Bacillus megaterium YK35 and application thereof

Abstract

The invention discloses bacillus megaterium YK35 and application thereof, and belongs to the technical field of microorganisms. The strain is preserved in the China General Microbiological Culture Collection Center on October 9, 2024, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.32158; the strain has efficient potassium dissolving capacity and IAA (indoleacetic acid) producing and phosphorus dissolving capacity, and can remarkably promote the growth of crops; excellent stress resistance including heavy metal stress resistance, acid and alkali stress resistance, salt stress resistance and flooding stress resistance is achieved, and the adaptability is wide; the invention also provides a microbial agent containing the strain. Pot experiment and field experiment verification show that the microbial agent can promote the growth of plants; the yield and quality of the potatoes can be remarkably improved, and the effects of high yield and income increase are achieved; the application of potash fertilizer can be reduced through the potassium dissolving function of the fertilizer; the invention provides a new strain resource for the microbial fertilizer industry, and shows a strong application value in the aspect of growth promotion and yield increase of rhizosphere.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and more specifically, relates to a strain of Bacillus megaterium YK35 and its application in crop stress resistance and fertilizer saving and growth promotion. Background Art

[0002] Potassium is one of the main nutrients necessary for plant growth and development, and has a vital impact on crop growth, yield and quality. However, the effective potassium content in the soil only accounts for 2% of the total potassium, and most of it exists in mineral form, which is extremely difficult for plants to absorb and utilize. In order to increase crop production, a large amount of chemical potassium fertilizer is applied to the soil, but excessive application of chemical fertilizers will not only increase costs and reduce potassium fertilizer utilization, but also destroy soil structure and aggravate environmental pollution.

[0003] Potassium-dissolving microorganisms can dissolve the insoluble potassium in the soil for plants to absorb and utilize. In addition, they can secrete some growth hormones required for plant growth, promote plant growth, and thus reduce the application of chemical potassium fertilizers, which is of great significance to the sustainable development of agriculture. Among them, Bacillus is widely used in microbial fertilizers due to its advantages such as spore production and long shelf life. Sun Jinfeng cultivated the selected potassium-dissolving bacteria in a shake flask test for 14 days. The strain can release 13.50 mg·L potassium from potassium feldspar. -1 (Sun Jinfeng et al., 2017); After 105 days of cultivation in soil, Bacillus pseudomycoides O-5 can dissolve 294.5 mg kg -1 Potassium, thereby promoting the absorption of potassium by tea plants; in addition, studies have shown that inoculation of Bacillus licheniformis with potassium-solubilizing ability under salt stress conditions can improve the morphological and physiological characteristics of tomato plants and enhance the salt stress resistance of plants (Muthuraja et al., 2022; Pramanik et al., 2019). These research results show that Bacillus has great potential to promote plant growth in soil potassium dissolution.

[0004] South China has heavy rainfall, and waterlogging and soil acidification are important factors that seriously threaten crop growth. However, the growth-promoting function of common Bacillus products on the market is usually inhibited under flooding and acidification conditions, which may be related to the colonization ability of functional strains under environmental stress. Bacillus agents are a type of product that can stimulate the natural growth process of crops, enhance crop nutrient absorption, fertilizer utilization efficiency, and tolerance to abiotic stress, thereby improving quality and yield. Therefore, it is necessary to screen out plant rhizosphere bacteria with significant growth-promoting effects under flooding and soil acidification conditions, which is of great significance for the development of new functional microbial resources. Summary of the invention

[0005] The purpose of the present invention is to address the problems existing in the prior art and disclose a strain of Bacillus megaterium YK35 and its application in crop stress resistance, fertilizer saving and growth promotion.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] The present invention screened and obtained a strain of Bacillus megaterium (B. megaterium) YK35, which was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on October 9, 2024, with the deposit number CGMCC No.32158.

[0008] A strain of Bacillus megaterium (B.megaterium) YK35, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 9, 2024, with the deposit number CGMCC No.32158.

[0009] Furthermore, the 16S rDNA nucleotide sequence of the above-mentioned Bacillus megaterium YK35 is shown in SEQ ID NO.1

[0010] Furthermore, the above-mentioned Bacillus megaterium YK35 is used as an indoleacetic acid (IAA) producing agent.

[0011] Furthermore, the above-mentioned Bacillus megaterium YK35 is used as a poorly soluble calcium phosphate dissolving agent.

[0012] Furthermore, the above-mentioned Bacillus megaterium YK35 is used as an agent for promoting plant growth under environmental stress; the environmental stress refers to heavy metal stress, acid-base stress, salt stress or flooding stress.

[0013] Furthermore, in the above-mentioned Bacillus megaterium YK35, the heavy metal in the heavy metal stress is one or a combination of Ni(II), Cu(II), and Cd(II); the acid-base stress is acid stress with a pH value of 5.5 to 6.5 or alkaline stress with a pH value of 7.5 to 9.5; and the salt stress is salt stress with a salinity of 1% to 9%.

