Climate adaptive microbial organic fertilizer and application thereof

Abstract

The invention relates to a climate-adaptive microbial organic fertilizer and application thereof, which can promote the absorption and utilization of nitrogen and phosphorus nutrient elements by plants and enhance the disease resistance of the plants by remarkably up-regulating the expression of specific functional genes in the plants, and has wide application prospects in agricultural production.

Description

Technical Field

[0001] This application belongs to the field of agricultural biotechnology, specifically relating to a climate-adaptive microbial organic fertilizer and its application. Background Technology

[0002] While traditional chemical fertilizer application can increase rice yields in the short term, long-term excessive application can lead to soil compaction, decreased fertility, and environmental pollution, hindering sustainable agricultural development. Therefore, exploring green, environmentally friendly, and efficient compound fertilizers (such as organic fertilizers and microbial fertilizers) is particularly urgent.

[0003] Traditional research focuses on the direct effects of compound fertilizers on plant growth phenotypes, often resulting in inconsistent effects depending on the application of compound fertilizers to crops under different growth conditions. Without understanding the intrinsic mechanisms of plant responses to fertilizers, such as which functional genes are regulated by fertilizers, it is difficult to identify the root causes of these differences and find quick solutions. However, studying the effects of compound fertilizers on plant gene regulation and combining this with phenotypic analysis can deepen our understanding of plant growth mechanisms, offering far-reaching application value and providing crucial theoretical support for precision agriculture, genetic engineering, and sustainable agricultural development.

[0004] Rice contains tens of thousands of functional genes. Among them, the genes OsMGD2 and OsMGD3 encode monogalactosylglycerol synthase (MGD), which mainly participates in the synthesis of galactolipases (MGDG and DGDG) in non-photosynthetic tissues (such as roots and flowers), thereby improving photosynthetic efficiency and biomass. The gene OsSAE1a is involved in the absorption, transport, and redistribution of phosphorus and nitrogen, and may play a key role in rhizosphere phosphorus activation and nitrogen assimilation. The gene OsSIZ2 regulates nitrogen metabolism-related genes (such as nitrate transporters and glutamine synthase), affecting nitrogen absorption, transport, and redistribution in the later stages of growth. The gene OsNR1 encodes nitrate reductase, a core enzyme in nitrogen metabolism, catalyzing the conversion of nitrate to nitrite and enhancing carbon-nitrogen co-operational efficiency. Summary of the Invention

[0005] To address the aforementioned technical problems, this application first provides a strain of *Pseudomonas* sp. SJA1, which was deposited on November 11, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26128. The address of the depository is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The inventors further performed whole-genome sequencing on SJA1, confirming that SJA1 is *Pseudomonas atacamensis*, and the constructed phylogenetic tree is as follows: Figure 12 As shown.

[0006] This application also provides a biological agent comprising one or more of the following: bacterial culture, bacterial suspension, bacterial powder, bacterial fermentation product (such as fermentation broth), bacterial culture or filtrate of the culture, or bacterial metabolites of the above-mentioned Pseudomonas sp. SJA1.

[0007] This application also provides a complex microbial community, including the aforementioned Pseudomonas sp. SJA1 and Bacillus megaterium JSY3; the JSY3 was deposited on August 10, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28153 (CN117586914B), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. In some embodiments, SJA1 and JSY3 are mixed in an equal ratio (e.g., 1:1), where the ratio can be a mass ratio or a volume ratio.

[0008] This application also provides a biological agent comprising the above-mentioned complex microbial community, and one or more mixtures of the microbial community's bacterial suspension, microbial metabolites, microbial fermentation products (such as fermentation broth), microbial culture or filtrate of the culture, or microbial metabolites.

[0009] In some embodiments, the biological agent can be a suitable formulation such as a microbial agent or fertilizer. In some embodiments, the microbial agent is an antibacterial agent (e.g., an antibacterial agent against bacterial blight); or the fertilizer is a compound fertilizer made from microbial fertilizer, organic fertilizer, and / or inorganic fertilizer. In some specific embodiments, the fertilizer is a compound fertilizer prepared by mixing organic fertilizer and microbial fertilizer. In some specific embodiments, the fertilizer is a compound fertilizer prepared by mixing inorganic fertilizer and microbial fertilizer. In some specific embodiments, the compound fertilizer is applied directly to the plant (e.g., rice). In some specific embodiments, the microbial fertilizer is applied to the plant (e.g., rice) sequentially with other fertilizers (e.g., organic fertilizer or inorganic fertilizer). In some specific embodiments, the application is performed at the root or above ground (e.g., foliar spraying).

[0010] This application also provides a microbial agent, including the aforementioned Pseudomonas or complex microorganisms and acceptable adjuvants.

[0011] In some embodiments, the adjuvant is, for example, a culture medium or microbial protectant suitable for the growth of the complex microbial community.

[0012] Examples of microbial protectants include alginate oligosaccharides, burdock oligosaccharides, vegetable oils, tetrahydropyrimidine, fumaric acid, etc.

[0013] This application also provides a method for upregulating the expression levels of OsMGD2, OsMGD3, OsSIZ2, OsSAE1a, and / or OsNR1 genes in plants, or a method for promoting plant growth, or a method for promoting nitrogen and phosphorus accumulation in plants, or a method for promoting plant resistance to bacterial blight, comprising applying a complex microbial community or applying a biological agent containing said complex microbial community; said complex microbial community includes a strain of Pseudomonas sp. SJA1 and Bacillus megaterium JSY3; said SJA1 was deposited on November 11, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26128, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; said JSY3 was deposited on August 10, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC. No. 28153, deposited at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0014] In some implementations, the OsMGD2, OsMGD3, OsSIZ2, OsSAE1a and / or OsNR1 genes are located in the leaves or roots of the plant.

