Novel microbial peptidase fertilizer
By using a mixed microbial fertilizer of Bacillus tekirii, Bacillus pleuropneumoniae, and Pseudomonas holmieae, the problems of long breeding cycles for disease-resistant varieties and chemical control have been solved, achieving efficient biological control of crop diseases and promoting growth.
Patent Information
- Application Number
- CN202511385189.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-26
- Publication Date
- 2025-12-02
AI Technical Summary
In existing technologies, the breeding cycle of disease-resistant varieties is long and the disease-resistant genes are singular. Chemical control leads to increased drug resistance and environmental pollution. The control effect of single microbial fertilizers is limited, making it difficult to effectively control crop diseases.
A novel microbial peptide fertilizer was prepared by mixing Bacillus tekirae, Bacillus pleuropneumoniae, and Pseudomonas hominis in a specific ratio. The fertilizer was activated by optimizing the fermentation medium to improve its inhibitory effect on pathogens causing diseases.
It enhances the prevention and control of crop diseases, realizes the combined functions of sterilization, disease resistance and growth promotion, and provides support for green biological control.
Smart Images
Figure SMS_1 
Figure SMS_2 
Figure SMS_3
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial fertilizer technology, specifically relating to a novel microbial peptide enzyme fertilizer. Background Technology
[0002] In the process of crop cultivation and production, the damage caused by diseases is inevitable. Currently, the main control measures for crop diseases include breeding disease-resistant varieties, chemical control, and biological control. However, the breeding cycle for disease-resistant varieties is long, and these varieties often possess only a single resistance gene, targeting specific pathogens, making them ill-equipped to cope with rapidly mutating diseases and limiting their ability to respond to sudden outbreaks.
[0003] Chemical control has advantages such as convenient application and rapid effect, and it is currently the main means of controlling field diseases, especially playing a key role in the control of large-scale outbreaks of diseases. However, long-term reliance on chemical pesticides can easily lead to the continuous enhancement of pathogen resistance, resulting in a gradual increase in the amount of pesticides used, forming a vicious cycle of "the more pesticides are used, the worse the control effect becomes"; moreover, long-term use of chemical pesticides can also cause problems such as excessive pesticide residues and damage to the ecological environment.
[0004] Compared to chemical control, biological control offers advantages such as high safety, strong sustainability, and environmental friendliness. The core of biological control lies in utilizing beneficial microorganisms and the peptides and enzymes they produce during their production and metabolism to inhibit or even kill pathogens, thereby achieving disease control. Furthermore, the growth and metabolism of beneficial microorganisms activate the soil, improve soil structure, and thus improve the crop growing environment, promoting crop growth.
[0005] Microbial fertilizers, as a key carrier for the green transformation of agriculture, contain beneficial microorganisms that can effectively control crop diseases, thus achieving biological control, and can also improve soil health problems caused by the irrational use of agricultural chemicals. However, microbial fertilizers containing only one beneficial microorganism have limited disease control effects, so it is necessary to research and develop microbial fertilizers containing multiple beneficial microorganisms.
[0006] There are currently no reports of microbial fertilizers containing Bacillus tekirae and Bacillus pleuropneumoniae, or microbial fertilizers containing Bacillus tekirae, Bacillus pleuropneumoniae and Pseudomonas hominis.
[0007] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention
[0008] The purpose of this invention is to provide a novel microbial peptide enzyme fertilizer to solve the problems existing in the prior art when cultivating disease-resistant varieties and chemically controlling crop diseases.
[0009] To achieve the above objectives, the present invention provides the following technical solution: The first objective of this invention is to provide a novel microbial peptide enzyme fertilizer, wherein the novel microbial peptide enzyme fertilizer is composed of Bacillus tekirae fertilizer and Bacillus hygroscopicus fertilizer in a mass ratio of 1-15:3-1, or is composed of Bacillus tekirae fertilizer, Bacillus hygroscopicus fertilizer and Pseudomonas holometaeus fertilizer in a mass ratio of 1-8:1:8-1.
