Disease-resistant growth-promoting yield-increasing strain and microbial agent and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a disease-resistant, growth-promoting and yield-increasing strain and a microbial agent and application thereof. The strain is a strain bacillus velezensis NB21 (CCTCC (China Center for Type Culture Collection) NO: M2024979) which is found autonomously; the strain can grow at low temperature, is strong in ecological environment adaptability, can be used for preparing a single microbial agent or a compound microbial agent, and can also be used for forming a composition with other functional organic and inorganic substances. The prepared microbial agent can significantly prevent and treat diseases, promote root development of crops such as rice and corn, enhance photosynthesis, improve system resistance, increase crop yield, enhance nutrient supply and improve soil micro-ecology. The strain is unique, strong in colonization ability and diversified in function, the prepared microbial agent fermentation production technology is mature, the cost is low, the application range is wide, the yield increasing and quality improving effects are outstanding, and the microbial agent has good application prospects.

Description

[0001] This application is a divisional application of the Chinese invention patent application. The original application date is March 26, 2025, the application number is: 2025103621543, the name of the invention is: A disease-resistant, growth-promoting and yield-increasing bacteria species and its composite microbial agent and application, publication number: CN119899776A; the applicant filed a divisional application. Technical Field

[0002] The present invention relates to the field of microbial technology, and in particular to a disease-resistant, growth-promoting, and yield-increasing bacterial strain, a microbial agent, and an application thereof. Background Art

[0003] In agricultural production, the high-intensity and excessive application of chemical fertilizers and pesticides has led to a decline in soil quality, degradation and even obstruction of its functions, water pollution and eutrophication, and a significant decline in crop quality and yield, affecting global food and food security and ecological and environmental security. To this end, there is an urgent need to continuously reduce the total amount of chemical pesticides and agricultural fertilizers used, and to replace old, high-risk chemical pesticides and inefficient chemical fertilizers with serious non-point source pollution through the research and development and application of innovative green inputs such as microbial pesticides and microbial fertilizers. Therefore, vigorously developing and promoting new green inputs (especially microbial sources) and reducing the input of pollution sources such as fertilizers and pesticides at the source are important paths to achieving sustainable global agricultural development.

[0004] Microorganisms are essential components of soil structure and function, the most active elements in the soil, and key determinants of soil fertility. Soil microorganisms drive soil fertility evolution, humus formation, material cycling, and energy conversion. They are both shapers and key determinants of healthy soils, crucial for maintaining agricultural production. Crop-associated microorganisms (rhizosphere microbes and endophytes) also originate from soil microbes. As a second genome of crops, crop-associated microorganisms participate in various physiological metabolism and growth and development processes, and are a key determinant of crop yield and stress tolerance. In particular, beneficial microorganisms that resist disease and promote growth have attracted considerable attention for their roles in stimulating seed germination, promoting plant growth, enhancing plant resistance, and preventing disease. Therefore, microbial agents and their related products (such as seed dressings, pesticides, fertilizers, and soil conditioners) are considered natural alternatives to chemical fertilizers and pesticides and are considered an environmentally friendly agricultural strategy with the potential to improve soil quality, promote crop growth, and enhance crop system resilience and productivity in the face of environmental change and soil degradation. Summary of the Invention

[0005] The present invention aims to provide a disease-resistant, growth-promoting and yield-increasing bacterial strain and its microbial agent and application.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] A disease-resistant, growth-promoting, and yield-increasing bacterial strain, the bacterial strain being Pseudomonas and / or Bacillus velezensis; wherein,

[0008] Pseudomonas sp.GM3 was deposited in China Center for Type Culture Collection on October 31, 2024, with the deposit number: CCTCC NO: M20242391; deposit address: Wuhan University, Wuhan, China;

[0009] Bacillus velezensis NB21 was deposited in the China Center for Type Culture Collection on May 17, 2024, with the deposit number: CCTCC NO: M2024979, and the deposit address is Wuhan University, Wuhan, China.

[0010] The Pseudomonas GM3 is characterized in that it is derived from rice roots, has a genome of approximately 6.85Mb circular chromosome, a GC content of 62.31%, and has the ability to synthesize Orfamide, Hydrogen cyanide, Pyoluteorin, Nantio-pyochelin, 3-Thiaglutamate, Pf-5pyoverdine, Azotobacti, 2,4-Diacetylphloroglucinol and a variety of novel RiPP-like compounds.

[0011] The Bacillus velezensis NB21 is characterized in that it is derived from deep-sea sediments in the South China Sea, has a genome of approximately 4.25Mb circular chromosome, a GC content of 45.97%, and has the ability to synthesize bacillibactin, surfactin, bacillomycin D, fengycin, macrolactin, bacillaene, difficidin, amyloliquecidin GF610 and a variety of terpene compounds.