[0015] In some implementations, promoting plant growth means promoting the biomass of both the aboveground and underground parts of the plant.

[0016] In some implementations, the aboveground and underground biomass includes one or more of plant height, fresh or dry weight of leaves, and fresh or dry weight of roots.

[0017] In some implementations, promoting nitrogen and phosphorus accumulation in plants means promoting nitrogen and phosphorus accumulation in plant leaves and roots.

[0018] In this field, obtaining bacterial solutions, suspensions, powders, fermentation products (such as fermentation broth), cultures or filtrates of single or complex bacterial groups is a conventional technique. Nevertheless, this application provides some examples: Single-cell bacterial solutions are prepared by inoculating activated strains SJA1 and JSY3 separately into liquid culture media; these solutions are centrifuged and resuspended in sterile water to obtain single-cell suspensions; the bacterial cells obtained after centrifuging these solutions or suspensions are dried and ground into powder to obtain single-cell powder; SJA1 and JSY3 are inoculated separately or simultaneously into fermentation media to obtain single-cell fermentation broth or fermentation broth of a complex bacterial group; single-cell bacterial solutions or suspensions of SJA1 and JSY3 are mixed in equal volumes, or single-cell powders of the two strains are mixed in equal masses to obtain a complex bacterial agent or biological preparation. The complex bacterial agent or biological preparation can be a solid or liquid product.

[0019] Microbial fertilizers include the aforementioned Pseudomonas or complex microbial communities.

[0020] This application also provides a compound fertilizer prepared using the above-mentioned biological agent.

[0021] This application also provides fertilizers prepared from the aforementioned Pseudomonas or complex microbial communities. In some embodiments, the fertilizer is a compound fertilizer. In some embodiments, the fertilizer includes the aforementioned microbial fertilizer, organic fertilizer, and / or inorganic fertilizer.

[0022] In some embodiments, the inorganic fertilizer is inorganic nitrogen fertilizer, inorganic phosphate fertilizer, and / or inorganic potassium fertilizer. In some embodiments, the inorganic nitrogen fertilizer includes urea and / or ammonium sulfate; in some embodiments, the inorganic phosphate fertilizer includes monoammonium phosphate and / or diammonium phosphate; in some embodiments, the inorganic potassium fertilizer includes potassium sulfate and / or potassium chloride.

[0023] This application also provides an antibacterial agent prepared using the above-mentioned biological agents, such as an antibacterial agent for combating bacterial blight.

[0024] Since the OsMGD2, OsMGD3, OsSIZ2, OsSAE1a, or OsNR1 genes are ubiquitous in plant tissues and have similar functions, the research results on the expression of these genes in this application are expected to be extended to more plant types besides rice. Plants can be monocotyledonous or dicotyledonous, herbaceous or woody, crops (e.g., rice), vegetable plants, or fruit plants, etc.

[0025] This application also provides the application of the above-mentioned bacteria, compound microbial groups, microbial agents, compound microbial agents, biological agents, compound fertilizers, or antibacterial agents in the field of agricultural planting, especially in the planting of crops (such as rice), vegetables, or fruits.

[0026] This application also provides a plant growth promoter prepared from the aforementioned bacteria, compound microbial communities, microbial agents, compound microbial agents, biological agents, the aforementioned compound fertilizers, or the aforementioned antibacterial agents. The plant growth promoter can be used, for example, to promote the biomass of the aboveground and underground parts of rice.

[0027] In some embodiments, the aboveground and underground biomass is at least one of the following: plant height, fresh or dry weight of leaves, and fresh or dry weight of roots.

[0028] This application also provides the use of the above-mentioned biological agent, the above-mentioned compound fertilizer, or the above-mentioned antibacterial agent in increasing the expression levels of the OsMGD2, OsMGD3, OsSIZ2, OsSAE1a and / or OsNR1 genes in rice leaves and roots.

[0029] This application also provides the use of the above-mentioned biological agent, the above-mentioned compound fertilizer, or the above-mentioned antibacterial agent in promoting the accumulation of nitrogen and phosphorus in rice leaves and roots.

[0030] This application also provides the use of the above-mentioned biological agent, the above-mentioned compound fertilizer, or the above-mentioned antibacterial agent in promoting rice resistance to bacterial blight.

[0031] This application also provides the use of the above-mentioned compound bacteria, biological agents, and compound fertilizer in promoting the cold or drought resistance of rice.

[0032] The compound bacteria, biological agents, and compound fertilizers of this application also have significant nitrogen emission reduction effects.

[0033] This application also provides the use of SJA1 or JSY3 single strains, for example, to promote rice growth; to increase the dry weight, fresh weight, nitrogen and phosphorus accumulation and nitrogen and phosphorus content of rice roots; and / or to increase the dry weight, fresh weight, nitrogen and phosphorus accumulation and nitrogen and phosphorus content of rice leaves; and / or to increase plant height; and / or to increase the expression levels of OsMGD2, OsMGD3, OsSIZ2 and / or OsSAE1a genes in rice (roots and leaves). Attached Figure Description

[0034] Figure 1 Rice plant height under different treatments.

