Microbial composition for realizing nitrogen reduction and synergism of flooded rice field as well as preparation method and application of microbial composition

Abstract

The invention provides a microbial composition for realizing nitrogen reduction and synergy of a flooded rice field as well as a preparation method and application of the microbial composition, and belongs to the technical field of rice field nitrogen reduction. The microbial composition disclosed by the invention comprises cyanobacteria microspheres and pseudomonas fluorescens microspheres, wherein the mass ratio of the cyanobacteria microspheres to the pseudomonas fluorescens microspheres is (4.8-5.2): (2.8-3.2); the cyanobacteria microspheres and the pseudomonas fluorescens microspheres are independently packaged. The cyanobacteria microspheres are spread on the water surface of a rice field 7-10 days before rice transplanting; in the early tillering stage of the rice, the pseudomonas fluorescens microspheres are applied to a 5-10cm soil layer at the rhizosphere of the rice, so that the nitrogen loss amount of the flooded rice field is reduced, the nitrogen fertilizer utilization rate and the rice yield of the rice are improved, and a technical support is provided for ensuring the stable yield and yield increase of the rice under the condition that the nitrogen fertilizer application amount of the flooded rice field is reduced.

Description

Technical Field

[0001] This invention relates to the field of composite microbial technology, and in particular to a microbial combination for reducing nitrogen and increasing efficiency in flooded paddy fields, its preparation method, and its application. Background Technology

[0002] With the development of industrialization, urbanization, and agricultural modernization, the quality of the water environment has deteriorated. While industrial point source pollution has been largely controlled, agricultural non-point source pollution, particularly the runoff of chemical fertilizers from farmland and its environmental impact, has gradually attracted attention. Fertilizer application and runoff lead to the deterioration of shallow groundwater quality and eutrophication of surface water bodies, severely impacting the water environment. In southern regions, most rainfall is concentrated between June and August, coinciding with the application of base fertilizer and topdressing during the tillering or jointing stages of rice, resulting in large amounts of ammonium bicarbonate and urea applied in the fields. Large quantities of nitrogen fertilizer are particularly susceptible to loss through rainfall runoff and leaching, polluting surface and shallow groundwater.

[0003] In the current "source reduction - process interception - end-of-pipe purification" technology system, engineering measures are costly and difficult to promote, chemical inhibitors pose ecological risks, and traditional microbial technologies such as rhizobium nitrogen fixatives are unsuitable for flooded rice paddies because they rely heavily on symbiosis with leguminous plants and their activity decreases sharply in flooded and oxygen-deficient environments. Therefore, there is an urgent need to develop a green nitrogen reduction technology that combines flood adaptability, multi-pathway control capabilities, and simplified agronomic practices. Summary of the Invention

[0004] In view of this, the present invention provides a microbial combination for reducing nitrogen and increasing efficiency in flooded paddy fields, its preparation method and application, in order to solve the above problems.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a microbial combination for reducing nitrogen and increasing efficiency in flooded paddy fields. The microbial combination includes cyanobacterial microspheres and fluorescent Pseudomonas microspheres, with a mass ratio of 4.8-5.2:2.8-3.2. The cyanobacterial microspheres and fluorescent Pseudomonas microspheres are packaged separately.

[0007] Preferably, the bacterial strain loading of the cyanobacterial microspheres is 1–9 × 10⁻⁶. 8 The strain loading of the fluorescent Pseudomonas microspheres was 1–9 × 10 CFU / g. 9 CFU / g.

[0008] Preferably, the carrier for the cyanobacterial microspheres and fluorescent Pseudomonas microspheres is sodium alginate-biochar; the particle size of the cyanobacterial microspheres and fluorescent Pseudomonas microspheres is independently 1-3 mm.

