Paenibacillus sp. feed NGT8 and its application in prevention and treatment of wheat basal stem rot

Abstract

The application discloses a feed bacillus NGT8 and application thereof in prevention and treatment of wheat stem base rot. Paenibacillus pabuli The feed bacillus (Bacillus NGT8) provided by the application is preserved in the Guangdong Microbial Culture Collection Center on May 7, 2025, and the preservation number is GDMCC No. 66267. The strain has good bacteriostatic effect on various plant pathogenic bacteria, has broad-spectrum bacteriostatic property, and can be used for preventing and treating plant diseases caused by the pathogenic bacteria. The NGT8 strain has good prevention and treatment effect when applied to the prevention and treatment of wheat stem base rot, and can reduce the incidence and disease index of wheat stem rot. The NGT8 strain provided by the application can be used for developing more plant pathogenic bacteria inhibitors or biocontrol agents, reducing the use of chemical pesticides and pesticide residues, and providing a new way for gradually replacing chemical control with biological control.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology and disease control technology, and more specifically, to a forage-type Bacillus NGT8 and its application in the control of wheat stem base rot. Background Technology

[0002] Wheat faces multiple diseases during its growth period, among which wheat stem rot has become a devastating soil-borne disease that has become increasingly prevalent in recent years, severely impacting wheat production. This disease is mainly caused by fungi of the genus *Fusarium* (such as *Fusarium pseudogranatum*). Fusarium pseudogramineaum Fusarium oxysporum ( Fusarium oxysporum Caused by pathogens such as mycelium or conidia, the pathogens overwinter in the soil, diseased plant debris, and seeds. After wheat is sown the following year, they invade through wounds at the base of the stem or the epidermis, and spread within the plant.

[0003] Wheat stem rot is characterized by its insidious and explosive nature. In the early stages, plants show no obvious symptoms, but later, brown rot spots appear at the base of the stem, damaging the vascular tissue and hindering nutrient and water transport. This results in symptoms such as lodging, withered white ears, and shriveled grains. In severely affected areas, yield losses can reach 10%-50%, or even total crop failure. With the expansion of wheat continuous cropping areas, the widespread adoption of straw return technology, and favorable temperature and humidity conditions due to global warming, the disease's incidence and severity are continuously increasing, making it a core bottleneck restricting high and stable wheat yields.

[0004] Currently, the main control methods for wheat stem base rot include chemical control, agricultural control, and biological control. Chemical control relies on fungicides such as triazoles for seed treatment or root drenching. Although it is effective in the short term, long-term use can easily lead to increased pathogen resistance, imbalance of soil microbiota, and pesticide residues pose potential risks to the ecological environment and food safety. Agricultural control, such as crop rotation and deep plowing, is difficult to promote due to limitations in planting structure and land resources, and its control effect is unstable. Biological control has become a research hotspot in the field of green control of agricultural diseases due to its environmental friendliness and strong sustainability. Among them, biocontrol bacteria inhibit pathogens through antagonistic effects, competition for nutrient space, and induction of plant resistance, and have the characteristics of no residue and low resistance. However, the biocontrol bacteria reported so far have problems such as weak strain specificity, control effect greatly affected by the environment, and weak colonization ability. Most strains only show antagonistic activity under laboratory conditions, and their field application effect is poor, which is difficult to meet the needs of production for efficient control of wheat stem base rot. Therefore, screening a biocontrol bacterium with strong antagonistic activity against the pathogen of wheat stem rot, good field colonization ability, and stable control effect, and developing its application technology, is of great practical significance for ensuring safe wheat production. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of existing biocontrol bacteria used to prevent and control wheat stem base rot. The present invention provides a feed-type Bacillus NGT8 and its application in the prevention and control of wheat stem base rot.

[0006] The first objective of this invention is to provide a feed-type Bacillus strain NGT8.

[0007] A second objective of this invention is to provide the application of the feed-grade Bacillus NGT8 strain.

[0008] A third objective of this invention is to provide an antibacterial agent.

