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

Abstract

The application discloses Bacillus velezensis and application thereof in prevention and treatment of stem base rot, the classification name of the Bacillus velezensis is GN-22, has been preserved in China typical culture preservation center CCTCC, and the preservation number is CCTCC NO: M2025566, and the preservation date is March 24, 2025.The Bacillus velezensis GN-22 of the application belongs to rhizosphere growth-promoting bacteria, has significant inhibiting effect on different pathogenic fungi such as fusarium graminearum, pseudofusarium graminearum, fusarium asiaticum, fusarium pseudotrichothecioides and fusarium oxysporum f.sp.cubense, plays an important role in biological control of corn, wheat stem base rot and other diseases; meanwhile, the strain has growth-promoting effect and can promote corn growth.The strain of the application has simple culture condition, is easy to preserve and easy to industrialize, and has good development and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of plant protection technology, specifically relating to a Bacillus belye and its application in the prevention and control of crop stem rot. Background Technology

[0002] Corn and wheat are major crops in my country, widely cultivated. However, they are susceptible to damage from pathogens at every stage of their growth, leading to decreased yield and quality, and toxin contamination. According to monitoring by the National Crop Pest and Disease Monitoring Network and expert analysis, major grain crops such as wheat, rice, corn, and potatoes will face severe pest and disease threats in 2024. The estimated affected area nationwide will reach 2.04 billion mu (approximately 133 million hectares), an increase of 15% compared to 2023 and 11% higher than the average from 2018 to 2022. Currently, the control of corn and wheat diseases mainly relies on chemical control. However, with the extensive use of chemical pesticides, their drawbacks are becoming increasingly apparent: chemical pesticides are difficult to degrade, easily leaving residues and causing environmental and biological hazards, disrupting the ecological balance. In recent years, biological control has received widespread attention due to its safety, and new biocontrol agents are constantly being discovered.

[0003] Biocontrol agents offer numerous advantages in the control of diseases in corn and wheat. Biological control methods utilize naturally occurring microorganisms, such as bacteria, fungi, and actinomycetes, to inhibit plant pathogens. These microorganisms not only effectively control diseases but also improve soil health, promote plant growth, and have a relatively small environmental impact, aligning with the development of sustainable agriculture. Furthermore, biocontrol agents differ from chemical pesticides in their mechanism of action, reducing the risk of pathogens developing resistance to fungicides, and possess broad-spectrum and persistent fungicidal activity. In addition to effectively controlling diseases, biocontrol agents can also enhance the plant's own immunity, helping to reduce disease occurrence. Summary of the Invention

[0004] To address the shortcomings of existing biocontrol bacteria applied to crops such as corn and wheat, this invention provides a Bacillus belye, which can act on a variety of plant fungi and promote plant growth.

[0005] This invention isolates a biocontrol strain, Bacillus velezensis GN-22, from a wheat-maize rotation field. This strain exhibits antagonistic activity against various fungi and promotes maize growth. The strain has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2025566, deposit date March 24, 2025, at Wuhan University, Wuhan, China.

[0006] The optimal fermentation conditions for Bacillus velezensis GN-22 of the present invention are as follows: LB medium with shaking culture at a shaking speed of 180 r / min to 220 r / min, a culture temperature of 28°C, an initial pH of 6 to 7, and a fermentation time of 24 to 60 h.

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

[0008] In one aspect, the present invention also provides the application of Bacillus velezensis GN-22 and its fermentation broth and fermentation supernatant containing metabolites in promoting plant growth. The fermentation broth is a bacterial culture containing a large amount of Bacillus velezensis GN-22 obtained by fermenting Bacillus velezensis GN-22 strain using a culture medium; the fermentation supernatant is the supernatant obtained by centrifuging the fermentation broth obtained after fermenting Bacillus velezensis 3-b strain. Those skilled in the art will 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, this invention also discloses a bacterial agent containing Bacillus velezensis GN-22. The bacterial agent can be a liquid formulation or a powder. For the liquid formulation, it is a bacterial solution containing Bacillus velezensis GN-22. The powder is prepared by fermenting the Bacillus velezensis GN-22 strain to obtain a fermentation broth, followed by freeze-drying. During the freeze-drying process of the fermentation broth, freeze-drying protectants, buffers, and other substances well-known in the art can also be added.

[0010] During plant growth, a microbial agent containing Bacillus velezensis GN-22 can be applied to the roots of the plant; preferably, a bacterial solution containing Bacillus velezensis GN-22 can be applied. In this invention, plant growth mainly involves the underground and above-ground parts, and the effect of the microbial agent on plant growth can be measured by indicators such as root length and leaf length.