[0035] Figure 2 Fresh weight and dry weight of rice leaves under different treatments.

[0036] Figure 3 Fresh and dry weight of rice roots under different treatments.

[0037] Figure 4 Nitrogen and phosphorus accumulation in rice leaves under different treatments.

[0038] Figure 5 Nitrogen and phosphorus accumulation in rice roots under different treatments.

[0039] Figure 6 Relative expression levels of the OsMGD2 gene in rice leaves and roots under different treatments.

[0040] Figure 7 Relative expression levels of the OsMGD3 gene in rice leaves and roots under different treatments.

[0041] Figure 8 Relative expression levels of the OsSIZ2 gene in rice leaves and roots under different treatments.

[0042] Figure 9 Relative expression levels of the OsSAE1a gene in rice leaves and roots under different treatments.

[0043] Figure 10 Relative expression levels of the OsNR1 gene in rice leaves and roots under different treatments.

[0044] Figure 11 Determination of the growth capacity of Bacillus subtilis after JS and MC treatment.

[0045] Figure 12 : SJA1 phylogenetic tree.

[0046] Figure 13 JavaScript processing promotes cold resistance in rice.

[0047] Figure 14 Under drought stress, JS treatment promotes rice growth and nutrient synthesis.

[0048] Figure 15 : Under drought stress, JS treatment promotes root development in rice.

[0049] Figure 16 SJA1 or JSY3 single bacteria can increase the dry weight and fresh weight of rice roots.

[0050] Figure 17 SJA1 or JSY3 single bacteria can increase nitrogen and phosphorus accumulation in rice roots.

[0051] Figure 18 SJA1 or JSY3 single bacteria can increase the nitrogen and phosphorus content in rice roots.

[0052] Figure 19 SJA1 or JSY3 single bacteria can increase the dry weight and fresh weight of rice leaves.

[0053] Figure 20 SJA1 or JSY3 single bacteria can increase nitrogen and phosphorus accumulation in rice leaves.

[0054] Figure 21 SJA1 or JSY3 single bacteria can increase the nitrogen and phosphorus content in rice leaves.

[0055] Figure 22 SJA1 or JSY3 single bacteria can increase plant height.

[0056] Figure 23 SJA1 or JSY3 single bacteria increased the expression of the OsSAE1a gene in rice roots and leaves.

[0057] Figure 24 SJA1 or JSY3 single bacteria increased the expression of the OsMGD3 gene in rice roots and leaves.

[0058] Figure 25SJA1 or JSY3 single bacteria increased the expression of the OsMGD2 gene in rice roots and leaves.

[0059] Figure 26 SJA1 or JSY3 single bacteria increased the expression of the OsSIZ2 gene in rice roots and leaves.

[0060] Figure 27 Data on the effects of various bacterial treatments on rice roots.

[0061] Figure 28 Data on the effects of various bacterial treatments on rice leaves. Detailed Implementation

[0062] To illustrate the universal design concept of this application, rice is used as an example below, but this should not be used as a reason to limit the scope of protection of this application.

[0063] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0064] Example 1 Experimental Design

[0065] I. Experimental Materials:

[0066] Rice variety: Huruan 1212 was selected, which is a common rice variety in this region and has a certain degree of representativeness and adaptability.

[0067] Soil: The soil used in the experiment was taken from paddy field soil of the control (CK) in the Qingpu Modern Agricultural Park to ensure a consistent soil background and reduce the impact of soil differences on the experimental results. The basic physicochemical properties of the soil before the experiment were: total nitrogen 1.70 g·kg⁻¹ -1 Total phosphorus 0.95 g·kg -1 0.11 g / kg of readily available potassium -1 Organic matter 29.60 g·kg -1 The pH value is 7.02.

[0068] Organic fertilizer: provided by Qingpu Modern Agricultural Park, rich in various organic matter and nutrients, providing basic nutrients for rice growth.

[0069] Microbial fertilizers: ① Compound microbial fertilizer: SJA1 Pseudomonas and JSY3 were used in combination to explore their synergistic effect. ② Commercial Bacillus (fertilizer) and commercial Pseudomonas (fertilizer): Commercially available products were used as a comparison to evaluate the relative advantages of compound microbial fertilizers.

[0070] II. Experimental Design: This study employed a pot experiment method, setting up 7 different treatment combinations, with 3 replicates for each treatment, for a total of 21 potted plants. Details are as follows:

[0071] Blank treatment (CK): Organic fertilizer from Qingpu Modern Agricultural Park was applied as a control group to evaluate the relative effects of other treatments.

[0072] Microbial fertilizer treatment (JS): Organic fertilizer from Qingpu Modern Agricultural Park was applied, and simultaneously, Bacillus megaterium microbial fertilizer JSY3 (CGMCC No. 28153) and Pseudomonas microbial fertilizer SJA1 (CGMCC No. 26128) were added to explore the growth-promoting effect of compound microbial fertilizer.

[0073] Microbial fertilizer treatment (JC): Organic fertilizer from Qingpu Modern Agricultural Park was applied, along with JSY3 Bacillus megaterium and commercial Pseudomonas aeruginosa. The differences between compound microbial fertilizer and commercial microbial fertilizer were compared.