[0009] The fourth objective of this invention is to provide a method for preventing and controlling plant pathogens or plant diseases caused by them.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a feed-type Bacillus strain ( Paenibacillus pabuli The NGT8 strain was deposited on May 7, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC NO:66267, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0011] This invention isolated and identified a forage-type Bacillus strain NGT8 from tobacco rhizosphere soil. Studies showed that this strain exhibited good inhibitory effects against various plant pathogens, including *Fusarium graminearum*, *Fusarium oxysporum* race 4 (Cuba specific type), rice sheath blight pathogen, tobacco downy mildew pathogen, litchi downy mildew pathogen, pepper gray mold pathogen, and cucurbit gray mold pathogen. It possesses broad-spectrum antibacterial activity, with a particularly strong inhibitory effect against *Fusarium graminearum*. This strain can be used to control plant diseases caused by these pathogens. Applying strain NGT8 to the control of wheat stem rot showed good efficacy, mitigating the harmful effects of the pathogen, reducing the incidence and disease index of wheat stem rot, with a control efficacy of 75.37%. The NGT8 strain provided by this invention, as a biocontrol bacterium, can be used to develop more plant pathogen inhibitors or biocontrol agents, reduce the use of chemical pesticides and pesticide residues, and provide a new way for biological control to gradually replace chemical control.

[0012] Therefore, the present invention provides the application of the feed-type Bacillus NGT8 strain in the prevention and control of plant pathogens.

[0013] This invention provides the application of the feed-type Bacillus NGT8 strain in the prevention and control of plant diseases caused by plant pathogens.

[0014] Preferably, the plant pathogen is *Fusarium graminearum* (…). Fusarium pseudogramineaum ), Fusarium oxysporum Cuban specialized physiological race 4 ( Fusarium oxysporum f.sp.cubense race 4 Foc4), Rhizoctonia solani ( Rhizoctonia solani Kühn ), Botrytis cinerea ( Botrytis cinerea ), Phytophthora indicum ( Phytophthora nicotianae ), Phytophthora lichee ( Peronophythora litchii One or more of the following.

[0015] Preferably, the plant disease is one or more of the following: wheat stem base rot, corn stem base rot, banana wilt, rice sheath blight, tobacco downy mildew, litchi downy mildew, pepper gray mold, and cucurbit gray mold.

[0016] This invention provides the application of the feed-type Bacillus NGT8 strain in the preparation of antibacterial agents.

[0017] The present invention provides an antibacterial agent containing Bacillus subtilis strain NGT8 and / or its fermentation broth.

[0018] Preferably, the concentration of the fermentation broth is not less than 1×10⁻⁶. 8 cfu / mL.

[0019] Further, the preparation method of the fermentation broth is as follows: Activated feed-type Bacillus is streaked in LB solid medium and cultured at 28 ℃ until single colonies grow; the cultured single colonies are transferred to LB liquid medium and shaken at 28 ℃ and 150-240 rpm to obtain seed culture; the seed culture is added to LB liquid medium and shaken again at 28 ℃ and 150-240 rpm to obtain feed-type Bacillus fermentation broth.

[0020] The present invention also provides a method for preventing and controlling plant pathogens or plant diseases caused by them, by treating with Bacillus subtilis strain NGT8 or the above-mentioned bacterial agents.

[0021] Preferably, the plant pathogen is *Fusarium graminearum* (…). Fusarium pseudogramineaum ), Fusarium oxysporum Cuban specialized physiological race 4 ( Fusarium oxysporum f.sp.cubense race 4 Foc4), Rhizoctonia solani ( Rhizoctonia solani Kühn ), Botrytis cinerea ( Botrytis cinerea Phytophthora indicum ( Phytophthora nicotianae ), Phytophthora lichee ( Peronophythora litchii One or more of the following.

[0022] Preferably, the plant disease is one or more of the following: wheat stem base rot, corn stem base rot, banana wilt, rice sheath blight, tobacco downy mildew, litchi downy mildew, pepper gray mold, and cucurbit gray mold.