[0009] The present invention also provides a method for preparing the aforementioned microbial assemblages, comprising the following steps:

[0010] (1) Inoculate the cyanobacterial seed culture into a nitrogen-free medium and ferment at 27–29℃ and 50–70 rpm for 72–96 h to obtain the cyanobacterial fermentation broth; the light conditions during cultivation are 30–50 μmol photons / (m²). 2 •s), with a light duration of 11-13 h / d, the cyanobacterial fermentation broth was centrifuged at 3500-4500 rpm for 14-16 min at 3-5℃, and the cyanobacterial cells were collected;

[0011] (2) Wash the cyanobacterial cells with PBS solution, then dehydrate them with ethanol of varying concentrations, collect the dehydrated cyanobacterial cells, mix the dehydrated cyanobacterial cells with cryoprotectant, and vacuum dry them.

[0012] (3) Inoculate the seed culture of Pseudomonas fluorescens into KB medium and ferment it at 28-32℃ and 160-200 rpm for 34-38 h to obtain the fermentation broth of Pseudomonas fluorescens; centrifuge the fermentation broth of Pseudomonas fluorescens at 3-5℃ and 7500-8500 rpm for 9-11 min to collect the cells of Pseudomonas fluorescens.

[0013] (4) After washing the Pseudomonas fluorescens cells with PBS solution, they were vacuum dried.

[0014] (5) Mix sodium alginate with biochar at a concentration of 2-3 w / v, homogenize, add CaCl2 solution at a concentration of 1.8-2.2 w / v, and solidify for 18-22 min to obtain microspheres with a diameter of 1-3 mm;

[0015] (6) The vacuum-dried bacterial cells and microspheres were mixed in PBS solution at a mass ratio of 1:9-11 and shaken at 3-5℃ and 40-60 rpm for 110-120 min to obtain cyanobacterial microspheres and fluorescent Pseudomonas microspheres.

[0016] (7) The cyanobacterial microspheres and fluorescent Pseudomonas microspheres are packaged separately in a mass ratio of 4.8-5.2:2.8-3.2 to obtain the microbial combination.

[0017] Preferably, the inoculum amount of cyanobacterial seed solution is 9-11%, and the inoculum amount of Pseudomonas fluorescens is 4-6%.

[0018] Preferably, the cryoprotectant solution comprises trehalose, monosodium glutamate, and ascorbic acid, wherein the concentrations of trehalose, monosodium glutamate, and ascorbic acid in the cryoprotectant solution are 8–12 w / v%, 0.8–1.2 w / v%, and 0.08–0.12 w / v, respectively, based on the bacterial cell mass, and the volume ratio of cyanobacterial cells to the cryoprotectant solution is 0.9–1.1:1.

[0019] Preferably, the vacuum drying temperature in steps (2) and (4) is -80 to -40°C, the vacuum degree is 0.08 to 0.12 mbar, and the vacuum drying time is 58 to 62 h.

[0020] The present invention also provides the application of the aforementioned microbial ensemble in reducing nitrogen runoff loss in paddy fields.

[0021] Preferably, the application of the microbial ensemble in reducing nitrogen runoff loss from paddy fields includes the following steps:

[0022] (1) 7-10 days before rice transplanting, apply the cyanobacterial microspheres from the microbial combination to the surface of the paddy field;

[0023] (2) In the early stage of rice tillering, fluorescent Pseudomonas microspheres from the microbial combination are applied to the soil layer of 5-10 cm in the rhizosphere of rice.

[0024] Preferably, the appropriate amount of cyanobacterial microspheres is 480-520 g / mu, and the amount of fluorescent Pseudomonas is 280-320 g / mu.

[0025] Cyanobacteria (Anabaena torulosa) can directly fix atmospheric nitrogen (N2) and convert it into ammonium (NH4). + Cyanobacteria increase soil nitrogen reserves and reduce reliance on chemical nitrogen fertilizers; they release oxygen through photosynthesis, improving the rhizosphere microenvironment of paddy fields, inhibiting the production of methane and hydrogen sulfide under strong reducing conditions, and indirectly optimizing nitrogen storage conditions; dead cyanobacterial cells release organic nitrogen and carbon, promoting soil microbial activity.