[0014] According to the classification of soil acidity and alkalinity in the prior art, soil acidity and alkalinity are divided into 9 levels: <4.5 very strong acidity, 4.5-5.5 strong acidity, 5.5-6.0 acidity, 6.0-6.5 weak acidity, 6.5-7.0 neutrality, 7.0-7.5 weak alkalinity, 7.5-8.5 alkalinity, 8.5-9.5 strong alkalinity, >9.5 very strong alkalinity. It can be seen that the Bacillus megaterium YK35 of the present invention has a relatively wide range of acid and alkali stress resistance.

[0015] Furthermore, the flooding stress includes flooding the culture dish with 5 mL and 10 mL of water; and flooding the potted plants with 200 mL of water every day and 200 mL of water every three days.

[0016] The second technical solution provided by the present invention is a microbial preparation, comprising the Bacillus megaterium YK35 strain or its fermentation liquid described in the first technical solution.

[0017] Furthermore, the fermentation liquid is obtained by inoculating Bacillus megaterium into a fermentation medium for fermentation culture.

[0018] Furthermore, the fermentation medium is LB medium.

[0019] Furthermore, the fermentation culture conditions are 30°C, 180r / min, and 24h.

[0020] Furthermore, the above-mentioned microbial preparation is used as a sprout and root growth promoter when corn seeds germinate; or the microbial preparation is used as a corn growth agent.

[0021] Furthermore, the above-mentioned microbial preparation is used as a potato seed dressing agent.

[0022] Furthermore, the above-mentioned microbial preparation, the potato seed dressing agent is used to promote the growth of potatoes and improve the yield and quality of potato tubers.

[0023] In addition, microbial preparations containing the above-mentioned Bacillus megaterium YK35 strain or its fermentation broth and their applications should also be within the protection scope of the present invention.

[0024] The present invention has the following beneficial effects:

[0025] The Bacillus megaterium YK35 strain provided by the present invention has a good growth-promoting effect. The strain not only has the ability to efficiently dissolve potassium, but also has the functions of dissolving phosphorus and producing IAA; it also has excellent stress resistance, can significantly resist the stress of heavy metals Ni, Cu and Cd, acid and alkali stress, and salt stress, and can still promote the germination and growth of crops under flooding stress conditions, and has a good application prospect. Field test tests show that the application of the potato seed dressing prepared by the strain can significantly improve the yield and quality of crops, and achieve the effect of high yield and increased income; in addition, the strain can also reduce the application of chemical potassium fertilizers through its own potassium dissolving function, thereby providing a strain source and a technical basis for obtaining an environmentally friendly microbial fertilizer that reduces the application of chemical potassium fertilizers, promotes high crop yields, and improves soil stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1It is the average potassium-solubilizing capacity of the screened potassium-solubilizing bacteria.

[0027] Figure 2 The figures are the Gram staining and spore staining results of Bacillus megaterium YK35 (a is the Gram staining picture, b is the spore staining picture).

[0028] Figure 3 This is the phylogenetic tree of Bacillus megaterium YK35.

[0029] Figure 4 This is a colorimetric diagram showing the IAA production capacity of Bacillus megaterium YK35.

[0030] Figure 5 This is a diagram showing the ability of Bacillus megaterium YK35 to resist heavy metal stress (scale bar is 1 cm).

[0031] Figure 6 This is a diagram showing the acid and alkali resistance of Bacillus megaterium YK35.

[0032] Figure 7 This is a diagram showing the effect of Bacillus megaterium YK35's ability to tolerate salt stress.

[0033] Figure 8 This is a diagram showing the effect of Bacillus megaterium YK35 on promoting corn seed germination under flooding conditions (A is a diagram showing the effect of adding 5 mL of water, applying the bacterial agent and the control group after flooding for 7 days; B is a diagram showing the effect of adding 10 mL of water, applying the bacterial agent and the control group after flooding for 7 days).

[0034] Fig. 9 This is a graph showing the growth-promoting effect of Bacillus megaterium YK35 in a corn pot experiment.

[0035] Fig.10 This is a graph showing the growth-promoting effect of Bacillus megaterium YK35 in a potato pot experiment.

[0036] Fig.11 This is the treatment distribution diagram of Bacillus megaterium YK35 in potato field trial. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0038] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0039] The organic fertilizers in the following examples were purchased from Huizhou Defeng Biotechnology Co., Ltd.

[0040] The compound fertilizers in the following examples were purchased from Guangzhou Omino Ecological Agriculture Technology Co., Ltd.