[0010] More specifically, the preparation method of the Bacillus tekirii fertilizer, Bacillus hygroscopicus fertilizer, or Pseudomonas hominis fertilizer is as follows: A single colony of Bacillus tekirii, Bacillus hygroscopicus, or Pseudomonas hominis is inoculated into NB liquid medium and cultured at 28°C with shaking at 180 rpm until OD... 600 The value is 0.8, which is recorded as the fermentation seed liquid; then, the fermentation seed liquid is inoculated into the fermentation nutrient solution at an inoculation rate of 8% by volume, and fermented at 28°C for 24 hours. During the fermentation, sterile air is introduced to maintain the dissolved oxygen concentration of the fermentation nutrient solution at a saturation of 25-40%; then, fresh fermentation nutrient solution is added to adjust the number of effective viable bacteria in the fermentation product, thus obtaining the Bacillus tekirae fertilizer, Bacillus hygroscopicus fertilizer, or Pseudomonas holmii fertilizer.
[0011] More specifically, the formula for the NB liquid culture medium is: 3g beef extract, 5g peptone, and 5g sodium chloride in 1L of distilled water, with a pH of 7.2.
[0012] More specifically, the fermentation nutrient solution formulas for the Bacillus tekirae fertilizer and the Bacillus alpinee fertilizer are as follows: 8g glucose, 30g corn flour, 12g wheat bran powder, 20g soybean meal powder, 4g yeast extract, 1.5g ammonium nitrate, 2.5g potassium dihydrogen phosphate, and 0.8g magnesium sulfate in 1L of distilled water, with a pH value of 7.2.
[0013] More specifically, the fermentation nutrient solution formula for the *Pseudomonas holmii* fertilizer is as follows: 1L of distilled water contains 5g of glucose, 20g of corn flour, 15g of soybean meal, 1g of yeast extract, 3g of potassium nitrate, 2g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, 0.2g of ferrous sulfate, and a pH value of 7.2.
[0014] More specifically, the effective viable count of the *Bacillus tegmentata* fertilizer, *Bacillus hygroscopicus* fertilizer, and *Pseudomonas hominis* fertilizer is all 2.0 × 10⁻⁶. 8 cfu / g.
[0015] The second objective of this invention is to provide a microbial fertilizer, characterized in that the microbial fertilizer includes the novel microbial peptide enzyme fertilizer.
[0016] The third objective of this invention is to provide the application of the novel microbial peptide enzyme fertilizer or the microbial fertilizer in promoting crop growth and controlling crop diseases.
[0017] Compared with the prior art, the present invention has the following beneficial effects: When the Bacillus tekirae microbial fertilizer and Bacillus hygroscopicus microbial fertilizer of the present invention are used together in a mass ratio of 1-15:3-1, or when Bacillus tekirae microbial fertilizer, Bacillus hygroscopicus microbial fertilizer and Pseudomonas holmieae microbial fertilizer are used together in a mass ratio of 1-8:1:8-1, they show a synergistic effect in inhibiting the mycelial growth of pathogenic fungi of crops. This indicates that the microbial peptidase of the present invention can improve the control effect on crop diseases and can be used as a compound microbial fertilizer for sterilization, disease resistance and growth promotion. It overcomes the problems existing in the cultivation of disease-resistant varieties and chemical control of crop diseases, and can provide support for the green biological control of crop diseases. Detailed Implementation
[0018] The technical solution of this invention patent will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.
[0019] 1. Materials and Methods 1.1 Beneficial microorganisms tested Bacillus tekirii: purchased from Guangdong Provincial Microbial Culture Collection Center Bacillus tequilaensis Number GDMCC NO. 813140: Highland Bacillus: Bacillus altitudinis purchased from Guangdong Provincial Microbial Culture Collection Center, number GDMCC NO. 813255; *Pseudomonas hootii*: purchased from Guangdong Provincial Center for Microbial Culture Collection Pseudomonas rhodesiae The number is GDMCC NO. 812979.
