Bacillus velezensis and application thereof in prevention and treatment of basal stem rot

Abstract

The invention discloses bacillus velezensis and application of the bacillus velezensis in prevention and treatment of basal stem rot, the classification name of the bacillus velezensis is bacillus velezensis GN-22, the bacillus velezensis is preserved in China Center for Type Culture Collection (CCTCC), the preservation number of the bacillus velezensis is CCTCC NO: M2025566, and the preservation date is March 24, 2025. The bacillus velezensis GN-22 disclosed by the invention belongs to rhizosphere growth-promoting bacteria, has a remarkable inhibition effect on different pathogenic fungi such as fusarium graminearum, fusarium pseudograminearum, fusarium asiaticum, fusarium verticillioides and fusarium oxysporum, and plays an important role in biological control of diseases such as corn and wheat stem rot; meanwhile, the strain has a growth promoting effect and can promote corn growth. The strain provided by the invention has the advantages of simple culture conditions, easiness in preservation, easiness in industrial production and good development and application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant protection, and specifically relates to a Bacillus velezensis and its application in the control of crop stem rot disease. Background Art

[0002] Maize and wheat are the main crops in China, with a wide planting range in the country. However, they are damaged by pathogenic bacteria at all growth stages, resulting in reduced yield and quality, as well as toxin pollution. According to the monitoring of the National Crop Pest and Disease Forecasting Network and expert analysis, major food crops such as wheat, rice, maize, and potato will face serious pest and disease threats in 2024. It is estimated that the area affected by pests and diseases across the country will reach 2.04 billion mu-times, an increase of 15% compared to 2023 and an increase of 11% compared to the average value from 2018 to 2022. Currently, the control of maize and wheat diseases mainly relies on chemical control. However, with the extensive use of chemical pesticides, the disadvantages of chemical control have gradually emerged. Chemical pesticides are not easily degraded, prone to residues and environmental and biological hazards, and can disrupt the ecological balance. In recent years, biological control has received extensive attention due to its safety, and new biocontrol agents have been continuously discovered.

[0003] Biocontrol agents have many advantages in the control of maize and wheat diseases. Biological control methods utilize microorganisms in nature, such as bacteria, fungi, and actinomycetes, to inhibit plant pathogenic bacteria. These microorganisms can not only effectively control diseases, but also improve soil health, promote plant growth, and have less impact on the environment, which is in line with the development direction of sustainable agriculture. In addition, the mechanism of action of biocontrol agents is different from that of chemical pesticides, which can reduce the risk of the development of resistance of pathogenic microorganisms to fungicides, and have broad-spectrum bactericidal activity and persistence. In addition to effectively controlling diseases, biocontrol agents can also enhance the immunity of plants themselves, helping to reduce the occurrence of diseases. Summary of the Invention

[0004] Aiming at the problem that the existing biocontrol bacteria applied to crops such as maize and wheat are still insufficient, the present invention provides a Bacillus velezensis, which can act on a variety of plant fungi and promote plant growth.

[0005] The present invention isolated a biocontrol strain Bacillus velezensis GN-22 from a wheat-maize rotation field, which has antagonistic effects on a variety of fungi and promotes the growth of maize. This strain has been deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M2025566, the deposit date of March 24, 2025, and the deposit address of Wuhan University, Wuhan, China.

[0006] The optimal fermentation conditions of Bacillus velezensis GN-22 of the present invention are as follows: Shake culture is carried out using LB medium, the shake culture speed is 180 r / min to 220 r / min, the culture temperature is 28 °C, the initial pH is 6 to 7, and the fermentation time is 24 to 60 h.

[0007] In one aspect of the present invention, the present invention provides the application of Bacillus velezensis GN-22 in the prevention and control of plant fungal diseases, and the fungal diseases include diseases caused by one or more of the pathogens such as Fusarium pseudograminearum, Fusarium asiaticum, Fusarium graminearum, Fusarium verticillioides, and Fusarium oxysporum. Preferably, the disease is maize stalk rot.

[0008] In one aspect of the present invention, the present invention also provides the application of Bacillus velezensis GN-22 and the fermentation broth and fermentation supernatant containing metabolites in promoting plant growth. The fermentation broth is a bacterial solution containing a large amount of Bacillus velezensis GN-22 obtained by fermenting the Bacillus velezensis GN-22 strain using a medium; the fermentation supernatant is the supernatant obtained by centrifuging the fermentation broth obtained after fermenting the Bacillus velezensis 3-b strain. Those skilled in the art can understand that both the fermentation broth and the fermentation supernatant contain various metabolites produced by the strain during fermentation, and these metabolites are the main components for the strain to perform its functions.