[0026] Pseudomonas fluorescens can secrete nitrification inhibitors (such as pyrroloquinoline quinone and antibiotics) to inhibit the activity of Nitrosomonas and slow down the production of ammonium (NH4)2. + ) to nitrate nitrogen (NO3) - The conversion of nitrate nitrogen into nitrogen reduces the risk of nitrate leaching; Pseudomonas fluorescens secretes growth hormones (such as IAA) and siderophores to promote rice root development and enhance nitrogen absorption capacity.

[0027] By adopting the above technical solution, the present invention has the following beneficial effects:

[0028] (1) This invention achieves a breakthrough synergistic benefit in green agricultural production in paddy field systems through the combined application of cyanobacteria and *Pseudomonas fluorescens*. In terms of environmental emission reduction, it resolves the inherent contradiction between high-yield fertilization and non-point source pollution: compared to conventional fertilization (CK1), total nitrogen runoff loss is sharply reduced by 70% (4.49 vs 15.3 kg·hm²). -1 (P<0.05), the loss of dissolved nitrogen, nitrate nitrogen and ammonium nitrogen decreased by 69.3% to 87.4% simultaneously, and the environmental efficiency coefficient reached 180.8, fundamentally breaking through the bottleneck of traditional technology that "increased production inevitably leads to increased pollution".

[0029] (2) This invention improves the nitrogen fertilizer utilization rate of rice. Under the same nitrogen input, it converts runoff nitrogen into crop-available nitrogen, resulting in a nitrogen fertilizer utilization rate increase of 87.3% (53.2% vs CK 128.4%, P<0.05) and a significant increase in grain nitrogen accumulation of 10.0% (98.2 vs 89.3 kg·hm²). -1 (P<0.05), the cumulative nitrogen efficiency coefficient (CIC) reached 91.3.

[0030] (3) This invention achieves increased rice yield, with a grain yield of 9360 kg·hm². -1 Compared with conventional fertilization, it significantly increased yield by 19.6% (P<0.05), and the yield increase and efficiency coefficient (CIC) reached 121.1. Detailed Implementation

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] The cyanobacterium (Anabaena torulosa) of this invention was purchased from Ningbo Taiste Biotechnology Co., Ltd., strain number: TISTR8014.

[0033] The Pseudomonas fluorescens strain was purchased from Ningbo Taisto Biotechnology Co., Ltd., strain number JCM5963.

[0034] The nitrogen-free culture medium consisted of: K₂HPO₄ 0.04 g / L, MgSO₄·7H₂O 0.075 g / L, CaCl₂·2H₂O 0.036 g / L, citric acid 0.006 g / L, ferric ammonium citrate 0.006 g / L, EDTANa₂ 0.001 g / L, Na₂CO₃ 0.02 g / L, and 1 mL of A5 trace element mixture, with a pH of 7.1.

[0035] A5 Trace Element Mixture: H3BO3 2.86 g / L, MnCl2·4H2O 1.81 g / L, ZnSO4·7H2O 0.222 g / L, Na2MoO4·2H2O 0.39 g / L, CuSO4·5H2O 0.079 g / L, CO(NO3)2·6H2O 0.0494 g / L.

[0036] KB medium: 20g peptone, 1.5g MgSO4·7H2O, 1.5g K2HPO4, 10mL glycerol, 1000mL water, pH=7.0.