[0023] The present invention has the following beneficial effects: This invention isolated and identified a forage-type Bacillus strain NGT8 from tobacco rhizosphere soil. Studies showed that this strain exhibited good inhibitory effects against various plant pathogens, including *Fusarium graminearum*, *Fusarium oxysporum* race 4 (Cuba specialized type), rice sheath blight pathogen, tobacco downy mildew pathogen, litchi downy mildew pathogen, pepper gray mold pathogen, and cucurbit gray mold pathogen. It possesses broad-spectrum antibacterial activity, with a particularly strong inhibitory effect against *Fusarium graminearum*. This strain can be used to control plant diseases caused by these pathogens. Application of the NGT8 strain in the control of wheat stem rot showed good efficacy, mitigating the harmful effects of the pathogen on plants, reducing the incidence and disease index of wheat stem rot, with a control efficacy of 75.37%. The NGT8 strain provided by this invention, as a biocontrol bacterium, can be used to develop more plant pathogen inhibitors or biocontrol agents, reduce the use of chemical pesticides and pesticide residues, and provide a new way for biological control to gradually replace chemical control. Attached Figure Description

[0024] Figure 1 This is a colony morphology diagram of strain NGT8.

[0025] Figure 2 This is a phylogenetic tree diagram of the 16S rDNA gene of strain NGT8.

[0026] Figure 3 This is a graph showing the inhibition of Fusarium pseudograss by the NGT8 strain.

[0027] Figure 4 This is a graph showing the inhibition of NGT8 strain against physiological race 4 of Fusarium oxysporum Cuban specialized type.

[0028] Figure 5 This is a graph showing the inhibition of Rhizoctonia solani by the NGT8 strain.

[0029] Figure 6 This is a graph showing the inhibition of Botrytis cinerea by the NGT8 strain.

[0030] Figure 7 This is a graph showing the inhibition of Phytophthora tobaccoii by the NGT8 strain.

[0031] Figure 8 This is a graph showing the inhibition of Phytophthora licheniformis by the NGT8 strain.

[0032] Figure 9 This is a diagram showing the control effect of the NGT8 strain on wheat stem base rot. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0034] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0035] The culture medium formulation used in the examples is as follows: (1) LB solid medium: 10.0 g / L tryptone, 10.0 g / L NaCl, 5.0 g / L yeast extract, 17.0 g / L agar, 1000 mL distilled water, autoclaved at 121℃ for 20 min.

[0036] (2) LB liquid culture medium: 10.0 g / L tryptone, 10.0 g / L NaCl, 5.0 g / L yeast extract, 1000 mL distilled water, autoclaved at 121℃ for 20 min.

[0037] (3) PDA solid culture medium: 200 g / L potato, 20 g / L glucose, 16 g agar, 1000 mL distilled water, autoclaved at 121℃ for 20 min.

[0038] (4) PDB liquid culture medium: 200 g / L potato, 20 g / L glucose, 1000 mL distilled water, autoclaved at 121℃ for 20 min.

[0039] Example 1 Isolation and Identification of Strains 1. Isolation of strains (1) The soil samples were obtained from tobacco rhizosphere soil in Shaoguan City, Guangdong Province. After collecting the tobacco rhizosphere soil samples, 1 g of tobacco rhizosphere soil was weighed into a sterile Erlenmeyer flask, 100 mL of sterile water was added, and the soil suspension was obtained by shaking and incubating at 28 ℃ and 180 rpm for 30 min.

[0040] (2) After centrifuging the soil suspension at 5000 r / min for 10 min, take the supernatant and spread it on LB solid medium. Incubate at 28℃ for 1 day until colonies grow.

[0041] (3) Pick a single colony and streak it onto a new B solid medium for purification. After the colony grows, as shown... Figure 1 As shown, its single colonies are nearly round, yellowish-white, and latex-like, with a smooth, viscous surface and abundant secondary metabolites. Single colonies were picked and cultured in LB liquid medium at 28 °C and 180 rpm for 24 h. The bacterial solution was then mixed with glycerol using standard methods and stored at -80 °C. This purified bacterium was named NGT8 strain.