[0009] In one aspect of the present invention, the present invention also discloses a bacterial agent containing Bacillus velezensis GN-22. The bacterial agent can be a liquid preparation or a powder preparation. For the liquid preparation, it is a bacterial solution containing Bacillus velezensis GN-22. The powder preparation is prepared by fermenting the Bacillus velezensis GN-22 strain to obtain a fermentation broth and then freeze-drying it. Substances well-known in the art such as freeze-drying protectants and buffers can also be added during the freeze-drying process of the fermentation broth.

[0010] During the growth period of plants, a bacterial agent containing Bacillus velezensis GN-22 can be applied to the roots of plants. Preferably, a bacterial solution containing Bacillus velezensis GN-22 is applied. In the present invention, plant growth mainly involves the underground and above-ground parts, and indicators such as root length and leaf length can be used to measure the effect of the bacterial agent on plant growth.

[0011] In one aspect of the present invention, due to the broad-spectrum antibacterial effect of Bacillus velezensis GN-22 of the present invention, it can also be used for the prevention and control of fungal diseases of one or more of a variety of other plants including wheat, rice, corn, rapeseed, and barley.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] In the present invention, healthy corn rhizosphere soil was collected from fields with different fertilization and straw returning treatments in Hefei by the five-point sampling method, and a biocontrol bacterium GN-22 with antagonistic effects against various pathogens of stalk rot was screened out. Its inhibition rate against Fusarium pseudograminearum reached 82.73%; the inhibition rate against Fusarium graminearum reached 81.47%; the inhibition rate against Fusarium verticillioides reached 69.67%; the inhibition rates against various pathogens such as Fusarium oxysporum and Fusarium asiativum reached more than 70%. The pot experiment showed that when comparing the effects of irrigating corn seedlings with GN-22 bacterial solution and irrigating corn seedlings with sterile water, it was found that the corn seedlings increased significantly (47%) under the treatment of the bacterial solution. It shows that GN-22 has a promoting effect on the growth of corn seedlings. Moreover, wheat grains with Fusarium graminearum were inoculated into the soil, and at the same time, corn seedlings were inoculated. The control effect of the 35% bacterial solution treatment group was the best, indicating that GN-22 has a protective effect on corn stalk rot at the seedling stage. Description of the Drawings

[0014] Figure 1 Bacillus velezensis GN-22

[0015] Figure 2 Confrontation culture diagram of Bacillus velezensis GN-22 and various pathogens; A: Fusarium pseudograminearum B: Sclerotinia sclerotiorum C: Fusarium verticilliooles D: Fusarium oxysporum E: Fusarium asiativum F: Fusarium graminearum

[0016] Figure 3 This is the phylogenetic tree diagram of Bacillus velezensis GN-22 in the present invention;

[0017] Figure 4 This is the diagram showing the growth effect of the fermentation filtrate of Bacillus velezensis on Fusarium graminearum in Example 2; (from left to right) CK, fermentation filtrate (50%)

[0018] Figure 5 The growth promotion effect of GN-22 on maize seedlings; (from left to right) 50% fermentation broth treatment, 40% fermentation broth treatment, 30% fermentation broth treatment, 20% fermentation broth treatment, 10% fermentation broth treatment, LB, blank

[0019] Figure 6 The control effect of GN-22 on maize stalk rot caused by F. graminearum; (from left to right) diseased group F. graminearum + 5% GN-22 fermentation broth F. graminearum + 15% GN-22 fermentation broth F. graminearum + 25% GN-22 fermentation broth F. graminearum + 35% GN-22 fermentation broth carbendazim blank group

[0020] Figure 7 The growth promotion effect of GN-22 on wheat seedlings; (from left to right in turn: water LB 10% fermentation broth treatment group 20% fermentation broth treatment group 30% fermentation broth treatment group 40% fermentation broth treatment group 50% fermentation broth treatment group)

[0021] Figure 8 The potted plant control effect of GN-22 on wheat stalk rot; (from left to right in turn: diseased group 5% bacterial liquid treatment group 15 bacterial liquid treatment group 25 bacterial liquid treatment group 35 bacterial liquid treatment group carbendazim water) Detailed implementation manners

[0022] The present invention will be further described below in conjunction with specific embodiments.

[0023] The test materials are as follows:

[0024] (1) Strains

[0025] The pathogen of the tested maize stalk rot (Fusarium graminearum) was provided by the strain bank of the College of Plant Protection, Anhui Agricultural University.

