A strain of Priestia megaterium YC5, microbial agent and its application

Abstract

The present invention discloses a Priestia megaterium strain YC5, a microbial agent thereof and applications thereof, belonging to the technical field of slow and controlled release fertilizers. For the first time, the present invention isolates and screens a Priestia megaterium ( Priestia megaterium Priestia megaterium ) YC5 from farmland soil, which can degrade and promote the nutrient release of nutrient polymeric slow release fertilizers. The strain has a good degradation effect on nutrient polymeric slow release fertilizers, and its nutrient release can be efficiently matched with crop requirements. Applying the strain to farmland soil where nutrient polymeric slow release fertilizers are applied, the degradation of nutrient polymeric slow release fertilizers by the strain can promote nutrient release, with the characteristics of high nutrient utilization efficiency, high matching degree between nutrient release and crop requirements, and environmental friendliness, which is of great significance for promoting the precise and wide application of nutrient polymeric slow release fertilizers in agriculture.

Description

Technical Field

[0001] The present invention relates to the technical field of controlled-release fertilizers, and specifically relates to a Priestia megaterium strain YC5, a bacterial agent thereof, and applications thereof. Background Art

[0002] Nutrient-polymerized slow-release fertilizers are fertilizers with a biodegradable high-molecular structure prepared by chemical methods from different nutrient elements required by crops. Due to the rich nutrient substances provided by such fertilizers, their application scope in agriculture is constantly expanding. However, during the preparation process of nutrient-polymerized slow-release fertilizers, a large amount of hydroxymethylurea fragments will be generated, and polycondensation reactions will continuously occur between the fragments, resulting in an increasing nutrient release period of the fertilizers, and even reaching several years. During the plant growth cycle, it is difficult to effectively degrade the fertilizers only relying on the microorganisms or enzymes existing in the soil, which affects the utilization efficiency of nutrient-polymerized slow-release fertilizers. At present, the existing nutrient-polymerized slow-release fertilizers can only meet the nutrient requirements of current-season food crops when applied in combination with traditional quick-acting fertilizers.

[0003] In the prior art, the main methods for regulating the release period of nutrient-polymerized slow-release fertilizers are through synthesis methods and preparation processes. The related patent CN116813404A discloses a method for preparing a controllable nutrient-polymerized slow-release fertilizer through copolymerization, cross-linking and other reactions by using a microbial agent configured from formaldehyde, urea, phosphoric acid, ammonium dihydrogen phosphate, maleic anhydride, locust bean gum, vinylamine, cross-linking catalyst, ammonium persulfate, Trichoderma harzianum and Bacillus megaterium. However, this method has complex raw material ratios and preparation processes, increasing the production cost of the fertilizers. The related patent CN113493360A uses a rotary flash drying production process to quickly produce nutrient-polymerized slow-release fertilizers. This method can quickly generate products during the reaction process and can control the release period of the fertilizers to a certain extent. However, it cannot adjust the fertilizer release performance for crops with different growth cycles, and does not truly achieve the matching of nutrient release and crop requirements.

[0004] Obtaining effective degrading bacteria that can be applied in combination with nutrient-polymerized slow-release fertilizers is an effective method to solve the problem of low nutrient utilization rate of this type of fertilizer. Priestia megaterium ( Priestia megaterium ) is a Gram-negative bacillus. In the prior art, the research on Priestia megaterium ( Priestia megaterium ) mainly focuses on inhibiting plant pathogenic fungi, repairing heavy metal environmental pollution, nitrogen fixation and promoting growth, etc. There is no relevant report on the degradation of nutrient-polymerized slow-release fertilizers by this strain. Summary of the Invention

[0005] In order to overcome the deficiencies of the above prior art, the present invention screened a Priestia megaterium ( Priestia megaterium) YC5, this strain shows great application prospects in degrading nutrient polymer - type slow - release fertilizers.

[0006] To achieve the above - mentioned purpose, the technical solution adopted in the present invention is as follows:

[0007] In the first aspect of the present invention, a strain of Priestia megaterium ( Priestia megaterium ) YC5 strain is provided. The Priestia megaterium ( Priestia megaterium ) YC5 strain was deposited at the China General Microbiological Culture Collection Center (abbreviated as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on March 26, 2024, and the deposit number is: CGMCC NO: 30135.

[0008] The Priestia megaterium ( Priestia megaterium ) YC5 strain was isolated from the corn field soil applied with nutrient polymer - type slow - release fertilizers in Qingxiang Community, Liutuan Town, Changyi City, Weifang City, Shandong Province (geographical coordinates N: 37º1′21″; E: 119º22′22″), and it has the following characteristics:

[0009] The colony is light yellow, opaque, round, and moist; observed under a scanning electron microscope, it has the morphology of rod - shaped bacteria; Gram - negative staining.

