Bacillus amyloliquefaciens and application thereof in disease resistance and growth promotion of peanuts
By screening Bacillus amyloliquefaciens BAZX-2 from peanut materials, the problems of environmental pollution and pathogen resistance in peanut disease control have been solved. It has achieved the dual effect of highly effective antibacterial activity and promotion of peanut seed germination, providing excellent strain resources for green control of peanut disease.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-28
- Publication Date
- 2026-04-24
AI Technical Summary
In existing technologies, the control of peanut diseases mainly relies on disease-resistant varieties and chemical pesticides, which leads to environmental pollution and increased pathogen resistance. Furthermore, there is a lack of highly efficient biological control strains, and the control effect is particularly poor for peanut fruit rot, white mold, and crown rot.
Bacillus amyloliquefaciens strain BAZX-2 was isolated and screened from peanut materials. Its taxonomic position was identified by 16S rDNA and rpoB gene sequence analysis. Its antibacterial activity against pathogenic bacteria was determined by confrontation culture method. Fermentation broth and cell treatment improved the germination index of peanut seeds.
The BAZX-2 strain showed an inhibition rate of 61.1%-77.11% against Aspergillus niger, Sclerotium sclerotiorum, and Fusarium oxysporum. The fermentation broth significantly improved peanut germination indicators, with germination potential, germination rate, and germination index increasing by 34.33%, 23.29%, and 27.47%, respectively, achieving the dual functions of disease resistance and growth promotion.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a Bacillus amyloliquefaciens and its application in peanut disease resistance and growth promotion. Background Technology
[0002] Peanuts are frequently affected by fruit rot caused by *Fusarium oxysporum*, white mold caused by *Sclerotium sclerotiorum*, and crown rot caused by *Aspergillus niger* during their growth. These diseases occur frequently or intermittently, causing significant losses to peanut yields in my country. Currently, the control of peanut fruit rot mainly relies on disease-resistant varieties and chemical control. However, long-term application of chemical pesticides not only pollutes the environment and causes pesticide residues, but also promotes drug resistance in pathogens and negatively impacts soil biodiversity. Biological control, with its significant advantages of high efficiency, eco-friendliness, and sustainable development, has become an important means of controlling harmful fungi and has received considerable attention in recent years. Current research indicates that antagonistic bacteria such as *Streptomyces* and *Bacillus* are highly effective in controlling small leaf spot, but the limited availability of suitable bacterial strains remains a challenge. Therefore, it is necessary to continue screening for highly effective biocontrol strains that antagonize multiple peanut diseases (peanut fruit rot, white mold, and crown rot). Studies by Guo Jing et al. have found that *Bacillus amyloliquefaciens* has a good inhibitory effect on eight pathogens of *Pinus sylvestris*, including *Colletotrichum gloeosporioides*, *Botrytis cinerea*, and *Botrytis cinerea* causal agent. Experiments by Yang Xue, Li Jianan, and others have demonstrated that *Bacillus amyloliquefaciens* can enhance the drought adaptability of oats and promote their growth. However, its application in peanuts is still somewhat lacking. Therefore, this study isolated a highly effective antagonistic strain, BAZX-2, from a large number of peanut materials from Shandong regional trials, and then conducted morphological and multi-gene phylogenetic analyses. The antibacterial properties of BAZX-2 bacterial suspension and sterile filtrate were tested using a confrontation culture method. The aim is to discover functional strains that effectively inhibit peanut diseases and provide technical support for green pest control in peanut cultivation. Summary of the Invention
[0003] This invention isolated and screened a biocontrol bacterium, BAZX-2, from peanut material HY25. Its taxonomic position was identified by 16S rDNA and rpoB gene sequence analysis. The antibacterial activity of the strain, fermentation broth, and sterile filtrate against three pathogenic bacteria was determined using a confrontation culture method. Its growth-promoting effect was evaluated through seed germination tests. The results showed that BAZX-2 is a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens). Bacillus amyloliquefaciensThe strain exhibited an inhibition rate of 61.1%-77.11% against three pathogenic bacteria, with the cell treatment showing significantly better antibacterial effects than the fermentation broth and sterile filtrate. BAZX-2 fermentation broth significantly improved the germination indices of three peanut varieties, with maximum increases in germination potential, germination rate, and germination index of 34.33%, 23.29%, and 27.47%, respectively. This invention confirms that BAZX-2 possesses both disease resistance and growth-promoting functions, providing excellent strain resources and technical support for the green control of peanut diseases.