[0012] An application of the disease-resistant, growth-promoting and yield-increasing bacterial strain, wherein the bacterial strain is used in nutrient supply, disease resistance and stress resistance, crop growth and yield promotion, and soil microecology improvement.

[0013] The nutrient supply refers to nitrogen fixation, phosphorus solubilization, and iron transport.

[0014] The disease resistance and stress resistance refer to resistance to bacteria, fungi, and enhancing the stress resistance of crop systems.

[0015] The said promotion of crop growth and increase in yield refers to promoting the development of crop root system, enhancing photosynthesis, increasing crop biomass, and improving the 100-grain weight or 1,000-grain weight and unit yield of crops.

[0016] A microbial agent containing the active bacterial species.

[0017] The bacterial agent is the culture, culture suspension or fermentation liquid of the active strain.

[0018] The microbial agent also contains auxiliary materials, which are one or more of biochar, humic acid, mushroom residue, amino acid, organic acid, vermiculite, peat, and diatomaceous earth.

[0019] When the above-mentioned auxiliary materials are biochar, mushroom residue, vermiculite, peat, and diatomaceous earth, the amount of addition is 1-20 times the mass of the microbial agent;

[0020] When the above-mentioned auxiliary materials are humic acid, amino acid, and organic acid, their added amount accounts for 0.001-1 times the mass of the microbial agent.

[0021] When the bacterial agent is a liquid preparation, the content of Pseudomonas GM3 is 2×10 8 CFU / mL or above, and / or the content of Bacillus velezensis NB21 is 2×10 8 CFU / mL or above;

[0022] When the bacterial agent is a solid preparation, the content of Pseudomonas GM3 is 2×10 8 CFU / g or above and / or the content of Bacillus Velezii NB21 is 5×10 8 CFU / g and above.

[0023] An application of the microbial agent, wherein the agent is used as a biological seed coating agent, a microbial pesticide, a microbial fertilizer, a biological organic fertilizer or a soil conditioner.

[0024] The advantages of the present invention are:

[0025] The Pseudomonas GM3 strain described in this invention is a self-discovered strain with nitrogen fixation, phosphate solubilization, antibacterial properties, iron transport, and crop growth promotion. The Bacillus Velezii NB21 strain, also discovered in this invention, exhibits antifungal properties, promotes seedling growth, and induces systemic resistance in crops. Both Pseudomonas GM3 and Bacillus Velezii NB21 are excellent strains for developing microbial agents.

[0026] The single or composite bacterial agents described in the present invention and their compositions composed of other functional organic and inorganic substances, the core microbial cells and bacterial liquid can be produced by liquid fermentation. The preparation method is mature and simple, the process is efficient, and the obtained cell content is high, which fully meets the needs of commercial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The bacterial morphology and genome circle map of Pseudomonas GM3 provided in the embodiment of the present invention; Figure 1A is the morphology of Pseudomonas GM3 bacteria, Figure 1 B is the genome circle map.

[0028] Figure 2 The present invention provides a phylogenetic tree of Pseudomonas GM3 based on the 16S rRNA gene sequence.

[0029] Figure 3 The bacterial morphology and genome circle map of Bacillus velezini NB21 provided in the embodiments of the present invention; Figure 3 A is the morphology of Bacillus velezensis NB21. Figure 3 B is the genome circle map.

[0030] Figure 4 The present invention provides a phylogenetic tree of Bacillus velez NB21 based on the 16S rRNA gene sequence.

[0031] Figure 5 This is the fingerprint of the lipopeptide compounds synthesized by Bacillus velezensis NB21 provided in the embodiments of the present invention.

[0032] Figure 6 This is a graph showing the results of the Bacillus Velez NB21 agent provided in an embodiment of the present invention promoting the growth of corn seedlings.

[0033] Figure 7 A comparison of corn ears harvested from the Changtu experimental field provided in an embodiment of the present invention after coating with the Bacillus Velez NB21 agent.

[0034] Figure 8 This is a diagram showing the effect of applying a composite microbial agent at the seedling stage on the growth promotion of rice seedlings.

[0035] Figure 9 This is a diagram showing the effect of applying a composite microbial agent at the booting stage to promote rice growth and stress resistance, as provided by an embodiment of the present invention.

[0036] Figure 10 This is a diagram showing the effect of applying a composite microbial agent to prevent and control rice false smut during the booting stage according to an embodiment of the present invention.

