A strain of bacteria for disease resistance, growth promotion and yield increase, its compound microbial inoculant and application

Abstract

The present invention relates to the field of microbial technology, and specifically to a strain for disease resistance, growth promotion and yield increase, its compound microbial agent and application. The strain is Pseudomonas sp. GM3 (CCTCC NO: M20242391) and / or Bacillus velezensis NB21 (CCTCC NO: M2024979) which are self-discovered strains; the above-mentioned strains can grow at low temperature and have strong ecological environment adaptability, and can be used to prepare single-agent or compound microbial agents, and can also form compositions with other functional organic and inorganic substances. The prepared microbial agent can significantly prevent and control diseases, promote the root development of crops such as rice and corn, enhance photosynthesis, improve systemic resistance, increase crop yield, and can also strengthen nutrient supply and improve soil microecology. The invention has good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and specifically relates to a strain with disease resistance, growth promotion and yield increase, a compound microbial agent thereof, and an application. Background Art

[0002] In agricultural production, the high-intensity and excessive application of chemical fertilizers and pesticides has led to problems such as the decline of soil quality, functional degradation or even obstacles, eutrophication of water body pollution, and significant decline in the quality and yield of crops, affecting global food and food safety as well as ecological environment safety. Therefore, it is urgent to continuously reduce the total application amount of chemical pesticides and agricultural fertilizers, and through the research and application of innovative green inputs such as microbial pesticides and microbial fertilizers, replace the old and high-risk chemical pesticides and replace the chemical fertilizers with low efficiency and serious non-point source pollution. Therefore, vigorously developing and promoting new green inputs (especially microbial sources) to reduce the input of pollution sources such as chemical fertilizers and pesticides from the source is an important path to achieve global agricultural sustainable development.

[0003] Microorganisms are an important part of the soil structure and function, and are also the most active and key factors determining the soil fertility in the soil. Soil microorganisms drive the evolution of soil fertility, the formation of humus, material cycling and energy conversion. They are both the shapers and important characterization factors of healthy soil, and are crucial for maintaining agricultural production. At the same time, crop-related microorganisms (rhizosphere microorganisms and endophytes) come from soil microorganisms. As the second genome of crops, crop-related microorganisms participate in various physiological metabolisms and growth and development processes of crops, and are also one of the key factors determining crop yield and stress tolerance. In particular, the beneficial microorganisms with disease resistance and growth promotion among them can also stimulate seed germination, promote plant growth, enhance plant stress resistance, prevent the occurrence of diseases, etc., and have attracted much attention. Therefore, microbial agents and their related products (such as seed coating agents, pesticides, fertilizers, and soil conditioners, etc.) as natural substitutes for chemical fertilizers and pesticides are considered an environmentally friendly agricultural strategy, and are expected to improve soil quality, promote crop growth and development, and improve crop system resistance and productivity in the case of environmental changes and soil degradation. Summary of the Invention

[0004] The purpose of the present invention is to provide a strain with disease resistance, growth promotion and yield increase, a compound microbial agent thereof, and an application.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] A strain with disease resistance, growth promotion and yield increase, the strain is Pseudomonas and / or Bacillus velezensis; wherein,

[0007] Pseudomonas Pseudomonassp. GM3, which was deposited with the China Center for Type Culture Collection on October 31, 2024, accession number: CCTCC NO: M20242391; deposit address: Wuhan University, Wuhan, China;

[0008] Bacillus velezensis Bacillus velezensis NB21, which was deposited with the China Center for Type Culture Collection on May 17, 2024, accession number: CCTCC NO: M2024979, deposit address: Wuhan University, Wuhan, China.

[0009] The described Pseudomonas GM3 is characterized in that it is derived from rice roots, has a circular chromosome of about 6.85 Mb, a GC content of 62.31%, and has the ability to synthesize Orfamide, Hydrogen cyanide, Pyoluteorin, Nantio-pyochelin, 3-Thiaglutamate, Pf-5 pyoverdine, Azotobacti, 2,4-Diacetylphloroglucinol and a variety of novel RiPP-like compounds.

[0010] The described Bacillus velezensis NB21 is characterized in that it is derived from deep-sea sediments in the South China Sea, has a circular chromosome of about 4.25 Mb, 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.

[0011] An application of the described disease-resistant, growth-promoting and yield-increasing bacterial strain, the application of the bacterial strain in nutrient supply, disease resistance and stress resistance, promoting crop growth and increasing yield, and improving soil microecology.

[0012] The described nutrient supply refers to nitrogen fixation, phosphorus solubilization, and iron transport.

[0013] The described disease resistance and stress resistance refer to antibacterial, antifungal, and enhancing the systemic stress resistance of crops.

