Method for promoting corn seed germination and resisting saline-alkali by pandoraea apista

Abstract

The present application relates to the technical field of corn seed germination and saline-alkali resistance, and discloses a method for promoting corn seed germination and saline-alkali resistance by pantoea ananatis. The method comprises the following steps: treating corn seeds with a bacterial liquid obtained by fermentation of pantoea ananatis, or coating or dressing corn seeds with a bacterial agent prepared from the pantoea ananatis; the pantoea ananatis colonizes on the surface of the seeds or root systems during the germination and growth of the corn seeds, promotes seed germination and improves the saline-alkali resistance of the corn by producing plant growth hormones and inducing systemic resistance. The pantoea ananatis ZK1-2025 bacterial agent has the potential of promoting growth and resisting saline-alkali stress, helps to maintain the health and stability of the ecological system, and has the application value of preparing microbial bacterial fertilizer.

Description

Technical Field

[0001] This invention relates to the field of corn seed germination and salt-alkali resistance technology, and discloses a method for promoting corn seed germination and salt-alkali resistance using Pantothecin pineapple. Background Technology

[0002] Nitrogen is an essential element for plant growth and development, but under natural conditions, most plants cannot directly utilize atmospheric nitrogen. Traditional agriculture relies primarily on chemical nitrogen fertilizers to meet crop nitrogen requirements; however, the long-term excessive application of chemical fertilizers has led to a series of serious environmental problems, including soil compaction, acidification, eutrophication of water bodies, and greenhouse gas emissions. Against this backdrop, nitrogen-fixing microorganisms have shown enormous application potential. It is estimated that up to 175 million tons of nitrogen enter the soil globally each year through biological nitrogen fixation. This process not only significantly reduces agriculture's dependence on chemical nitrogen fertilizers but also makes a crucial contribution to maintaining soil fertility and ecological balance.

[0003] Salinization is another major problem hindering global agricultural production. High pH levels and high sodium ion (Na+) concentrations in saline-alkali soils contribute to this problem. + Salt concentrations, along with osmotic stress and ion toxicity, severely affect the normal growth and development of plants. Salt stress not only hinders nutrient absorption and reduces photosynthetic efficiency but also leads to the accumulation of reactive oxygen species, causing oxidative damage. Salinization disrupts soil microbial community structure and reduces soil ecological function. Rhizosphere microorganisms have shown significant effects in alleviating plant salt stress. Their mechanisms of action include regulating ion balance, synthesizing osmotic regulators, enhancing antioxidant defense systems, and improving the rhizosphere microenvironment, providing new pathways for the bioremediation and agricultural utilization of saline-alkali land.

[0004] Pineapple pancreatitis ( Pantoea ananatis This is an excellent rhizosphere growth-promoting bacterium that promotes crop growth and enhances plant health through multiple mechanisms. The strain possesses nitrogen-fixing and phosphorus-solubilizing capabilities, converting atmospheric nitrogen and insoluble phosphorus in the soil into forms that plants can directly absorb and utilize; it can synthesize plant hormones such as auxins, directly stimulating root development; and it inhibits plant pathogens, induces broad-spectrum plant resistance, and alleviates abiotic stresses such as heavy metal pollution and salt stress, thereby comprehensively improving crop growth performance and health. Summary of the Invention

[0005] To address the aforementioned problems, this invention discloses a method for promoting corn seed germination and salt tolerance using Pantothecin pineapple, the method comprising the following steps: The bacterial solution obtained from the fermentation of Pantotheca pineapple is used to soak corn seeds, or the bacterial agent prepared by Pantotheca pineapple is used to coat or mix corn seeds. Pantotheca pineapple colonizes on the surface of seeds or roots during the germination and growth of corn seeds, and promotes seed germination and improves the salt and alkali tolerance of corn by producing plant growth hormones and inducing systemic resistance.

[0006] Preferably, the preservation number of the Pantotheca pineapple is CGMCC No. 35263.

[0007] Preferably, the method for preparing the pineapple pancreatic bacteria broth includes: inoculating pineapple pancreatic bacteria into LB liquid medium and shaking and culturing at 25-37°C and 150-200 r / min for 12-36 hours to obtain the fermentation broth.