[0041] Example 1 Screening, separation and purification of strains

[0042] The potato rhizosphere soil from the farm of South China Agricultural University in Guangzhou was used as the screening soil sample. 10.0 g of soil sample was weighed and placed in a 250 mL Erlenmeyer flask (containing 5–7 glass beads) containing 90 mL of sterile water. The soil sample was dispersed by shaking at 30 °C and 180 rpm for 30 min. 5 mL of the supernatant was placed in a test tube and heated in a water bath at 90 °C for 10 min. 250 μL of the supernatant after the water bath was added to a 100 mL Erlenmeyer flask containing 25 mL of potassium-dissolving liquid medium (glucose 5.0 g, MgSO4·7H2O 0.5 g, CaCO3 0.1 g, FeCl3 0.005 g, Ca3(PO4) 22.0 g, potassium feldspar 2.0 g, distilled water 1000 mL, pH 7.0-7.5, sterilized at 121 °C for 20 min) and placed at 30 °C and 180 rpm for enrichment culture. After 7 days, the enriched culture solution was diluted to 10 -2 ~10 -5 Concentration gradient, take 100 μL and spread it on LB medium, invert it and culture it in a constant temperature incubator at 30℃ for 12 hours, and purify the strains with different morphologies by streaking on the plate, repeat the process until pure strains are obtained, and number them YK1~YK102, and store them in a refrigerator at 4℃ for use.

[0043] Drawing of potassium standard curve: Absorb 100ug·mL -1 2, 5, 10, 20, 40, and 60 mL of K standard solution are placed in 100 mL volumetric flasks respectively, and the same amount of reagent components as the test solution are added to make the ion components in the standard solution similar to the test solution, and dilute to 100 mL with water. Use the galvanometer reading on the flame photometer as the ordinate and the potassium concentration as the abscissa to draw a standard curve.

[0044] Use an inoculation loop to pick up the purified numbered strains and activate them overnight at 37°C, 180 rpm for 12 h, then transfer them to 5 mL of fresh LB medium and culture until the OD 600 =0.4, inoculated into potassium-dissolving liquid medium at a 1% inoculum volume ratio, and the control group CK was inoculated with an equal amount of LB medium, cultured at 30°C, 180 rpm. After 3, 5, and 7 days of culture, 10 mL of bacterial solution was drawn and rotated at 10000 r·min -1 The supernatant was centrifuged at 5000 rpm for 5 minutes, and the soluble potassium content of the transformed strain was determined by a flame spectrophotometer after removing the potassium content in CK. The experiment was repeated three times. Figure 1As shown in the figure, a large number of strains were screened from the soil, among which YK24, YK35, and YK78 had higher potassium solubilization capacity. The strains with higher potassium solubilization capacity were inoculated into LB liquid medium and cultured overnight until OD 600 =0.8~1.2, the bacterial solution was mixed with 50% sterile glycerol at a volume ratio of 1:1 and stored in a -80℃ refrigerator for further study. Combining the growth ability and potassium solubilization ability, YK35 was selected as a suitable strain from a large number of strains as the subsequent test material.

[0045] Example 2 Characterization of YK35 strain

[0046] (1) Colony morphology characteristics

[0047] The colonies formed by YK35 after culturing on nutrient agar medium (peptone 10.0 g, beef extract 3.0 g, NaCl 5.0 g, agar 18.0 g, distilled water 1000 mL, pH 7.0-7.2) for 24 h were round or oval, white, with a moist and smooth surface, not sticky and easy to pick up.

[0048] (2) Growth characteristics

[0049] A single colony on the plate was picked up and placed in LB liquid medium. After culturing in a shaker at 180 rpm and 37 °C for 12 h, it was inoculated into fresh LB liquid medium at a 1% inoculum volume ratio and cultured for 12 h. The number of viable bacteria in the fermentation broth was measured by the plate count method and was 3.4 × 10 8 cfu / mL.

[0050] (3) Gram staining

[0051] Pick a single colony of YK35 in liquid LB medium, culture it in a shaker at 37°C and 180rpm for 12h, draw 10μL of sterile water on a slide, draw 1μL of bacterial solution into the added sterile water, mix well, dry and fix on a flame; initially stain with crystal violet for 1min, wash with water, and dry naturally; mordant with iodine solution for 1min, wash with water, and dry; decolorize with 95% ethanol for 30s, wash with water, and dry; counterstain with 0.5% safranin for 1min, wash with water, and dry, and observe under a 100x oil microscope. The results are as follows: Figure 2 As shown in Figure a, the bacteria are blue-purple, indicating that the YK35 strain is a Gram-positive bacterium.

[0052] (4) Spore staining

[0053] Pick the colonies formed by YK35 after 48 hours of culture on nutrient agar medium and place them on a glass slide. Pipette 20 μL of sterile water on the glass slide and mix with the strain, dry naturally, fix by flame heating, add 5% malachite green staining solution, heat, make it steam 3-4 times within 30 seconds, rinse with tap water for 30 seconds after cooling. Add 0.5% safranin counterstaining solution, wash with water after 30 seconds, wait until dry, and observe the results under a microscope. The results are as follows Figure 2 As shown in Figure b, the spores are green and the bacteria are red, indicating that the YK35 strain has the ability to produce spores.