[0074] Microbial fertilizer treatment (SC): Organic fertilizer from Qingpu Modern Agricultural Park was applied, combined with commercially available Bacillus subtilis fertilizer and SJA1 Pseudomonas aeruginosa, to further compare the effects of different microbial fertilizer combinations.

[0075] Microbial fertilizer treatment (MC): Apply organic fertilizer from Qingpu Modern Agricultural Park, and add commercially purchased Bacillus spp. fertilizer and commercially purchased Pseudomonas spp. as a representative treatment of commercial microbial fertilizer.

[0076] Microbial fertilizer + bacterial blight treatment (JS+B): Based on the microbial fertilizer treatment (JS), bacterial blight pathogens were inoculated to evaluate the disease resistance effect of the compound microbial fertilizer.

[0077] Microbial fertilizer + bacterial blight treatment (MC+B): Based on the microbial fertilizer treatment (MC), bacterial blight pathogens were inoculated, and the disease resistance of different microbial fertilizer treatments was compared.

[0078] Microbial fertilizer treatment (S): Organic fertilizer from Qingpu Modern Agricultural Park was applied, and SJA1 was added to examine the effect of single-strain SJA1.

[0079] Microbial fertilizer treatment (J): Organic fertilizer from Qingpu Modern Agricultural Park was applied, and JSY3 was added to investigate the effect of single-strain JSY3.

[0080] Example 2 Experimental Method

[0081] The collected fresh soil was manually removed of residue and then air-dried. It was then sieved through a 20-mesh sieve to remove larger particles and impurities. White plastic buckets were selected as potting containers, with a bottom diameter of 13.5 cm and a height of 16 cm. Before rice transplanting, according to the treatment groups, each bucket was filled with 1.5 kg of sieved dry soil and thoroughly mixed with organic fertilizer. The application rate of organic fertilizer was calculated based on the standard of 240 kg·hm⁻² N to ensure consistent nutrient supply across treatments. After filling the pots, the soil was watered one day in advance to allow it to fully absorb water and reach a suitable moisture level. For rice transplanting, healthy rice seedlings with uniform growth were selected, with three seedlings per hole per pot. Care was taken to maintain the integrity of the seedling root system during transplanting, avoiding damage to facilitate rapid recovery and growth in the pots.

[0082] The activated strains JSY3 and SJA1 were inoculated into LB liquid medium and cultured on a shaker at 140 rpm for 24 hours. Next, 3 liters of LB liquid medium were prepared and sterilized to ensure sterility of the culture environment. Then, 50 ml of the bacterial culture was added to the 3 liters of LB liquid medium and transferred to a fermenter for further shaking culture. In the fermenter, the bacterial culture was cultured under suitable conditions for three days to promote the proliferation of the strains. After fermentation, the bacterial culture was poured into sterile bottles for later use. JSY3 and SJA1, JSY3 and commercial Pseudomonas, and SJA1 and commercial Bacillus were mixed in a 1:1 ratio to prepare a solution. 25 ml of the mixed bacterial solution was applied to each rice plant twice, once on the first day and once on the seventh day after transplanting, for a total of two applications.

[0083] For disease inoculation, when the rice reaches the appropriate growth stage (about two weeks after transplanting), the JS+B and MC+B treatments are inoculated by spraying. The bacterial blight pathogen solution with OD600=1.5 is evenly sprayed onto the rice leaves. After three days, observations are made and samples are taken to determine the bacterial growth capacity.

[0084] Throughout the growing season, do not drain the water; maintain a water level of 1-3 cm in the pot. Regularly check the water level of the potted plants and replenish water as needed to meet the water requirements for rice growth. Regularly record the growth status of the rice and check for the occurrence of rice diseases, and take photos of the leaf morphology for subsequent analysis of rice growth and development. Calculate the disease index based on the severity of disease occurrence to evaluate the disease resistance effects of different treatments.

[0085] The experiment concluded at the tillering stage of the rice potted plants, during which rice samples were collected. Rice leaves and roots were used to analyze the nitrogen and phosphorus content in the plants and to detect the expression of nitrogen and phosphorus-related genes using qPCR technology, providing fundamental data for further research.

[0086] The physicochemical properties of roots and leaves were determined as follows: total phosphorus was determined by the molybdenum-antimony colorimetric method; total nitrogen was determined by the Kjeldahl method.

[0087] To study the expression of functional genes in roots and leaves: Total RNA was extracted from rice leaves and roots using a plant rapid extraction kit from Beijing Bestgen Biotechnology Co., Ltd. RNA concentration was detected using a NanoDrop 2000 spectrophotometer, and the quality of total RNA was assessed by agarose gel electrophoresis. 0.5–1 µg of total RNA was reverse transcribed into cDNA. Using cDNA as a template, qRT-PCR was performed to determine the relative expression levels of nitrogen and phosphorus-related genes. The reaction volume was 20 µl, and the reaction program was: 95℃ for 30 s; 95℃ for 10 s; 60℃ for 30 s; 40 cycles. Each sample underwent qRT-PCR in triplicate. The relative gene expression levels were calculated using the 2-ΔΔCt method. OsActin and OsUbq were used as internal control genes.