[0026] (2) Main reagents

[0027] Agar, yeast extract, tryptone, sodium chloride, glucose, sucrose, 95% ethanol, glacial acetic acid, calcium carbonate, sodium hydroxide, hydrochloric acid, sodium bicarbonate, sodium dihydrogen carbonate, congo red, gelatin, potassium iodide, dipotassium hydrogen phosphate, beef extract, diphenylamine, soluble starch.

[0028] (3) Culture medium

[0029] Caseinase medium, tryptone medium, nitrate reduction medium, nitrite reduction medium, malonate utilization medium, pyocyanin detection medium, fluorescent pigment medium, starch hydrolysis medium, cellulose hydrolysis medium, LB medium, PDA medium, citrate medium.

[0030] Example 1

[0031] Screening and identification of antagonistic bacteria against maize stalk rot

[0032] Collect the surface soil (soil at 10 cm - 15 cm) from the maize stalk rot disease field. After passing through a 2 mm stainless steel sieve, weigh 10 g of the soil sample and put it into a triangular flask containing 90 mL of sterile water. Shake it at 200 rpm for 1 h to make a soil suspension. Pipette 1 mL of the soil suspension and dilute it 10 1 -10 7 times with sterile water. Subsequently, pipette 0.1 mL of each diluted soil suspension and spread it evenly on the LB solid medium plate, and incubate it at 28 °C for 2 d - 3 d. After the colonies grow out, continuously streak culture to obtain pure cultures and store them at 4 °C. Pick the actinomycetes and inoculate them into the LB liquid medium, and culture them in a shaker at 28 °C and 180 r / min for 3 d.

[0033] Inoculate the activated F. graminearum fungal block at the center position of the PDA plate, place it in an incubator at 22 °C, and after culturing for 1 d, place sterile filter papers (Φ = 6 mm) 2.5 cm on both sides of the center position of the plate. Pipette 1 μL of the bacterial liquid onto the filter paper and place it in an incubator at 25 °C for culture. Measure the colony diameter after 3 d, repeat each experiment 3 times, and calculate the inhibition rate.

[0034] Inhibition rate calculation formula:

[0035]

[0036] An experimental separation and screening yielded a strain of Bacillus velezensis, numbered GN-22. Through a plate confrontation experiment, the antibacterial rate of Bacillus velezensis against Fusarium pseudograminearum reached 82.73%; the antibacterial rate against Fusarium graminearum reached 81.47%; the antibacterial rate against Fusarium verticillioides reached 69.67%; and the antibacterial rate against various pathogenic fungi such as Fusarium oxysporum and Fusarium asiaticum exceeded 70%( Figure 2 ). BLAST alignment and homology analysis were performed on the 16S rDNA, gyrA, rpoB, purH, and groEL genes of strain GNN22 to retrieve the above genes of other strains in the species, genus, and cluster with the highest homology similarity to strain GN-22. Using the Neighbor-joining Method (NJ) in the software MEGA X, a phylogenetic topology tree for multilocus sequence analysis of strain GN-22 was constructed based on the gyrA, rpoB, purH, and groEL genes. The results showed that in the Bacillus amyloliquefacies group, strain GN-22 clustered with Bacillus velezensis, which confirmed that strain GN-22 belongs to Bacillus velezensis( Figure 3 ).

[0037] Comparative test: Bacillus velezensis 3-b (CN202410740581.6), preservation number: CCTCC NO: M2022618, provided by the culture collection of the College of Plant Protection, Anhui Agricultural University) Through a plate confrontation experiment, the antibacterial rate of Bacillus velezensis against Fusarium pseudograminearum was 70.71%; the antibacterial rate against Fusarium graminearum reached 80.32%; the antibacterial rate against Fusarium verticillioides was 51.80%; it can be seen that the biological activity of the newly screened Bacillus velezensis GN-22 of the present invention is significantly superior to that of Bacillus velezensis 3-b.

[0038] Example 2

[0039] Effect of the fermentation broth and sterile fermentation broth of Bacillus velezensis GN-22 on the growth of the pathogen causing maize stalk rot

[0040] Pick the activated Bacillus velezensis GN-22 and inoculate it into LB medium, and culture it in a shaker at 28°C and 180 r / min for 3 days. Then, aliquot the fermentation broth into centrifuge tubes, place them in a low-temperature high-speed centrifuge (4°C, 10,000 r / min), take the supernatant and discard the precipitate. Filter it with a sterile bacterial filter (Φ = 0.22 μm) to obtain the sterile fermentation broth of strain GN22.