[0042] 2. Sequence homology analysis and phylogenetic tree construction Genomic DNA of strain NGT8 was extracted using a boiling water bath method with bacterial suspension. Using the extracted NGT8 genomic DNA as a template, PCR amplification was performed using universal bacterial 16S rDNA primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR reaction mixture consisted of 1 μL DNA template, 0.5 μL each of forward and reverse primers, 10 μL Taq polymerase, and ddH2O to a final volume of 20 μL. The PCR program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 1 min, for a total of 35 cycles; and a final extension at 72℃ for 5 min.

[0043] The obtained 16S rDNA PCR amplification product was then detected by 1% agarose gel electrophoresis, and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results were submitted to the NCBI database for BLAST homology analysis, and then MEGA 5.0 software was used for sequence alignment and phylogenetic tree construction.

[0044] The results showed that the 16S rDNA sequence of strain NGT8 was 1442 bp in size, as shown in SEQ ID NO. 1. BLAST alignment analysis of the 16S rDNA sequence of strain NGT8 showed that strain NGT8 was similar to *Bacillus subtilis* (a type of basalt). Paenibacillus pabuli The homology among them reached 98%, and their phylogenetic tree results are as follows: Figure 2 As shown, it can be seen that the NGT8 strain is related to feed-type Bacillus ( Paenibacillus pabuli Within the same branch, the kinship is the closest.

[0045] Based on the morphological and molecular biological characteristics of strain NGT8, strain NGT8 is taxonomically classified as a forage bacillus (Bacillus subtilis). Paenibacillus pabuli The strain was named NGT8 and deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 7, 2025, with accession number GDMCC No: 66267. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0046] Example 2 Preparation of fermentation broth for NGT8 strain The NGT8 strain stored at -80 ℃ was activated, and single colonies of the activated strain were streaked on LB solid medium and incubated at 28 ℃ for 48 h. Single colonies of the incubated strain were then transferred to LB liquid medium and shaken at 28 ℃ and 180 rpm for 24 h to obtain the NGT8 strain seed culture.

[0047] NGT8 strain seed culture was added at 1% to LB liquid medium and incubated at 28 ℃ and 180 rpm for 12 h to obtain NGT8 strain fermentation broth for later use. The concentration of this fermentation broth was determined to be approximately 1×10⁻⁶. 8 cfu / mL.

[0048] Example 3: Antibacterial effect of NGT8 strain Plant pathogens preserved in this laboratory: Fusarium graminearum ( Fusarium pseudogramineaum ), Fusarium oxysporum Cuban specialized physiological race 4 ( Fusarium oxysporum f.sp.cubense race 4 Foc4), Rhizoctonia solani ( Rhizoctonia solani Kühn ), Botrytis cinerea ( Botrytis cinerea Phytophthora indicum ( Phytophthora nicotianae ), Phytophthora lichee ( Peronophythora litchii As indicator bacteria, the four-point confrontation method was used. Inoculations of the indicator bacteria were made by placing bacterial discs in the center of PDA plates. Fermentation broth of the NGT8 strain prepared in Example 2 was dropped onto four points approximately 2.5 cm from the bacterial discs, with 5 μL of NGT8 fermentation broth at each point. An equal amount of ddH2O was added to the control group, and three replicates were set up. The plates were incubated at 28°C until the control group reached 2 / 3 of the plate's length. The inhibition status and the size of the inhibition zone were observed. Colony diameters were recorded, and the inhibition rate was calculated and statistically analyzed according to the inhibition rate calculation formula.

[0049] Inhibition rate (%) = (Control colony diameter - Treatment colony diameter) / Control colony diameter × 100%.