[0008] Preferably, the bacterial cell concentration OD of the *Plasmodium pineapple* bacterial solution is... 600 The value is 0.5–2.0, or the effective viable count is 1 × 10⁻⁶. 7 CFU / mL ~ 1×10 9 CFU / mL.

[0009] Preferably, the specific steps of the seed soaking treatment are as follows: after cleaning and disinfecting the corn seeds, soak them in diluted pineapple pancreatic bacteria solution for 4 to 24 hours at room temperature, then take them out, rinse them with clean water and air dry them.

[0010] Preferably, the soaking time is 8 to 12 hours and the soaking temperature is 25 to 30°C.

[0011] Preferably, the salt-alkali resistance treatment refers to sowing corn seeds in saline-alkali soil with a salt content of 0.1% to 0.6% after the corn seeds have been treated with Panax notoginseng, or irrigating them with saline-alkali water after sowing.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: The *Pantoinella pineapple* strain ZK1-2025 of this invention possesses beneficial properties such as nitrogen fixation, IAA secretion, promotion of seed rooting, and resistance to salt and alkali stress. *Pantoinella pineapple* strain ZK1-2025 can promote corn seed germination, specifically showing a 22.72% increase in root length and a 40.34% increase in shoot length, resulting in a more robust root system; it also enhances the resistance of corn seeds to salt and alkali stress, specifically showing an 81.68% increase in root length and a 75.99% increase in shoot length under saline-alkali conditions. The *Pantoinella pineapple* strain ZK1-2025 has the potential to promote growth and improve resistance to salt and alkali stress, contributing to the maintenance of ecosystem health and stability, and has application value in the preparation of microbial fertilizers. Attached Figure Description

[0013] Figure 1The growth status of Pantotheca pineapple ZK1-2025 on LB medium on day 3.

[0014] Figure 2 The image shows ZK1-2025 on day 3 of growth under a 100× optical microscope.

[0015] Figure 3 Electrophoresis image of nucleic acid identification for ZK1-2025 PCR.

[0016] Figure 4 This is the phylogenetic tree of ZK1-2025.

[0017] Figure 5 Image showing the IAA effect of ZK1-2025.

[0018] Figure 6 The growth status of ZK1-2025 on Ashby solid medium.

[0019] Figure 7 Images showing the effect of ZK1-2025 on corn seed germination.

[0020] Figure 8 This refers to the root and bud growth of corn seeds.

[0021] Figure 9 Images showing the effects of ZK1-2025 on maize germination under saline-alkali stress.

[0022] Figure 10 The results show the root and shoot growth of maize seeds under saline-alkali stress. Detailed Implementation

[0023] Please refer to Figures 1 to 9 The purpose of this invention is to provide an application of Pantothecin pineapple in promoting corn seed germination and improving corn seed resistance in saline-alkali environments.

[0024] The present invention provides a Pantoea ananatis ZK1-2025 strain isolated from soil and deposited at the China General Microbiological Culture Collection Center on July 16, 2025, with accession number CGMCC No. 35263.

[0025] The Pantothecin ZK1-2025 pineapple strain provided by this invention has beneficial properties such as nitrogen fixation, IAA secretion, promotion of maize seed germination, and improvement of maize seed resistance to salt and alkali stress.

[0026] The applications include promoting plant growth, specifically including growth indicators such as the bud length and root length of corn seeds, and the bud length and root length of corn seeds under salt and alkali stress.

[0027] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0028] Example 1: Isolation and identification of Pantothecin ZK1-2025 in pineapple Separation of ZK1-2025 This strain was isolated from the rhizosphere soil of maize.

[0029] Weigh 10g of soil sample and add it to 90ml of sterile water. Place the sample on a shaker at 150r / min for 20min, let it stand for 10min, and take the supernatant as a 1×10⁻¹ dilution. Dilute the solution to 1×10⁻¹ using a gradient concentration method. -2 1×10 -3 1×10 -4 1×10 -5 Take 100 μL of each serial dilution and spread it onto Ashby agar. Incubate at 28°C for 4-5 days. Pick representative single colonies, purify them three times on LB agar, and store them at -80°C with 25% glycerol for later use.