[0010] The 16S rDNA sequence of the Priestia megaterium ( Priestia megaterium ) YC5 strain is shown in SEQ ID NO.1.

[0011] In the second aspect of the present invention, a microbial agent is provided, and the microbial agent uses Priestia megaterium ( Priestia megaterium ) YC5 as the active ingredient.

[0012] The Priestia megaterium ( Priestia megaterium ) YC5 strain in the microbial agent exists in the form of cultured live bacteria or live bacteria fermentation broth.

[0013] The live bacteria fermentation broth is prepared by the following method:

[0014] Inoculate the seed liquid of the Priestia megaterium ( Priestia megaterium ) YC5 strain into LB liquid medium, and carry out fermentation culture under the conditions of 25 - 30 °C and 140 - 180 r / min until the cell concentration OD600 = 0.6 - 0.8.

[0015] Preferably, the volume ratio of the seed liquid of the Priestia megaterium ( Priestia megaterium ) YC5 strain to the LB liquid medium is 1:10 - 30.

[0016] The bacterial agent also includes components such as nitrogen decomposer, phosphorus decomposer, cellulose decomposer, caseinase producer, amylase producer, urease producer, lipase producer, and phosphatase producer.

[0017] Preferably, the dosage form of the bacterial agent is liquid or solid, and the solid form includes powder or granule.

[0018] In the third aspect of the present invention, there is provided the use of the above-mentioned Priestia megaterium ( Priestia megaterium ) YC5 strain or bacterial agent in the nutrient degradation of nutrient polymerized slow-release fertilizer.

[0019] Preferably, the nutrient polymerized slow-release fertilizer is a slow-release fertilizer with different molecular weights prepared by addition reaction and polycondensation reaction of formaldehyde, urea, phosphate, and potassium salt.

[0020] Preferably, the nutrient polymerized slow-release fertilizer is any one of high molecular weight nutrient polymerized slow-release fertilizer, medium molecular weight nutrient polymerized slow-release fertilizer, or low molecular weight nutrient polymerized slow-release fertilizer.

[0021] Preferably, the synthesis steps of the nutrient polymerized slow-release fertilizer are as Figure 1 shown.

[0022] The basic structural feature of the nutrient polymerized slow-release fertilizer is hydroxymethylurea or dihydroxymethylurea.

[0023] Preferably, for the application method of the nutrient polymerized slow-release fertilizer, by applying nutrient polymerized slow-release fertilizers with different molecular weights to crops in different growth cycles, and then inoculating the Priestia megaterium ( Priestia megaterium ) YC5 strain or the bacterial agent containing the Priestia megaterium ( Priestia megaterium ) YC5 strain into the corresponding soil;

[0024] Or directly applying the large particle nutrient polymerized biological slow-release fertilizer prepared by extrusion granulation of nutrient polymerized slow-release fertilizers with different molecular weights and the Priestia megaterium ( Priestia megaterium ) YC5 bacterial powder to the soil.

[0025] The Priestia megaterium ( Priestia megaterium ) YC5 strain has a preservation number of: CGMCC NO: 30135. When added to the soil of the corresponding crop in the form of a live bacterial fermentation broth, the cell concentration of the fermentation broth is OD 600 = 0.6 - 0.8.

[0026] The crops in different growth periods include long growth cycle crops, medium growth cycle crops, or short growth cycle crops. Among them, long growth cycle crops include apples, pears, peaches, citrus fruits, etc., medium growth cycle crops include wheat, corn, rice, peanuts, etc., and short growth cycle crops include leafy vegetables, etc.

[0027] Preferably, the high molecular weight nutrient polymerized slow-release fertilizer is suitable for crops with long growth cycles, the medium molecular weight nutrient polymerized slow-release fertilizer is suitable for crops with medium growth cycles, and the low molecular weight nutrient polymerized slow-release fertilizer is suitable for crops with short growth cycles.

[0028] Preferably, the inoculation ratio of the Priestia megaterium ( Priestia megaterium ) YC5 strain in short growth cycle crops is 5-10%, in medium growth cycle crops is 4-8%, and in long growth cycle crops is 1-5%.

[0029] The large granular nutrient polymerized biological slow-release fertilizer is composed of a nutrient polymerized slow-release fertilizer, Priestia megaterium ( Priestia megaterium ) YC5 bacterial powder, a carrier, and a synergist.

[0030] In the large granular nutrient polymerized biological slow-release fertilizer, the mass ratio of the nutrient polymerized slow-release fertilizer, the carrier, and the synergist is 1:(1.5-3):(0.3-0.6). The addition amount of Priestia megaterium ( Priestia megaterium ) YC5 bacterial powder in each gram of the large granular nutrient polymerized biological slow-release fertilizer is (2-4)×10 8 CFU / g.

