Disease-resistant growth-promoting yield-increasing strain and compound 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 compound microbial agent and application thereof. The strain is a strain pseudomonas GM3 (CCTCC (China Center For Type Culture Collection) NO: M20242391) and / or a strain bacillus velezensis NB21 (CCTCC NO: M2024979) which are / is found autonomously; the strains can grow at low temperature, are high 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, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a disease-resistant, growth-promoting and yield-increasing bacterial strain and a composite microbial agent and application thereof. Background Art

[0002] In agricultural production, the high-intensity and excessive application of fertilizers and pesticides has led to the decline of soil quality, functional degradation and even obstruction, water pollution and eutrophication, and a significant decline in crop quality and yield, which have affected global food and food security and ecological and environmental security. To this end, it is urgent to continue to reduce the total amount of chemical pesticides and agricultural fertilizers used, and replace old and 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 from the source is an important path to achieve sustainable development of global agriculture.

[0003] Microorganisms are an important part of soil structure and function, and are also the most active and key factors in determining soil fertility. Soil microorganisms drive the evolution of soil fertility, humus formation, material circulation and energy conversion. They are not only the shapers of healthy soil but also important characterization factors, which are crucial to 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 metabolism and growth and development processes of crops, and are also one of the key factors determining crop yield and stress tolerance. In particular, beneficial microorganisms that resist disease and promote growth can also stimulate seed germination, promote plant growth, enhance plant stress resistance, and prevent and control the occurrence of diseases, which have attracted much attention. Therefore, microbial agents and their related products (such as seed dressing agents, pesticides, fertilizers and soil conditioners, etc.) are considered to be an environmentally friendly agricultural strategy as natural alternatives to chemical fertilizers and pesticides, which are expected to improve soil quality, promote crop growth and development, and improve crop system resistance and productivity under environmental changes and soil degradation. Summary of the invention

[0004] The present invention aims to provide a disease-resistant, growth-promoting and yield-increasing bacterial strain and a composite microbial agent and application thereof.

[0005] To achieve the above purpose, the present invention adopts the following technical solution: A disease-resistant, growth-promoting and yield-increasing bacterial strain, the bacterial strain is Pseudomonas and / or Bacillus Velezii; wherein, Pseudomonas Pseudomonas sp. GM3, 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; Bacillus Velez Bacillus velezensis NB21, deposited in China Center for Type Culture Collection on May 17, 2024, with the deposit number: CCTCC NO: M2024979, and the deposit address: Wuhan University, Wuhan, China.

[0006] The Pseudomonas GM3 is characterized in that it is derived from the rice root system, the genome is a circular chromosome of about 6.85Mb, the GC content is 62.31%, and it 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.

[0007] The Velez Bacillus NB21 is characterized in that it originates from deep-sea sediments of the South China Sea, has a genome of about 4.25Mb and a 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.

[0008] 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.

[0009] The nutrient supply refers to nitrogen fixation, phosphorus solubilization, and iron transport.

[0010] The disease resistance and stress resistance refer to resistance to bacteria, fungi, and enhancing the stress resistance of crop systems.

[0011] The said promotion of crop growth and increase of 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.

[0012] A microbial agent containing the active bacterial species.

[0013] The bacterial agent is the culture, culture suspension or fermentation liquid of the active strain.

[0014] 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.

[0015] When the above auxiliary materials are biochar, mushroom residue, vermiculite, peat, and diatomaceous earth, the amount added is 1-20 times the mass of the microbial agent; When the above 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.

[0016] 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 Velezii NB21 is 2×10 8 CFU / mL or above; 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.

[0017] 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.

[0018] The advantages of the present invention are: 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 crop growth promotion. The Bacillus Velezii NB21 is a self-discovered strain, and has the functions of antifungal, seedling strengthening and growth promotion, and inducing crop systemic resistance. The Pseudomonas GM3 and Bacillus Velezii NB21 described in the present invention are excellent strains for the development of microbial agents.

[0019] The single or composite bacterial agent and the composition composed of the same with other functional organic and inorganic substances described in the present invention, the core microbial cells and bacterial liquid can be produced by liquid fermentation, the preparation method is mature and simple, the process is efficient, the obtained cell content is high, and the needs of commercial production are fully met. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The bacterial morphology and genome circle diagram of Pseudomonas GM3 provided in the embodiment of the present invention; Figure 1 A is the morphology of Pseudomonas GM3 bacteria. Figure 1 B is the genome circle map.