[0014] The described promotion of crop growth and increase in yield refers to promoting crop root development, enhancing photosynthesis, increasing crop biomass, increasing the 100-grain weight or 1000-seed weight of crops, and unit yield.

[0015] A microbial inoculant, the inoculant contains the described active bacterial strain.

[0016] The described inoculant is a culture, culture suspension or fermentation broth of the active strain.

[0017] The microbial agent also contains excipients, which are one or more of biochar, humic acid, mushroom residue, amino acid, organic acid, vermiculite, peat, and diatomite.

[0018] When the above excipients are biochar, mushroom residue, vermiculite, peat, or diatomite, their addition amount is 1 - 20 times the mass of the microbial agent;

[0019] When the above excipients are humic acid, amino acid, or organic acid, their addition amount is 0.001 - 1 times the mass of the microbial agent.

[0020] When the microbial agent is a liquid preparation, the content of Pseudomonas GM3 is 2×10 8 CFU / mL or more, and / or the content of Bacillus velezensis NB21 is 2×10 8 CFU / mL or more;

[0021] When the microbial agent is a solid preparation, the content of Pseudomonas GM3 is 2×10 8 CFU / g or more and / or the content of Bacillus velezensis NB21 is 5×10 8 CFU / g or more.

[0022] An application of the microbial agent as described above, the application of the microbial agent as a biological seed coating agent, microbial pesticide, microbial fertilizer, bio - organic fertilizer, or soil conditioner.

[0023] Advantages of the present invention:

[0024] The Pseudomonas GM3 described in the present invention is a self - discovered strain, and has the functions of nitrogen fixation, phosphorus solubilization, antibacterial, iron transport, and promoting crop growth. Bacillus velezensis NB21 is a self - discovered strain, which has the functions of antifungal, promoting strong seedlings and growth, and inducing systemic resistance of crops. The Pseudomonas GM3 and Bacillus velezensis NB21 described in the present invention are excellent strains for developing microbial agents.

[0025] For the single or compound microbial agent described in the present invention and the composition composed of it and other functional organic and inorganic substances, the core microbial cells and cell liquid can be produced by liquid fermentation. Its preparation method is mature, simple, the process is efficient, the obtained cell content is high, and it fully meets the requirements of commercial production. Description of the Drawings

[0026] Figure 1 It is the cell morphology of Pseudomonas GM3 and its genomic circular map provided by the embodiment of the present invention; Figure 1 A is the cell morphology diagram of Pseudomonas GM3, Figure 1 B is the genomic circular map.

[0027] Figure 2The phylogenetic tree of Pseudomonas GM3 based on 16S rRNA gene sequence provided by the embodiments of the present invention.

[0028] Figure 3 The cell morphology and genomic circular map of Bacillus velezensis NB21 provided by the embodiments of the present invention; Figure 3 A is the cell morphology diagram of Bacillus velezensis NB21, Figure 3 B is the genomic circular map.

[0029] Figure 4 The phylogenetic tree of Bacillus velezensis NB21 based on 16S rRNA gene sequence provided by the embodiments of the present invention.

[0030] Figure 5 The fingerprint of lipopeptide compounds synthesized by Bacillus velezensis NB21 provided by the embodiments of the present invention.

[0031] Figure 6 The result diagram of the promotion of maize seedling growth by the bacterial agent of Bacillus velezensis NB21 provided by the embodiments of the present invention.

[0032] Figure 7 The comparison diagram of maize ears harvested after coating with the bacterial agent of Bacillus velezensis NB21 in the Changtu experimental field provided by the embodiments of the present invention.

[0033] Figure 8 The situation diagram of promoting strong seedling growth of rice seedlings after applying the compound microbial bacterial agent at the seedling stage provided by the embodiments of the present invention.

[0034] Figure 9 The effect diagram of promoting rice growth and stress resistance by applying the compound microbial bacterial agent at the booting stage provided by the embodiments of the present invention.

[0035] Figure 10 The effect diagram of preventing and controlling false smut of rice by applying the compound microbial bacterial agent at the booting stage provided by the embodiments of the present invention.

[0036] Figure 11 The influence diagram of the compound microbial bacterial agent on the weight of single rice grains provided by the embodiments of the present invention.