[0031] The carrier is one or more of sawdust, corn straw, humic acid, and rice straw, and the synergist is one or more of aluminite powder and magnesium stearate.

[0032] Preferably, the working parameters of the extrusion granulator are: feeding time 5-10 seconds, extrusion time 1-3 seconds, and pressure 50-100T.

[0033] Compared with the prior art, the beneficial effects of the present invention are:

[0034] 1. A strain of nutrient polymerized slow-release fertilizer degrading bacteria was isolated and screened from the corn field soil where the nutrient polymerized slow-release fertilizer was applied, and it was named Priestia megaterium ( Priestia megaterium ) YC5. This strain has a good degradation effect on the nutrient polymerized slow-release fertilizer. The nutrient polymerized slow-release fertilizer treated with the YC5 strain will have a certain degree of mass loss. At 3300-3500 cm -1 , 1600-1650 cm -1 , a significant decrease in amide groups can be observed, and a significant increase in nutrient ions required for crop growth is converted, improving the nutrient absorption and utilization rate of crops.

[0035] 2. The strains provided by the present invention are expected to be used in agricultural production in combination with nutrient - polymerized slow - release fertilizers. By adjusting the inoculation ratio of the strains, the nutrient requirements of crops in different growth cycles can be met. On the one hand, it can avoid the fertilizer pollution problem caused by untimely degradation during the crop growth cycle. On the other hand, it can also improve the nutrient utilization rate of fertilizers. The present invention provides a new method and idea for the efficient utilization of nutrient - polymerized slow - release fertilizers, which is not only environmentally friendly, low - cost, but also easy to operate, and has a broader application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is the synthesis steps of the nutrient - polymerized slow - release fertilizer.

[0037] Figure 2 It is the colony morphology of Priestia megaterium ( Priestia megaterium ) YC5 on the LB medium.

[0038] Figure 3 It is the Priestia megaterium ( Priestia megaterium ) YC5 observed under a 20,000 - fold field - emission electron scanning electron microscope, showing the bacterial morphological characteristics.

[0039] Figure 4 It is the growth curve of Priestia megaterium ( Priestia megaterium ) YC5, a degrading bacterium of the nutrient - polymerized slow - release fertilizer.

[0040] Figure 5 It is the clear zone produced by Priestia megaterium ( Priestia megaterium ) YC5 after degrading the nutrient - polymerized slow - release fertilizer after staining.

[0041] Figure 6 It is the degradation effect of Priestia megaterium ( Priestia megaterium ) YC5 on the nutrient - polymerized slow - release fertilizer. Among them, Figure 6 in A, the degradation of three groups of treatments is analyzed by the gravimetric method, Figure 6 and in B, the infrared analysis results before and after the degradation of treatment two are shown. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0042] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0043] As mentioned above, the nutrient - polymerized slow - release fertilizer has a high - molecular structure resistant to degradation. After being applied to the soil, it will be hydrolyzed under the catalysis of biological conditions such as microorganisms and enzymes, thereby releasing the nutrient elements required by crops. However, relying solely on the microorganisms or enzymes existing in the soil, it is difficult to achieve the effective degradation of the fertilizer during the plant growth cycle, which affects the utilization efficiency of the nutrient - polymerized slow - release fertilizer.

[0044] In view of the above problems, the present invention has achieved original technological breakthroughs from the following aspects:

[0045] (1) To achieve the precise and wide application of nutrient-polymerized slow-release fertilizers in agriculture, the present invention continuously attempts to isolate and screen new degrading strains from the field soil where nutrient-polymerized slow-release fertilizers have been applied. Finally, a Priestia megaterium ( Priestia megaterium ) YC5 strain was isolated from the corn field soil in Qingxiang Community, Liutuan Town, Changyi City, Weifang City, Shandong Province. The present invention first discovers that this strain has the ability to degrade nutrient-polymerized slow-release fertilizers.

[0046] (2) The present invention discovers that the Priestia megaterium ( Priestia megaterium ) YC5 strain can significantly degrade nutrient-polymerized slow-release fertilizers under short-term culture conditions (2 weeks), which can increase the degradation rate of nutrient-polymerized slow-release fertilizers by 15%; after 5 weeks, the degradation rate of nutrient-polymerized slow-release fertilizers can reach 80 - 90%, and the degradation effect and speed are very significant. And by adjusting the inoculation ratio of the Priestia megaterium ( Priestia megaterium ) YC5 strain, the nutrient requirements of crops in different growth cycles can be met. On the one hand, it can avoid the fertilizer pollution problem caused by untimely degradation during the crop growth cycle, and on the other hand, it can also improve the nutrient utilization rate of fertilizers. It is not only environmentally friendly, but also has low application costs and is easy to operate. The present invention is a major breakthrough in the functional exploration of Priestia megaterium ( Priestia megaterium ).