[0037] Figure 11 This is a graph showing the effect of the composite microbial agent provided in an embodiment of the present invention on the weight of rice grains per plant.

[0038] Figure 12 This is a diagram of the MetagenomeSeq analysis results of the composite bacterial agent treatment group and the control group provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0039] In order to better understand the content of the present invention, further description will be given below in conjunction with specific embodiments, but the protection content of the present invention is not limited thereto.

[0040] The independently discovered Pseudomonas sp. GM3 and Bacillus velezensis NB21 provided by the present invention can both efficiently colonize in the rhizosphere and phyllosphere of various crops, have strong environmental adaptability, and have multiple functions such as strengthening seedlings, promoting growth, resisting diseases, and increasing yields, and have very broad application prospects.

[0041] Example 1 Identification of Pseudomonas sp. GM3

[0042] Pseudomonas GM3 was isolated from rice roots in Shenyang, Liaoning, and deposited in the China Center for Type Culture Collection on October 31, 2024, with the deposit number: CCTCC NO: M20242391.

[0043] The bacteria forms round light yellow colonies on TSA medium. The surface of the colonies is flat and dry. Figure 1 A), Gram staining shows red rods. It grows well at 15-35°C. Total DNA of Pseudomonas GM3 was extracted and the genome was sequenced and assembled using Oxford Nanopore third-generation sequencing technology combined with Illumina NovaSeq second-generation sequencing technology. The complete genome map was obtained. The genome is a circular chromosome of approximately 6.85Mb with a GC content of 62.31% ( Figure 1 B) Contains 6 copies of 5S rRNA, 5 copies of 16S rRNA, 5 copies of 23S rRNA, and 74 tRNAs. The 16S rRNA gene sequence is 1534 bp long, and the nucleotide sequence is as follows:

[0044] >GM3_16S rDNA

[0045]

[0046] Based on the 16S rRNA gene sequence of Pseudomonas GM3, its closely related standard strains were retrieved using EZBioCloud, and the Neighbour-joining tree was constructed using MEGA 6.0 software. The results showed that Pseudomonas GM3 and the model strain Pseudomonas protegens CHA0 T (CP003190), Pseudomonas saponiphila DSM 9751 T (FNTJ01000001) and Pseudomonas sesami SI-P133 T (EU912472) clustered on an independent larger branch, with 16S rRNA gene sequence homologies of 100.0%, 99.52%, and 98.94%, respectively. Figure 2 ). Especially Pseudomonas GM3 and Pseudomonas protegens CHA0 T The highest homology was found, clustering on the same evolutionary branch, indicating that Pseudomonas GM3 had the closest evolutionary relationship with strains of the P. protegens species. Furthermore, the JSpeciesWS online analysis tool was used to calculate the Average Nucleotide Identity (ANI) between Pseudomonas GM3 and its closest strains using the obtained whole genome data. The results are shown in Table 1. Pseudomonas GM3 showed no significant difference in homology with the model strain CHA0 of Pseudomonas protegens. T The ANI values ​​of other strains of this species were all less than 93%. According to the gold standard of the whole genome ANI value greater than 95% for the same species, it was proved that Pseudomonas GM3 was a species different from Pseudomonas protegens.

[0047] Table 1 ANI values ​​between Pseudomonas GM3 and its closest evolutionary strains

[0048]

[0049] Example 2 Identification of Bacillus velezensis NB21

[0050] Bacillus velezensis NB21 was isolated from sediments at a depth of 3622 meters in the South China Sea and deposited in the China Center for Type Culture Collection on May 17, 2024, with the deposit number: CCTCC NO: M2024979. The bacterium forms round, fleshy white colonies on LB plates, with irregular circles and dense wrinkles on the surface. On TSA medium, the edges are wrinkled and convex, and the center is concave ( Figure 3 A), Gram staining shows blue bacteria. It can grow well at 15-40°C and can use citrate, D-xylose, L-arabinose, D-mannitol, etc. as sole carbon sources for growth. Total DNA of Bacillus velezinis NB21 was extracted and the genome was sequenced and assembled using Oxford Nanopore third-generation sequencing technology combined with Illumina NovaSeq second-generation sequencing technology to obtain a complete genome map. Its genome is a circular chromosome of approximately 4.25Mb with a GC content of 45.97% ( Figure 3 B) Contains 9 copies of 5S rRNA, 9 copies of 16S rRNA, 9 copies of 23S rRNA, and 89 tRNAs. The 16S rRNA gene sequence is 1547 bp long, and the nucleotide sequence is as follows:

[0051] >NB21_16S rDNA

[0052]