[0041] Add the sterile fermentation broth to PDA medium to prepare a plate containing 50% fermentation metabolites. Use a puncher with an inner diameter of 6 mm to punch out the edge mycelial blocks of the activated Fusarium graminearum, and transfer them to the center position of the PDA plate. Use the plate without adding the sterile fermentation broth as a blank control, and incubate it at a constant temperature of 22°C. After 3 days, record the data and calculate the inhibition rate.

[0042] As can be seen from Example 1, strain GN-22 has a strong inhibitory effect on Fusarium graminearum. The results show that both the fermentation broth and the sterile fermentation broth of Bacillus velezensis GN-22 have obvious antagonistic effects on Fusarium graminearum. Among them, the inhibition rate of the fermentation broth reaches 81.47%, and the inhibition rate of the sterile fermentation broth reaches 62.28% (Table 1; Figure 4 )

[0043] Table 1 Inhibition rates of GN22 fermentation broth and sterile fermentation broth on Fusarium graminearum

[0044] Strain Colony diameter (cm) Inhibition rate (%) Fermentation broth of GN-22 0.67 81.47 Sterile fermentation broth of GN-22 6.03 62.28

[0045] Example 3

[0046] Study on the effect of GN-22 on the growth of maize seedlings

[0047] Pick a single colony of strain GN-22 and inoculate it into LB medium, and culture it in a shaker at 28°C and 180 r / min for 3 days. After disinfecting and germinating maize seeds, plant them, and pour the GN-22 bacterial suspension onto the roots of maize. The control group is irrigated with sterile water.

[0048] The results show that the bacterial suspension of GN-22 has a growth-promoting effect on the growth of maize seedlings during the growth process of maize. For the maize plants irrigated with the bacterial suspension, the plant height of a single maize plant is on average 5 cm longer than that of the untreated maize, and the average root length of a single maize plant increases by 0.3 cm (as shown in Table 2). During the growth process of maize, irrigating a certain amount of the bacterial suspension has a certain growth-promoting effect. The plants are sturdier than the maize plants not irrigated with the bacterial suspension. Under the same sample conditions, the plants irrigated with the bacterial suspension have more tillers and grow more vigorously (as Figure 5 shown)

[0049] Table 2 Growth-promoting effect of GN-22 on maize seedlings

[0050] Treatment group Plant height (plants / cm) Root length (plants / cm) CK 20.3167±2.13613b 15.150±1.7783abc LB 14.3667±2.30157c 11.625±3.3254d 30% treatment group 25.3167±2.52251a 15.425±0.6188ab

[0051] Example 4

[0052] Study on the Pot Control Effect of GN-22 against Maize Stalk Rot

[0053] After disinfecting the maize seeds, they were sterilized by moist heat at 121 °C for 45 min. Then, 5 to 6 pieces of Fusarium graminearum fungal cakes were inoculated and cultured at 25 °C for 7 d. The maize seeds were germinated after disinfection and then planted. The soil substrate was sterilized at 121 °C for 50 min. First, 1 / 3 of the substrate was filled into each pot (diameter 10 cm), a layer of infected wheat grains was spread, a layer of substrate was spread, and the treated maize seeds were placed in the center and covered with soil. There were 45 pots for each treatment, divided into 3 groups. Management was carried out in the conventional manner, and the number of diseased plants and the control effect of the diseased plant rate were observed and recorded.

[0054] The results showed that: for the maize seedlings inoculated with Fusarium graminearum, the leaves turned yellow and withered, and the base of the stem was soft-rotted. For the maize seedlings treated with GN-22 by root irrigation, with the increase in the concentration of the GN-22 fermentation broth, the control effect against Fusarium graminearum was better, the diseased plant rate decreased, and the control effect of the diseased plant rate was significantly improved (Table 3; Figure 6 )

[0055] Table 3 Control Effect of GN22 against Maize Stalk Rot Caused by F. graminearum

[0056] Treatment group Diseased plant rate (%) Control efficiency (%) F.graminearum + 5% GN22 fermentation broth 92.22±4.157a 4.76±3.366e F.graminearum + 15% GN22 fermentation broth 73.33±5.442b 21.43±5.830d F.graminearum + 25% GN22 fermentation broth 48.89±3.140c 47.62±3.366c F.graminearum + 35% GN22 fermentation broth 37.78±3.144d 59.52±3.371b F.graminearum + carbendazim 35.55±3.144d 61.91±3.371b F.graminearum 93.33±0.000a 0e

[0057] Example 5

[0058] Study on the Effect of GN-22 on the Growth of Wheat Seedlings

[0059] A single colony of strain GN-22 was picked and inoculated into LB medium, and cultured in a shaker at 28 °C and 180 r / min for 3 d. After disinfecting and germinating maize, it was planted. The GN-22 bacterial suspension was watered onto the roots of wheat, and the control group was watered with sterile water.