[0050] The antibacterial results are shown in Table 1 below. The NGT8 strain exhibits good inhibitory effects against all six plant pathogens. The plate inhibition results against *Fusarium graminearum*, *Fusarium oxysporum* race 4 (specific type), *Rhizoctonia solani*, *Botrytis cinerea*, *Phytophthora nicotinica*, and *Phytophthora licoricei* are shown below. Figures 3-8 As shown, strain NGT8 exhibits significant inhibitory effects against Fusarium graminearum and Botrytis cinerea. This indicates that strain NGT8 possesses broad-spectrum antibacterial activity, capable of inhibiting various plant pathogens. As a biocontrol agent, it can control a variety of plant pathogens and the plant diseases they cause, thus broadening the antibacterial spectrum of strain NGT8.

[0051] Table 1 Antibacterial effect of NGT8 strain

[0052] Example 4: Control effect of NGT8 strain on wheat 1. Preparation of Fusarium spore suspension The *Fusarium graminearum* strain preserved on slant culture was activated. Mycelial blocks were picked and transferred to PDA solid medium and cultured in the dark at 28°C for 5 days. Four 5 mm diameter mycelial discs from the activated *Fusarium graminearum* were picked and cultured in 100 mL PDB liquid medium at 180 rpm for 3 days in the dark at 28°C. After filtering the mycelium, the number of spores was counted using a hemocytometer, and the concentration was adjusted to 1 × 10⁻⁶ using sterile water. 5 per mL.

[0053] 2. Wheat Cultivation Select brightly colored and plump wheat grains, soak them in 2% sodium hypochlorite for 1 minute, then soak them in 75% ethanol for 1 minute, and rinse them three times with sterile water. Place them in petri dishes containing sterile water and incubate at 25℃ for 24 hours until the wheat grains germinate and show white sprouts. Sterilize the soil at high temperature. Mix vermiculite and sterilized soil in a 1:3 ratio, fill plastic bowls with a diameter of 14 cm and a height of 6 cm to one-third of the bowl's height, and sow 10 wheat grains in each bowl.

[0054] 3. Determination of the control effect of wheat stem base rot A control group and a treatment group were set up. The treatment group was watered with 7.5 mL of NGT8 bacterial solution (concentration of 1×10⁻⁶). 8 (cfu / mL) and 7.5 mL of *Fusarium graminearum* spore suspension (concentration 1×10⁻⁶). 5 The control group was treated with 7.5 mL of sterile water and 7.5 mL of *Fusarium graminearum* spore suspension (concentration 1×10⁻⁶). 5 The treatment frequency was 3 days / time, with each treatment repeated 3 times. The plants were cultured at 25℃ under alternating light and dark conditions for 12 h / 12 ​​h. After 14 days, samples were taken and photographed to record the seedling height and the length of brown discoloration at the stem base. The incidence rate was then calculated using the following formula. And the disease index.

[0055] The methods for calculating incidence and disease index are as follows:

[0056]

[0057] Disease index = ∑ (number of diseased plants at each level × corresponding disease level) / (total number of plants surveyed × highest disease level) × 100; The disease severity grading criteria are as follows: 0, asymptomatic; 1, mild root / stem browning (0-25%); 2, moderate browning (25%-50%); 3, severe browning (over 50%).

[0058] The results are as follows Figure 9 As shown, in the control group, wheat plants infected with wheat stem base rot fungus exhibited obvious symptoms of stunting and browning at the stem base; compared with the control group, wheat seedlings in the treatment group that were simultaneously inoculated with wheat stem base rot fungus and NGT8 bacterial solution showed significantly fewer lesions and better growth.

[0059] The statistical results of incidence and disease index are shown in Table 2. The results show that the use of NGT8 bacterial solution significantly reduced the incidence and disease index of wheat stem rot, alleviated the effect of the pathogen on wheat seedlings, and effectively prevented and controlled wheat stem base rot.

[0060] Table 2. Control efficacy of strain NGT8 against wheat stem rot.

[0061] Note: The data in the table are mean ± standard deviation; different letters in the column distribution indicate that there is a significant difference at the P<0.01 level.