[0037] Example 1

[0038] 1. Preparation of cyanobacterial microspheres

[0039] The cyanobacterium *Anabaena torulosa* TISTR8014 was inoculated into nitrogen-free medium and placed under white LED light (40 μmol photons / (m²)). 2 •s), light-dark cycle 12h:12h), cultured in shake flasks at 28℃ and 50rpm. When cultured to the logarithmic growth phase (OD... 600 ≈0.5), inoculated at a rate of 10% into nitrogen-free culture medium, and fermented at 28℃ and 50 rpm for 96 h to obtain cyanobacterial fermentation broth. The fermentation broth was centrifuged at 4℃ and 4000 rpm for 15 min to collect cyanobacterial cells. The cyanobacterial cells were washed three times with sterile 10 mM phosphate buffer (pH = 7.2). The cyanobacterial cells were dehydrated with ethanol of varying concentrations: 30% ethanol (v / v) suspension, incubated at 4℃ for 30 min, centrifuged at 4000 rpm for 5 min, and the cells were collected and placed in 50% ethanol. The above operation was repeated. Then, the cells were suspended in 70% ethanol and the above operation was repeated. Finally, the cells were suspended in anhydrous ethanol, incubated at 4℃ for 30 min, and centrifuged at 4000 rpm for 5 min. After centrifugation, the bacterial sludge was collected, and the water content of the fresh nitrogen-fixing cyanobacteria was measured to be 83.77–86.76%. Fresh nitrogen-fixing cyanobacteria were dehydrated and mixed with a cryoprotectant solution at a volume ratio of 1:1. The cryoprotectant consisted of 10 w / v trehalose + 1 w / v monosodium glutamate + 0.1 w / v ascorbic acid. The mixture was vacuum-dried at -40°C and 0.1 mbar for 60 h. Sodium alginate (3 w / v) and biochar were mixed at a mass ratio of 1:1 and homogenized to obtain a mixture. This mixture was then dripped into a 2% CaCl2 solution using a syringe for 20 min to solidify the mixture, adjusting the drip rate with a 20G syringe tip to crosslink it into 1–3 mm microspheres. The dried bacterial cells and microspheres were then mixed in sterile PBS (bacteria:microspheres = 1:10, w / w) and shaken at 4°C (50 rpm, 2 h) to load the cyanobacteria cells onto a sodium alginate-biochar carrier at a loading capacity of 10.8 CFU / g.

[0040] 2. Preparation of fluorescent Pseudomonas microspheres

[0041] Pseudomonas fluorescens JCM5963 was inoculated into KB medium and cultured in shake flasks at 30°C and 180 rpm. When the culture reached the logarithmic growth phase (OD500), the culture was... 600 ≈0.7), inoculated at a 5% inoculum into KB medium, and fermented at 30℃ and 180 rpm for 36 h. The fermentation broth was centrifuged at 8000 rpm for 10 min at 4℃ to collect *Pseudomonas fluorescens* cells. The *P. fluorescens* cells were washed three times with sterile phosphate buffer (pH = 7.0). The water content of the fresh *P. fluorescens* sludge obtained by centrifugation was 85%. Fresh *P. fluorescens* were dehydrated under vacuum at -40℃ and then loaded onto a sodium alginate-biochar carrier (loading method same as for cyanobacteria) to form microspheres with a particle size of 1–3 mm, with a loading of 10... 9 CFU / g.

[0042] Example 2. Flooded Paddy Field Experiment

[0043] The experimental area is located at the Dali Comprehensive Experimental Station of the Environmental Protection Research Institute of the Ministry of Agriculture and Rural Affairs, Dali Bai Autonomous Prefecture, Yunnan Province (N: 25°50′01″, E: 100°07′42″, altitude: 1974.49m). The area has a northern subtropical low-latitude plateau monsoon climate, with an average annual temperature of 15.1℃, an average temperature of 8.8℃ in the coldest month, and 20.1℃ in the hottest month. The frost-free period is 230 days per year, with distinct dry and wet seasons. The rainy season is mainly concentrated from May to October, with an average annual rainfall of 1078.9 mm and an average of 136 rainy days per year. Conventional rice cultivation and management practices are followed according to local customs.