[0088] Method for determining bacterial growth capacity: The bacterial suspension was resuspended in 10 mM MgCl2 and diluted to an OD600 of 0.2. The OD600=0.2 suspension was then diluted 1000-fold. Rice leaves were placed in 1.2 ml of 10 mM MgCl2 solution with three sterile steel balls added. The sample was then ground in an automated sample grinder for 30 seconds at a frequency of 50 Hz, repeated six times. After grinding, 40 µl of the bacterial suspension was taken and diluted in 160 µl of 10 mM MgCl2 solution. This serial dilution was repeated until the bacterial concentration was suitable for counting. 10 µl of each concentration of bacterial suspension was added to NA medium. After the medium was clear of visible liquid, it was sealed and incubated at 28°C for 40 h. Bacterial numbers were expressed as colony forming units (CFU). Counts were performed at concentration gradients within the CFU range of 20-100, with three replicates for each concentration gradient.

[0089] Rice disease statistics method: 15 days after spray inoculation, the diseased area of ​​rice leaves was counted, with at least 15 leaves counted per rice plant. The leaves were graded and recorded according to the severity of the diseased area. Based on the survey results, the rice disease index was calculated as follows: Disease Index = [(Number of diseased leaves at each grade * Relative grade value) / (Total number of leaves surveyed * 11)] * 100.

[0090] Example 3: Rice growth and the response of functional genes OsMGD2 and OsMGD3

[0091] I. Effects on functional genes:

[0092] Relative expression levels of the OsMGD2 gene under different treatments: from Figure 6As can be seen, in leaves, the relative expression level of the OsMGD2 gene was higher in both the JSY3+SJA1 treatment and the SJA1+commercial bacteria treatment, significantly higher than that in the commercial microbial fertilizer treatment. In roots, the relative expression level of the OsMGD2 gene was also highest in the JSY3+SJA1 treatment, and the relative expression level in the SJA1+commercial bacteria treatment was also significantly higher than that in the commercial microbial fertilizer treatment.

[0093] Relative expression levels of the OsMGD3 gene under different treatments: from Figure 7 As can be seen, the expression level of the OsMGD3 gene in the leaves was highest in the JSY3+SJA1 treatment, which was significantly higher than that in the commercial microbial fertilizer treatment. The relative expression levels of JSY3+commercial bacteria and SJA1+commercial bacteria were also significantly higher than those in the commercial microbial fertilizer treatment. Similarly, in the roots, the relative expression level of the JSY3+SJA1 treatment was the highest, which was significantly higher than that of other treatments.

[0094] II. Impact on rice growth:

[0095] Rice plant height under different treatments: such as Figure 1 As shown, among all treatments, the JS treatment had the longest plant height (36.4 cm), and both the JS and SC treatments were significantly taller than the other groups. The JC treatment had the shortest plant height (22.9 cm), slightly shorter than the CK treatment. The JS treatment significantly promoted rice seedling growth, while JC, SC, and MC treatments did not increase plant height as much as the JS treatment. The JS treatment showed a highly significant difference compared to the MC treatment, indicating that the JS treatment was significantly more effective than the MC treatment in promoting rice seedling growth.

[0096] Fresh weight and dry weight of rice leaves under different treatments: from Figure 2 As can be seen, the JS treatment showed significantly higher fresh and dry weights of rice leaves than other groups, indicating that the JS treatment had a significant growth-promoting effect on rice leaves. The CK treatment had the lowest fresh and dry weights of rice leaves and served as a control group. The JC, SC, and MC treatments all showed higher fresh and dry weights of rice leaves than the CK treatment, but lower than the JS treatment, indicating that commercial microbial fertilizers were less effective at promoting plant growth than the JS treatment. There was a significant difference between the JS treatment and the MC treatment.

[0097] Fresh weight and dry weight of rice roots under different treatments: from Figure 3 The results show that the JS treatment had higher root fresh weight and dry weight than other groups, and significantly higher than the CK treatment, indicating that the JS treatment had the best effect on promoting root growth in rice. The JC treatment had a similar root fresh weight to the CK treatment, but a significantly higher dry weight. The SC treatment had both higher root fresh weight and dry weight than the CK treatment, but lower than the JS treatment. The MC treatment had root fresh weight and dry weight similar to the CK treatment, indicating that commercial microbial fertilizers had no significant effect on promoting root growth in rice. Applying JSY3 or SJA1 bacteria in combination with commercial bacteria could still show a growth-promoting effect, and there was a highly significant difference between the JS treatment and the MC treatment.

[0098] Example 4: Nitrogen and phosphorus accumulation in rice and the response of functional genes OsSIZ2, OsSAE1a, and OsNR1.

[0099] I. Effects on functional genes:

[0100] Relative expression levels of the OsSIZ2 gene under different treatments: from Figure 8 As can be seen from the data, in the leaves, the relative expression level of the OsSIZ2 gene was the highest in the JSY3+ commercial microbial fertilizer treatment, which was significantly higher than that in the commercial microbial fertilizer treatment. The relative expression level of the JSY3+SJA1 treatment was also significantly higher than that in the commercial microbial fertilizer treatment. In the roots, the expression level of the OsSIZ2 gene was the highest in the JSY3+SJA1 treatment, which was significantly higher than that in other treatments.

[0101] Relative expression levels of the OsSAE1a gene under different treatments: from Figure 9 As can be seen from the data, in the leaves, the relative expression level of the OsSAE1a gene was the highest in the JSY3+SJA1 treatment and the SJA1+commercial bacteria treatment, which was significantly higher than other treatments. In the roots, the relative expression level of the OsSAE1a gene in the JSY3+commercial bacteria treatment was significantly higher than other treatment groups. There was no significant difference between the JSY3+SJA1 treatment and the commercial microbial fertilizer treatment.