[0054] (5) Molecular identification

[0055] Pick a single YK35 colony and put it into a centrifuge tube containing 100 μL sterile water. After pipetting and beating evenly, heat it in a 95°C water bath for 15 min to rupture the cells and release the DNA. Finally, pipette out 1 μL and add it to the PCR system (25 μL PCR reaction system: 1 μL DNA template, 1 μL primer F1 (1 mM) (5'-AGAGTTTGATCCTGGCTCAG-3'), 1 μL primer R1 (1 mM) (5'-TACGGCTACCTTGTTACGACTT-3'), 9.5 μL ddH2O, 12.5 μL 2×Taq PCR Mix.) for amplification and gel running verification.

[0056] PCR reaction amplification system: pre-denaturation at 95°C for 3 min, enter thermal cycle; denaturation at 95°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 1 min, a total of 35 cycles; extension at 72°C for 5 min, stop at 4°C.

[0057] The length and concentration of PCR products were detected by 1.5% agarose gel electrophoresis. The PCR amplification products with bands were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The obtained 16S rDNA sequence was entered into the NCBI website of the National Center for Biological Information of the United States for Blast comparison. All sequences in the database were compared and analyzed using the Blast program. Mega7.0 software was used for sequence analysis and phylogenetic tree construction. The results are as follows Figure 3 shown.

[0058] In summary, the strain YK35 was identified as Bacillus megaterium and was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on October 9, 2024, with the deposit number CGMCC No.32158; the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The 16S rRNA nucleotide sequence of the bacterium is shown in SEQ ID NO: 1.

[0059] Example 3 Determination of the ability of YK35 to produce indoleacetic acid

[0060] Qualitative determination of IAA production by strains: Conduct a qualitative IAA test on the isolated and preserved strains. Pick a single colony on the LB plate and place it in 3mL of LB medium containing 100mg / L tryptophan. Incubate at 30℃ and 180rpm for 24h. Pipette 200μL of bacterial solution and an equal amount of Salkowski colorimetric solution (mixed with 50mL 35% HClO4 and 1mL 0.5 mol / L FeCl3, prepared and used immediately) into a white porcelain plate. Use 200μL of uninoculated LB medium containing 100mg / L tryptophan and 200μL of 25mg / L IAA solution as blank control and positive control, respectively. Place in a dark environment for 30min for color development. The color development results of the YK35 strain are as follows: Figure 4 As shown, the mixed solution turned red, indicating that the strain had the ability to produce IAA.

[0061] Quantitative determination of IAA production by strains: prepare 0, 2.5, 5, 10, 15, 20, and 25 mg / L IAA solutions, take 2 mL of each concentration of IAA solution and mix them with an equal amount of Salkowski colorimetric solution. After color development for 30 min in a dark environment, use a spectrophotometer to determine the OD value of the mixed solution at a wavelength of 530 nm. Draw a standard curve with OD value as the ordinate and IAA solution concentration as the abscissa.

[0062] The strains with better color development were inoculated in LB medium for 24 h and their OD values ​​were measured. 600 The supernatant was mixed with an equal amount of Salkowski colorimetric solution and placed in the dark for 30 minutes. The OD value of the mixture at a wavelength of 530 nm was measured by a spectrophotometer. The amount of IAA produced by different strains was calculated by comparing the standard curve. The YK35 strain had a high OD value at OD 600 =1.0, the amount of IAA produced is 28.54 mg / L.

[0063] Example 4 Determination of phosphorus dissolving capacity of YK35

[0064] Activate strain YK35 on LB solid medium plate and culture at 37℃ for 12h. Pick a single colony of YK35 on the plate and inoculate it into a test tube containing LB liquid medium and culture it in a shaker at 37℃, 180rpm for 12h. Transfer the culture solution to fresh LB liquid medium and shake it in a shaker at 37℃, 180rpm until OD 600=0.4. 0.2 mL of bacterial solution (according to the amount of 1% culture medium) was inoculated into 20 mL of phosphate-dissolving medium (glucose 10.0 g, iron phosphate 5.0 g, MgCl2·6H2O 5.0 g, MgSO4·7H2O0.25 g, KCl 0.2 g, (NH4)2SO40.1 g, distilled water 1000 mL, pH 7.0), and cultured in a shaking table at 30°C, 180 rpm. After 7 days of culture, 2 mL of culture solution was centrifuged at 8000 rpm for 5 min, and the supernatant was taken. The water-soluble phosphorus content was determined by the molybdenum antimony anti-spectrophotometric method in the National Environmental Protection Standard of the People's Republic of China (HJ 632-2011). The control was set as the control, and the phosphorus content of the control treatment was removed, which was the phosphorus dissolving amount of the strain. Each treatment was repeated 3 times. The phosphorus dissolving amount of YK35 after 7 days of culture in calcium phosphate as the phosphorus source medium was 201.88 mg / L.

[0065] Example 5 Effect test of YK35 on heavy metal, acid and salt stress

[0066] (I) Determination of the heavy metal stress tolerance of YK35

[0067] A single colony of YK35 was picked from the LB plate and incubated in LB liquid medium at 37°C, 180 rpm, overnight. Then, the colony was transferred to 4 mL LB liquid medium at a 1% inoculum volume ratio and incubated at 37°C, 180 rpm until OD 600 =0.4.