[0011] In one aspect, due to the broad-spectrum antibacterial activity of Bacillus vesiculosus GN-22 of the present invention, it can also be used for the prevention and control of one or more fungal diseases in 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] This invention collected healthy maize rhizosphere soil from fields in Hefei City with different fertilization and straw return treatments using a five-point sampling method. A biocontrol bacterium, GN-22, exhibiting antagonistic effects against multiple pathogens causing stem rot, was screened. Its inhibition rate against *Fusarium pseudograminearum* reached 82.73%; against *Fusarium graminearum*, 81.47%; against *Fusarium verticillioides*, 69.67%; and against multiple pathogens such as *Fusarium oxysporum* and *Fusarium asiativum*, exceeding 70%. Pot experiments showed that comparing the effects of irrigating maize seedlings with GN-22 bacterial solution versus irrigating with sterile water, the seedlings grew significantly faster (47%) under the bacterial solution treatment. This indicates that GN-22 promotes the growth of maize seedlings. Furthermore, when wheat grains infected with Fusarium graminearum were inoculated into the soil, and maize seedlings were simultaneously inoculated, the 35% bacterial solution treatment group showed the best control effect, indicating that GN-22 has a protective effect against maize stalk rot during the seedling stage. Attached Figure Description

[0014] Figure 1 Bacillus vesiculosus GN-22

[0015] Figure 2 Culture diagrams of *Bacillus belyeis* GN-22 confronting various pathogens; A: *F. pesedogra minearum*; B: *S. sclerotiorum*; C: *F. verticilliooles*; D: *F. oxysporum*; E: *F. asiaticum*; F: *F. graminearum*.

[0016] Figure 3 This is a phylogenetic tree diagram of Bacillus belyssus GN-22 in this invention;

[0017] Figure 4 The graph shows the effect of Bacillus belyss fermentation filtrate on the growth of Fusarium graminearum in Example 2; (from left to right) CK, fermentation filtrate (50%)

[0018] Figure 5 The growth-promoting 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 F. graminearum maize stalk rot; (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 control group.

[0020] Figure 7 The growth-promoting effect of GN-22 on wheat seedlings; (from left to right: LB water, 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 efficacy of GN-22 against wheat stem base rot in potted plants; (from left to right: disease group, 5% bacterial solution treatment group, 15% bacterial solution treatment group, 25% bacterial solution treatment group, 35% bacterial solution treatment group, carbendazim solution) Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments.

[0023] The test materials are as follows:

[0024] (1) Microbial strains

[0025] The pathogen of 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, Disodium Bicarbonate, Sodium Dihydrogen Carbonate, Congo Red, Gelatin, Potassium Iodide, Dipotassium Hydrogen Phosphate, Beef Extract, Diphenylamine, Soluble Starch.

[0028] (3) Culture medium

[0029] Casein medium, tryptone medium, nitrate-reducing medium, nitrite-reducing medium, malonic acid utilization medium, pyocyanin detection medium, fluorescent dye 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] Topsoil samples (10-15 cm deep) were collected from fields affected by maize stalk rot. After passing through a 2 mm stainless steel sieve, 10 g of soil sample was weighed and placed in an Erlenmeyer flask containing 90 mL of sterile water. The flask was shaken at 200 rpm for 1 hour to prepare a soil suspension. 1 mL of the soil suspension was then serially diluted 10 times with sterile water. 1 -10 7 The soil suspension was then divided into several dilutions. 0.1 mL of each dilution was evenly spread onto LB agar plates and incubated at 28°C for 2-3 days. After colonies appeared, the culture was streaked continuously to obtain pure cultures, which were then stored at 4°C. Actinomycetes were then picked and inoculated into LB liquid medium and cultured at 28°C and 180 rpm for 3 days.

[0033] The activated *F. graminearum* bacterial clumps were inoculated into the center of a PDA plate and placed in an incubator at 22°C for 1 day. After incubation, sterile filter paper discs (Φ = 6 mm) were placed 2.5 cm to either side of the center of the plate, and 1 μL of bacterial suspension was transferred onto the filter paper discs and placed in an incubator at 25°C for further incubation. The colony diameter was measured after 3 days. Each experiment was repeated 3 times, and the inhibition rate was calculated.