[0102] Relative expression levels of the OsNR1 gene under different treatments: from Figure 10 As can be seen from the data, in the leaves, the relative expression level of the OsNR1 gene was the highest in the SJA1+ commercial bacteria treatment, which was significantly higher than that of other treatments. The relative expression level of the gene in the JSY3+SJA1 treatment was significantly higher than that in the commercial microbial fertilizer treatment. In the roots, the relative expression level of the gene in the SJA1+ commercial bacteria treatment was the highest, which was significantly higher than that in the commercial microbial fertilizer treatment and the CK treatment.

[0103] II. Nitrogen and phosphorus accumulation in rice:

[0104] Nitrogen and phosphorus accumulation in rice leaves under different treatments: From Figure 4 The results showed that the JS treatment had the highest nitrogen and phosphorus accumulation in leaves, significantly higher than other treatments. The CK treatment had the lowest nitrogen and phosphorus accumulation and served as a control group. The MC treatment showed higher nitrogen accumulation in leaves than the CK treatment; while the JC, SC, and MC treatments showed a more significant increase in phosphorus accumulation in leaves than the CK treatment. This indicates that commercial organic microbial fertilizers promote nitrogen and phosphorus accumulation in rice leaves, but not as much as the JS treatment, which significantly increased nitrogen and phosphorus accumulation in rice leaves.

[0105] Nitrogen and phosphorus accumulation in rice roots under different treatments: From Figure 5It was observed that the JS treatment resulted in the highest nitrogen and phosphorus accumulation in rice roots, showing a significant difference. The CK and MC treatments showed lower nitrogen and phosphorus accumulation in rice roots. The JC and SC treatments showed higher nitrogen and phosphorus accumulation in rice roots than the CK and MC treatments, but lower than the JS treatment. This indicates that commercial microbial fertilizers may have a significant effect on nitrogen and phosphorus accumulation in rice roots, but their effect on increasing root nitrogen and phosphorus accumulation is not significant. The JS treatment showed significant differences from other treatments, while the JC and SC treatments may have shown an ability to increase nitrogen and phosphorus accumulation in rice roots due to the combination of commercial microorganisms with the JSY3 or SJA1 microorganisms studied in this study.

[0106] Example 5: Disease resistance of rice

[0107] The experiment involved inoculating *Bacillus thuringiensis*, the causal agent of bacterial blight, and the growth capacity of *Bacillus thuringiensis* after treatment with JS and MC was determined. The results are shown in Table 1. Figure 11 The results show that the bacterial blight pathogen grew in significantly higher numbers in the MC treatment than in the JS (JSY3+SJA1) treatment. This indicates that compound microbial fertilizer has a significant effect on enhancing the disease resistance of rice.

[0108] Table 1 Results of the White Leaf Blight Disease Index

[0109] deal with Disease index JSY3+SJA1 5.32 MC 8.25

[0110] In summary, the combined application of organic fertilizer and microbial fertilizer can significantly improve the growth indicators and nutrient content of rice, while also enhancing its disease resistance. The JS treatment group, in particular, showed significant advantages in promoting rice growth and increasing nutrient content.

[0111] Example 6: Rice Cold Resistance Induction Experiment

[0112] 1. Experimental Design and Materials: This embodiment aims to verify the effect of the microbial organic fertilizer (hereinafter referred to as JS) described in this application on inducing low-temperature resistance in crops. Experimental Groups: Three treatment groups were set up: Treatment Group (JS): inoculated with the self-made compound microbial agent described in this application + organic fertilizer; Control Group 1 (MC): inoculated with a similar commercial compound microbial agent + organic fertilizer; Control Group 2 (CK): only applied with an equal amount of organic fertilizer. Replication Setup: Each treatment was set up with 4 replicates.

[0113] 2. Test methods:

[0114] Seedling raising and transplanting: Seedlings are raised in artificial climate incubators. After the rice grows to the "three leaves and one heart" stage, they are transplanted into 3L pots.

[0115] Low-temperature stress treatment: After transplanting, the plants were first acclimatized to the lower environment for 7 days at 15°C. Then, all plants were transferred to a low-temperature stress environment at 10°C for 7 days.

[0116] Index determination: After the stress treatment ended, the plant's growth indicators (leaf fresh weight, root fresh weight, plant height, root length), physiological indicators (SPAD value) and phenotypic traits were comprehensively evaluated.

[0117] 3. Experimental Results and Analysis:

[0118] Table 2:

[0119] CK JS MC Fresh leaf weight (g) 0.32±0.03 0.40±0.08 0.37±0.03 Fresh root weight (g) 0.17±0.02 0.29±0.03 0.23±0.02 Plant height (cm) 21.1±2.1 27.4±2.46 26±1.25 Root length (cm) 4.47±0.25 5.06±0.25 4.57±0.35 SPAD value 8.37±0.31 7.87±0.42 8.13±1.13

[0120] The results showed a significant improvement in growth indicators: the JS treatment significantly promoted plant growth under low-temperature stress. Compared with the CK group, the leaf fresh weight, root fresh weight, plant height, and root length of the JS treatment group increased by approximately 25%, 70%, 30%, and 13%, respectively; the leaf fresh weight, root fresh weight, root length, and plant height of the MC treatment group were also lower than those of the JS treatment group. Especially in terms of root development, the root fresh weight of the JS treatment group increased by 70%, which was significantly better than the MC and CK treatments. This indicates that the compound microbial agent described in this application can significantly enhance root biomass accumulation and improve root vitality under low temperatures, which is key to the plant's climate adaptability. In addition, under continuous stress at 10°C, the control group (CK) and the commercial microbial fertilizer group (MC) plants showed obvious leaf wilting and growth stagnation; while the JS treatment group plants showed less wilting, full growth, and significant low-temperature tolerance. Although the SPAD values ​​(relative chlorophyll content) did not differ significantly among the different treatments, the overall biomass indicators demonstrated that the JS treatment effectively alleviated the inhibition of photosynthetic product allocation by low temperature (see Table 2 and ...). Figure 13 ).