[0030] The identification of ZK1-2025: 1. Morphological identification of ZK1-2025 Strain strain ZK1-2025 was inoculated onto LB solid medium, and the morphology of single colonies was observed after 3 days. The results are as follows: Figure 1 As shown, the colonies of strain ZK1-2025 are round, with neat edges, slightly yellow, transparent, and easy to pick up.

[0031] 2. ZK1-2025 Gram staining identification: Prepare a smear of the bacterial culture using standard methods, dry and fix it. Stain with crystal violet for 2 minutes, then wash with water. Rinse off any remaining water with iodine solution, cover with iodine solution for about 1 minute, then wash with water. Blot off any remaining water on the slide with filter paper, decolorize with 95% ethanol for 25 seconds, and immediately wash with water. Counterstain with safranin solution for about 2 minutes, then wash with water. After drying, observe under an oil immersion microscope. Figure 2 As can be seen, ZK1-2025 turns red after Gram staining, indicating it is a Gram-negative bacterium.

[0032] 3. Molecular identification of ZK1-2025: Using universal primers 27F (AGAGTTGATCMTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT) for bacterial 16S rDNA, PCR amplification products were sequenced using conventional methods. Sequence homology was analyzed by NCBI BLAST alignment, and a phylogenetic tree was constructed using MEGA12.0. ZK1-2025 was found to be on the same branch as *Ustilago maydis*. Colony morphology, Gram staining, and molecular identification results indicated that ZK1-2025 is *Ustilago maydis* (or *Ustilago maydis*). Pantoea ananatis ).

[0033] Example 2: Analysis of beneficial characteristics of Pantothecin ZK1-2025 in pineapple: 1. ZK1-2025 IAA Secretion Assay: The activated bacterial strain was inoculated into LB liquid medium containing 1 g / L L-tryptophan using an inoculation loop and cultured at 28°C with shaking at 180 rpm for 3 days. The fermentation broth was centrifuged at 5000 rpm for 10 min, and 200 μL of the supernatant was mixed with an equal volume of Salkowski colorimetric reagent. A 100 mg / L indoleacetic acid standard solution was used as a positive control. The mixture was incubated in the dark for 30 min, and the color of the reaction solution was observed to turn red; a red color indicated the production of IAA. The indoleacetic acid standard was diluted to concentrations of 100 mg / L, 50 mg / L, 25 mg / L, 12.5 mg / L, and 6.25 mg / L, respectively. The above reaction solutions were then cultured at OD00. 530nm The absorbance was measured, and a standard curve for indoleacetic acid (IAA) solution was plotted. Based on the standard curve, the IAA yield in the fermentation broth was calculated. The IAA production effect of ZK1-2025 is as follows: Figure 4 As shown, the reaction solution turns red, indicating that the supernatant contains IAA. This means that ZK1-2025 has the ability to secrete IAA, with a secretion level of 32 mg / L.

[0034] 2. Determination of nitrogen fixation capacity of ZK1-2025: The ZK1-2025 strain was inoculated onto Ashby solid medium and cultured at 28°C for 3 days. The ability of the strain to grow on nitrogen-fixing medium was observed. If it could grow, it would demonstrate that the strain possesses the corresponding nitrogen-fixing ability. Results are as follows: Figure 5 As shown, ZK1-2025 can grow on nitrogen-fixing medium, meaning that ZK1-2025 has nitrogen-fixing ability.

[0035] Ashby nitrogen-fixing medium: mannitol 10.0g, KH2PO4 0.2g, CaCO3 5.0g, MgSO4·7H2O 0.2g, NaCl 0.3g, CaSO4·2H2O 0.1g, agar 18g, distilled water to a final volume of 1L, pH 7.0, sterilized at 121°C for 25min.