[0004] The first objective of this invention is to provide a Bacillus amyloliquefaciens (Bacillus) Bacillus amyloliquefaciens The strain number is BAZX-2. The strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 9, 2026. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The accession number is CGMCCNo. 37345.
[0005] Furthermore, the aforementioned Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens The 16S rDNA sequence of BAZX-2 is shown in SEQ ID NO.1.
[0006] Furthermore, the aforementioned Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens The rpob sequence of BAZX-2 is shown in SEQ ID NO.2.
[0007] A second objective of the present invention is to provide the above-mentioned Bacillus amyloliquefaciens (BAM) Bacillus amyloliquefaciens Application of BAZX-2 in the control of flowering plant diseases caused by Aspergillus niger, Sclerotium sclerotiorum and / or Fusarium oxysporum.
[0008] A third objective of this invention is to provide the aforementioned Bacillus amyloliquefaciens (BAM). Bacillus amyloliquefaciens Application of BAZX-2 in peanut disease resistance and growth promotion.
[0009] Furthermore, the aforementioned resistance includes resistance to peanut diseases caused by Aspergillus niger, Sclerotium sclerotiorum, and / or Fusarium oxysporum.
[0010] The present invention also provides a biological agent, the biological agent comprising Bacillus amyloliquefaciens (Bacillus amyloliquefaciens). Bacillus starch liquefier Fermentation broth, sterile filtrate, or cells of BAZX-2.
[0011] Furthermore, the aforementioned biological agents are applied in the disease resistance and growth promotion of peanuts.
[0012] Furthermore, the aforementioned peanut disease resistance includes peanut diseases caused by Aspergillus niger, Sclerotium truncatum, and / or Fusarium oxysporum.
[0013] Furthermore, the present invention also provides Bacillus amyloliquefaciens (BAM) Bacillus amyloliquefaciens Application of BAZX-2 bacterial cells in resisting flowering plant diseases caused by Aspergillus niger, Sclerotium sclerotiorum and / or Fusarium oxysporum.
[0014] Furthermore, the present invention also provides Bacillus amyloliquefaciens (BAM) Bacillus amyloliquefaciens BAZX-2 fermentation broth is used to improve the germination indicators, germination potential, germination rate and germination index of peanut varieties.
[0015] The beneficial effects of this invention are as follows: the strain of this invention has an inhibition rate of 61.1%-77.11% against three pathogenic bacteria, namely Aspergillus niger, Sclerotium sclerotiorum, and / or Fusarium oxysporum, with the antibacterial effect of the bacterial cell treatment being significantly better than that of the fermentation broth and sterile filtrate; the BAZX-2 fermentation broth can significantly improve the germination index of three peanut varieties, with the maximum increase in germination potential, germination rate, and germination index reaching 34.33%, 23.29%, and 27.47%, respectively; this invention confirms that BAZX-2 has both disease resistance and growth promotion functions, and can simultaneously control multiple peanut diseases, providing excellent strain resources and technical support for the green prevention and control of peanut diseases. Attached Figure Description
[0016] Figure 1 To decipher the colony diagram of Bacillus amyloliquefaciens; Figure 2 Phylogenetic tree of BAZX-2 constructed using primer A as the 16S primer sequence and Figure 2 A phylogenetic tree of BAZX-2 constructed using the rpoB primer sequence; Figure 3 Antibacterial spectrum of strain BAZX-2; In the figure, A represents inhibition against Aspergillus niger, B represents inhibition against Sclerotinia sclerotiorum, and C represents inhibition against Fusarium oxysporum. Figure 4 The antibacterial effects of different treatments of strain BAZX-2 on three pathogenic bacteria are shown in the figure. Detailed Implementation
[0017] Biocontrol strain: Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens) Code name BAZX-2.
[0018] Target pathogenic fungi: Sclerotium sclerotiorum ( Sclerotium rolfsii Sacc. Fusarium oxysporum ( Fusarium oxysporum ), Aspergillus niger ( Aspergillus niger ).