[0068] Take 100 μL OD 600 =0.4 bacterial solution is added to 4mL LB medium (mass volume ratio of agar powder is 0.75%) at 50-60℃, mixed, poured on a 15mL LB solid medium (mass volume ratio of agar powder is 1.5%) plate, and blown on a clean bench for 20 minutes. Take the sterilized filter paper and place it in the center of the plate, and drop 10μL of 50mM, 100mM, 200mM, 300mM, 400mM Cu(II), 20mM, 40mM, 60mM, 80mM, 100mM Zn(II), 10mM, 20mM, 30mM, 40mM, 50mM Cd(II) on the filter paper respectively. After drying, place it in a 37℃ biochemical incubator and culture it for 24h. Use the Bacillus subtilis model strain NCIB 3610 as a control, record the diameter of the transparent circle, and the test results are as follows: Figure 5 shown.

[0069] The test results showed that under the stress of Zn(II) heavy metal, the diameter of the transparent zone produced by YK35 was smaller than that of the model strain NCIB 3610, indicating that the bacterium showed resistance to Zn(II) heavy metal. Compared with the model strain NCIB 3610, YK35 showed obvious resistance to Cu(II) and Cd(II) heavy metal stress. These test results provide a working basis for the application of the bacterium in soils with heavy metal residues in the future.

[0070] (II) Determination of acid-base stress resistance of YK35

[0071] Prepare LB gradient medium with pH values ​​of 4.0 to 12.0 (gradient of 0.5). Inoculate the bacterial colony (strain YK35) on the plate into a test tube containing LB liquid medium and culture overnight at 37°C, 180 rpm on a shaker. Transfer the bacterial liquid to fresh LB liquid medium and shake at 37°C, 180 rpm until OD 600 =0.4. Dispense the culture media with different pH values ​​into 24-well plates, with 2 mL of LB liquid culture medium in each well. Take 20 μL of OD 600 = 0.4 bacterial solution (based on 1% of the amount of culture medium) in a 24-well plate, 37 ° C, 180 rpm culture for 24 h, the growth of the strain was determined by an ELISA instrument, and the experiment was repeated three times.

[0072] The growth curve of the strain cultured in LB medium with different pH values ​​for 24 h showed that YK35 could grow well in medium with a pH value of 5.5 to 9.5 (the growth curve in the range of pH values ​​that can grow is shown in Figure 2). Figure 6 The results show that YK35 has good acid and alkali stress resistance and can be used in acidic and alkaline soils.

[0073] (III) Determination of salt stress tolerance of YK35

[0074] Prepare LB solid medium containing 1%, 3%, 5%, 7%, 9% and 11% NaCl and 3 mL LB liquid medium, inoculate the strain into the solid plate and liquid medium respectively, incubate the solid plate at 37°C for 24 h, observe the growth of the strain, and incubate the liquid medium at 37°C and 180 rpm for 24 h, and measure its OD 600 value.

[0075] The results are as follows Figure 7As shown in Figure 2, strain YK35 can still grow at 7% NaCl concentration, but the activity is low. At 11% NaCl concentration, the growth of strain YK35 is inhibited. In liquid culture medium containing 1%, 3%, 5%, 7%, 9%, 11% and 13% NaCl, the OD 600 The values ​​can all reach 1.0, but the strain grows slowly in the liquid medium containing 7% NaCl and is inhibited in the liquid medium containing 11% NaCl, which is consistent with the plate results. This shows that the strain has good salt tolerance and can be applied to moderate saline-alkali land, including severe saline-alkali land.

[0076] Example 6 Effect of YK35 on bud length and root length during germination of corn seeds

[0077] (1) Preparation of bacterial suspension:

[0078] ① After activating the strain, inoculate it into LB liquid medium. After overnight culture, transfer it to fresh LB liquid medium and shake it until OD 600 =1.0;

[0079] ② Centrifuge the bacterial solution in a centrifuge at 8000 rpm for 5 minutes;

[0080] ③ Dilute the bacterial solution to a concentration of 10 7 cfu·mL -1 spare.

[0081] (2) Seed surface disinfection: Soak in 75% ethanol for 2 min, rinse twice with sterile water, soak in 2% sodium hypochlorite solution for 2 min, rinse 2-3 times with sterile water, place on sterile filter paper to dry naturally, and germinate for later use.

[0082] (3) The effect comparison test was conducted using YK35 and blank control, with two treatment groups (the first group of bacterial agent treatment used 5 mL YK35 bacterial suspension + 0.025 g potassium feldspar, denoted by T1; the control treatment used 5 mL sterile water + 0.025 g potassium feldspar, denoted by CK1, and each treatment was watered 5 mL per day; the second group used 10 mL YK35 bacterial suspension + 0.025 g potassium feldspar as bacterial agent treatment, denoted by T2; 10 mL sterile water + 0.025 g potassium feldspar as blank control, denoted by CK2, and each treatment was watered 5 mL per day). Each treatment was replicated three times, and each replicate contained 20 germinated corn seeds of uniform size. The bacterial suspension was added to the filter paper, and the corn seeds were placed on the filter paper. After culturing in a biochemical incubator at 25°C for 7 days, the sprout length and root length of the seeds were measured.