[0047] (3) The Priestia megaterium ( Priestia megaterium ) YC5 has a strong ability to degrade nutrient-polymerized slow-release fertilizers and can survive with nutrient-polymerized slow-release fertilizers as the sole nitrogen source. And Priestia megaterium YC5 has not been genetically modified, has strong bacterial vitality, and the bacterial cells themselves originate from farmland soil. When it is released into the natural environment, it is harmless to humans, animals and plants and does not pollute the environment.

[0048] Therefore, the Priestia megaterium ( Priestia megaterium ) YC5 of the present invention has great application value in the degradation of nutrient-polymerized slow-release fertilizers, and thus the present invention is proposed.

[0049] The following combines examples to further describe in detail the specific implementation manners of the present invention. The following detailed descriptions are all illustrative and are intended to provide further explanations for the present application rather than limiting the scope of the present invention.

[0050] The nutrient polymerized slow-release fertilizer used in the present invention is prepared in the laboratory. The preparation method is as follows: First, urea and formaldehyde undergo a cross-linking polymerization reaction in an alkaline environment to obtain a hydroxymethylurea intermediate product. According to different ratios of urea to formaldehyde, the polymerization reaction continues to further obtain dihydroxymethylurea. The length, number, and type of the molecular chain of dihydroxymethylurea are affected by factors such as the molar ratio of urea to formaldehyde, reaction time, and reaction temperature. The longer the length, the larger the molecular weight.

[0051] The specific preparation steps are as follows: Add 83.33 g of formaldehyde and 70 ml of deionized water to a round-bottom flask that has already been pre-weighed with 36 g of urea, and adjust the pH to 8.0 using 5% potassium hydroxide solution and 1% phosphoric acid solution. Subsequently, stir and react for 1.5 hours at a temperature of 25 °C to carry out the cross-linking polymerization reaction to obtain a hydroxymethylurea intermediate product and further obtain dihydroxymethylurea. Then, raise the temperature to 50 °C and slowly add 27.73 g of diammonium hydrogen phosphate, and continue to stir and react for 30 minutes. After that, add 1.5 g of polyphosphoric acid for curing reaction. Stop the reaction when the reaction system shows a milky white state, remove it and dry it to obtain the nutrient polymerized slow-release fertilizer, and store it for later use.

[0052] All the culture media used in the present invention need to be sterilized at 121 °C for 20 min. The formula of the enrichment liquid medium with the nutrient polymerized slow-release fertilizer as the sole nitrogen source is: K 2 HPO 4 2.4 g, KH 2 PO 4 1.2 g, MgSO 4 ·7H 2 O 0.2 g, CaCl 2 ·2H 2 O 0.025 g, Fe 2 (SO 4 ) 3 0.008 g, 0.1% D-glucose, 1 ml of inorganic salt solution, 7.5 ml of vitamin solution, 2 g of nutrient polymerized slow-release fertilizer, 1000 ml of sterile distilled water. The solid screening medium is prepared by adding 20 g of agar powder to the enrichment liquid medium.

[0053] The formula of the inorganic salt solution used in the enrichment liquid medium and the solid screening medium is: FeCl 2 ·4H 2 O 1.8 g, CoCl 2 ·6H 2 O 0.25 g, NiCl 2 ·6H 2 O 0.01 g, CuCl 2 ·2H 2O 0.01 g, MnCl 2 ·4H 2 O 0.70 g, ZnCl 2 0.1 g, H 3 BO 3 0.5 g, Na 2 MoO 4 ·2H 2 O 0.03 g, Na 2 SeO 3 ·5H 2 O 0.01 g, 1000 ml of sterile distilled water; After preparation, store it in the refrigerator at 4 °C. When using, take 1 ml and make up the volume to 1 L as the inorganic salt solution for use.

[0054] The difference between the preparation method of the nutrient polymerized slow-release fertilizer degrading bacteria screening solid medium and the enrichment liquid medium is that the "2 g of nutrient polymerized slow-release fertilizer" in the enrichment liquid medium formula is increased to "6 g of nutrient polymerized slow-release fertilizer", and 20 g of agar powder is added.

[0055] The preparation method of the initial nutrient polymerized slow-release fertilizer medium is the same as that of the enrichment liquid medium.