[0121] 4. Experimental conclusions: The microbial organic fertilizer described in this application shows a better mitigation effect under low temperature stress. It can significantly enhance the cold resistance of plants by promoting root development and overall biomass accumulation, and has excellent climate adaptability characteristics.

[0122] Example 7: Rice drought resistance induction experiment

[0123] 1. Experimental Design and Materials: This embodiment aims to verify the drought resistance induction effect of the microbial organic fertilizer (hereinafter referred to as JS) described in this application on crops.

[0124] Experimental Groups: The treatments were consistent with the cold resistance test. Three treatment groups were set up: Treatment Group (JS): inoculated with the self-made compound microbial agent described in this invention + organic fertilizer; Control Group 1 (MC): inoculated with a similar commercial compound microbial agent + organic fertilizer; Control Group 2 (CK): only applied with an equal amount of organic fertilizer. Replication Setup: Each treatment was set up with 4 replicates.

[0125] 2. Test methods:

[0126] Seedling raising and transplanting: Seedlings are raised in artificial climate incubators. After the rice grows to the "three leaves and one heart" stage, they are transplanted into 3L pots.

[0127] Drought stress treatment: After transplanting, the plants were first cultivated at 25°C for 7 days to allow them to regrow and adapt to the environment, maintaining a water level of 2 cm during cultivation. After 7 days, the water was allowed to dry naturally, and the plants were kept dry for another week before relevant indicators were measured.

[0128] Index determination: After the stress treatment ended, the plant's growth indicators (leaf fresh weight, root fresh weight, plant height, root length), physiological indicators (SPAD value) and phenotypic traits were comprehensively evaluated.

[0129] 2. Experimental Results and Analysis:

[0130] The results showed a highly significant increase in biomass and photosynthetic indicators: Under continuous drought stress, the JS treatment group significantly promoted plant growth and nutrient synthesis. Compared with the CK group, the leaf fresh weight, root fresh weight, plant height, and SPAD value (relative chlorophyll content) of the JS treatment group increased significantly by approximately 136%, 200%, 43%, and 256%, respectively. Of particular note was the highly significant 200% increase in root fresh weight in the JS treatment group. This indicates that the microbial fertilizer described in this application can greatly stimulate the developmental potential of the root system under drought and water scarcity conditions, promoting the formation of a larger root network and thus significantly improving water absorption efficiency to cope with drought stress. Meanwhile, phenotypic observation confirmed that under the same drought conditions, plants in the CK and MC groups (commercial microbial fertilizer) showed obvious wilting; while the JS treatment group showed significantly lower wilting, with leaves not only maintaining good extension but also a deeper leaf color (consistent with high SPAD values), demonstrating excellent physiological dehydration resistance (see Table 3). Figure 14 , Figure 15 ).

[0131] 3. Experimental Conclusion: The microbial organic fertilizer described in this application has significant advantages in alleviating growth inhibition caused by water deficit. Its core strain can effectively promote root expansion and maintain the stability of the photosynthetic system, giving plants excellent drought resistance and adaptability, and its technical effect is significantly better than existing conventional commercial microbial fertilizers.

[0132] Table 3:

[0133] CK JS MC Fresh leaf weight (g) 0.59±0.12 1.39±0.3 0.94±0.11 Fresh root weight (g) 0.22±0.05 0.66±0.17 0.38±0.04 Plant height (cm) 25.5±4.77 36.47±1.22 30.5±3.5 Root length (cm) 5.13±0.21 6.13±0.35 6±0.26 SPAD value 9.72±3.21 34.62±6.91 16.13±3.64

[0134] Example 8 Field Nitrogen Reduction Trial

[0135] 1. Experimental Design and Methods: This embodiment aims to verify the ammonia and nitrous oxide emission reduction effects of the microbial organic fertilizer (JS) described in this application under actual field production conditions. Experimental Groups: This study set up 4 treatment groups, namely: blank control group (CK): no fertilizer was applied throughout the entire growth period; conventional fertilizer group (CT): conventional chemical fertilizer was applied according to the local farmers' habits at the experimental site; commercial microbial fertilizer group (MC): based on the nitrogen application amount of CT, an equal amount of "organic fertilizer + commercial compound microbial agent" was applied; the treatment group of this application (JS): based on the nitrogen application amount of CT, an equal amount of "organic fertilizer + self-made compound microbial agent of this application" was applied.

[0136] Monitoring methods: High-frequency gas monitoring was conducted after three key fertilization periods throughout the rice growth cycle. After each fertilization, ammonia (NH3) volatilization was collected and measured for 9 consecutive days using a closed intermittent ventilation method. At the same time, nitrous oxide (N2O) emission flux was monitored every other day after fertilization using a static chamber method.