[0037] Figure 12 The MetagenomeSeq analysis result diagram of the compound bacterial agent treatment group and the control group provided by the embodiments of the present invention. Detailed implementation manners

[0038] For better understanding of the content of the present invention, further explanations are given below in combination with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0039] The self-discovered Pseudomonas strains provided by the present invention PseudomonasPseudomonas sp. GM3 and Bacillus velezensis strains Bacillus velezensis NB21, the said Pseudomonas species Pseudomonas Pseudomonas sp. GM3 and Bacillus velezensis strains Bacillus velezensis Both NB21 can efficiently colonize in the rhizosphere and phyllosphere of various crops, have strong environmental adaptability, and have various effects such as promoting seedling growth, disease resistance, and yield increase, and have very broad application prospects.

[0040] Example 1 Strain Pseudomonas Pseudomonas Identification of Pseudomonas sp. GM3

[0041] Pseudomonas GM3 was isolated from the roots of rice in Shenyang, Liaoning Province, and was deposited in the China Center for Type Culture Collection on October 31, 2024, with the deposit number: CCTCC NO: M20242391.

[0042] On TSA medium, this bacterium forms round light yellow colonies with a flat and dry colony surface ( Figure 1 A), and Gram staining shows red bacilli. It grows well at 15 - 35°C. The total DNA of Pseudomonas GM3 was extracted, and genome determination and assembly were carried out using the third-generation Oxford Nanopore sequencing technology combined with the second-generation Illumina NovaSeq sequencing technology to obtain its complete genome map. Its genome is a circular chromosome of about 6.85 Mb with a GC content of 62.31% ( Figure 1 B). It contains 6 copies of 5S rRNA, 5 copies of 16S rRNA, 5 copies of 23S rRNA, and 74 tRNAs. The length of the 16S rRNA gene sequence is 1534 bp, and the nucleotide sequence is as follows:

[0043] >GM3_16S rDNA

[0044]

[0045] Based on the 16S rRNA gene sequence of Pseudomonas GM3, its closely related type strain was retrieved using EZBioCloud, and a Neighbour-joining tree was constructed using MEGA 6.0 software. The results showed that Pseudomonas GM3 and the type strains 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 ). In particular, Pseudomonas GM3 and Pseudomonas protegens CHA0 T had the highest homology and clustered on the same evolutionary branch, indicating that Pseudomonas GM3 had the closest evolutionary relationship with the strains of P. protegens species. However, further using the JSpeciesWS online analysis tool, the Average Nucleotide Identity (ANI) between Pseudomonas GM3 and the closest strains was calculated using the obtained whole-genome data. The results are shown in Table 1. Pseudomonas GM3 had an ANI value less than 93% whether with Pseudomonas protegens the type strain CHA0 of T or other strains of this species. According to the gold standard that the whole-genome ANI value of the same species is greater than 95%, it was proved that Pseudomonas GM3 was a bacterial species different from Pseudomonas protegens .

[0046] Table 1 ANI values between Pseudomonas GM3 and the strains with the closest evolution

[0047]

[0048] Example 2 Identification of Bacillus velezensis Bacillus velezensis Identification of NB21

[0049] Bacillus velezensis NB21, isolated from sediments at a depth of 3622 meters in the South China Sea, was deposited at the China Center for Type Culture Collection on May 17, 2024, with the deposit number: CCTCC NO: M2024979. This bacterium formed round, flesh-white colonies on LB plates, irregularly round, with a wrinkled and dense surface. On TSA medium, the edge was wrinkled and raised, and the center was sunken ( Figure 3A), Bacilli that stain blue by Gram staining. It can grow well at 15-40 °C and can use citrate, D-xylose, L-arabinose, D-mannitol, etc. as the sole carbon source for growth. The total DNA of Bacillus velezensis NB21 was extracted, and the genome was sequenced and assembled using the third-generation Oxford Nanopore sequencing technology combined with the second-generation Illumina NovaSeq sequencing technology to obtain the complete genome map. Its genome is a circular chromosome of about 4.25 Mb, with a GC content of 45.97% ( Figure 3 B). It contains 9 copies of 5S rRNA, 9 copies of 16S rRNA, 9 copies of 23S rRNA, and 89 tRNAs. The length of the 16S rRNA gene sequence is 1547 bp, and the nucleotide sequence is as follows:

[0050] >NB21_16S rDNA

[0051]

[0052] Based on the 16S rRNA gene sequence of Bacillus velezensis NB21, the closely related type strains were retrieved using EZBioCloud, and a Neighbour-joining tree was constructed using MEGA 6.0 software. The results showed that Bacillus velezensis NB21 and the type strains Bacillus siamensis KCTC 13613 T (AJVF01000043), Bacillus velezensis CR-502 T , NRRL B-41580 T (AY603658), Bacillus velezensis FZB42 T (CP000560) and Bacillus amyloliquefaciens DSM 7 T (FN597644) were grouped on an independent larger branch, and the 16S rRNA gene sequence homology was 99.86%, 99.86%, 99.81% and 99.59% respectively ( Figure 4 ). It can be seen that Bacillus velezensis NB21 belongs to the group closely related to Bacillus velezensis.