[0021] Figure 2 The present invention provides a phylogenetic tree of Pseudomonas GM3 based on 16S rRNA gene sequence.

[0022] Figure 3 The bacterial morphology and genome circle map of Bacillus Velez NB21 provided in the embodiments of the present invention; Figure 3A is the morphology of Bacillus velez NB21. Figure 3 B is the genome circle map.

[0023] Figure 4 A phylogenetic tree of Bacillus velez NB21 based on 16S rRNA gene sequence provided in an embodiment of the present invention.

[0024] Figure 5 The fingerprint of lipopeptide compounds synthesized by Bacillus velezensis NB21 provided in the embodiment of the present invention.

[0025] Figure 6 This is a graph showing the results of promoting the growth of corn seedlings using the Bacillus Velez NB21 agent provided in an embodiment of the present invention.

[0026] Figure 7 A comparison diagram of corn ears harvested from the Changtu test field provided in an embodiment of the present invention after coating with the Bacillus Velez NB21 bacterial agent.

[0027] 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 according to an embodiment of the present invention.

[0028] Fig. 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 provided by an embodiment of the present invention.

[0029] Fig.10 This is a diagram showing the effect of applying a composite microbial agent to control rice false smut during the booting stage provided by an embodiment of the present invention.

[0030] Fig.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.

[0031] Fig.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

[0032] In order to better understand the content of the present invention, further description is given below in conjunction with specific embodiments, but the protection content of the present invention is not limited thereto.

[0033] The independently discovered Pseudomonas species provided by the present invention Pseudomonas sp. GM3 and Bacillus velez strains Bacillus velezensis NB21, the Pseudomonas species Pseudomonas sp. GM3 and Bacillus velez strains Bacillus velezensisNB21 can efficiently colonize in the rhizosphere and leaf zone of a variety of crops, has strong environmental adaptability, and has multiple functions such as strengthening seedlings, promoting growth, resisting diseases, and increasing yields. It has a very broad application prospect.

[0034] Example 1 Bacterial species Pseudomonas Pseudomonas Identification of sp. GM3 Pseudomonas GM3 was isolated from rice roots in Shenyang, Liaoning, and was deposited in the China Center for Type Culture Collection on October 31, 2024, with the deposit number: CCTCC NO: M20242391.

[0035] The bacteria forms round light yellow colonies on TSA medium. The surface of the colonies is flat and dry. Figure 1 A), Gram staining is red bacilli. It grows well at 15-35℃. The total DNA of Pseudomonas GM3 was extracted, and the genome was determined and assembled using Oxford Nanopore third-generation sequencing technology combined with Illumina NovaSeq second-generation sequencing technology to obtain its complete genome map. Its genome is a circular chromosome of about 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 tRNA. The 16S rRNA gene sequence is 1534 bp long, and the nucleotide sequence is as follows: >GM3_16S rDNA

[0036] Based on the 16S rRNA gene sequence of Pseudomonas GM3, 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 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, clustered in the same evolutionary branch, indicating that Pseudomonas GM3 and P. protegens The strains of the same species have the closest evolutionary relationship. However, the JSpeciesWS online analysis tool was further used to calculate the Average Nucleotide Identity (ANI) between Pseudomonas GM3 and the closest strains using the obtained whole genome data. The results are shown in Table 1. Pseudomonas protegens The model strain CHA0 T The ANI values ​​of other strains of the same species were all less than 93%. According to the gold standard of ANI value of the whole genome of the same species greater than 95%, it was proved that Pseudomonas GM3 was different from Pseudomonas protegens A species of bacteria.

[0037] Table 1 ANI values ​​between Pseudomonas GM3 and the closest evolutionary strains

[0038] Example 2 Bacillus Velez Bacillus velezensis Identification of NB21 Bacillus Velezii 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 3A), Gram staining is blue. It can grow well at 15-40℃, and can use citrate, D-xylose, L-arabinose, D-mannitol, etc. as the sole carbon source for growth. The total DNA of Bacillus velez NB21 was extracted, and the genome was determined and assembled using Oxford Nanopore third-generation sequencing technology combined with Illumina NovaSeq second-generation sequencing technology to obtain its complete genome map. Its genome is a circular chromosome of about 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 tRNA. The 16S rRNA gene sequence is 1547 bp long, and the nucleotide sequence is as follows: >NB21_16S rDNA

[0039] Based on the 16S rRNA gene sequence of Bacillus Velezii 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 Velezii NB21 and the model strain 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) clustered on an independent larger branch, with 16S rRNA gene sequence homologies of 99.86%, 99.86%, 99.81% and 99.59% respectively ( Figure 4 ). It can be seen that Bacillus Velezii NB21 belongs to a group similar to Bacillus Velezii.