[0083] The test results show that YK35 can promote the growth of buds and roots of corn seeds (see Figure 8), the germination rate of corn seeds in the first group of CK treatment group was 85.00%. After applying strain YK35, the average germination rate of corn seeds was 93.33%, which was 9.80% higher than that of CK; the bud length of the bacteria treatment was 35.73% higher than that of CK; and the main root length was 38.70% higher. The germination rate of corn seeds in the second group of CK treatment group was 82.02%. After applying strain YK35, the germination rate of corn seeds was 92.00%, which was 12.02% higher than that of CK; the bud length of the bacteria treatment was 37.62% higher than that of CK; and the main root length was 41.17% higher (see Table 1). This shows that strain YK35 can still significantly improve the germination rate of corn seeds and significantly promote the elongation of corn buds and roots under waterlogging stress conditions.

[0084] Table 1 Effect of fungus application on corn seed germination

[0085]

[0086] Example 7 Test on the effect of YK35 on the growth of potted corn

[0087] (1) Preparation of bacterial suspension:

[0088] ① After activating the strain, inoculate it into LB liquid medium. After overnight culture, transfer it to fresh LB liquid medium and shake it until OD 600 =1.0;

[0089] ② Centrifuge the bacterial solution in a centrifuge at 8000 rpm for 5 minutes;

[0090] ③ Resuspend the cells in an equal amount of sterile water and dilute to 10 8 cfu·mL -1 spare.

[0091] (2) Corn seedling cultivation:

[0092] Wash corn seeds for germination, soak them with gauze to preserve moisture, place them in a 25℃ biochemical incubator, and move them to a 50-hole seedling pot when the seed buds are about 1 cm long, with one seed in each hole. Water them appropriately every day, and when the corn seedlings grow 3 leaves, they are ready for transplanting.

[0093] (3) Experimental design:

[0094] Two treatments were set up (the first group was watered 200 mL per day, and the bacterial agent treatment was added with YK35 bacterial suspension, represented by T1, and the blank control was added with the same amount of sterile water, represented by CK1; the second group was watered once every three days, 200 mL each time, and the bacterial agent treatment was added with YK35 bacterial suspension, represented by T2, and the blank control was added with the same amount of sterile water, represented by CK2). Each treatment was repeated 3 times. Each pot of each treatment was filled with 2.5 kg of soil, and chemical fertilizers urea and superphosphate were applied, with the application amounts of 87 mg urea and 92 mg superphosphate per kilogram of soil, respectively.

[0095] Take photos and record on the 14th and 21st days after transplanting ( Fig. 9 ). Samples were collected on the 21st day, and the height, stem diameter, number of leaves and fresh weight of the aboveground parts of the corn plants were measured. The aboveground parts after harvest were sterilized at 105°C for 30 min, dried at 65°C to constant weight, and then the dry weight was weighed.

[0096] The results are as follows Fig. 9 As shown in Table 2, on the 14th day of corn planting, the plant height, leaf number and stem diameter of the treatments with strain YK35 and CK were 40.22 and 35.45 cm, 4.68 and 4.1 leaves, 3.63 and 4.2 mm, respectively. The plant height, leaf number and stem diameter of the treatment with strain YK35 were significantly higher than those of the blank treatment without bacteria, increasing by 13.46%, 14.15% and 15.70%, respectively, indicating that the application of this strain can promote the growth of corn. On the 21st day of corn planting, the results are shown in Table 2: the plant height, stem diameter and aboveground fresh weight and dry weight of the treatment with strain YK35 were 61.46 cm, 9.12 mm, 47.37 g and 5.32 g, respectively; the plant height, stem diameter and underground fresh weight and dry weight of the treatment with CK were 52.37 cm, 8.30 mm, 33.67 g and 3.98 g, respectively. Compared with CK, the plant height, stem diameter, and aboveground fresh weight and dry weight of corn plants treated with strain YK35 increased by 17.36%, 9.88%, 40.69% and 33.67%, respectively, indicating that the addition of Bacillus megaterium YK35 obtained in the present invention can significantly promote the growth of corn plants.

[0097] Table 2 Comparison of growth indicators of potted corn plants under different treatments (21st day)

[0098]

[0099] Note: The values ​​in the figure are mean ± standard error. The same letters in the same column indicate no significant variance (Duncan, p < 0.05), the same below.

[0100] Example 8 Test on the effect of YK35 on promoting potato growth

[0101] (1) Preparation of seed dressing agent: streak five plates of YK35 strain on LB solid medium plates by coating, incubate in a 37°C incubator for 24 h, scrape the bacterial cells on the surface of the plate with a sterile cardboard, place in 20 g of diatomaceous earth and mix well to make a plate containing 10 colonies. 8 cfu / g of seed dressing, set aside, CK is 20g diatomaceous earth without adding fungicides.