[0019] Bacillus amyloliquefaciens used in strain testing ( Bacillus amyloliquefaciensBAZX-2 was isolated and preserved from peanut variety HY25. Peanut Fusarium strain, peanut Aspergillus niger strain, and Sclerotium sclerotiorum were all isolated from regional trial materials and preserved at the Shandong Peanut Research Institute.
[0020] Plant materials: Huayu 25, Liaohua 3, and Nongda R271, bred by Shandong Peanut Research Institute.
[0021] Test culture media: LB and PDA culture media used in the experiment were provided by Beijing Solarbio Science & Technology Co., Ltd. Example 1
[0022] Isolation and screening of biocontrol bacteria Following the method of Photolo et al. (2020) with slight modifications, endophytic bacteria were isolated from peanut seeds of Healthy Flower No. 25. The seed surface disinfection steps were as follows: soaking in 1% sodium hypochlorite (NaClO) for 3 minutes, soaking in 75% ethanol for 1 minute, and finally rinsing three times with sterile distilled water.
[0023] The seeds were longitudinally split into four pieces and transferred to a 50 mL Erlenmeyer flask containing 30 mL of LB liquid medium. The flask was placed on a rotary shaker and incubated at 150 rpm for 3 hours. The suspension was then transferred to a 10 mL centrifuge tube and centrifuged at 15,000 rpm for 10 minutes. 150 μL of the supernatant was evenly spread onto LB agar plates and incubated at 28°C in the dark for 1–2 days. Single colonies with distinct morphological differences were picked for purification and then stored in glycerol tubes at -80°C.
[0024] Identification of strain BAZX-2 The plate confrontation method (Kumari et al., 2024) was used to screen all isolated strains for antagonistic activity against Fusarium oxysporum, Aspergillus niger, and Sclerotium rolfsii. One strain (strain number ZX-1) showed good antifungal activity and was selected for further research.
[0025] Molecular identification of the strain was performed. BAZX-2 was streaked onto LB agar plates and incubated at 28°C in the dark for 72 hours. Colony morphology was then observed. Figure 1As shown. Genomic DNA was extracted using a bacterial genomic DNA extraction kit (Solarbio, Beijing) as a template. PCR amplification was performed using the universal 16S rDNA primer 27F (5'-AGAGTTTGATCMTGGCTCAG-3' / 1492R (5'-TACGGYTACCTTGTTACGACTT-3') and the rpoB gene primer 2292F / 3354R (5-AGGTCAACTAGTTCAGTATG-GAC-3'5'-AAGAACCGTAACCGGCAACTT-3) [P3]. The target fragment was amplified by PCR. The PCR reaction system (30 μL) consisted of: 1 μL DNA, 1 μL each of forward and reverse primers (10 μmol / L), 15 μL 2×TaqPlus PCR Mix (TIANGEN), and 12 μL ddH2O. PCR reaction cycles: 95℃ for 5 min; 95℃ for 30 s, 55.8℃ for 30 s, 72℃ for 1.5 min, 35 cycles; 72℃ for 5 min. After passing 2% agarose gel electrophoresis, the reaction products were sent to Qingdao NIO Science & Technology Biotechnology Co., Ltd. for sequencing. The sequencing results were analyzed and compared with the 16S rDNA and rpob sequences of standard strains in the NCBI database, and a multigene phylogenetic tree was constructed using the 16S rDNA and rpob sequences. The bootstrap value was set to 1000 replicates to determine its taxonomic position.
[0026] The 16S rDNA and rpoB gene fragments of BAZX-2 were obtained by PCR amplification, and the two gene sequences were obtained by sequencing and splicing. After alignment of the sequenced sequences with NCBI, 10-15 sequence files of type strains with similar lengths to the sample sequence were selected and downloaded. Multiple sequence alignment was performed between these files and the sample sequence using MEGA software, and a phylogenetic tree (Bootstrap ≥ 1000) was constructed using the neighbor-joining (NJ) method. BLAST analysis was performed, and the results showed that BAZX-2 and... Bacillus amyloliquefaciens (KF803276.1) clustered on the same branch ( Figure 2 China A and Figure 2 As shown in B, Figure 2 The BAZX-2 phylogenetic tree constructed using the 16S primer sequence (A). Figure 2 The phylogenetic tree of BAZX-2 is constructed using the rpoB primer sequence (B in the middle). In summary, based on the morphological characteristics and multigene phylogenetic analysis of strain BAZX-2, it is confirmed that BAZX-2 is a type of Bacillus amyloliquefaciens (Bacillus). Bacillus amyloliquefaciens ).