[0053] Based on the 16S rRNA gene sequence of Bacillus velezini NB21, EZBioCloud was used to retrieve its closely related standard strains, and MEGA 6.0 software was used to construct a neighbor-joining tree. The results showed that Bacillus velezini NB21 and the model strain Bacillus siamensis KCTC 13613 T (AJVF01000043), Bacillusvelezensis CR-502 T 、NRRL B-41580 T (AY603658), Bacillus velezensis FZB42 T (CP000560) and Bacillus amyloliquefaciens DSM 7 T (FN597644) clustered on an independent larger branch, with 16S rRNA gene sequence homologies of 99.86%, 99.86%, 99.81% and 99.59% ( Figure 4 ). It can be seen that Bacillus Velezii NB21 belongs to a group related to Bacillus Velezii.

[0054] However, due to the large number of Bacillus species and their diverse and complex evolution, classification results based solely on 16S rRNA gene sequences are unreliable. Therefore, the JSpeciesWS online analysis tool was further used to calculate the ANI value between Bacillus velezensis NB21 and its closest strains using the obtained whole genome data. The results are shown in Table 2. Bacillus velezensis NB21 and the model strain NRRL B-41580 T The ANI value was 98.25%, which was greater than 95%, and thus the Bacillus velezensis NB21 was identified as Bacillus velezensis NB21.

[0055] Table 2 ANI values ​​between Bacillus velezensis NB21 and its closest evolutionary strains

[0056]

[0057] In addition, Bacillus velez NB21 was isolated from deep-sea sediments in the South China Sea. It exists in a very unique ecological environment with high pressure, low temperature and high salinity, and therefore has evolved its unique genetic information. Further comparison of the secondary metabolite synthesis gene cluster sequence of the closest Bacillus velez preserved in the GenBank database revealed that the genome of Bacillus velez NB21 encodes Surfactin (357012nt~422419nt), Macrolactin

[0058] The biosynthetic gene clusters of compounds such as (1486317nt~1574541nt), Fengycin (2789498nt~2859109nt), Bacillomycin D (2859386nt~2927314nt), Bacillibactin (3325499nt~3377287nt) and Bacilysin (3910979nt~3952397nt) have approximately 97% homology with the highest gene in the database, and the highest homology with the biosynthetic gene cluster of Difficidin (2240778nt~2346951nt) in the database is 98%. In particular, the highest homology found in the genome, encoding an unknown non-ribosomal peptide located between nt 2049040 and nt 2094568, was only 83%, while the highest homology found in the genome, encoding an amyloliquecidin GF610-type thiopeptide compound located between nt 671601 and nt 701016, was only 73%. These findings indicate that Bacillus velezensis NB21 has evolved a unique genetic sequence, making it a distinct strain of Bacillus velezensis from any known strain.

[0059] Example 3 Function of Pseudomonas sp.GM3

[0060] AntiSMASH analysis and manual correction revealed that Pseudomonas GM3 harbors more than 16 secondary metabolite gene clusters, with the potential to produce orfamide, hydrogen cyanide (HCN), 3-thiaglutamate, pyoluteorin, nantio-pyochelin, pf-5pyoverdine, azotobactin, and 2,4-diacetylphloroglucinol (DAPG) backbone compounds, as well as the potential to synthesize a variety of novel RiPP-like compounds (Table 3). This suggests that Pseudomonas GM3 is an excellent strain for producing a variety of bioactive lead compounds.

[0061] Table 3 Potential of Pseudomonas GM3 to synthesize diverse secondary metabolites

[0062]

[0063] Studies have found that Orfamide has been shown to have antifungal and biosurfactant effects, affecting the swarming movement of Pseudomonas and lysing the zoospores produced by oomycete plant pathogens; Pyoluteorin has a killing effect on oomycetes and some bacteria; DAPG has an antibacterial effect on a variety of plant pathogenic fungi; hydrogen cyanide HCN is volatile and can help plants inhibit various diseases. Strain antagonism experiments have shown that Pseudomonas GM3 has strong antibacterial activity (such as Staphylococcus aureus, Escherichia coli and Acinetobacter baumannii, etc.) and significant antifungal activity (such as rice sheath blight). In addition, the stability constant of Pseudomonas siderophores such as Pyoverdine binding to iron is 10 32 , which is much higher than the stability constant of iron binding between iron carriers of some pathogens such as Fusarium wilt, which is 10 29 , which is very important for the efficient colonization of Pseudomonas, as well as for promoting plant growth and combating various plant diseases. Pseudomonas GM3 was inoculated on a chrome azure (CAS) plate and cultured at 28°C for 72 hours, showing a moderate ability to secrete siderophores (Table 4). In addition, Pseudomonas GM3 was inoculated on an inorganic phosphate agar medium and cultured at 28°C for 72 hours, showing good phosphate solubilization activity (Table 4). It was also inoculated in a nitrogen-free liquid medium and cultured at 28°C for 7 days, showing nitrogen fixation activity (Table 4). It can colonize in the rhizosphere and aboveground plants of rice and corn, promoting their root development and seedling growth. The above shows that Pseudomonas GM3 can exert antibacterial and antifungal effects through multiple actions, and also has good phosphate solubilization, nitrogen fixation and iron carrier secretion functions, making it an excellent plant growth-promoting bacterium and biocontrol microorganism.