[0020] 1.2 Tested microbial fertilizer 1.2.1 Bacillus tekiria fertilizer Single colonies of Bacillus tekirae were inoculated into NB liquid medium and cultured at 28°C with shaking at 180 rpm until OD reached. 600 The value was 0.8, recorded as the fermentation seed liquid; then, the fermentation seed liquid was inoculated into the fermentation nutrient solution at an inoculation rate of 8% by volume, and fermented at 28°C for 24 hours. During fermentation, sterile air was introduced to maintain the dissolved oxygen concentration of the fermentation nutrient solution at 40% saturation; then, fresh fermentation nutrient solution was added to adjust the effective viable cell count of the fermentation product to 2.0 × 10⁻⁶. 8CFU / g yields Bacillus tekirae bacterial fertilizer; among which... NB liquid culture medium formula: 1L of distilled water contains 3g beef extract, 5g peptone, and 5g sodium chloride, with a pH of 7.2; The fermentation nutrient solution formula is as follows: 1L of distilled water contains 8g of glucose, 30g of corn flour, 12g of wheat bran powder, 20g of soybean meal powder, 4g of yeast extract, 1.5g of ammonium nitrate, 2.5g of potassium dihydrogen phosphate, and 0.8g of magnesium sulfate, with a pH value of 7.2.
[0021] 1.2.2 Highland Bacillus Fertilizer Single colonies of *Bacillus hygroscopicus* were inoculated into NB liquid medium and cultured at 28°C with shaking at 180 rpm until OD reached. 600 The value was 0.8, recorded as the fermentation seed liquid; then, the fermentation seed liquid was inoculated into the fermentation nutrient solution at an inoculation rate of 8% by volume, and fermented at 28°C for 24 hours. During fermentation, sterile air was introduced to maintain the dissolved oxygen concentration of the fermentation nutrient solution at 40% saturation; then, fresh fermentation nutrient solution was added to adjust the effective viable cell count of the fermentation product to 2.0 × 10⁻⁶. 8 CFU / g yields Bacillus subtilis fertilizer; among which... NB liquid culture medium formula: 1L of distilled water contains 3g beef extract, 5g peptone, and 5g sodium chloride, with a pH of 7.2; The fermentation nutrient solution formula is as follows: 1L of distilled water contains 8g of glucose, 30g of corn flour, 12g of wheat bran powder, 20g of soybean meal powder, 4g of yeast extract, 1.5g of ammonium nitrate, 2.5g of potassium dihydrogen phosphate, and 0.8g of magnesium sulfate, with a pH value of 7.2.
[0022] 1.2.3 *Pseudomonas hootii* bacterial fertilizer Single colonies of *Pseudomonas hominis* were inoculated into NB liquid medium and cultured at 28°C with shaking at 180 rpm until OD reached. 600 The value was 0.8, recorded as the fermentation seed liquid; then, the fermentation seed liquid was inoculated into the fermentation nutrient solution at an inoculation rate of 8% by volume, and fermented at 28°C for 24 hours. During fermentation, sterile air was introduced to maintain the dissolved oxygen concentration of the fermentation nutrient solution at 35% saturation; then, fresh fermentation nutrient solution was added to adjust the effective viable cell count of the fermentation product to 2.0 × 10⁻⁶. 8 CFU / g yields *Pseudomonas hootii* bacterial fertilizer; among which... NB liquid culture medium formula: 1L of distilled water contains 3g beef extract, 5g peptone, and 5g sodium chloride, with a pH of 7.2; Fermentation nutrient solution formula: 1L of distilled water contains 5g glucose, 20g corn flour, 15g soybean meal powder, 1g yeast extract, 3g potassium nitrate, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 0.2g ferrous sulfate, and pH value 7.2.
[0023] 1.3 Effects of different microbial fertilizers on crop diseases 1.3.1 Test pathogens Fusarium graminearum ( Fusarium pseudograminearum The 1,000 stalks were isolated from wheat plants infected with stem rot in the suburbs of Zhengzhou, Henan Province, and preserved on PDA medium.