[0040] However, due to the large number of Bacillus and their diverse and complex 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 Velezii NB21 and the closest strain using the obtained whole genome data. The results are shown in Table 2. Bacillus velezensis The type strain NRRL B-41580 T The ANI value of was 98.25%, which was greater than 95%, so Bacillus velez NB21 was identified as Bacillus velez Bacillus velezensis NB21.

[0041] Table 2 ANI values ​​between Bacillus velez NB21 and the closest evolutionary strains

[0042] In addition, Bacillus Velezii 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 salt, and therefore has also evolved its unique genetic information. Further comparison of the secondary metabolite synthesis gene cluster sequences of the closest Bacillus Velezii stored in the GenBank database revealed 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 Velezii NB21 had a homology of about 97% with the highest gene in the database, and a highest homology of 98% with the biosynthetic gene cluster of Difficidin (2240778nt~2346951nt) in the database. In particular, the highest homology of the unknown non-ribosomal peptide located between 2049040nt and 2094568nt in its genome is only 83%, and the highest homology of the thiopeptide compound Amyloliquecidin GF610 located between 671601nt and 701016nt is only 73%. The above shows that Bacillus Velezii NB21 has evolved a unique genetic sequence and is a unique Bacillus Velezii strain different from any known strain.

[0043] Example 3 Pseudomonas Pseudomonas Function of sp. GM3 Using antiSMASH analysis and manual correction, it was found that Pseudomonas GM3 contained more than 16 secondary metabolite gene clusters, with 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 GM3 is an excellent strain for producing a variety of bioactive lead compounds.

[0044] Table 3 Potential of Pseudomonas GM3 to synthesize diverse secondary metabolites

[0045] 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) 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 carrier binding to iron 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 72h, 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 72h, showing good phosphate solubilization activity (Table 4). It was 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 effects, and has good phosphate solubilization, nitrogen fixation and iron carrier secretion functions, making it an excellent plant growth-promoting bacterium and biological control microorganism.

[0046] Table 4 Comparison of functional evaluation of Pseudomonas GM3 and other laboratory strains

[0047] Note: ― represents inactivity, ┼ represents weak activity, ┼┼ represents moderate activity, ┼┼┼ represents strong activity, and ┼┼┼┼ represents extremely strong activity.

[0048] Example 4 Bacillus Velez Bacillus velezensis NB21 Features Using antiSMASH analysis and manual correction, it was found that Bacillus velez NB21 contained more than 15 secondary metabolite gene clusters, with the potential to produce antifungal and plant resistance-inducing Surfactin, Fengycin and Bacillomycin D lipopeptides, Bacillibactin iron carriers that improve the colonization ability of strains, antibacterial Macrolactin, Bacillaene and Difficidin polyketides, thiopeptide antibiotic Amyloliquecidin GF610 and other backbone compounds, as well as the potential to synthesize some novel PKS-NRPS hybrids and terpene compounds (Table 5). This shows 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 velez NB21 has strong inhibitory activity against the pathogen of corn leaf blight, Helicospora macrospora, 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 velez NB21 can produce signal molecule-like lipopeptides Surfactins that affect multicellular behaviors such as the motility, colony morphology and biofilm formation of Bacillus, as well as Fengycins and Bacillomycins D lipopeptide compounds that induce plant systemic resistance and anti-pathogenic fungi ( Figure 5 ). In addition, in the pot experiment, Bacillus Velez NB21 showed a strong effect in promoting the growth of corn seedlings. It can be seen that Bacillus Velez NB21 has significant antifungal, seedling-strengthening and growth-promoting effects, and inducing crop systemic resistance. This strain is also an excellent plant growth-promoting bacterium and biological control microorganism.