[0137] 2. Experimental Results and Analysis:

[0138] The test results showed that the conventional fertilizer treatment (CT) had the highest levels of ammonia volatilization and nitrous oxide emissions among all treatments, indicating that the traditional pure fertilizer model suffers from serious nitrogen loss and greenhouse gas emissions. The significant advantage of this application's emission reduction effect (another core innovation) is that while the addition of microbial agents in the organic fertilizer substitution system generally alleviated nitrogen loss, the self-made microbial fertilizer in this application showed unexpectedly superior results. Compared with the application of similar commercial compound microbial fertilizer (MC), the ammonia volatilization of the JS treatment in this application was further reduced by approximately 19%, and nitrous oxide emissions were further reduced by approximately 6%. This significant difference indicates that the specific strains in this application can more efficiently regulate soil carbon and nitrogen cycles and inhibit greenhouse gas and atmospheric ammonia emissions during nitrification / denitrification processes in complex field environments (see Table 4).

[0139] 3. Experimental Conclusions: Field trial results fully demonstrate that the climate-adaptive microbial organic fertilizer described in this application can not only replace chemical fertilizers and reduce agricultural non-point source pollution, but also achieve deeper levels of greenhouse gas emission reduction and nitrogen conservation based on existing commercial organic fertilizers. This technical solution demonstrates outstanding technical value in mitigating climate change (emission reduction) while improving fertilizer utilization.

[0140] Table 4. Cumulative Ammonia Volatilization Emissions from Rice under Different Models in Qingpu Paddy Fields in 2025 (Unit: kg NH) 3 / N2O hm -2 )

[0141] deal with Total ammonia volatilization Total nitrous oxide emissions Ammonia volatilization rate (%) CK 18.64±0.31b 0.89±0.04c — CT 29.58±1.23a 2.77±0.27a 4.56±0.51a MC 24.42±2.20ab 1.99±0.01b 2.41±0.92b JS 19.68±1.23b 1.88±0.03b 0.87±0.51c

[0142] Example 9: Experimental Results of Single Bacteria SJA1 and JSY3

[0143] The inventors repeated the experiments according to the methods in Examples 1-4 to demonstrate the effects of single-strain SJA1 and JSY3. The results showed that, compared with the control (CK), both SJA1 and JSY3 single-strain strains significantly increased the dry weight, fresh weight, nitrogen and phosphorus accumulation, and nitrogen and phosphorus content of rice roots (see...). Figures 16-18 , Figure 27 Both methods significantly increased the dry weight, fresh weight, nitrogen and phosphorus accumulation, and nitrogen and phosphorus content of rice leaves (see...). Figures 19-21 , Figure 28 Both methods significantly increased plant height (see...). Figure 22 Both significantly increased the expression levels of OsMGD2, OsMGD3, OsSIZ2, and / or OsSAE1a genes in rice roots and leaves (see...). Figures 23-26 In particular, the single bacterium SJA1 (S) significantly promoted the accumulation of nitrogen and phosphorus in rice roots and increased nitrogen and phosphorus content, even exceeding the effect of the compound bacterium JS (see [link to article]). Figures 17-18 , Figure 27 Those skilled in the art will understand that the results of repeated experiments cannot be exactly the same as those of previous experiments; slight differences are normal, but the overall trend is consistent.

[0144] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of this application. Therefore, if such modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include such modifications and variations.

Claims

1. A strain of Pseudomonas sp. SJA1, characterized in that, The SJA1 was deposited on November 11, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 26128.

2. A biological agent, characterized in that, It includes one or more of the following: bacterial culture, bacterial suspension, bacterial powder, bacterial fermentation product, bacterial culture or filtrate of the culture, or bacterial metabolites of SJA1 as described in claim 1.

3. The biological agent as described in claim 2, characterized in that, The biological agent is a microbial agent or fertilizer.

4. The biological agent as described in claim 3, characterized in that, The microbial agent is an antibacterial agent; or the fertilizer is a compound fertilizer made from microbial fertilizer, organic fertilizer and / or inorganic fertilizer.

5. A complex microbial community comprising SJA1 as described in claim 1 and Bacillus megaterium JSY3; wherein JSY3 was deposited on August 10, 2023, at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 28153.

6. The complex microbial community as described in claim 5, characterized in that, The ratio of SJA1 to JSY3 is 1:1; the ratio is a mass ratio or a volume ratio.

7. A biological agent, characterized in that: It includes the complex microbial community as described in claim 5 or 6, and one or more mixtures of the microbial community's bacterial suspension, bacterial metabolites, bacterial fermentation broth, bacterial culture or filtrate of the culture, or bacterial metabolites.

8. The use of the SJA1 of claim 1, the biological agent of any one of claims 2-4, the complex microbial community of any one of claims 5-6, or the biological agent of claim 7 in increasing the expression levels of the OsMGD2, OsMGD3, OsSIZ2, OsSAE1a and / or OsNR1 genes in rice leaves and / or roots.

9. The use of the SJA1 of claim 1, the biological agent of any one of claims 2-4, the complex microbial community of any one of claims 5-6, or the biological agent of claim 7 in increasing the dry weight, fresh weight, nitrogen and phosphorus accumulation and / or nitrogen and phosphorus content of rice leaves and / or roots.

10. The use of the complex microbial community according to any one of claims 5-6, or the biological agent according to claim 7, in promoting rice resistance to cold, drought, or bacterial blight.

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