[0044] The experimental treatment is as follows:

[0045] CK0: No nitrogen fertilizer applied;

[0046] CK1: Local conventional fertilization (225 kg N·hm) -1 );

[0047] CK2: Reduce fertilizer application by 30% (157.5 kg N·hm) -1 );

[0048] Treatment 1: Cyanobacteria were applied only on top of CK2;

[0049] Treatment 2: Apply only fluorescent Pseudomonas aeruginosa to the CK2 treatment regimen;

[0050] Treatment 3: Cyanobacteria and fluorescent Pseudomonas were applied simultaneously on the basis of CK2.

[0051] Application method of cyanobacteria: 7 days before rice transplanting, apply the cyanobacterial microspheres prepared in Example 1 evenly to the paddy field surface at a rate of 500 g / mu to establish the initial population. Application method of Pseudomonas fluorescens: In the early stage of rice tillering, apply the Pseudomonas fluorescens microspheres prepared in Example 1 at a rate of 300 g / mu to the soil layer 5-10 cm deep around the rice rhizosphere.

[0052] The experimental paddy fields were divided into several plots, each with an area of ​​25m². 2 Each treatment was repeated three times, with each cell having one repeat. A 2-meter-wide protective zone was set up between adjacent cells. Each cell was equipped with an inlet and an outlet, and runoff monitoring devices were installed at both the inlet and outlet. Water samples were collected from the outlet regularly to determine the nitrogen content.

[0053] The following indicators were measured:

[0054] 1. Measurement of nitrogen runoff loss and total nitrogen loss rate in paddy fields

[0055] Drainage outlets are set up on one side of each community and connected to the collection pool. The height difference between the drainage outlets is 8cm. When it rains, if the water level in the community exceeds the drainage outlet, it is considered that one runoff is generated. The runoff flows into the collection pool of each community. The water level in the pool is measured and the runoff loss of the community is determined by volume method. At the same time, the runoff water sample is taken in a 500mL plastic bottle, stored at low temperature, and the water sample index analysis is completed within 24 hours.

[0056] Total nitrogen (TN) content in runoff samples was determined using the alkaline potassium persulfate oxidation-ultraviolet spectrophotometric method. After filtration through a 0.45 μm microporous membrane, dissolved nitrogen (DN) content in the runoff samples was determined using the alkaline potassium persulfate oxidation-ultraviolet spectrophotometric method. Nitrate nitrogen (NO3) content in the filtered runoff samples was determined using a Smart Chem200 fully automated discontinuous chemical analyzer. - -N) and ammonium nitrogen (NH4) + The content of (-N) was measured and recorded. Rainfall during the rice growing season was measured and recorded.

[0057] N runoff loss (kg·hm) -1 = Runoff volume (m³) 3 ·hm -1 ) × Nitrogen concentration in runoff (kg·m -3 ).

[0058] N runoff loss rate (%) = [N runoff loss (kg) / total nitrogen application (kg)] × 100%.

[0059] The synergistic effect coefficient (CIC value) of the microbial combinations in treatment 3 was calculated using the following formula:

[0060] The microbial consortium (CM) consists of cyanobacterium At and Pseudomonas fluorescens Ps. Each microorganism has its own effect on improving the nitrogen reduction and yield increase related indicators (the reduction or increase value compared with CK1 without applying microorganisms). The improvement index of At is set as the standard value of 100;

[0061] The improvement index of Ps = the improvement effect of Ps / the improvement effect of At × 100;

[0062] The actual improvement index of CM = the improvement effect of CM / the improvement effect of At × 100;

[0063] The theoretical improvement index of CM = the improvement index of At + the improvement index of Ps;

[0064] The synergistic coefficient CIC value = the actual improvement index of CM / the theoretical improvement index of CM × 100;

[0065] CIC ≥ 120 shows a synergistic effect; CIC ≤ 80 shows an antagonistic effect; 80 < CIC < 120 shows an additive effect.

[0066] The data of N runoff loss, N runoff loss rate, N apparent utilization rate and rice yield index measured below are subjected to multiple comparisons using the LSD test method.