[0056] Example 1: Isolation and identification of the nutrient polymerized slow-release fertilizer degrading bacteria Priestia megaterium ( Priestia megaterium ) YC5

[0057] 1. Strain isolation

[0058] Collect the field soil applied with nutrient polymerized slow-release fertilizer from the corn field in Qingxiang Community, Liutuan Town, Changyi City, Weifang City, Shandong Province. After removing the surface soil, use a sterile instrument to collect the soil sample of the 15 - 35 cm soil layer containing nutrient polymerized slow-release fertilizer. Take 10 g of the field soil and add it to 90 ml of the enrichment liquid medium containing 1% nutrient polymerized slow-release fertilizer, and perform enrichment culture on a constant temperature shaker at 25 °C and 150 r / min for 7 days; After 7 days, transfer it successively according to an inoculation amount of 1% to the liquid enrichment medium containing 2%, 4%, and 6% nutrient polymerized slow-release fertilizer for 3 days of enrichment culture to obtain a further enriched culture solution; After culturing in the above enrichment medium for a certain period of time, the obtained culture solution is first made up with the medium without nutrient polymerized slow-release fertilizer according to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7After dilution, 100 μl was taken and evenly spread on a solid screening medium containing 6% nutrient polymer slow-release fertilizer, and cultured in an inverted position at 25 °C for 5 days; the "plate-clear zone method" was used to observe the colony situation, and iodine solution (I 2 -KI) was used for staining and observation. A colony inoculation loop was used to pick colonies with different colony morphologies and large clear zones, and further streak isolation and purification were carried out on a solid screening medium containing 6% nutrient polymer slow-release fertilizer; after the above isolation and purification, the changes in the colony and the size of the clear zone were observed. If there were still many colonies, colonies with larger clear zones were continuously selected and further streak isolation and purification were carried out on a solid screening medium containing 6% nutrient polymer slow-release fertilizer until colonies with relatively large clear zones were selected; the finally selected colonies were inoculated into a test tube containing 6% nutrient polymer slow-release fertilizer, and an equal volume of 40% glycerol was added at the same time. After numbering, it was stored in a -80 °C ultra-low temperature refrigerator for standby.

[0059] 2. Strain identification

[0060] (1)Gram staining and morphological identification

[0061] The purified strain YC5 was streaked in parallel on an LB solid medium and cultured at 25 °C for 24 h, and then Gram stained. Under an optical microscope, the strain showed blue-violet after Gram staining, indicating that it was a Gram-positive bacterium. The colony of this bacterium was light yellow and opaque, round ( Figure 2 ). At the same time, under a scanning electron microscope, the rod-shaped bacterial morphology could be clearly observed ( Figure 3 ). The growth curve of strain YC5 in the LB medium was as shown in Figure 4 . During the 48-h culture period, the strain showed a trend of first increasing and then decreasing, and the maximum growth rate was reached at the 30th h.

[0062] (2)Molecular identification

[0063] To determine the phylogenetic status of strain YC5, bacterial primers shown in SEQ ID NO.2 - SEQ ID NO.3 were used to perform PCR amplification on the single strain obtained by the above isolation.

[0064] SEQ ID NO.2: 5-AGAGTTTGATCCTGGCTCAG-3 (27F);

[0065] SEQ ID NO.3: 5-GGTTACCTTGTTACGACTT-3 (1492R).

[0066] The conserved 16S rDNA sequence of the isolated strain was analyzed, and the 16S rDNA sequence of this strain is as shown in SEQ ID NO. The obtained sequence results were retrieved and aligned by BLAST at the National Center for Biotechnology Information (NCBI) in the United States, and it was found that the conserved sequence of strain YC5 had the highest similarity with Priestia megaterium strain NBRC 15959, which was 95.62%. Thus, it was determined that the isolated strain was Priestia megaterium YC5.

[0067] This strain was deposited, and the deposit information is as follows:

[0068] Name of the strain: Priestia megaterium ( Priestia megaterium ) strain YC5;

[0069] Taxonomic naming: Priestia megaterium Priestia megaterium ;

[0070] Deposit institution: General Microbiology Center of China Committee for Culture Collection of Microorganisms;

[0071] Abbreviation of the deposit institution: CGMCC;

[0072] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences;

[0073] Deposit date: March 26, 2024;

[0074] Deposit number: CGMCC NO: 30135.

[0075] The laboratory deposit method of the obtained Priestia megaterium ( Priestia megaterium ) strain YC5 is as follows:

[0076] The short-term deposit method is: Inoculate strain YC5 on an LB solid medium, culture it at 25 °C for 2 days, and after the strain grows well, store it in a refrigerator at 4 °C. This deposit method does not exceed 1 month;

[0077] The long-term deposit method is: Mix the grown strain YC5 with glycerol at a concentration of 20% and store it in a -80 °C refrigerator.