[0053] However, due to the large number of Bacillus strains and the complex and diverse evolution, the classification results based solely on the 16S rRNA gene sequence are unreliable. Therefore, the JSpeciesWS online analysis tool was further used to calculate the ANI value between Bacillus velezensis NB21 and the closest strain using the obtained whole-genome data. The results are shown in Table 2. The ANI value between Bacillus velezensis NB21 and Bacillus velezensis the type strain NRRL B-41580 of T was 98.25%, which is greater than 95%. Therefore, Bacillus velezensis NB21 was identified as Bacillus velezensis Bacillus velezensis NB21.

[0054] Table 2 ANI values between Bacillus velezensis NB21 and the most closely related strains in evolution

[0055]

[0056] In addition, Bacillus velezensis NB21 was isolated from deep-sea sediments in the South China Sea. It exists in very unique ecological environments such as high pressure, low temperature, and high salinity, and thus has also evolved its unique genetic information. Further comparing the secondary metabolite biosynthetic gene cluster sequences of the closest Bacillus velezensis stored in the GenBank database, it was found that the biosynthetic gene clusters encoding compounds such as Surfactin (357012nt - 422419nt), Macrolactin (1486317nt - 1574541nt), Fengycin (2789498nt - 2859109nt), Bacillomycin D (2859386nt - 2927314nt), Bacillibactin (3325499nt - 3377287nt), and Bacilysin (3910979nt - 3952397nt) in the genome of Bacillus velezensis NB21 had a maximum gene homology of approximately 97% with those in the database, and the maximum homology of the biosynthetic gene cluster of Difficidin (2240778nt - 2346951nt) in the database was 98%. In particular, the highest homology of the unknown non-ribosomal peptide encoded between 2049040nt and 2094568nt in its genome was only 83%, and the highest homology of the amyloliquecidin GF610-like lanthipeptide compound encoded between 671601nt and 701016nt was only 73%. The above indicates that Bacillus velezensis NB21 has evolved a unique genetic sequence and is a unique strain of Bacillus velezensis different from any known strain.

[0057] Example 3 Pseudomonas Pseudomonas Function of sp. GM3

[0058] Using antiSMASH analysis and manual correction, it was found that Pseudomonas sp. GM3 contains more than 16 secondary metabolite gene clusters and has the potential to produce Orfamide, Hydrogen cyanide (HCN), 3-Thiaglutamate, Pyoluteorin, Nantio-pyochelin, Pf-5 pyoverdine, 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 shows that Pseudomonas sp. GM3 is an excellent strain for producing a variety of bioactive lead compounds.

[0059] Table 3 Potential of Pseudomonas sp. GM3 to synthesize diverse secondary metabolites

[0060]

[0061] Research findings have shown that Orfamide has been proven to have antifungal and biosurfactant effects, affecting the swarming motility of Pseudomonas and lysing zoospores produced by oomycete plant pathogens; Pyoluteorin has a killing effect on oomycetes and some bacteria; DAPG has antibacterial effects against a variety of phytopathogenic fungi; hydrogen cyanide (HCN) is volatile and can help plants inhibit various diseases. The strain antagonism experiment showed that Pseudomonas GM3 has strong antibacterial activities (such as Staphylococcus aureus, Escherichia coli, and Acinetobacter baumannii, etc.) and significant antifungal activity (such as Rhizoctonia solani of rice). In addition, the stability constant of the siderophore Pyoverdine of Pseudomonas with iron binding is 10 32 , which is much higher than the stability constant of 10 29 of the siderophore of some pathogenic bacteria such as Fusarium oxysporum with iron binding. It 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 Chrome Azurol S (CAS) plates and incubated at 28 °C for 72 h, showing a medium ability to secrete siderophores (Table 4). In addition, Pseudomonas GM3 was inoculated on inorganic phosphorus agar medium and incubated at 28 °C for 72 h, showing good phosphorus-solubilizing activity (Table 4). When inoculated in nitrogen-free liquid medium and incubated at 28 °C for 7 days, it also showed nitrogen-fixing activity (Table 4). It can colonize in the rhizosphere and above-ground plants of rice and maize, promoting the development of their roots and the growth of seedlings. The above indicates that Pseudomonas GM3 can play antibacterial and antifungal roles through multiple functions, and also has good functions of phosphorus solubilization, nitrogen fixation, and secretion of siderophores, making it an excellent plant growth-promoting bacterium and biocontrol microorganism.