[0060] The results showed that the bacterial suspension of GN-22 had a growth-promoting effect on the growth of maize seedlings during the growth process of wheat. For the wheat watered with the bacterial suspension, the plant height of a single wheat plant was on average 3 cm longer than that of the untreated wheat, and the average root length of a single maize plant increased by 0.7 cm (as shown in Table 4). During the growth process of maize, watering with a certain amount of the bacterial suspension had a certain growth-promoting effect, and the plants grew more vigorously than the wheat without watering with the bacterial suspension (as Figure 7 )

[0061] Table 4 Growth-Promoting Effect of GN-22 on Wheat Seedlings

[0062] Treatment group Plant height (plants / cm) Root length (plants / cm) CK 16.28±0.224d 8.58±0.749b LB 17.97±0.376bcd 12.23±0.573a 30% treatment group 21.18±1.587a 13.01±1.032a

[0063] Example 6

[0064] Study on the Control Effect of GN-22 on Wheat Crown Rot in Pot Experiments

[0065] After disinfecting wheat seeds, they were sterilized by moist heat at 121 °C for 45 min. Then, 5 to 6 pieces of Fusarium pseudograminearum agar discs were inoculated, and the seeds were cultured at 25 °C for 7 d. After disinfection, the wheat seeds were germinated and then planted. The soil substrate was sterilized at 121 °C for 50 min. First, 1 / 3 of the substrate was filled into each pot (10 cm in diameter), a layer of infected wheat grains was spread, then a layer of substrate was laid, and the treated wheat seeds were placed in the center and covered with soil. There were 45 pots for each treatment, divided into 3 groups. Management was carried out in the conventional way, and the number of diseased plants and the control effect of the diseased plant rate were observed and recorded.

[0066] The results showed that: for the maize seedlings inoculated with Fusarium pseudograminearum, the leaves turned yellow and withered, and the stem bases showed soft rot. For the wheat seedlings treated with GN-22 by root irrigation, with the increase in the concentration of the GN-22 fermentation broth, the control effect on Fusarium pseudograminearum was better, the diseased plant rate decreased, and the control effect of the diseased plant rate increased significantly (Table 5; Figure 8 )

[0067] Table 5 Control Effect of GN22 on Wheat Crown Rot Caused by F.graminearum

[0068] Treatment group Diseased plant rate (%) Control efficiency (%) F.pseudograminearum + 5% GN22 fermentation broth 64.20%±8.34ab 17.33％±10.73c F.pseudograminearum + 15% GN22 fermentation broth 57.67%±3.30bc 25.75%±4.24bc F.pseudograminearum + 25% GN22 fermentation broth 46.67%±9.43cd 39.92％±12.13b F.pseudograminearum + 35% GN22 fermentation broth 41.67％±6.13d 46.35％±7.89b F.pseudograminearum + carbendazim 22.07％±6.41e 71.59％±8.25a F.pseudograminearum 79％±3.30a \

[0069] The above content further elaborates on the present invention in combination with specific implementation manners. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention pertains, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, and all should be regarded as falling within the protection scope determined by the claims submitted for the present invention.

Claims

1. A biocontrol bacterium, characterized in that, The biocontrol bacterium is Bacillus velezensis GN-22, which is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M2025566, the deposit date is March 24, 2025, and the deposit address is Wuhan University, Wuhan, China.

2. A bacterial agent, characterized in that, The microbial agent contains the biocontrol bacterium Bacillus velezensis GN-22 described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The microbial agent is a liquid preparation or a powder preparation.

4. The microbial agent according to claim 2, wherein The liquid preparation is a fermentation broth containing the biocontrol bacterium Bacillus velezensis GN-22.

5. Use of the biocontrol bacterium according to claim 1 or the microbial agent according to claim 2 in the prevention and control of plant diseases, characterized in that, The diseases are Fusarium graminearum, Fusarium pseudograminearum, Fusarium asiaticum, Fusarium verticillioides, Fusarium oxysporum.

6. The application according to claim 5, characterized in that, The diseases are maize stalk rot or wheat stalk rot.

7. Use of the biocontrol bacterium according to claim 1 or the microbial agent according to claim 2 in promoting plant growth.

8. The application according to claim 7, wherein The plants include maize, wheat, rice, rape, barley.

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