[0024] PDA medium formula: 200g peeled potato, 20g glucose, 15g agar, distilled water to a final volume of 1L, natural pH.
[0025] 1.3.2 Test reagent solution The microbial fertilizer (100% by mass) in "1.2 Test Microbial Fertilizer" was diluted with sterile water to obtain single-agent stock solutions. Multiple formulations were prepared, and each single-agent stock solution was mixed with each formulation and then diluted with sterile water to create five mass concentration gradients, which are the test solutions. All the above solutions were prepared and used immediately.
[0026] 1.3.3 Test Methods The toxicity of individual agents and mixtures against *Fusarium graminearum*, the causal agent of wheat stem rot, was determined using the mycelial growth rate method. Specifically, 1 mL of the test agent solution was mixed thoroughly with 9 mL of pre-melted and cooled PDA medium, poured into a 9 cm diameter petri dish, and allowed to cool and solidify to form a drug-containing plate. A treatment with an equal volume of sterile water served as a blank control. Each treatment was replicated in triplicate, with three plates per replicate.
[0027] The tested pathogen was inoculated onto PDA medium and cultured at 25°C for 3 days. A 5mm diameter mycelial disc was punched along the edge of the colony and inoculated into the center of the plate (1 disc / plate). The plate was then capped and incubated at 25°C. When the diameter of the control colony reached approximately 6cm, the colony diameter was measured using the cross-sectional method, and the inhibition rate of mycelial growth on the tested pathogen by different treatments was calculated.
[0028]
[0029] 1.3.4 Data Analysis Regression analysis was performed on the logarithmic values of the agent concentration and the corresponding mycelial growth inhibition probability values to derive the virulence regression equation and the EC50 of the agent against the tested pathogen. 50 The measured toxicity index (ATI), theoretical toxicity index (TTI), and co-toxicity coefficient (CTC) of the mixture were calculated using the Sun Yunpei method.
[0030] The combined effects of the drugs were evaluated based on the co-toxicity coefficient (CTC): CTC ≤ 80 indicates antagonistic effect, 80 < CTC < 120 indicates additive effect, and CTC ≥ 120 indicates synergistic effect. The results are shown in Table 1-3.
[0031]
[0032] Table 1 shows the EC50 toxicity of Bacillus tegleria, Bacillus pleuropneumoniae, and Pseudomonas hominis fertilizer against Fusarium graminearum, the causal agent of wheat stem rot. 50 The concentrations were 134.5582 mg / L, 112.7639 mg / L, and 183.1947 mg / L, respectively.
[0033]
[0034] Table 2 shows that when *Bacillus tekirae* fertilizer and *Bacillus hygroscopicus* fertilizer are compounded at a certain mass ratio, the co-toxicity coefficient of the compound combination in inhibiting the mycelial growth of *Fusarium graminearum* within the mass ratio range of 1-15:3-1 is greater than 120, demonstrating a synergistic effect. Therefore, the combined application of *Bacillus tekirae* fertilizer and *Bacillus hygroscopicus* fertilizer in this invention can improve the control effect on wheat stem rot.
[0035]
[0036] Table 3 shows that when *Bacillus tekirii* fertilizer, *Bacillus pleuropneumoniae* fertilizer, and *Pseudomonas hookieri* fertilizer are compounded in a certain mass ratio, the co-toxicity coefficient of the compound combination in inhibiting the mycelial growth of *Fusarium graminearum* within the mass ratio range of 1-8:1:8-1 is greater than 120, demonstrating a synergistic effect. Therefore, the combined application of *Bacillus tekirii* fertilizer, *Bacillus pleuropneumoniae* fertilizer, and *Pseudomonas hookieri* fertilizer in this invention can improve the control effect on wheat stem rot.