[0062] Table 4 Comparison of functional evaluations between Pseudomonas GM3 and other laboratory strains

[0063]

[0064] Note: ― indicates no activity, ┼ indicates weak activity, ┼┼ indicates medium activity, ┼┼┼ indicates strong activity, ┼┼┼┼ indicates extremely strong activity.

[0065] Example 4 Bacillus velezensis Bacillus velezensis Functions of NB21

[0066] Using antiSMASH analysis and manual correction, it was found that Bacillus velezensis NB21 contains more than 15 secondary metabolite gene clusters, and has the potential to produce antifungal and plant resistance-inducing lipopeptides such as Surfactin, Fengycin, and Bacillomycin D, the iron carrier Bacillibactin that improves the colonization ability of the strain, antibacterial polyketides such as Macrolactin, Bacillaene, and Difficidin, and the potential to synthesize some novel PKS-NRPS hybrid and terpene compounds (Table 5). This indicates that Bacillus velezensis NB21 is an excellent strain for producing a variety of bioactive lead compounds. Research has shown that lipopeptide compounds such as Surfactins and Fengycins are the main components for the multicellular behavior of Bacillus, inducing 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 extremely strong inhibitory activity against Exserohilum turcicum, the pathogen of northern corn leaf blight, and Fusarium graminearum, the pathogen of corn stalk rot. In the plate confrontation method, the diameter of the inhibition zone is greater than 10 cm. Metabolic fingerprint analysis showed that Bacillus velezensis NB21 can produce signal molecule-like lipopeptides such as Surfactins that affect the multicellular behaviors of Bacillus, such as motility, colony morphology, and biofilm formation, as well as Fengycins and Bacillomycins D lipopeptide compounds that induce plant systemic resistance and antifungal effects ( Figure 5 ). In addition, in the pot experiment, Bacillus velezensis NB21 showed a strong effect on promoting the growth of corn seedlings. It can be seen that Bacillus velezensis NB21 has significant antifungal, seedling-strengthening and growth-promoting, and crop systemic resistance-inducing effects. This strain is also an excellent plant growth-promoting bacterium and biocontrol microorganism.

[0067] Table 5 Potential of Bacillus velezensis NB21 to synthesize diverse secondary metabolites

[0068]

[0069] Example 5 Preparation of a single or compound bacterium agent and its composition for disease resistance, growth promotion, and yield increase

[0070] After streaking and activating the preserved Pseudomonas sp. GM3 on TSA medium, a single colony was inoculated into TSB liquid medium and cultured overnight. Then, it was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of TSB liquid medium at an inoculation amount of 2% by volume. It was placed in a shaker at 200 rpm and cultured in the flask at 28 °C for 24 h. Then, a small amount of the bacterial solution was taken for microscopic examination. After the microscopic examination was correct, it was transplanted into a 3000 mL flask containing 1000 mL of TSB or King's B liquid medium at a volume ratio of 3%. It was placed in the shaker again at 200 rpm and cultured in the flask at 28 °C for 70 h. Then, the fermentation broth was collected, and the content of Pseudomonas sp. GM3 was 2×10 9 CFU / mL or 9×10 9 CFU / mL. This fermentation broth can be directly used as a single Pseudomonas sp. GM3 liquid bacterial agent.

[0071] The Bacillus velezensis NB21 bacterial solution stored in a glycerol tube was inoculated into LB medium at an inoculation amount of 0.05% by volume and activated overnight. Then, the freshly activated strain was inoculated into a 500 mL Erlenmeyer flask containing 100 mL at an inoculation amount of 3% by volume. It was placed in a shaker at 200 rpm and cultured in the flask at 35 °C for 24 h. Then, it was transplanted into a 3000 mL flask containing 1000 mL at the same volume ratio. It was placed in the shaker again at 200 rpm and cultured in the flask at 35 °C for 36 h. After the fermentation was completed, the content of Bacillus velezensis NB21 in the fermentation broth was 5×10 8 CFU / mL, and it can be used as a single Bacillus velezensis NB21 liquid bacterial agent.

[0072] The above-fermented Pseudomonas sp. GM3 bacterial solution and Bacillus velezensis NB21 bacterial solution were mixed at a volume ratio of 1:1, and leucine, isoleucine, polyglutamic acid, acetic acid, lactic acid, or / and mineral source humic acid, etc. with a mass ratio of 0.5% were added to prepare a liquid compound microbial liquid bacterial agent.