[0049] Table 5 Potential of Bacillus velez NB21 to synthesize diverse secondary metabolites

[0050] Example 5 Preparation of single or composite bacterial agents for disease resistance, growth promotion and yield increase and their compositions After the preserved Pseudomonas GM3 was streaked and activated on TSA medium, a single colony was inoculated into TSB liquid medium and cultured overnight, and then inoculated into a 500mL triangular shake flask with a liquid volume of 100mL TSB liquid medium according to a volume ratio of 2%. Place it in a shaker at 200rpm, shake the flask for 24 hours at 28℃, take a small amount of bacterial liquid for microscopic examination, and after the microscopic examination is correct, transfer it to a 3000mL shake flask with a liquid volume of 1000mL TSB or King's B liquid medium according to a volume ratio of 3%, place it in a shaker at 200rpm again, shake the flask for 70 hours at 28℃, and collect the fermentation liquid. The contents of Pseudomonas GM3 are 2×10 9 CFU / mL or 9×10 9 CFU / mL, the fermentation broth can be directly used as a single Pseudomonas GM3 liquid inoculant.

[0051] The Bacillus Velez NB21 culture liquid stored in the glycerol tube was inoculated into LB medium at a volume ratio of 0.05% for overnight activation. Then the freshly activated strain was inoculated into a 500mL triangular shake flask with a liquid volume of 100mL at a volume ratio of 3%. It was placed in a shaker at 200rpm and cultured at 35℃ for 24h, and then transplanted into a 3000mL shake flask with a liquid volume of 1000mL at the same volume ratio, and placed in a shaker at 200rpm again, and cultured at 35℃ for 36h. After the fermentation was completed, the content of Bacillus Velez NB21 in the fermentation broth was 5×10 8 CFU / mL, it can be used as a single Bacillus Velezii NB21 liquid inoculant.

[0052] The fermented Pseudomonas GM3 bacterial solution and the Bacillus Velez NB21 bacterial solution are mixed in a volume ratio of 1:1, and 0.5% by mass of leucine, isoleucine, polyglutamic acid, acetic acid, lactic acid or / and mineral humic acid are added to prepare a liquid composite microbial liquid inoculant.

[0053] The fermentation broth of Pseudomonas GM3 or Bacillus Velezii NB21 was centrifuged at 4000 rpm for 30 min using a large-capacity centrifuge, and the supernatant was discarded to collect the bacteria. The wet weight of each bacteria was mixed with lactose at a ratio of 1:1, stirred evenly into a paste, dehydrated using a freeze dryer, and crushed by a grinder (200-600 mesh) to prepare a single strain of microbial powder. The content of Pseudomonas GM3 powder was 5×10 11 CFU / g, the content of Bacillus Velez NB21 powder is 1×10 11 CFU / g.

[0054] 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.

[0055] Example 6 Growth-promoting and yield-increasing effects of Bacillus velez NB21 bacterial agent 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.

[0056] 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.

[0057] The prepared single Bacillus Velezii NB21 powder was used in the same manner as the seed coating agent for coating seeds. 8Seeds were mixed with the proportion of CFU live bacteria content, spread out in the cool warehouse to air dry, and sowed within a week. The corn plants of each treatment were harvested by taking one plant ear every 4 plants, with 15 plants in a group and three parallel groups in one treatment as samples for seed testing and yield measurement. The average ear thickness and average ear length of corn in each treatment sample were measured and calculated respectively, the grain weight and 100-grain weight were weighed, and the unit yield was estimated. For example, the average wet weight of each group of corn cobs mixed with Bacillus Velez NB21 in the Gongzhuling experimental field was 4.807kg, which was a relative increase of 7.05% compared with the control group (Table 6). After the corn cobs were dried, they were threshed, the weight of 45 corn kernels (corn kernel weight) was weighed, and 100 corn kernels were randomly taken and weighed, and the average value was calculated by repeating the weighing three times. Comparison found that the corn kernel weight and 100-grain weight of corn applied with Bacillus Velez NB21 were 10.835kg and 44.06g, respectively, which were significantly higher than 10.25kg and 43.89g of the control group. In the Changtu experimental field, the relative yield increase of corn was more significant after seed treatment with Bacillus Velez NB21, with a relative yield increase of up to 13.79% (Table 6, Figure 7 ).