[0036] Example 3: Analysis of the effect of Pantothecin ZK1-2025 on promoting maize seed germination and the resistance of maize seeds to salt and alkali stress: 1. ZK1-2025 promotes corn seed germination: The activated bacterial strain was inoculated into LB liquid medium and cultured at 28°C and 180 rpm for 48 h. The medium components were removed by centrifugation to obtain ZK1-2025 bacterial cells. The cells were resuspended in sterile distilled water, and the bacterial concentration was adjusted to 1×10⁻⁶. 9 CFU / mL was prepared for use. Corn seeds were sterilized with sodium hypochlorite and then soaked in bacterial solution for 6 hours to coat them. Ten coated and ten uncoated corn seeds were placed in each petri dish, and incubated at 28℃ for 5 days to observe germination.

[0037] Results analysis: The results showed that ZK1-2025 had a significant promoting effect on maize seed germination. Figure 6 , Figure 7 Compared with the control group, the root length increased by 22.72% (P<0.05) and the shoot length increased by 40.34% (P<0.01), and the roots of the coated seeds were more robust, indicating that ZK1-2025 has a significant promoting effect on the germination of maize seeds.

[0038] 2. Effect of ZK1-2025 on maize seed germination resistance to salt-alkali stress: The activated bacterial strain was inoculated into LB liquid medium and cultured at 28°C and 180 rpm for 48 h. The medium components were removed by centrifugation to obtain ZK1-2025 bacterial cells. The cells were resuspended in sterile distilled water, and the bacterial concentration was adjusted to 1×10⁻⁶. 9 CFU / mL was prepared for use. Corn seeds were sterilized with sodium hypochlorite and then soaked in bacterial solution for 6 hours to coat them. Coated and uncoated corn seeds were placed in petri dishes containing 100 mM NaHCO3 and 150 mM NaCl, respectively, with 10 seeds per dish. The dishes were incubated at 28°C for 5 days, and germination was observed.

[0039] Results analysis: The results show that ZK1-2025 has a salt-alkali stress resistance effect on maize seed germination ( Figure 8 , Figure 9 Compared with the control group, root length increased by 81.88% (P<0.0001) and shoot length increased by 75.99% (P<0.05). This indicates that ZK1-2025 has a salt-alkali stress resistance effect on maize seed germination.

Claims

1. A method for promoting corn seed germination and salt tolerance using Pantothecin pineapple, characterized in that, The method includes the following steps: The bacterial solution obtained from the fermentation of Pantotheca pineapple is used to soak corn seeds, or the bacterial agent prepared by Pantotheca pineapple is used to coat or mix corn seeds. Pantotheca pineapple colonizes on the surface of seeds or roots during the germination and growth of corn seeds, and promotes seed germination and improves the salt and alkali tolerance of corn by producing plant growth hormones and inducing systemic resistance.

2. The method according to claim 1, characterized in that, The preservation number of the pineapple pancreatic fungus is CGMCC No. 35263.

3. The method according to claim 1, characterized in that, The preparation method of the pineapple pancreatic bacteria broth includes: inoculating pineapple pancreatic bacteria into LB liquid medium, and shaking and culturing at 25-37℃ and 160 r / min for 12-36 hours to obtain the fermentation broth.

4. The method according to claim 1, characterized in that, The bacterial cell concentration OD of the *Pantheraquinone* pineapple culture 600 The value is 0.5–2.0, or the effective viable count is 1 × 10⁻⁶. 7 CFU / mL ~ 1×10 9 CFU / mL.

5. The method according to claim 1, characterized in that, The specific steps of the seed soaking treatment are as follows: after cleaning and disinfecting the corn seeds, soak them in diluted pineapple pancreatic bacteria solution for 4 to 24 hours at room temperature, then take them out, rinse them with clean water and air dry them.

6. The method according to claim 5, characterized in that, The soaking time is 8 hours, and the soaking temperature is 25-30℃.

7. The method according to claim 1, characterized in that, It also includes methods for salt and alkali resistance treatment: after corn seeds have been treated with Panadol pineapple, they are sown in saline-alkali soil with a salt content of 0.1% to 0.6%, or irrigated with saline-alkali water after sowing.

Images