[0064] Table 4 Comparison of functional evaluation of Pseudomonas GM3 and other laboratory strains

[0065]

[0066] Note: Inactive, Weakly active, Moderately active, For strong activity, It is extremely active.

[0067] Example 4 Function of Bacillus velezensis NB21

[0068] AntiSMASH analysis and manual correction revealed that Bacillus velez NB21 harbors more than 15 secondary metabolite gene clusters, showing potential for producing antifungal and plant resistance-inducing lipopeptides such as surfactin, fengycin, and bacillomycin D; the siderophore bacillibactin, which enhances colonization; antibacterial polyketides such as macrolactin, bacillaene, and difficidin; and backbone compounds such as the thiopeptide antibiotic amyloliquecidin GF610. Furthermore, the strain also has the potential to synthesize novel PKS-NRPS hybrids and terpene compounds (Table 5). This suggests that Bacillus velez NB21 is an excellent strain for producing a variety of bioactive lead compounds. Studies have shown that lipopeptide compounds such as Surfactins and Fengycins are the main components of Bacillus multicellular behavior, induction of plant systemic resistance and antifungal activity (doi: / 10.1111 / 1462-2920.13405 and doi: 10.1111 / 1751-7915.12238). Studies have shown that Bacillus velezensis NB21 has strong inhibitory activity against the pathogen of corn leaf blight, Helicoverpa macrophyte, and the pathogen of corn stem rot, Fusarium graminearum. The diameter of the inhibition zone in the plate confrontation method is greater than 10 cm. Metabolic fingerprint analysis shows that Bacillus velezensis NB21 can produce signal molecule-like lipopeptides Surfactins that affect the motility, colony morphology and biofilm formation of Bacillus multicellular behavior, as well as Fengycins and Bacillomycins D lipopeptide compounds that induce plant systemic resistance and anti-pathogenic fungi ( Figure 5 In addition, in potted experiments, Bacillus Velez NB21 showed a strong effect in promoting the growth of corn seedlings. This shows that Bacillus Velez NB21 has significant antifungal, seedling-strengthening and growth-promoting effects, and can induce crop systemic resistance. This strain is also an excellent plant growth-promoting bacterium and biocontrol microorganism.

[0069] Table 5 Potential of Bacillus velezensis NB21 to synthesize diverse secondary metabolites

[0070]

[0071] Example 5 Preparation of single or composite bacterial agents for disease resistance, growth promotion and yield increase and their compositions

[0072] After the preserved Pseudomonas GM3 was streaked and activated on TSA medium, a single colony was inoculated into TSB liquid medium and cultured overnight. Then, the inoculum volume ratio was 2% and inoculated into a 500mL triangular shake flask containing 100mL TSB liquid medium. After being placed in a shaker at 200rpm and cultured at 28°C for 24 hours, a small amount of bacterial liquid was taken for microscopic examination. After the microscopic examination was confirmed, the inoculation volume ratio was 3% and transferred into a 3000mL shake flask containing 1000mL TSB or King's B liquid medium. After being placed in a shaker at 200rpm again and cultured at 28°C for 70 hours, the fermentation broth was collected. The Pseudomonas GM3 content was 2×10 9 CFU / mL or 9×10 9 CFU / mL, the fermentation broth can be directly used as a single Pseudomonas GM3 liquid inoculum.

[0073] The Bacillus velezensis NB21 liquid stored in the glycerol tube was inoculated into LB medium at a volume ratio of 0.05% for overnight activation. The freshly activated strain was then inoculated into a 500mL triangular shake flask with a liquid volume of 100mL at a volume ratio of 3%. The culture was placed in a shaker at 200rpm and cultured at 35℃ for 24h. Then, the culture was transplanted into a 3000mL shake flask with a liquid volume of 1000mL at the same volume ratio, and the culture was placed in a shaker at 200rpm and cultured at 35℃ for 36h. After the fermentation was completed, the content of Bacillus velezensis NB21 in the fermentation broth was 5×10 8 CFU / mL, it can be used as a single Bacillus Velezii NB21 liquid inoculum.

