 49.12%, and the control effect reaches 48.65%; compared with CK-2, the disease incidence rate of the SZ-T8 treatment is higher, but the disease index and the control effect are obviously better than those of the CK-2, the disease index is reduced by 34.72%, and the control effect is improved by 27.18%.

TABLE 6 control of root rot of inbred S745-1211 seedling stage by SZ-T8 bacterial suspension treatment

As shown in Table 7, at the same time, the germination vigor, rooting rate and survival seedling rate of the seeds treated by the SZ-T8 bacterial suspension are not significantly different from those of the seeds treated by CK-1, which indicates that the seed soaking by the SZ-T8 bacterial suspension has no adverse effect on the viability and the vigor of the seeds.

TABLE 7 influence of SZ-T8 bacterial suspension treatment on seed germination and seedling formation of inbred S745-1211

In addition, as shown in Table 8, the seedling height and root length after the SZ-T8 bacterial suspension treatment are not significantly different from those of CK-1, but the fresh weight of the lower part and the fresh weight of the upper part are significantly improved, which indicates that the seed soaking of the SZ-T8 viable bacterial suspension can prevent corn root rot and promote the healthy growth of corn seedlings.

TABLE 8 influence of SZ-T8 bacterial suspension treatment on the growth of inbred S745-1211 seedlings

Example 4 broad-spectrum bacteriostatic Activity of biocontrol bacteria SZ-T8

To further investigate whether biocontrol bacteria have an inhibitory effect on other plant pathogenic fungi and thereby evaluate their potential as biocontrol agents, in this example SZ-T8 was subjected to a plate counter test with pathogenic bacteria isolated from six different plant diseases, such as cucumber target leaf spot, tomato early blight, alligator leaf spot, cotton anthocyanin mould, rice sheath blight and peanut southern blight, in the same way as in example 1 above.

As shown in FIG. 5 and Table 9, the biocontrol strain SZ-T8 has inhibition effect on 6 pathogenic bacteria separated from different plants, wherein the average inhibition rate effect on two pathogenic bacteria of Alternaria solani which causes early blight of tomatoes and Rhizoctonia solani which causes southern blight of peanuts is best, and the average inhibition rate respectively reaches 98.34% and 97.47%, which shows that the SZ-T8 has inhibition effect on three Fusarium bacteria which cause corn root rot and also has certain biocontrol potential on biological control of other plant diseases.

TABLE 9 inhibition of different plant pathogenic bacteria by SZ-T8 Strain

Claims

1. A biocontrol strain SZ-T8, characterized in that it is classified and named Bacillus velezensis SZ-T8, the preservation unit is China Center for Type Culture Collection, the preservation number is CCTCC NO: M20241102, and the preservation date is May 30, 2024.

2. A biocontrol agent, characterized in that the active ingredient of the biocontrol agent is the biocontrol strain SZ-T8 according to claim 1.

3 . The biocontrol agent according to claim 2 , wherein the concentration of the biocontrol strain SZ-T8 in the biocontrol agent is 1×10 ⁷ to 1×10 ⁹ CFU·mL ^-1 .

4. Use of the biocontrol strain SZ-T8 according to claim 1, or the biocontrol agent according to any one of claims 2 to 3, in antagonizing one or more pathogenic bacteria selected from Fusarium graminearum, Fusarium pseudoverticillium, and Fusarium erythrorhizium.

5. Use of the biocontrol strain SZ-T8 according to claim 1, or the biocontrol agent according to any one of claims 2 to 3 in controlling corn root rot at the seedling stage.

6. The use according to claim 5, characterized in that the corn seedling root rot is caused by one or more pathogenic bacteria of Fusarium graminearum, Fusarium pseudoverticillium, and Fusarium erythrorhizium infecting corn.

7. Use of the biocontrol strain SZ-T8 according to claim 1, or the biocontrol agent according to any one of claims 2 to 3 in promoting the survival of corn seedlings or promoting the aboveground growth of corn seedlings.

8. A method for preventing corn root rot at the seedling stage, comprising:

Before sowing corn seeds, the biocontrol agent according to any one of claims 2 to 3 is used to soak the seeds.

9. The method according to claim 8, characterized in that the seed soaking time is 4 to 12 hours.

10. Use of the biocontrol strain SZ-T8 according to claim 1 in controlling cucumber target spot disease, tomato early blight, alligator leaf spot disease, cotton blue mold disease, rice sheath blight or peanut white rot.