[0102] (2) Preparation of seed potato tubers: Select Feureta F9 seed potatoes, select seed potatoes with obvious buds, cut the seed potatoes into tubers of uniform size, weighing 30±5g, and each tuber has only one bud. The cut tubers are surface disinfected and sterilized, washed with 75% ethanol for 2 minutes, washed twice with clean water, treated with 3% sodium hypochlorite for 1 minute, washed twice with clean water, and blown dry.

[0103] (3) Seed dressing: Mix the prepared seed dressing agent with seed potato tubers in a plastic basin disinfected with alcohol to ensure that the cut surface of each tuber is evenly coated with the prepared seed dressing agent. Use diatomaceous earth without the agent as a blank control. Each treatment has a total of 4 tubers. The prepared tubers are first stored in a 4°C refrigerator.

[0104] (4) Potting: A round flower pot with an inner diameter of 12.5 cm and a height of 11.5 cm was selected. The soil volume of each pot was 0.9 kg. The nitrogen and phosphorus fertilizers were the same for each treatment, with the nitrogen fertilizer dosage of 150 mg per kg of soil and the phosphorus fertilizer (P2O5) dosage of 100 mg per kg of soil. Potassium feldspar powder (K2O 9.2%) was used as a potassium source in each treatment group. One potato tuber was placed in each pot, and the sowing depth was about 6 cm.

[0105] After 30 days of potato plant germination, the plant height, stem diameter, aboveground fresh weight, underground fresh weight, aboveground dry weight, underground dry weight and root length of potato plants were measured. The results are shown in Table 3. The results show that the potato plants treated with YK35 inoculant have a significant growth-promoting effect compared with the blank control CK. YK35 increased the plant height, stem diameter, aboveground fresh weight, underground fresh weight, aboveground dry weight, underground dry weight and root length of potato plants by 41.55%, 37.92%, 54.33%, 51.61%, 16.01%, 41.92%, 65.00% and 26.41%, respectively. The growth-promoting effect of YK35 in the potato pot experiment is shown in Table 3. Fig.10 As shown in the figure, it can be seen that the number of leaves of potato plants treated with YK35 bacterial agent is significantly more than that of CK.

[0106] Table 3 Growth characteristics of potato on the 30th day

[0107]

[0108] Example 9: Test on the effect of YK35 on promoting potato yield

[0109] (1) Experimental materials and planting conditions

[0110] Cutting seed potatoes into pieces: Cut the seed potatoes into pieces, each piece weighs 25-30g, and each piece of seed potatoes contains 1-2 bud points.

[0111] Planting specifications: Double-row ridge planting is adopted, with ridge width of 1.2 meters (including ridge furrows), ridge height of 25-30cm, plant spacing of 20cm, row spacing within ridges of 25-30cm, and planting depth of 5-6cm.

[0112] Conventional fertilization: 400kg / mu of commercial organic fertilizer + 120kg / mu of high-potassium compound fertilizer (15-5-25). After tilling the land, spread commercial organic fertilizer and compound fertilizer, then make ridges and cover with fertilizer.

[0113] (2) Experimental design

[0114] Plot area: The plot area is arranged according to the actual plot, e.g. Fig.11 As shown, each cell is about 15.84m 2 , as follows: three ridges in total, 3×1.2m×4.4m=15.84m 2 Two rows were planted per ridge, 20 tubers were planted per row, and 6×20=120 tubers were planted per plot. This was repeated 4 times, and a total of 4×120=480 tubers were prepared per treatment.

[0115] Seed dressing ingredients and treatment: Mix the bacterial agent YK35 and 250g diatomaceous earth thoroughly to prepare a seed dressing with a bacterial content of 10 9 cfu / g, no bacterial agent was used as the blank control (CK1), and a conventional bacterial agent on the market (70% thiophanate-methyl, wettable powder, dosage 1000g / 100kg potato) was used as the positive control (CK2). The yield was measured on the 100th day after planting.

[0116] Potatoes were harvested and measured 100 days after planting. The results are shown in Table 4. Compared with CK1 and CK2, the treatment with YK35 increased the yield of large potatoes by 21.52% and 12.94% respectively; compared with CK1, the treatment with YK35 increased the yield of medium potatoes by 28%; compared with no application of bacteria and conventional treatment with methyl thiophanate, the yield of commercial potatoes treated with YK35 increased by 23.17% and 11.15% respectively. According to the market price of 1.6 yuan / kg, the seed dressing made with YK35 and diatomaceous earth can increase the income by 400-500 yuan per mu compared with the conventional methyl thiophanate on the market. Under conventional fertilization conditions, the seed dressing made with YK35 and diatomaceous earth can replace the conventional methyl thiophanate on the market, which can increase the yield of commercial potatoes and increase farmers' income.