[0027] Example 2 Fermentation broth preparation Preparation of fermentation broth, sterile filtrate, and bacterial cells: 5 mL of BAZX-2 seed culture was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of culture medium and cultured at 30 °C and 180 r / min for 24 h with shaking to obtain fermentation broth; the fermentation broth was centrifuged at 4 °C and 4000 r / min for 1 h, and the precipitate obtained by centrifugation was the bacterial cells; the supernatant was filtered through a 0.22 µm pore size filter membrane, and centrifuged again at 4 °C overnight to obtain sterile filtrate.
[0028] Inhibitory effects of fermentation broth, sterile filtrate, and cell cells on pathogenic hyphae growth Aspergillus niger, Fusarium oxysporum, and Sclerotium sclerotiorum were inoculated into the center of each PDA plate containing different fermentation broths, sterile filtrate, and bacterial cells. PDA plates containing sterile liquid culture medium were used as blank controls. The plates were incubated in the dark at 23°C. Each treatment was repeated 3 times. When the pathogenic fungal colonies reached 2 / 3 of the control plate, the colony diameter was measured and the inhibition rate was calculated.
[0029] Strain ZX-1's inhibition rate against three pathogens The antibacterial effect of strain ZX-1 was determined using three pathogens, including Aspergillus niger, as target bacteria. The results showed that strain ZX-1 exhibited good inhibitory activity against all three pathogens, indicating that strain ZX-1 has good inhibitory activity against common peanut pathogens. The antibacterial effect is shown in [see attached figure]. Figure 3 The antibacterial rate is shown in Table 1. Figure 3 In the diagram, A represents the antibacterial effect of Aspergillus niger, B represents the effect of Sclerotinia sclerotiorum, and C represents the antibacterial effect of Fusarium oxysporum.
[0030] Table 1. Inhibition rate of strain BAZX-2 against three pathogens.
[0031] Inhibitory effects of bacterial strains, fermentation broth, and sterile filtrate on pathogens Depend on Figure 4 As shown in Table 2, the sterile filtrate had a poor inhibitory effect on *Sclerotium riberi*, while the bacterial cells had the best inhibitory effect.
[0032] The bacteria showed good efficacy against Aspergillus niger and Fusarium oxysporum, with an inhibition rate of up to 71% against Aspergillus niger and up to 75% against Fusarium oxysporum, effectively inhibiting its growth.
[0033] Table 2. Inhibition rate and colony diameter of strain BAZX-2 against three pathogenic bacteria.
[0034] Example 3 Effects on peanut seed germination Peanut seeds were surface-sterilized with 75% ethanol for 30 seconds, rinsed thoroughly with sterile water, and dried with filter paper. 180 seeds each of HY25, Liaohua 3, and Nongda 09123 were randomly selected, with 30 seeds placed in each 50mL beaker. The treatment group beakers contained 30mL of BAZX-2 fermentation broth with an OD600 of 0.6. The control group was treated with sterile water and soaked at room temperature (approximately 25℃) for 3 hours. To prevent mold contamination and interference with test results, all experimental tools, including distilled water, filter paper, petri dishes, and tweezers, were sterilized using an autoclave. Two layers of cut filter paper were placed in a 90mm petri dish and moistened with sterile distilled water. The treated seeds were then evenly arranged in the petri dish and labeled. Each experiment was repeated three times, and the seeds were incubated at 25℃. The germination rate was observed and recorded every 24 hours. The germination period begins on day 1 of the seedbed. Based on previous experiments, peanut germination takes 7 days to complete one cycle. The germination standard is that the germinated seed develops a normal radicle, and a seed with a radicle longer than half the length of the seed is considered a normally germinated seed.
[0035] Germination potential GE = (Number of germinated seeds within 3 days / Total number of seeds tested) × 100%; Germination rate GP = (Number of germinated seeds within 7 days / Total number of seeds tested) × 100%; Germination index GI = ∑(Gt / Dt), where Gt refers to the number of germinated plants on day t; Dt refers to the corresponding number of germination days. SPSS 26.0 software was used for significance analysis, and P < 0.05 was considered significant.