[0034] Inhibition rate calculation formula:

[0035]

[0036] A strain of *Bacillus belyss*, designated GN-22, was isolated and screened in the experiment. After plate confrontation experiments, *Bacillus belyss* showed an inhibition rate of 82.73% against *Fusarium pseudograminearum*, 81.47% against *Fusarium graminearum*, 69.67% against *Fusarium verticillioides*, and over 70% against various pathogens including *Fusarium oxysporum* and *Fusarium asiativum*. 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 from other strains in the same species, genus, and cluster with the highest homology similarity to strain GN-22. A phylogenetic topology tree for strain GN-22 was constructed using the Neighbor-joining Method (NJ) based on the gyrA, rpoB, purH, and groEL genes in MEGA X software. The results showed that strain GN-22 clustered with Bacillus velezensis in the Bacillus amyloliquefacies group, confirming 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 strain bank of the College of Plant Protection, Anhui Agricultural University) showed an inhibition rate of 70.71% against Fusarium pseudograminearum, 80.32% against Fusarium graminearum, and 51.80% against Fusarium verticillioides after plate confrontation experiments. It can be seen that the bioactivity of the newly screened Bacillus velezensis GN-22 in this invention is significantly better than that of Bacillus velezensis 3-b.

[0038] Example 2

[0039] Effects of Bacillus vesiculosus GN-22 fermentation broth and aseptic fermentation broth on the growth of *Bacillus vesiculosus* var. *vesiculosus*, the pathogen causing corn stalk rot.

[0040] Activated Bacillus belye GN-22 was inoculated into LB medium and cultured for 3 days at 28°C and 180 rpm in a shaker. The fermentation broth was then aliquoted into centrifuge tubes and centrifuged at 4°C and 10,000 rpm. The supernatant was collected and the precipitate discarded. The broth was filtered through a sterile bacterial filter (Φ = 0.22 μm) to obtain the sterile fermentation broth of strain GN22.

[0041] Aseptic fermentation broth was added to PDA medium to prepare plates containing 50% fermentation metabolites. Activated mycelial blocks of *C. corn stalk rot* were extracted from the edge of the plates using a 6mm inner diameter punch and transferred to the center of the PDA plates. Plates without aseptic fermentation broth served as blank controls. All plates were incubated at 22°C. After 3 days, data were recorded and the inhibition rate was calculated.

[0042] As shown in Example 1, strain GN-22 has a strong inhibitory effect on Fusarium graminearum. The results indicate that both the fermentation broth and sterile fermentation broth of Bacillus belyssioides GN-22 have significant antagonistic effects on Fusarium graminearum, with the fermentation broth showing an inhibition rate of 81.47% and the sterile fermentation broth showing an inhibition rate of 62.28% (Table 1). Figure 4 ).

[0043] Table 1. Inhibition rates of GN22 fermentation broth and sterile fermentation broth against *Corn stalk rot*.

[0044] strain Colony diameter (cm) Inhibition rate (%) GN-22 fermentation broth 0.67 81.47 GN-22 aseptic fermentation broth 6.03 62.28

[0045] Example 3

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

[0047] Single colonies of strain GN-22 were inoculated into LB medium and cultured for 3 days at 28℃ and 180 rpm. After sterilization and germination, corn was planted, and a suspension of GN-22 bacteria was applied to the roots of the corn. The control group was watered with sterile water.

[0048] The results showed that the GN-22 bacterial solution promoted the growth of maize seedlings. Maize plants irrigated with the bacterial solution were on average 5 cm taller and 0.3 cm longer in root length than untreated plants (as shown in Table 2). Irrigation with a certain amount of bacterial solution during maize growth had a certain growth-promoting effect; the plants were more robust than those not irrigated. Under the same sample conditions, plants irrigated with the bacterial solution produced more tillers and grew more vigorously (e.g., ...). Figure 5 (As shown).

[0049] Table 2 shows the growth-promoting effect of GN-22 on maize seedlings.

[0050] Processing group Plant height (plants / cm) Root length (per plant / 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 potted control efficacy of GN-22 against maize stalk base rot

[0053] After disinfecting the corn seeds, they were sterilized by moist heat at 121℃ for 45 minutes. Then, 5-6 Fusarium graminearum mycelium cakes were inoculated and cultured at 25℃ for 7 days. The corn seeds were then germinated after disinfection before planting. The soil substrate was sterilized at 121℃ for 50 minutes. In each pot (10cm in diameter), 1 / 3 of the substrate was filled, followed by a layer of infected wheat grains, then another layer of substrate. The treated corn seeds were placed in the center and covered with soil. Each treatment consisted of 45 pots, divided into 3 groups. Management was carried out according to standard methods, and the number of diseased plants and the disease incidence rate were observed and recorded to determine the control efficacy.