[0117] Table 4 Comparison of field potato yield indicators under different treatments

[0118]

[0119] Note: Extra-large potato production refers to tubers ≥175g, first-grade potatoes refer to tubers between 75 and 175g, commercial potatoes refer to tubers ≥75g, and small potatoes refer to tubers <75g. Potatoes are calculated based on the market purchase price of 1.6 yuan / kg.

[0120] Example 10 Test on the effect of YK35 on improving potato quality

[0121] When the field experiment was harvested, 30 potatoes were taken from each treatment and brought back to the laboratory to determine their quality elements. Determination of nitrogen, phosphorus and potassium content of potato tubers: After cutting the potato tubers, they were sterilized at 105℃ for 30 minutes, dried at 65℃ to constant weight, weighed and crushed. 0.5000g of the sample was placed in a 100mL Kelvin flask, 1mL of distilled water was added to moisten it, 4mL of concentrated H2SO4 was added and shaken well, 2mL of hydrogen peroxide (H2O2) was added twice, shaken well, and placed on an electric stove for digestion (an exhaust device must be provided). At this time, the plant sample and the acid will react violently. After the violent reaction is over, the solids disappear and become a basically transparent solution. Digest for 10 minutes. During the digestion process, H2SO4 is concentrated and white SO2 gas is decomposed. When the solution is brown, cool it (the bottle wall is not hot), and then add 2mL of H2O2 in batches. The total amount of H2O2 is 8-10mL. After the solution is completely clear, boil for 10 minutes to fully drive off the H2O2. After cooling, transfer the liquid in the Kelvin flask to a 100mL volumetric flask without loss and dilute with water for testing. Total nitrogen is determined by distillation, total phosphorus is determined by molybdenum antimony colorimetry, and total potassium is determined by flame photometry. Tuber N, P, K accumulation (kg·hm -2 ) = tuber N, P, K content (%) × tuber dry matter mass (kg·hm -2 ).

[0122] The results are shown in Table 5. The commercial bacterial agent (CK2) can increase the nitrogen accumulation by 1.49%, the phosphorus accumulation by 13.69% and the potassium accumulation by 13.28%; YK35 can increase the nitrogen accumulation by 1.40%, the phosphorus accumulation by 15.03% and the potassium accumulation by 16.03%.

[0123] Table 5 Comparison of quality element indicators of field potatoes under different treatments

[0124]

[0125] Method for determining potato quality elements: The starch content of potatoes was determined by referring to the iodine colorimetric method in the potato test research method, the reducing sugar content was determined by referring to the 3,5-dinitrosalicylic acid colorimetric method in the Fertilizer Field Test Guide (National Agricultural Technology Extension Service Center, 2018), the vitamin C content was determined by the national standard method (GB / T5009.159-2003), and the protein was determined by the biuret method in the industry standard method (NY / T 1678-2008).

[0126] As shown in Table 6, the market agent (CK2) increased the vitamin C content by 18.06%, the protein content by 62.66%, and the starch content by 16.30%, while YK35 increased the vitamin C content by 23.40%, the protein content by 63.60%, and the starch content by 21.97%, indicating that the microbial agent YK35 in the present invention can significantly improve the quality of potatoes compared with the market agent.

[0127] Table 6 Comparison of quality and flavor indexes of field potatoes under different treatments

[0128]

[0129] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A strain of Bacillus megaterium YK35, characterized in that: The bacteria was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 9, 2024, with the deposit number CGMCC No.32158.

2. The Bacillus megaterium YK35 according to claim 1, characterized in that The 16S rDNA nucleotide sequence is shown in SEQ ID NO.

1.

3. The Bacillus megaterium YK35 according to claim 1, characterized in that The Bacillus megaterium YK35 is used as an indoleacetic acid (IAA) producing agent.

4. The Bacillus megaterium YK35 according to claim 1, characterized in that The Bacillus megaterium YK35 is used as a poorly soluble calcium phosphate dissolving agent.

5. The Bacillus megaterium YK35 according to claim 1, characterized in that The Bacillus megaterium YK35 is used as a plant growth promoting agent under environmental stress; the environmental stress refers to heavy metal stress, acid-base stress, salt stress or flooding stress.

6. The Bacillus megaterium YK35 according to claim 5, characterized in that The heavy metal in the heavy metal stress is one or a combination of Ni(II), Cu(II), and Cd(II); the acid-base stress is acid stress with a pH value of 5.5 to 6.5 or alkaline stress with a pH value of 7.5 to 9.5; and the salt stress is salt stress with a salinity of 1% to 9%.

7. A microbial preparation, characterized in that: The invention comprises the Bacillus megaterium YK35 strain or its fermentation liquid as claimed in claim 1.

8. Use of the microbial preparation according to claim 7 as a sprout and root growth promoter during germination of corn seeds; or use of the microbial preparation as a corn growth promoter.

9. Use of the microbial preparation according to claim 7 as a potato seed dressing agent.

10. Use of the microbial preparation according to claim 9 as a potato seed dressing agent, characterized in that: The potato seed dressing agent is used for promoting the growth of potatoes and improving the yield and quality of potato tubers.

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