[0073] The fermentation broth of the above Pseudomonas sp. GM3 or Bacillus velezensis NB21 was centrifuged at 4000 rpm for 30 min using a large-capacity centrifuge, and the supernatant was discarded to collect the bacterial cells. Then, the wet weight of each bacterial cell was mixed with lactose at a ratio of 1:1 and stirred evenly into a paste, and dehydrated using a freeze dryer and pulverized (200 - 600 mesh) using a pulverizer to prepare a microbial bacterial powder of a single strain. The content of Pseudomonas sp. GM3 bacterial powder was 5×10 11 CFU / g, and the content of Bacillus velezensis NB21 bacterial powder was 1×10 11 CFU / g.

[0074] Pseudomonas GM3 powder and Bacillus Velez NB21 powder are mixed in a mass ratio of 1:1 to obtain composite microbial powder. On this basis, biochar with a particle size of 100-500 mesh and 1 times the mass of the biochar, humic acid, amino acid or / and mushroom residue, vermiculite, peat or diatomaceous earth with 10 times the mass of the biochar are added to form different compositions, which can be used as biological organic fertilizers.

[0075] Example 6 Growth-promoting and yield-increasing effects of Bacillus velez NB21 bacterial agent

[0076] Select corn seeds of uniform size and full kernels, disinfect them with NaClO solution, rinse them with sterile water three times, and air dry them naturally. Then mix the corn seeds with the prepared single Bacillus Velez NB21 bacterial solution at a ratio of 1:40 (fermentation volume mL: seed mass g), mix the fermentation solution and seeds thoroughly, and the control group (CK) is treated with LB liquid culture medium. The corn seeds are air-dried and set aside.

[0077] Sterilize the soil and vermiculite in a mass ratio of 1:1 and mix them evenly, then put them into plastic pots of 13cm×13cm. Sow the corn seeds treated with the seed dressing in the pots, 6 seeds per pot, and 10 pots per treatment. The temperature was controlled within the range of 24~28℃, the humidity was 70%, and the light was 16L / 8D. When the seedling stage was cultivated to about the 30th day, 20 corn seedlings were randomly selected in the treatment and the plant height of the corn seedlings was measured. Figure 6 As shown, the average plant height of plants treated with Bacillus Velezii NB21 increased by 10.37% compared with the control group (CK1), showing significant growth-promoting and seedling-strengthening effects.

[0078] The prepared single Bacillus Velez NB21 powder was used in the same manner as the seed coating agent for coating seeds. 8Mix seeds with the proportion of viable CFU bacteria content, spread them out after mixing and air-dry in a cool place in the warehouse, and sow within one week. Harvest the corn plants of each treatment by taking one ear from every 4th plant, with 15 plants in a group and three parallels for each treatment, which serve as samples for seed inspection and yield measurement. Measure and calculate the average ear diameter and average ear length of the corn in each treatment sample respectively, weigh the grain weight and 100-grain weight, and estimate the unit yield. For example, the average wet weight of each group of corn cobs treated with Bacillus velezensis NB21 in the Gongzhuling experimental field was 4.807 kg, and the relative corn yield increase was 7.05% compared with the control group (Table 6). After the corn cobs are dried, thresh them, weigh the weight of the corn kernels of 45 plants (kernel weight), and randomly take 100 corn kernels to weigh, repeating three times and calculating the average value. It was found that the kernel weight and 100-grain weight of the corn treated with Bacillus velezensis NB21 were 10.835 kg and 44.06 g respectively, which were significantly higher than 10.25 kg and 43.89 g of the control group. In the Changtu experimental field, after the treatment of mixing seeds with Bacillus velezensis NB21, the relative corn yield increase effect was more significant, and the relative corn yield increase was as high as 13.79% (Table 6, Figure 7 )

[0079] Table 6 Results of seed inspection and yield measurement of corn in Gongzhuling and Changtu experimental fields

[0080]

[0081] Example 7 Seedling strengthening, stress resistance, disease resistance, growth promotion and yield increase effects of compound microbial inoculant

[0082] Use the compound microbial liquid inoculant prepared according to Example 5 with Pseudomonas GM3, Bacillus velezensis NB21 and added 0.5% mineral source humic acid, spray it when the rice seedlings have three leaves and one heart (5 - 10 days before transplanting), or directly irrigate the compound microbial inoculant 24 hours before and after rice transplanting, applying 1 L per mu, and taking the non-application of the compound microbial inoculant as the control; during the growth process of rice, it can significantly enhance the resistance of rice seedlings to low-temperature stress and the seedling recovery speed is fast. Two weeks after the application of the compound microbial inoculant, it can significantly promote the growth of rice seedlings, especially promote the development of rice roots. Compared with the control group, after the application of the compound microbial inoculant, the average root weight (g), root length (cm) and lateral root number of each rice 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 of each plant increased by 14.02%. Malondialdehyde (MDA) is the final decomposition product of membrane lipid peroxidation, and its content can reflect the degree of adversity damage suffered by plants. After the application of the compound inoculant, the MDA content decreased by 4.55%, which can be considered that the stress resistance of the crop system is enhanced and the damage of the environment to the crop is reduced ( Figure 8 )