[0054] The results showed that maize seedlings inoculated with Fusarium graminearum had yellowing and death of leaves and soft rot at the base of the stem. Maize seedlings treated with GN-22 root irrigation showed better control effect against Fusarium graminearum as the concentration of GN-22 fermentation liquid increased, and the disease incidence rate decreased accordingly, with a significant improvement in the control effect (Table 3). Figure 6 ).

[0055] Table 3. Control efficacy of GN22 against F. graminearum maize stalk rot.

[0056] Processing group Disease incidence rate (%) Protection 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 wheat seedling growth

[0059] Single colonies of strain GN-22 were inoculated into LB medium and cultured for 3 days at 28℃ and 180 rpm. After corn was sterilized and germinated, it was planted. A suspension of GN-22 bacteria was applied to the roots of wheat, while the control group was watered with sterile water.

[0060] The results showed that the GN-22 bacterial solution promoted the growth of maize seedlings during wheat growth. Wheat plants irrigated with the bacterial solution were on average 3 cm taller than untreated wheat, and maize root length increased by an average of 0.7 cm per plant (as shown in Table 4). Irrigating maize with a certain amount of the bacterial solution during its growth process had a certain growth-promoting effect, and the plants grew more vigorously than wheat plants that were not irrigated with the bacterial solution (e.g., ...). Figure 7 (As shown).

[0061] Table 4. Growth-promoting effect of GN-22 on wheat seedlings

[0062] Processing group Plant height (plants / cm) Root length (per plant / 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 pot control efficacy of GN-22 against wheat stem base rot

[0065] After disinfecting the wheat seeds, they were sterilized by moist heat at 121℃ for 45 minutes. Then, 5-6 pieces of *Fusarium graminearum* mycelium were inoculated and cultured at 25℃ for 7 days. The wheat seeds were then disinfected again, germinated, and then planted. The soil substrate was sterilized at 121℃ for 50 minutes. In each pot (10cm in diameter), 1 / 3 of the substrate was filled, followed by a layer of infected wheat grains, then another layer of substrate. The treated wheat seeds were placed in the center and covered with soil. Each treatment consisted of 45 pots, divided into 3 groups. Management was carried out according to standard methods, and the number of diseased plants, disease incidence rate, and control efficacy were observed and recorded.

[0066] The results showed that maize seedlings inoculated with Fusarium graminearum had yellowing and death of leaves and soft rot at the base of the stem. Wheat seedlings treated with GN-22 root irrigation showed better control effect against Fusarium graminearum with increasing concentration of GN-22 fermentation broth, and the disease incidence rate decreased accordingly, with a significant improvement in the control efficacy (Table 5). Figure 8 ).

[0067] Table 5 shows the control effect of GN22 on F. graminearum wheat stem base rot.

[0068] Processing group Disease incidence rate (%) Protection 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% ± 9.43 cd 39.92％±12.13b F. pseudograminearum + 35% GN22 fermentation broth 41.67％±6.13d 46.35％±7.89b F. peudograminearum + carbendazim 22.07％±6.41e 71.59％±8.25a F. pseudograminearum 79％±3.30a \

[0069] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A biocontrol bacterium, characterized in that, The biocontrol bacteria is Bacillus vesiculus GN-22 ( Bacillus velezensis GN-22), deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2025566, deposited on March 24, 2025, at Wuhan University, Wuhan, China.

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

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

4. The microbial agent according to claim 3, characterized in that, The liquid preparation is a fermentation broth containing the biocontrol bacterium Bacillus vesiculosus GN-22.

5. The application of the biocontrol bacteria according to claim 1 or the microbial agent according to claim 2 in the control of plant diseases, characterized in that, The disease is caused by Fusarium graminearum. Fusarium graminearum, Fusarium pseudograss Fusarium pseudograminearum Fusarium anaeroides Fusarium asiativum Fusarium pseudoverticum Fusarium verticillioides Fusarium scutellatus Caused by Fusarium oxysporum .

6. The application as described in claim 5, characterized in that, The disease is corn stem base rot or wheat stem base rot.

7. The application of the biocontrol bacteria according to claim 1 or the microbial agent according to claim 2 in promoting the growth of corn, wheat, rice, rapeseed, and barley.

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