[0058] Table 6 Results of corn seed testing and yield measurement in Gongzhuling and Changtu experimental fields

[0059] Example 7 Effects of composite microbial agent on seedling strengthening, stress resistance, disease resistance, growth promotion and yield increase The composite microbial liquid agent prepared according to Example 5, Pseudomonas GM3, Bacillus Velez NB21 and 0.5% mineral humic acid were used for spraying when the rice seedlings had three leaves and one heart (5-10 days before transplanting), or the composite microbial agent was directly irrigated 24 hours before and after rice transplanting, and 1L was applied per mu. At the same time, the control was taken without the composite microbial agent. During the growth of rice, the resistance of rice seedlings to low temperature stress was significantly enhanced, and the seedling recovery speed was fast. After 2 weeks of application of the composite microbial agent, the growth of rice seedlings, especially the development of rice root system, was significantly promoted. Compared with the control group, the average root weight (g), root length (cm) and lateral root number of rice per plant after the composite microbial agent was applied were increased by 38.16%, 30.94% and 12.68%, respectively, the chlorophyll content of rice leaves was increased by 48.5%, and the average fresh weight per plant was 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 fungicide, the MDA content decreased by 4.55%, which can be considered that the stress resistance of the crop system is enhanced and the damage to the crop by the environment is reduced ( Figure 8 ).

[0060] The above-mentioned composite microbial liquid inoculant was applied at the rice booting stage, with an application rate of 15L per mu, and no composite microbial inoculant was used as the 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, the proline (Pro) content was significantly increased by 31.8%, the MDA content was significantly reduced by 17.8%, and the superoxide dismutase (SOD) and peroxidase (POD) activities were significantly enhanced, increasing by 87.7% and 105.2%, respectively, with P values ​​of 0.052 and 0.104, respectively, close to the significant level ( Fig. 9 After applying the compound 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% ( Fig.10 ).

[0061] 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 evaluation. Among them, Ciyutuo 1 and Mengjiatai 1 were only applied with composite microbial agents in the booting stage, and Mengjiatai 2 and Daomeng Space were applied with composite microbial agents in both 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 booting stage, the weight of rice grains per plant increased significantly. The weight of rice grains per plant in the application of the compound microbial agent was 55.24-73.05 g, while that of the control was 50.71-58.40 g. The average yield increase was 9-25% ( Fig.11 ).

[0062] Then, 1,000 rice ears were randomly selected from the rice grains taken from each cluster of rice and weighed to calculate the thousand-grain weight. The thousand-grain weight of rice in Ciyutuo 1 and Mengjiatai 1 fields, where the compound fungicide was applied only at the booting stage, increased by 1.8% and 5.9%, respectively, while the thousand-grain weight of rice in Mengjiatai 2 and Daomeng Space fields, where the compound fungicide was applied at both the seedling and booting stages, increased by 2.3% and 15.5%, respectively.

[0063] In addition, whether a single microbial agent is applied alone or a composite microbial agent is applied in addition to the booting stage, it can significantly promote the growth and development of rice and greatly improve the stress resistance of crops. In general, the composite microbial agent is more effective than a single microbial agent in promoting growth, resisting diseases and increasing yield.

[0064] Example 8 Microecological Optimization Effect of Composite Microbial Agent 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).

[0065] Table 7 Changes in relative abundance of main bacterial classes in rice rhizosphere after treatment with composite microbial agents

[0066] MetagenomeSeq analysis was further completed, and the results were as follows Fig.12 As 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 disease-resistant, growth-promoting and yield-increasing bacterial strain, characterized in that: The bacterial species is Pseudomonas Pseudomonas sp. GM3), or, Pseudomonas and Bacillus velezensis ( Bacillus velezensis NB21); among which, Pseudomonas Pseudomonas sp. GM3, deposited in China Center for Type Culture Collection on October 31, 2024, with the deposit number: CCTCC NO: M20242391; Bacillus Velez Bacillus velezensis NB21 was deposited in the China Center for Type Culture Collection on May 17, 2024, with the deposit number: CCTCC NO: M2024979.

2. An application of the disease-resistant, growth-promoting and yield-increasing bacterial strain according to claim 1, characterized in that: The bacterial species Pseudomonas, or Pseudomonas and Bacillus Velezii are used in nutrient supply, 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 nutrient supply refers to nitrogen fixation, phosphorus solubilization, and iron transport.

4. 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.

5. 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 of yield refers to promoting the development of crop root system, enhancing photosynthesis, increasing crop biomass, and improving the crop 100-grain weight or 1,000-grain weight and unit yield.

6. A microbial agent, characterized in that: The bacterial agent contains the single or composite active bacterial strain as described in claim 1.

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

8. The microbial agent according to claim 7, 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.

9. The microbial agent according to claim 6, characterized in that: 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 Velezii NB21 is 2×10 8 CFU / mL or above; 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.

10. An application of the microbial agent according to claim 6, characterized in that: The bacterial agent is used as a biological seed coating agent, a microbial pesticide, a microbial fertilizer, a biological organic fertilizer or a soil conditioner.

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