[0083] During the booting stage of rice, apply the above compound microbial liquid inoculant at a rate of 15 L per mu, and use the non-application of the compound microbial inoculant as a control; two weeks after applying the inoculant, it can significantly increase the chlorophyll content by 12.8%, significantly increase the leaf area of the flag leaf of rice by 7.1%; it can also significantly improve the systemic stress resistance of rice, the content of proline (Pro) increases significantly by 31.8%, the content of MDA decreases significantly by 17.8%, and the activities of superoxide dismutase (SOD) and peroxidase (POD) increase significantly, by 87.7% and 105.2% respectively, and the P values are 0.052 and 0.104 respectively, close to the significant level ( Figure 9 ). After applying the compound microbial inoculant, the disease index of false smut of rice decreases significantly, from 2.50% in the control group to 1.21% (the control effect is 85.1%); the diseased panicle rate decreases significantly, from 10.61% in the control group to 6.93% ( Figure 10 ).

[0084] Meanwhile, in the experimental area (Ciyutuo 1, Mengjiatai 1, Mengjiatai 2 and Daomeng Space), set up 4 experimental plots for yield increase evaluation. Among them, only the compound microbial inoculant is applied at the booting stage in Ciyutuo 1 and Mengjiatai 1, and the compound microbial inoculant is applied at both the seedling stage and the booting stage in Mengjiatai 2 and Daomeng Space, and the application rate is as described above. After the rice is completely mature, the five-point sampling method is used to evaluate the rice yield. Specifically, samples are taken from the above compound inoculant treatment group and the blank control without applying the inoculant respectively. Five points are taken from each sample plot, and three clusters of rice are taken from each point. Count the number of panicles of each cluster of rice, and remove and weigh the spikelets on each panicle of rice to obtain the spikelet weight of each cluster of rice. At 4 different experimental sites, after applying the compound inoculant at the seedling stage and / or the booting stage, the weight of single-plant rice grains increases significantly. The weight of single-plant applying the compound inoculant is 55.24 - 73.05 g, while the control is 50.71 - 58.40 g, and the average yield increase range is 9 - 25% ( Figure 11 ).

[0085] Subsequently, randomly take 1000 rice grains from the rice grains taken from each cluster of rice for weighing to calculate the 1000-grain weight. In the fields of Ciyutuo 1 and Mengjiatai 1 where only the compound microbial inoculant is applied at the booting stage, the 1000-grain weight of rice increases by 1.8% and 5.9% respectively, while in the fields of Mengjiatai 2 and Daomeng Space where the compound microbial inoculant is applied at both the seedling stage and the booting stage, the 1000-grain weight of rice increases by 2.3% and 15.5% respectively.

[0086] In addition, whether applying a single inoculant alone or adding a compound microbial inoculant during the booting stage can significantly promote the growth and development of rice and greatly improve the stress resistance of crops. Generally speaking, the compound microbial inoculant is more prominent than the single inoculant in terms of promoting growth, disease resistance and yield increase.

[0087] Example 8 Microecological optimization effect of compound microbial inoculant

[0088] In Example 7, after treating rice with a composite microbial agent, rice roots and rhizosphere soil were collected during the rice filling and graining period, and high-fidelity rice root surface soil (containing microorganisms) was obtained by ultrasonic treatment; then total DNA of the samples was extracted, and the diversity and differences of the rhizosphere microbiome were compared based on the 16S rRNA gene sequence using Illumina NovaSeq high-throughput sequencing technology. The results showed that about 4 / 5 of the bacterial abundance of Liaoning rice rhizosphere microorganisms was mainly distributed in the Proteobacteria ( Proteobacteria )、Green Bay Fungi ( Chloroflexi ), Acidobacteria ( Acidobacteriota ), Bacteroidetes ( Bacteroidota )、Desulfobacteria( Desulfobacterota ), Actinobacteria ( Actinobacteria ), Firmicutes ( Firmicutes ) and Myxomycetes ( Myxococcota After the composite bacterial agent treatment, 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%. At the class level, the relative abundance of the main bacteria also changed significantly (Table 7). In particular, the most important Chlorobacteria, the Anaerobic Fungus class ( Anaerolineae ) increased by 3.33% from 8.84% in the control to 12.06%. Vicinamibacteria The relative abundance of the class of fungi increased by 0.74% compared with the control, reaching 4.01%. The study found that some microorganisms in the class of anaerobic fungi have the function of light energy nutrition and can decompose complex organic matter. Vicinamibacteria The bacteria of the group have the potential to dissolve phosphorus in the soil. In addition, compared with the control group, the relative abundance of the uncultivated group MBNT15 increased by 1.5%, and the relative abundance of KD4-96 increased by 0.91%. It can be considered that the mutualism in the soil has been strengthened, which has enhanced the circulation and utilization of special and complex substrates (uncultivable means that it has not been cultivated yet, but it exists and can be detected, and there is only a group code. These microorganisms need special nutrition to reproduce better, indicating that mutualism has been strengthened).