[0078] Example 2: Fermentation culture of Priestia megaterium ( Priestia megaterium ) strain YC5

[0079] Priestia megaterium ( Priestia megateriumThe seed liquid of strain YC5 was inoculated into LB liquid medium at a volume ratio of 1:10, and fermented and cultured at 25 °C and 140 r / min until the cell concentration OD600 = 0.6 - 0.8 to obtain Priestia megaterium ( Priestia megaterium ) the fermentation broth of strain YC5.

[0080] Example 3: Detection of the clear zone of Priestia megaterium ( Priestia megaterium ) strain YC5

[0081] To more intuitively observe the degradation ability of Priestia megaterium ( Priestia megaterium ) strain YC5 to nutrient polymer slow-release fertilizer, the clear zone method was used in this experiment, and iodine solution (I 2 -KI) was used as the color developer. The specific operation steps were as follows: One drop of the bacterial liquid of Priestia megaterium ( Priestia megaterium ) strain YC5 was added dropwise to the middle of the four positions of the solid medium for screening nutrient polymer slow-release fertilizer-degrading bacteria. After culturing in a biochemical incubator at 25 °C for 3 days, the medium was stained with iodine solution (I 2 -KI). As Figure 5 shown, after staining, it could be clearly observed that a clear zone with a significant difference from the surrounding iodine solution could be formed outside Priestia megaterium YC5, indicating that this strain has an obvious ability to degrade nutrient polymer slow-release fertilizer.

[0082] Example 4: Determination of the degradation ability of Priestia megaterium ( Priestia megaterium ) strain YC5 to nutrient polymer slow-release fertilizer

[0083] A total of three treatments were set in the experiment: Treatment 1 (nutrient polymer slow-release fertilizer), Treatment 2 (nutrient polymer slow-release fertilizer + YC5), Treatment 3 (nutrient polymer slow-release fertilizer + YC5 + root exudates). Among them, root exudates are substances secreted or released by different parts of the roots into the growth medium during the growth process of plants. Therefore, to provide a simulated root ecological environment in actual planting and observe the degradation effect of its nutrient polymer slow-release fertilizer, Treatment 3 was set.

[0084] The collection method of the root exudates (maize root exudates) added to Treatment Group 3 was as follows: One maize seedling was planted at a depth of 5 mm through a soil culture experiment. The plant was grown at a temperature of 25 °C and a light of 14 h / day (photosynthetically active radiation intensity 300 μmol·m -2 ·s -1)(Grow) for 28 days. By weighing the rhizobox every day, the soil water content was maintained at 60% of the field water holding capacity with distilled water. After 28 days, the soil in the rhizobox was destructively sampled. According to the previous research method, the rhizosphere soil was removed by hand shaking, and the residual soil on the roots was rinsed with sterilized water. Then the corn plants were transferred to 200 mL of sterilized distilled water for cultivation, and the roots were kept in the dark. After 10 h, the root exudates were filtered through a 0.45 mm filter paper. The 200 ml of root exudates from each replicate were freeze-dried into powder and weighed.

[0085] The treatment group was a culture medium of 1 L of initial nutrient polymeric slow-release fertilizer; the second treatment was to use the fermentation broth of Priestia megaterium ( Priestia megaterium )YC5 prepared in Example 2 as an inoculant and inoculated into a 1 L culture medium of initial nutrient polymeric slow-release fertilizer at an inoculation amount of 5% by volume for degradation test; the third treatment group was to add 20 µl of 10 µM corn root exudates on the basis of the second treatment group. The three groups of treatments were cultured on a shaker at 25 °C and 150 r / min for 5 weeks. The degradation of the nutrient polymeric slow-release fertilizer was detected every day in the first week, and the degradation was detected once a week from the second week to the sixth week. As Figure 6 shown, the weight method and infrared spectroscopy were used to analyze its degradation.

[0086] The results showed that adding Priestia megaterium ( Priestia megaterium )YC5 could significantly improve the degradation of the nutrient polymeric slow-release fertilizer. Compared with the first treatment, the degradation rates of the second and third treatments could be increased by 114.97% and 188.08% respectively. It could be seen that on the basis of adding Priestia megaterium ( Priestia megaterium )YC5, adding root exudates could increase the degradation rate of the nutrient polymeric slow-release fertilizer by 34.02%, which would significantly improve the utilization efficiency of the nutrient polymeric slow-release fertilizer. The detection results of the third treatment also showed that in actual planting, the root exudates secreted by the crop itself would interact with Priestia megaterium ( Priestia megaterium )YC5, thus promoting the efficient utilization of the nutrient polymeric slow-release fertilizer in the crop.