[0074] The fermented Pseudomonas GM3 bacterial solution and the Bacillus Velezii NB21 bacterial solution were mixed in a volume ratio of 1:1, and 0.5% by mass of leucine, isoleucine, polyglutamic acid, acetic acid, lactic acid and / or mineral humic acid were added to prepare a liquid composite microbial liquid inoculum.

[0075] The fermentation broth of Pseudomonas GM3 or Bacillus Velezii NB21 was centrifuged at 4000 rpm for 30 minutes using a large-capacity centrifuge, and the supernatant was discarded to collect the cells. The cells were then mixed with lactose at a ratio of 1:1 by wet weight, stirred evenly into a paste, dehydrated using a freeze dryer, and crushed in a grinder (200-600 mesh) to produce a single-species microbial powder. The Pseudomonas GM3 powder contained 5×10 11 CFU / g, the content of Bacillus velezensis NB21 powder is 1×10 11 CFU / g.

[0076] Pseudomonas GM3 powder and Bacillus Velezella NB21 powder are mixed in a mass ratio of 1:1 to obtain a composite microbial powder. To this powder, biochar (100-500 mesh, equivalent to 100% by mass), humic acid, amino acids, and / or mushroom residue, vermiculite, peat, or diatomaceous earth (100-500 mesh, equivalent to 100% by mass) are added to form different compositions, which can be used as bio-organic fertilizers.

[0077] Example 6 Growth-promoting and yield-increasing effects of the Bacillus velezensis NB21 agent

[0078] Corn seeds of uniform size and full kernels were selected and disinfected with a NaClO solution. After removal, they were rinsed three times with sterile water and allowed to air dry. The corn seeds were then treated with a single culture of the prepared Bacillus velezensis NB21 at a ratio of 1:40 (fermentation volume mL: seed mass g). The fermentation liquid and seeds were thoroughly mixed. A control group (CK) was treated with LB liquid medium. The corn seeds were air-dried and then used for further processing.

[0079] Sterilize the soil and vermiculite in a mass ratio of 1:1 and mix them evenly. Pour the mixture into plastic pots of 13cm×13cm. Sow the corn seeds that have been treated with seed dressing in the pots, 6 seeds per pot, and sow 10 pots for each treatment. The temperature is controlled within the range of 24-28℃, the humidity is 70%, and the light intensity is 16L / 8D. When the seedling stage is about 30 days, randomly select 20 corn seedlings in the treatment and measure the height of the corn seedlings. Figure 6 As shown, the average plant height of plants treated with Bacillus velezensis NB21 increased by 10.37% compared with the control group (CK1), showing a significant growth-promoting and seedling-strengthening effect.

[0080] The prepared single Bacillus Velezii NB21 powder was used to coat seeds according to the method of seed coating agent. 8Seeds were dressed according to the ratio of CFU viable bacterial content. After dressing, the seeds were spread out in a cool, shady warehouse to air-dry and sowed within a week. Corn plants from each treatment were harvested by taking one ear from every four plants. Each group of 15 plants, with three replicates per treatment, served as samples for seed and yield testing. The average ear diameter and ear length of each treatment were measured and calculated, and kernel weight and 100-kernel weight were measured, and specific yield was estimated. For example, in the Gongzhuling experimental field, the average wet weight of corn cobs treated with Bacillus Velez-Bacillus NB21 was 4.807 kg per cob, a 7.05% relative yield increase compared to the control group (Table 6). After drying, the cobs were threshed, and the kernels from 45 plants were weighed (kernel weight). 100 kernels were randomly selected and weighed three times, and the average value was calculated. Comparison revealed that the kernel weight and 100-kernel weight of corn treated with Bacillus Velez-Bacillus NB21 were significantly higher than the control group's 10.25 kg and 43.89 g, respectively. In the Changtu experimental field, the relative yield increase of corn was more significant after seed dressing with Bacillus velezensis NB21, with a yield increase of up to 13.79% (Table 6, Figure 7 ).