[0036] Peanut seed germination after treatment with strain BAZX-2 As shown in Table 3, the number of seeds germinated after treatment with the fermentation broth of strain BAZX-2 was higher than that of seeds treated with sterile water. Compared with the control, the fermentation broth of strain BAZX-2 promoted the germination of peanut seeds to varying degrees and improved the various germination indicators of different seeds.
[0037] Table 3. Germination indicators of three peanut varieties after soaking for 3 hours.
[0038] As shown in Table 3, the number of seeds germinated after treatment with the fermentation broth of strain BAZX-2 was higher than that of seeds treated with sterile water, showing an overall upward trend: the treatment groups of all varieties were significantly higher than the control group in terms of germination potential, germination rate and germination index, indicating that soaking for 3 hours has a general promoting effect on the germination performance of these three peanut varieties.
[0039] Germination potential improvement was relatively stable: the germination potential of the three varieties increased by 28.13%-34.33%, indicating that the treatment can effectively accelerate the germination initiation speed of seeds and enable them to enter the germination stage earlier.
[0040] The germination index showed a relatively balanced improvement: the germination index combines germination speed and uniformity, and the improvement for the three varieties ranged from 21.83% to 27.47%, indicating a good promoting effect.
[0041] This invention isolates and screens Bacillus amyloliquefaciens BAZX-2 from peanut material HY25 in Shandong regional trials. Its inhibition rate against three major pathogenic bacteria in peanuts reaches 61.1%-77.11%, significantly higher than some previously reported Bacillus amyloliquefaciens strains (such as Wu Fengkang).
[21] The reported strains showed an inhibition rate of approximately 50%-60% against cotton Verticillium wilt, with the bacterial cells exhibiting the best antibacterial effect.
[0042] BAZX-2 fermentation broth significantly improved the germination index of three peanut varieties. Among them, HY25, as the strain isolation source, showed a more significant improvement in germination index, which may indicate a host adaptability advantage and provides a reference for subsequent targeted applications.
[0043] This invention confirms that BAZX-2 has both disease resistance and growth promotion functions, overcoming the limitations of single biocontrol bacteria and laying the foundation for the development of peanut-specific biological agents.
Claims
1. A type of Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), characterized by: Its strain number is BAZX-2. The strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 9, 2026. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the accession number is CGMCC No. 37345.
2. The Bacillus amyloliquefaciens as described in claim 1 ( Bacillus amyloliquefaciens ), characterized by: Its 16S rDNA sequence is shown in SEQ ID NO.
1.
3. The Bacillus amyloliquefaciens as described in claim 1, characterized in that: Its rpob sequence is shown in SEQ ID NO.
2.
4. The Bacillus amyloliquefaciens as described in claim 1 ( Bacillus amyloliquefaciens Application in the control of flowering plant diseases caused by Aspergillus niger, Sclerotium sclerotiorum and / or Fusarium oxysporum.
5. The Bacillus amyloliquefaciens as described in claim 1 ( Bacillus amyloliquefaciens It is used in the disease resistance and growth promotion of peanuts.
6. The application as described in claim 5, characterized in that: The aforementioned resistance includes resistance to peanut diseases caused by Aspergillus niger, Sclerotium sclerotiorum, and / or Fusarium oxysporum.
7. A biological agent, characterized in that: The biological agent comprises Bacillus amyloliquefaciens as described in claim 1 (… Bacillus amyloliquefaciens Fermentation broth, sterile filtrate, or cells of BAZX-2.
8. The application of the biological agent as described in claim 7 in the disease resistance and growth promotion of peanuts.
9. The Bacillus amyloliquefaciens as described in claim 1 ( Bacillus amyloliquefaciens The application of its mycelium in resisting the flowering diseases caused by Aspergillus niger, Sclerotium sclerotiorum and / or Fusarium oxysporum.
10. The Bacillus amyloliquefaciens as described in claim 1 ( Bacillus amyloliquefaciens Its fermentation broth is used to improve the germination indicators, germination potential, germination rate and germination index of peanut varieties.