[0087] From the infrared analysis results before and after the degradation of the second treatment, it can be seen that the characteristic peak near 3330 cm -1 belongs to the stretching vibration absorption peak of the terminal amide group of the nutrient polymeric slow-release fertilizer molecule. With the progress of degradation, the absorbance of the characteristic peak decreased significantly. The characteristic peak of the -CH -1 - group was observed near 1630 cm 2 , and the absorbance of the nutrient polymeric slow-release fertilizer decreased significantly. Generally, the breakage of the polymer chain is usually accompanied by a decrease in the absorbance of the -CH 2 - group. Therefore, it can be proved that Priestia megaterium (Priestia megaterium 1) YC5 can promote the degradation of nutrient-polymerized slow-release fertilizers.

[0088] Example 5: Application of Priestia megaterium ( Priestia megaterium ) YC5 in crops with different growth cycles

[0089] To verify the application potential of Priestia megaterium ( Priestia megaterium ) YC5 in crops with different growth cycles, short-growth-cycle crops (pakchoi), medium-growth-cycle crops (corn), and long-growth-cycle crops (apple) were selected for experiments in the experiment. When the crops were harvested, the corresponding indicators were measured to analyze the utilization efficiency of nutrient-polymerized slow-release fertilizers.

[0090] Among them, the detection method of nitrogen use efficiency is as follows: At the time of crop harvest, the yield was immediately measured by the weighing method. Then, the fresh plants were taken back to the laboratory, dried, weighed, ground, and passed through a 100-mesh sieve for standby. The nitrogen cumulative uptake of the crops was measured by the Kjeldahl method. At the same time, according to the formula: Nitrogen use efficiency (%) = [(Y i ×C N1 ) - (Y 0 ×C N2 )] / F N × 100% to calculate the nitrogen use efficiency. In the formula, "Y i ", "C N1 ", "Y 0 ", "C N2 ", "F N " represent the yield of fertilized crops, the nitrogen content of fertilized crops, the yield of non-fertilized crops, the nitrogen content of non-fertilized crops, and the nitrogen content in the fertilizer, respectively.

[0091] (1) Pakchoi (Shanghaiqing)

[0092] Three groups of treatments were set up in the experiment, with 6 plants in each group. The blank control group did not apply nutrient-polymerized slow-release fertilizers and Priestia megaterium ( Priestia megaterium ) YC5; the group only applying nutrient-polymerized slow-release fertilizers applied nutrient-polymerized slow-release fertilizers at a nutrient ratio of 15-8-10 kg / hm 2 before sowing; the group applying nutrient-polymerized slow-release fertilizers + YC5 first applied nutrient-polymerized slow-release fertilizers at a nutrient ratio of 15-8-10 kg / hm 2 before sowing. After the pakchoi emerged, a YC5 bacterial solution of Priestia megaterium ( Priestia megaterium ) corresponding to 6% of the nutrient-polymerized slow-release fertilizer was inoculated near the roots. Other management was carried out according to the conventional management of farmers. The experimental period lasted for 28 days in total. When the pakchoi was harvested, the corresponding indicators were measured.

[0093] (2) Corn (Xinrui 25)

[0094] Three groups of treatments were set up in the experiment, with each group covering an area of 1 mu. In the blank control group, no nutrient polymer slow-release fertilizer and Priestia megaterium ( Priestia megaterium ), YC5, were applied. In the group only applying nutrient polymer slow-release fertilizer, the nutrient polymer slow-release fertilizer was applied at a nutrient ratio of 250 - 90 - 60 kg / hm 2 before sowing. For the group applying nutrient polymer slow-release fertilizer + YC5, the nutrient polymer slow-release fertilizer was first applied at a nutrient ratio of 250 - 90 - 60 kg / hm 2 before sowing. After the corn emerged, a suspension of Priestia megaterium ( Priestia megaterium ), YC5, equivalent to 5% of the nutrient polymer slow-release fertilizer was inoculated near the roots. Other management practices followed the conventional practices of farmers. The experimental period lasted for a total of 96 days, and corresponding indicators were measured at the time of corn harvest.

[0095] (3) Apple trees (Yantai Red Fuji)

[0096] Three groups of treatments were set up in the experiment, with each group consisting of 1 tree. In the blank control group, no nutrient polymer slow-release fertilizer and Priestia megaterium ( Priestia megaterium ), YC5, were applied. In the group only applying nutrient polymer slow-release fertilizer, the nutrient polymer slow-release fertilizer was applied at a ratio of 400 - 200 - 400 kg / hm 2 after each annual apple harvest. For the group applying nutrient polymer slow-release fertilizer + YC5, the nutrient polymer slow-release fertilizer was first applied at a ratio of 400 - 200 - 400 kg / hm 2 after each annual apple harvest. During the budding period of the apple tree, a suspension of Priestia megaterium ( Priestia megaterium ), YC5, equivalent to 3% of the nutrient polymer slow-release fertilizer was inoculated near the roots. Other management practices followed the conventional practices of farmers. The experimental period lasted for 1 year, and corresponding indicators were measured at the time of apple harvest.