[0081] Table 6 Results of corn seed testing and yield measurement in Gongzhuling and Changtu experimental fields

[0082]

[0083] Example 7: Effects of composite microbial agents on seedling growth, stress resistance, disease resistance, growth promotion and yield increase

[0084] The composite microbial liquid inoculant prepared according to Example 5, comprising Pseudomonas GM3, Bacillus Velezii NB21, and 0.5% mineral humic acid, was sprayed on rice seedlings when they had three leaves and one heart (5-10 days before transplanting), or directly irrigated 24 hours before and after transplanting. 1 L of the composite microbial inoculant was applied per mu, and a control group without the composite microbial inoculant was used. The results showed that the rice seedlings were significantly more resistant to low temperature stress and had a faster recovery rate during growth. Two weeks after application, the composite microbial inoculant significantly promoted the growth of rice seedlings, especially the development of the rice root system. Compared with the control group, the average root weight (g), root length (cm), and number of lateral roots per plant increased by 38.16%, 30.94%, and 12.68%, respectively, the chlorophyll content of rice leaves increased by 48.5%, and the average fresh weight per plant increased by 14.02%. Malondialdehyde (MDA) is the final decomposition product of membrane lipid peroxidation, and its content can reflect the degree of damage suffered by plants under adverse conditions. After the application of the compound microbial agent, the MDA content decreased by 4.55%, which can be considered that the crop system's resistance to stress is enhanced and the damage to the crop caused by the environment is reduced ( Figure 8 ).

[0085] The above-mentioned composite microbial liquid inoculant was applied at 15 L per mu during the rice booting stage, and no composite microbial inoculant was used as a control. After 2 weeks of application, the chlorophyll content was significantly increased by 12.8%, and the leaf area of ​​the rice flag leaf was significantly increased by 7.1%. It also significantly improved the systemic stress resistance of rice, with the proline (Pro) content significantly increased by 31.8%, the MDA content significantly decreased by 17.8%, and the superoxide dismutase (SOD) and peroxidase (POD) activities significantly enhanced, increasing by 87.7% and 105.2%, respectively, with P values ​​of 0.052 and 0.104, respectively, which were close to the significant level ( Figure 9 After the application of the composite microbial agent, the disease index of rice false smut was significantly reduced from 2.50% in the control group to 1.21% (the control effect was 85.1%); the diseased ear rate was significantly reduced from 10.61% in the control group to 6.93% ( Figure 10 ).

[0086] At the same time, four experimental plots were set up in the experimental area (Ciyutuo 1, Mengjiatai 1, Mengjiatai 2 and Daomeng Space) for yield increase evaluation. Among them, Ciyutuo 1 and Mengjiatai 1 were only applied with composite microbial agents during the booting stage, while Mengjiatai 2 and Daomeng Space were applied with composite microbial agents during the seedling and booting stages, and the application amount was as described above. After the rice was fully mature, the rice yield was evaluated by the five-point sampling method. Specifically, the above-mentioned composite microbial agent treatment group and the blank control without microbial agent application were sampled separately, five points were taken from each plot, and three clusters of rice were taken from each point. The number of panicles of each cluster of rice was measured, and the panicle grains on each panicle of rice were removed and weighed to obtain the panicle grain weight of each cluster of rice. At four different experimental sites, after the application of the compound microbial agent at the seedling stage and / or the booting stage, the weight of rice grains per plant increased significantly. The weight of rice grains per plant treated with the compound microbial agent ranged from 55.24 to 73.05 g, while that of the control group was 50.71 to 58.40 g. The average yield increase was 9 to 25% ( Figure 11 ).

[0087] Then, 1,000 rice ears were randomly selected from each rice cluster and weighed to calculate the thousand-grain weight. In plots Ciyutuo 1 and Mengjiatai 1, where the compound inoculant was applied only during the booting stage, the thousand-grain weight increased by 1.8% and 5.9%, respectively. In plots Mengjiatai 2 and Daomeng Space, where the compound inoculant was applied at both the seedling and booting stages, the thousand-grain weight increased by 2.3% and 15.5%, respectively.

[0088] Furthermore, whether a single microbial agent is applied alone or supplemented with a composite microbial agent during the booting stage, it significantly promotes rice growth and development and significantly improves the crop's stress resistance. Overall, composite microbial agents are more effective than single agents in promoting growth, resisting diseases, and increasing yields.

[0089] Example 8 Microecological Optimization Effect of Composite Microbial Agent