[0062] In summary, this invention isolated and identified a forage-type Bacillus strain NGT8. Studies show that this strain exhibits good inhibitory activity against various plant pathogens, including *Fusarium graminearum*, *Fusarium oxysporum* race 4 (Cuba specialized type), *Rhizoctonia solani*, *Botrytis cinerea*, *Phytophthora tobaccoii*, and *Phytophthora litchifolia*, demonstrating broad-spectrum antibacterial activity. It shows particularly strong inhibitory effects against *Fusarium graminearum*, making it suitable for controlling plant diseases caused by these pathogens. In the control of wheat stem rot, strain NGT8 showed good efficacy, mitigating the pathogen's effects on plants, reducing the incidence and disease index of wheat stem rot, with a control efficacy of 75.37%. The NGT8 strain provided by this invention, as a biocontrol agent, can be used to develop more plant pathogen inhibitors or biocontrol agents, reducing the use and residues of chemical pesticides, and providing a new approach for biological control to gradually replace chemical control.

[0063] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of feed-grade Bacillus ( Paenibacillus pabuli NGT8 strain, characterized in that, This strain was deposited on May 7, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO:66267, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The application of the feed-type Bacillus NGT8 strain according to claim 1 in the control of plant pathogens, characterized in that, The plant pathogen is *Fusarium graminearum* (…). Fusarium pseudogramineaum ), Fusarium oxysporum Cuban specialized physiological race 4 ( Fusarium oxysporumf.sp.cubense race 4 Foc4), Rhizoctonia solani ( Rhizoctonia solani Kühn ), Botrytis cinerea ( Botrytis cinerea ), Phytophthora indicum ( Phytophthora nicotianae ), Phytophthora lichee ( Peronophythora litchii One or more of the following.

3. The application of the feed-type Bacillus NGT8 strain according to claim 1 in the prevention and control of plant diseases caused by plant pathogens, characterized in that, The plant pathogen is *Fusarium graminearum* (…). Fusarium pseudogramineaum ), Fusarium oxysporum Cuban specialized physiological race 4 ( Fusarium oxysporum f.sp.cubense race 4 Foc4), Rhizoctonia solani ( Rhizoctonia solani Kühn ), Botrytis cinerea ( Botrytis cinerea ), Phytophthora indicum ( Phytophthora nicotianae ), Phytophthora lichee ( Peronophythora litchii One or more of the following.

4. The application according to claim 3, characterized in that, The plant diseases mentioned are one or more of the following: wheat stem base rot, corn stem base rot, banana wilt, rice sheath blight, tobacco Phytophthora blight, lychee downy mildew, pepper gray mold, and cucurbit gray mold.

5. The use of the feed-type Bacillus NGT8 strain according to claim 1 in the preparation of antibacterial agents.

6. An antibacterial agent, characterized in that, Contains the feed-grade Bacillus NGT8 strain as described in claim 1 and / or its fermentation broth.

7. The antibacterial agent according to claim 6, characterized in that, The concentration of the fermentation broth is not less than 1×10⁻⁶. 8 cfu / mL.

8. A method for preventing and controlling plant pathogens or plant diseases caused by them, characterized in that, The feed-grade Bacillus NGT8 strain as described in claim 1 or the bacterial agent as described in claim 6 is used for treatment.

9. The method according to claim 8, characterized in that, The plant pathogen is *Fusarium graminearum* (…). Fusarium pseudogramineaum ), Fusarium oxysporum Cuban specialized physiological race 4 ( Fusarium oxysporum f.sp.cubense race 4 Foc4), Rhizoctonia solani ( Rhizoctonia solani Kühn ), Botrytis cinerea ( Botrytis cinerea ), Phytophthora indicum ( Phytophthora nicotianae ), Phytophthora lichee ( Peronophythora litchii One or more of the following.

10. The method according to claim 8, characterized in that, The plant diseases mentioned are one or more of the following: wheat stem base rot, corn stem base rot, banana wilt, rice sheath blight, tobacco Phytophthora blight, lychee downy mildew, pepper gray mold, and cucurbit gray mold.

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