[0097] The experimental results showed (Table 1) that inoculating the suspension of Priestia megaterium ( Priestia megaterium ), YC5, under the condition of applying nutrient polymer slow-release fertilizer could significantly improve crop yield and nitrogen use efficiency. In the short-term crop Chinese cabbage, after inoculating the suspension of Priestia megaterium ( Priestia megaterium ), YC5, the yield of Chinese cabbage increased by 19.24% and the nitrogen use efficiency increased by 25.44%; the corn yield increased by 15.14% and the nitrogen use efficiency increased by 18.60%; the apple yield increased by 14.33% and the nitrogen use efficiency increased by 11.83%. The experimental results indicated that: by adjusting the inoculation ratio of crop strains at different growth stages in the present invention, the nutrient polymer slow-release fertilizer was degraded under the action of the strains, thereby regulating the release performance of the fertilizer, enabling the full exertion of fertilizer efficiency, meeting the nutrient requirements of crops at different growth stages during growth, and truly achieving the matching of nutrient release and crop requirements.

[0098] Table 1: Application and effects of Priestia megaterium ( Priestia megaterium ) YC5 in crops at different growth stages

[0099]

[0100] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various modifications and changes can be made to the present application. Any modification

[0101] equivalent replacement, improvement, etc. should all be included within the protection scope of the present application.

Claims

1. A strain of Priesteria gigantea ( Priestia megaterium )YC5 strain, characterized in that The Priesteria gigantea ( Priestia megaterium )The YC5 strain was deposited in the China General Microbiological Culture Collection Center on March 26, 2024, with the deposit number: CGMCC NO: 30135.

2. A bacterial agent, characterized in that The bacterial agent is Priesteria gigantea described in claim 1 ( Priestia megaterium )YC5 is the active ingredient.

3. The bacterial agent according to claim 2, characterized in that The bacterial agent contains Priesteria gigantea ( Priestia megaterium ) The YC5 strain exists in the form of cultured live bacteria or live bacteria fermentation broth.

4. The bacterial agent according to claim 3, characterized in that The live bacteria fermentation liquid in the bacterial agent is prepared by the following method: Priesteria gigantea Priestia megaterium ) The seed liquid of YC5 strain was inoculated into LB liquid medium and fermented at 25-30°C and 140-180r / min until the bacterial concentration OD 600 =0.6-0.

8.

5. The Priesteria gigantea of ​​claim 1 ( Priestia megaterium ) Use of the YC5 strain or the bacterial agent according to claim 4 in degrading nutrient polymerized slow-release fertilizers whose basic structure is hydroxymethylurea or dihydroxymethylurea; The nutrient polymerized slow-release fertilizer whose basic structure is hydroxymethyl urea or dihydroxymethyl urea is any one of high molecular weight nutrient polymerized slow-release fertilizer, medium molecular weight nutrient polymerized slow-release fertilizer and low molecular weight nutrient polymerized slow-release fertilizer.

6. The use according to claim 5, characterized in that: The nutrient polymerized slow-release fertilizer with a degradation base structure of hydroxymethylurea or dihydroxymethylurea is prepared by introducing Priesteria gigantea ( Priestia megaterium )YC5 strain or Priesteria gigantea ( Priestia megaterium ) The bacterial agent of the YC5 strain was physically mixed with the nutrient polymerized slow-release fertilizer and inoculated into the soil; Alternatively, a nutrient-polymerizing slow-release fertilizer can be combined with Priesteria gigantea ( Priestia megaterium ) YC5 bacterial powder is extruded and granulated to obtain large-particle nutrient-polymerized biological slow-release fertilizer, which is directly applied to the soil; The Priesteria gigantea ( Priestia megaterium ) The inoculation ratio of the YC5 strain in short-growth cycle crops is 5-10%, the inoculation ratio in medium-growth cycle crops is 4-8%, and the inoculation ratio in long-growth cycle crops is 1-5%; The large-particle nutrient-polymerized biological slow-release fertilizer is composed of nutrient-polymerized slow-release fertilizer, Priesteria gigantea ( Priestia megaterium )YC5 bacterial powder, carrier and synergist; The mass ratio of the nutrient polymerized slow-release fertilizer, the carrier, and the synergist in the large-particle nutrient polymerized biological slow-release fertilizer is 1:(1.5-3):(0.3-0.6), and the amount of Priesteria gigantea ( Priestia megaterium )The amount of YC5 powder added is (2-4)×10 8 CFU / g.

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