[0067] The field nitrogen runoff loss amount, total nitrogen loss rate and the synergistic coefficient of the microbial consortium are shown in Table 1.

[0068] Table 1 Nitrogen runoff loss amount and TN loss rate in paddy fields

[0069]

[0070] Note: The values are the average of three replicates. Different letters in the same column indicate significant differences between treatments (P < 0.05).

[0071] The results in Table 1 show that different fertilization treatments have a significant impact on nitrogen runoff loss in paddy fields. The conventional fertilization treatment (CK1) results in the highest nitrogen loss, with a total nitrogen (TN) loss of 15.3 kg·hm -1 ,and the losses of dissolved nitrogen (DN), nitrate nitrogen (NO3 - -N) and ammonium nitrogen (NH4 + -N) are also the highest, with a total nitrogen loss rate as high as 6.80%. In contrast, the nitrogen losses of the unfertilized control (CK0) are extremely low.

[0072] Treatments 1, 2, and 3 can all significantly reduce nitrogen loss. Treatment 1 reduces the TN loss to 9.21 kg·hm -1 ,a reduction of about 40% compared with CK1, and Treatment 2 further reduces it to 8.77 kg·hm-1 Compared to CK1, it decreased by approximately 43%, and also showed improvement in reducing nitrate nitrogen loss (0.45 kg·hm). -1 Treatment 3 was superior to treatment 1. Treatment 3 showed the most significant effect, with a TN loss of 4.49 kg·hm². -1 The levels were significantly lower than all other fertilization treatments, decreasing by approximately 70% compared to CK1. Meanwhile, treatment 3 showed lower levels of DN and NO3. - -N, NH4 + The loss of -N was also the lowest, with the total nitrogen loss rate dropping to a minimum of 2.85%.

[0073] It is evident that Treatment 3 can effectively reduce the total nitrogen runoff loss and the loss of nitrogen in various forms in paddy fields, significantly reducing the total nitrogen loss rate, thereby mitigating environmental risks and improving nitrogen use efficiency. Furthermore, the combined application of cyanobacteria and *Pseudomonas fluorescens* in Treatment 3 showed a significant synergistic effect with a CIC of 180.8.

[0074] 2. Measurement of nitrogen fertilizer utilization rate and yield of rice

[0075] After the rice matured, the grains, straw, and roots were threshed and collected separately. After blanching at 105℃ for 30 minutes, they were dried at 65℃ to constant weight, and the dry matter mass of different parts was recorded. The yield per hectare was calculated based on the harvested grain mass from each plot. The samples were then crushed, passed through a 100-mesh sieve, and the nitrogen content of each part of the plant (grain and straw) was measured using the H₂SO₄-H₂O₂-distillation method. The sum of the nitrogen content in the grains, straw, and roots was the cumulative nitrogen (N), and the cumulative N in the straw was the sum of the nitrogen content in the straw and roots.

[0076] The nitrogen fertilizer utilization rate of rice was calculated, and the results are shown in Table 2. The rice yield is shown in Table 3.

[0077] Rice yield per hectare = (grain weight harvested in the plot / plot area) × 10000;

[0078] Apparent nitrogen utilization rate (%) = [(cumulative nitrogen amount of crop in nitrogen application area (kgN·hm)] -1 - Accumulated N in crops in blank areas (kgN·hm) -1 )) / Nitrogen application rate (kg N·hm) -1 )]×100%.

[0079] Table 2 Nitrogen fertilizer utilization rate of rice

[0080]

[0081] Note: The values ​​are the average of three replicates. Different letters in the same column indicate statistically significant differences between treatments (P<0.05).