[0037] In summary, the combined use of Bacillus tekirii fertilizer and Bacillus hygroscopicus fertilizer in a mass ratio of 1-15:3-1, or the combined use of Bacillus tekirii fertilizer, Bacillus hygroscopicus fertilizer, and Pseudomonas holmii fertilizer in a mass ratio of 1-8:1:8-1, exhibits a synergistic effect in inhibiting the mycelial growth of pathogenic fungi of crops. This indicates that the microbial peptidase of the present invention can improve the control effect on crop diseases and can be used as a compound microbial fertilizer for sterilization, disease resistance, and growth promotion. It helps to overcome the problems existing in the cultivation of disease-resistant varieties and chemical control of crop diseases, and thus can provide support for the green biological control of crop diseases.
Claims
1. A novel microbial peptide enzyme fertilizer, characterized in that, The novel microbial peptide enzyme fertilizer is composed of Bacillus tekirae fertilizer and Bacillus hygroscopicus fertilizer in a mass ratio of 1-15:3-1, or is composed of Bacillus tekirae fertilizer, Bacillus hygroscopicus fertilizer and Pseudomonas holometaeus fertilizer in a mass ratio of 1-8:1:8-1.
2. The novel microbial peptide enzyme fertilizer according to claim 1, characterized in that, The preparation method of the *Bacillus tekirae* fertilizer, *Bacillus hygroscopicus* fertilizer, or *Pseudomonas hominis* fertilizer is as follows: A single colony of *Bacillus tekirae*, a single colony of *Bacillus hygroscopicus*, or a single colony of *Pseudomonas hominis* is inoculated into NB liquid medium and cultured at 28°C with shaking at 180 r / min until OD... 600 The value is 0.8, which is recorded as the fermentation seed liquid; then, the fermentation seed liquid is inoculated into the fermentation nutrient solution at an inoculation rate of 8% by volume, and fermented at 28°C for 24 hours. During the fermentation, sterile air is introduced to maintain the dissolved oxygen concentration of the fermentation nutrient solution at a saturation of 25-40%; then, fresh fermentation nutrient solution is added to adjust the number of effective viable bacteria in the fermentation product, thus obtaining the Bacillus tekirae fertilizer, Bacillus hygroscopicus fertilizer, or Pseudomonas holmii fertilizer.
3. The novel microbial peptide enzyme fertilizer according to claim 2, characterized in that, The NB liquid culture medium is formulated as follows: 1L of distilled water contains 3g of beef extract, 5g of peptone, and 5g of sodium chloride, with a pH of 7.
2.
4. The novel microbial peptide enzyme fertilizer according to claim 2, characterized in that, The fermentation nutrient solution formulas for the Bacillus tekirae inoculum fertilizer and the Bacillus alpineus inoculum fertilizer are as follows: 1L of distilled water contains 8g of glucose, 30g of corn flour, 12g of wheat bran powder, 20g of soybean meal powder, 4g of yeast extract, 1.5g of ammonium nitrate, 2.5g of potassium dihydrogen phosphate, and 0.8g of magnesium sulfate, with a pH value of 7.
2.
5. The novel microbial peptide enzyme fertilizer according to claim 2, characterized in that, The fermentation nutrient solution formula for the *Pseudomonas holmii* inoculant fertilizer is as follows: 1L of distilled water contains 5g glucose, 20g corn flour, 15g soybean meal, 1g yeast extract, 3g potassium nitrate, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 0.2g ferrous sulfate, and has a pH of 7.
2.
6. The novel microbial peptide enzyme fertilizer according to claim 2, characterized in that, The effective viable counts of the *Bacillus tekirii* fertilizer, *Bacillus hygroscopicus* fertilizer, and *Pseudomonas hominis* fertilizer were all 2.0 × 10⁻⁶. 8 cfu / g.
7. A microbial fertilizer, characterized in that, Includes the novel microbial peptide enzyme fertilizer as described in claim 1.
8. The application of the novel microbial peptide enzyme fertilizer of claim 1 or the microbial fertilizer of claim 7 in promoting crop growth and controlling crop diseases.