[0090] In Example 7, after treating rice with a composite microbial agent, rice roots and rhizosphere soil were collected during the rice filling and grain setting period, and high-fidelity rice root surface soil (containing microorganisms) was obtained by ultrasonic treatment. Total DNA from the samples was then extracted, and the diversity and differences of the rhizosphere microbiome were compared based on 16S rRNA gene sequences using Illumina NovaSeq high-throughput sequencing technology. The results showed that approximately 4 / 5 of the bacterial abundance in the Liaoning rice rhizosphere microbiome was mainly distributed in Proteobacteria, Chloroflexi, Acidobacteriota, Bacteroidota, Desulfobacterota, Actinobacteria, Firmicutes, and Myxococcota. After treatment with the composite inoculant, the average relative abundance of Chlorobacteria increased from 13.41% to 17.51%, Acidobacteria from 9.43% to 10.67%, Desulfobacteria from 6.83% to 7.15%, and Actinobacteria from 4.71% to 5.22%. The relative abundance of major bacterial groups also changed significantly at the class level (Table 7). In particular, the abundance of the Anaerolineae, the most dominant class within Chlorobacteria, increased by 3.33% to 12.06%, compared to 8.84% in the control. The relative abundance of the Vicinamibacteria, a class within the Acidobacteria, increased by 0.74% to 4.01%. The study found that some microorganisms within the Anaerolineae class possess phototrophic properties and can decompose complex organic matter, while bacteria from the Vicinamibacteria group have the potential to solubilize phosphorus in the soil. In addition, compared with the control group, the relative abundance of the uncultivable group MBNT15 increased by 1.5%, and the relative abundance of KD4-96 increased by 0.91%. It can be considered that the symbiotic metabolism in the soil has been strengthened, and the circulation and utilization of special and complex substrates have been enhanced (uncultivable means that it has not been cultured yet, but it exists and can be detected, and it only has a group code. These microorganisms require special nutrition to reproduce better, indicating that symbiotic metabolism has been strengthened).

[0091] Table 7 Changes in relative abundance of major bacterial classes in rice rhizosphere after treatment with composite microbial agents

[0092]

[0093] MetagenomeSeq analysis was further completed, and the results were as follows Figure 12As shown, the main groups with significant differences between the two groups after the composite inoculum treatment compared to the control group were Acidobacteria, Actinobacteria, Armored Bacteroidetes, Bacteroidetes, Chlorobacteria, Desulfobacteria, Firmicutes, Myxomycetes, Nitrospira, and Proteobacteria. In particular, the relative abundance of Thiobacillus, Mefhylomonas, and Aminicenantales increased significantly, indicating that the inoculum treatment significantly improved the metabolism of sulfur, one-carbon compounds such as methane and methanol, and recalcitrant organic compounds such as phenols and N-heterocyclic compounds in the rice rhizosphere. Furthermore, the genus-level groups of unculturable Subgroup 7, SB-5, BSV26, MBNT15, 4-29-1, OM190, and Ellin6067 increased significantly, indicating that the inoculum stimulated interactive nutrient metabolism in the rice rhizosphere and enhanced the turnover efficiency of soil nutrients and specific elements. In summary, it can be seen that composite microbial agents enhance the circulation of soil substances and elements and improve the efficiency of crop nutrient utilization by regulating the structure and function of rhizosphere microbial communities.

Claims

1. A disease-resistant, growth-promoting, and yield-increasing bacterial strain characterized by: The strain is Bacillus velezensis NB21; Bacillus velezensis NB21 was deposited in the China Center for Type Culture Collection on May 17, 2024, with the deposit number: CCTCC M2024979.

2. A use of the disease-resistant, growth-promoting, and yield-increasing bacterial strain according to claim 1, characterized in that: The bacterial strain is used in disease resistance and stress resistance, promoting crop growth and yield, and improving soil microecology.

3. The use of the disease-resistant, growth-promoting and yield-increasing bacterial strain according to claim 2, characterized in that: The disease resistance and stress resistance refer to resistance to bacteria, fungi, and enhancing the stress resistance of crop systems.

4. The use of the disease-resistant, growth-promoting and yield-increasing bacterial strain according to claim 2, characterized in that: The said promotion of crop growth and increase in yield refers to promoting the development of crop root system, enhancing photosynthesis, increasing crop biomass, and improving the 100-grain weight or 1,000-grain weight and unit yield of crops.

5. A microbial agent, characterized in that: The microbial agent contains the active bacterial species described in claim 1.

6. The microbial agent according to claim 5, characterized in that: The bacterial agent is the culture, culture suspension or fermentation liquid of the active strain.

7. The microbial agent according to claim 6, characterized in that: The microbial agent also contains auxiliary materials, which are one or more of biochar, humic acid, mushroom residue, amino acid, organic acid, vermiculite, peat, and diatomaceous earth.

8. The microbial agent according to claim 5, characterized in that: When the bacterial agent is a liquid preparation, the content of Bacillus velezensis NB21 is 2×10 8 CFU / mL or above; When the bacterial agent is a solid preparation, the content of Bacillus velezensis NB21 is 5×10 8 CFU / g and above.

9. A use of the microbial agent according to claim 5, characterized in that: The bacterial agent is used as a biological seed coating agent, microbial pesticide, microbial fertilizer, biological organic fertilizer or soil improver.

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