[0082] The results in Table 2 show that different fertilization treatments significantly affected nitrogen fertilizer use efficiency and nitrogen accumulation in rice. The apparent nitrogen use efficiency of the conventional fertilization treatment (CK1) was 28.4%, and the total nitrogen accumulation in the aboveground parts was 134.5 kg·hm². -1 (Grain 89.3 kg·hm) -1 45.2 kg·hm of rice straw -1 The unfertilized control (CK0) had significantly lower values ​​for all indicators.

[0083] Improved fertilization treatments significantly increased nitrogen fertilizer use efficiency: the total nitrogen accumulation in treatments 1 and 2 was 139.1 kg·hm², respectively. -1 and 134.2 kg·hm -1 Treatment 3 showed no significant difference from CK1, but its apparent nitrogen use efficiency increased dramatically to 43.6% and 40.4%, respectively, representing increases of 53.5% and 42.3% compared to CK1. Treatment 3 showed the most outstanding effect, with a total nitrogen accumulation of 154.3 kg·hm² in the aboveground parts. -1 The cumulative nitrogen content of the grains was 98.2 kg·hm. -1 The cumulative nitrogen content of rice straw was 56.1 kg·hm². -1 The nitrogen apparent use efficiency (NFU) of treatment 3 was significantly higher than all other fertilization treatments, jumping to 53.2%, an increase of 87.3% compared to CK1. In treatment 3, the combined application of cyanobacteria and Pseudomonas fluorescens showed a synergistic effect (CIC) of 91.3, indicating an additive effect. Therefore, treatment 3 demonstrates its superior effectiveness in promoting nitrogen uptake and accumulation in rice and improving fertilizer use efficiency.

[0084] Table 3 Rice yield

[0085] deal with <![CDATA[Grain yield / (kg·hm -1 )]]> Production increase rate / % CIC (Cost-Increase Ratio) CK0 6280e - - CK1 8130d 29.5 - Process 1 8870b 41.2 - Process 2 8410bc 33.9 - Process 3 9360a 49.0 121.1

[0086] Note: The values ​​are the average of three replicates. Different letters in the same column indicate statistically significant differences between treatments (P<0.05).

[0087] The data in Table 3 show that different fertilization treatments had a significant impact on rice yield. The grain yield of the conventional fertilization treatment (CK1) was 8130 kg·hm². -1 Compared with the unfertilized control (CK0, 6280 kg·hm) -1 Yield increased by 29.5%; in the improved fertilization treatment, treatment 1 yielded 8870 kg·hm². -1 Compared to CK1, the yield increased by 9.1%, with a yield increase rate of 41.2%. The yield of treatment 2 was 8410 kg·hm². -1 Compared to CK1, the yield increased by 3.4%, with a yield increase rate of 33.9%. Treatment 3 performed best, with a grain yield of 9360 kg·hm². -1The yield was significantly higher than other treatments, with a 19.6% increase compared to CK1 and a 49.0% increase compared to CK0. The synergistic coefficient (CIC) of treatment 3 (combined application of cyanobacteria and fluorescent Pseudomonas) was 121.1, showing a significant synergistic effect.

[0088] As can be seen from the above embodiments, the present invention provides a microbial combination for reducing nitrogen and increasing efficiency in flooded paddy fields, its preparation method, and its application. The present invention significantly increases rice yield while achieving environmental emission reduction and efficient nitrogen utilization.

[0089] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A microbial ensemble for reducing nitrogen and increasing efficiency in flooded paddy fields, characterized in that, The microbial assemblages comprise cyanobacterial microspheres and fluorescent Pseudomonas microspheres, wherein the mass ratio of the cyanobacterial microspheres to the fluorescent Pseudomonas microspheres is 4.8–5.2:2.8–3.2; the cyanobacterial microspheres and the fluorescent Pseudomonas microspheres are packaged separately.

2. The microbial assemblages according to claim 1, characterized in that, The bacterial strain loading of the cyanobacterial microspheres is 1–9 × 10⁻⁶. 8 The strain loading of the fluorescent Pseudomonas microspheres was 1–9 × 10 CFU / g. 9 CFU / g.