[0089] Table 7 Changes in relative abundance of main bacterial classes in rice rhizosphere after treatment with composite microbial agents

[0090]

[0091] MetagenomeSeq analysis was further completed, and the results were as follows Figure 12As shown in the figure, compared with the control group, the main groups with large differences among the groups were Acidobacteria, Actinobacteria, Armored Bacteroidetes, Green Bay Bacteria, Desulfobacteria, Firmicutes, Myxomycetes, Nitrospira and Proteobacteria after the composite bacterial agent treatment. In particular, the number of Thiobacillus ( Thiobacillus ), Methylomonas ( Mefhylomonas )、 Aminicenantales The relative abundance of the above data showed that the metabolism of sulfur, single-carbon compounds such as methane and methanol, and refractory organic matter such as phenols and N-heterocyclic compounds in the rice rhizosphere was significantly improved after the treatment with the microbial agent. In addition, the genus-level groups such as uncultivable Subgroup 7, SB-5, BSV26, MBNT15, 4-29-1, OM190 and Ellin6067 increased significantly, indicating that the microbial agent stimulated the interactive nutrient metabolism in the rice rhizosphere and enhanced the turnover efficiency of soil nutrients and special elements. In summary, the composite microbial agent can enhance the circulation of soil materials and elements and improve the utilization efficiency of nutrients by crops by regulating the structure and function of the rhizosphere microbial community.

Claims

1. A strain of bacteria that resists diseases, promotes growth and increases yields, characterized in that: The strain is Pseudomonas Pseudomonas sp. GM3, or, Pseudomonas Pseudomonas sp. GM3 and Bacillus velezensis ( Bacillus velezensis ); wherein, Pseudomonas Pseudomonas sp. GM3, was deposited at the China Center for Type Culture Collection on October 31, 2024, with the deposit number: CCTCC NO: M20242391; Bacillus velezensis Bacillus velezensis NB21, was deposited at the China Center for Type Culture Collection on May 17, 2024, with the deposit number: CCTCC NO: M2024979.

2. Use of the strain for disease resistance, growth promotion and yield increase according to claim 1, characterized in that: The application of the strain Pseudomonas in nutrient supply or disease resistance; The nutrient supply is nitrogen fixation, phosphorus solubilization, and iron transport; The disease resistance is against Rhizoctonia solani of rice.

3. Use of the strain for disease resistance, growth promotion and yield increase according to claim 1, characterized in that: The application of Pseudomonas and Bacillus velezensis in disease and stress resistance, promoting crop growth and yield increase, or improving soil microecology; The disease and stress resistance are against false smut of rice and enhancing the systemic stress resistance of crops; The promotion of crop growth and yield increase is to promote crop root development, enhance photosynthesis, increase crop biomass, increase the 100-grain weight, 1000-grain weight, or unit yield of crops; The crop is rice.

4. A microbial inoculant, characterized in that: The microbial agent contains the single or compound active strains described in claim 1.

5. The microbial inoculant according to claim 4, wherein: The microbial agent is a culture, culture bacterial suspension, or fermentation broth of the active strain.

6. The microbial inoculum according to claim 5, wherein: The microbial agent also contains auxiliary materials, and the auxiliary materials are one or several of biochar, humic acid, mushroom residue, amino acid, organic acid, vermiculite, peat, and diatomite.

7. The microbial inoculant according to claim 4, wherein: When the microbial agent is a liquid preparation, the content of Pseudomonas sp. GM3 is 2×10 8 CFU / mL or more, and / or the content of Bacillus velezensis NB21 is 2×10 8 CFU / mL or more; When the microbial agent is a solid preparation, the content of Pseudomonas GM3 is 2×10 8 CFU / g or more, and / or the content of Bacillus velezensis NB21 is 5×10 8 CFU / g or more.

8. Use of the microbial inoculant according to claim 4, characterized in that: The application of the microbial agent as a biological seed coating agent, microbial pesticide, microbial fertilizer, bio-organic fertilizer, or soil conditioner.

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