3. The microbial assemblages according to claim 1, characterized in that, The carriers for the cyanobacterial microspheres and fluorescent Pseudomonas microspheres are sodium alginate-biochar; the particle sizes of the cyanobacterial microspheres and fluorescent Pseudomonas microspheres are independently 1–3 mm.

4. The method for preparing the microbial assemblage according to claim 1, characterized in that, Includes the following steps: (1) Inoculate the cyanobacterial seed culture into a nitrogen-free medium and ferment at 27-29℃ and 50-70 rpm for 72-96 h to obtain cyanobacterial fermentation broth; the light conditions during culture are 30-50 μmol photons / (m2·s) and the light time is 11-13 h / d. Centrifuge the cyanobacterial fermentation broth at 3-5℃ and 3500-4500 rpm for 14-16 min to collect cyanobacterial cells; (2) Wash the cyanobacterial cells with PBS solution, then dehydrate them with ethanol of varying concentrations, collect the dehydrated cyanobacterial cells, mix the dehydrated cyanobacterial cells with cryoprotectant solution, and vacuum dry them. (3) Inoculate the seed culture of Pseudomonas fluorescens into KB medium and ferment it at 28-32℃ and 160-200 rpm for 34-38 h to obtain the fermentation broth of Pseudomonas fluorescens; centrifuge the fermentation broth of Pseudomonas fluorescens at 3-5℃ and 7500-8500 rpm for 9-11 min to collect the cells of Pseudomonas fluorescens. (4) After washing the Pseudomonas fluorescens cells with PBS solution, they were vacuum dried. (5) Mix sodium alginate with biochar at a concentration of 2-3 w / v, homogenize, add CaCl2 solution at a concentration of 1.8-2.2 w / v, and solidify for 18-22 min to obtain microspheres with a diameter of 1-3 mm; (6) The vacuum-dried bacterial cells and microspheres were mixed in PBS solution at a mass ratio of 1:9-11 and shaken at 3-5℃ and 40-60 rpm for 110-120 min to obtain cyanobacterial microspheres and fluorescent Pseudomonas microspheres. (7) The cyanobacterial microspheres and fluorescent Pseudomonas microspheres are packaged separately in a mass ratio of 4.8-5.2:2.8-3.2 to obtain the microbial combination.

5. The preparation method according to claim 4, characterized in that, The inoculum size for cyanobacteria seed culture is 9–11%, and for Pseudomonas fluorescens, it is 4–6%.

6. The preparation method according to claim 4, characterized in that, The cryoprotectant solution includes trehalose, monosodium glutamate, and ascorbic acid. The concentrations of trehalose, monosodium glutamate, and ascorbic acid in the cryoprotectant solution are 8–12 w / v%, 0.8–1.2 w / v%, and 0.08–0.12 w / v, respectively, based on the bacterial cell mass. The volume ratio of cyanobacterial cells to the cryoprotectant solution is 0.9–1.1:

1.

7. The preparation method according to claim 4, characterized in that, The vacuum drying temperature in steps (2) and (4) is -80 to -40°C, the vacuum degree is 0.08 to 0.12 mbar, and the vacuum drying time is 58 to 62 h.

8. The application of the microbial ensemble according to any one of claims 1 to 3 in reducing nitrogen runoff loss from paddy fields.

9. The application according to claim 8, characterized in that, Includes the following steps: (1) 7-10 days before rice transplanting, apply the cyanobacterial microspheres from the microbial assemblage described in claim 1 to the surface of the paddy field; (2) In the early stage of rice tillering, fluorescent Pseudomonas microspheres from the microbial combination described in claim 1 are applied to the soil layer of 5-10 cm in the rhizosphere of rice.

10. The application according to claim 9, characterized in that, The appropriate amount of cyanobacterial microspheres is 480-520 g / mu, and the amount of fluorescent Pseudomonas is 280-320 g / mu.

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