A strain of Agrobacterium tumefaciens DTI9 and its application

Abstract

This invention discloses a strain of Agrobacterium spp. ( Agrobacterium rubi This invention discloses Agrobacterium tumefaciens DTI9, deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 30, 2026, with accession number CGMCC No. 38525. The invention also discloses the preparation of the inoculum. Furthermore, this invention discloses the application of Agrobacterium tumefaciens DTI9 and its preparation in promoting growth and resilience in chili seedlings under salt-low nitrogen combined stress, as well as its application in promoting chili seedling growth and enhancing stress resistance reserves under normal cultivation conditions. Agrobacterium tumefaciens DTI9 of this invention possesses the dual advantages of promoting growth under normal cultivation conditions and protecting seedlings against salt-low nitrogen combined stress. It exhibits strong ecological adaptability and stable function, and has significant application value and broad prospects for promotion in the fields of green improvement of secondary saline-alkali soils and green, high-efficiency chili cultivation.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, specifically to a strain of Agrobacterium tumefaciens DTI9 and its applications. Background Technology

[0002] In agricultural production, the long-term excessive application of chemical fertilizers in pursuit of high crop yields has become a common phenomenon. This unreasonable fertilization pattern easily leads to soil nutrient imbalance and salt accumulation, which in turn causes the continuous degradation of arable land quality, with secondary salinization being particularly prominent.

[0003] Secondary soil salinization poses a dual threat to agricultural production, damaging the soil environment and inhibiting crop growth. At the soil level, excessive salt disrupts soil structure, leading to soil compaction and disrupting microbial balance, thus causing a continuous decline in soil fertility. At the crop level, the increased osmotic pressure in saline soil directly inhibits water absorption, with seedlings being particularly sensitive. The seedling stage is a critical window for salt stress; high salinity can also damage the physiological functions of seedling roots, causing wilting, seedling burn, growth stagnation, and even death, severely reducing survival rates and thus hindering early establishment and later yield formation.

[0004] Currently, production mainly employs physical amendments (such as irrigation and salt leaching) and chemical amendments (such as gypsum application). However, these methods generally suffer from high costs, short-lasting effects, and unstable results. Furthermore, irrigation and salt leaching can exacerbate water and fertilizer loss and secondary soil damage, while chemical amendments may also cause new soil pollution.

[0005] Microbial inoculants, as a green, low-carbon, and sustainable biological improvement technology, are considered an ideal alternative to traditional improvement methods. However, existing commercial microbial agents have significant technical shortcomings in their application to saline-alkali farmland: First, most functional strains have poor salt tolerance, making it difficult to effectively colonize and proliferate in saline-alkali soils, resulting in unstable field growth-promoting and stress-resistance effects; second, functional strains often have single functions and cannot simultaneously address combined stresses such as salt stress and low nitrogen deficiency; third, their mitigation effect on salt damage during the seedling stage is limited, making it difficult to establish an effective rhizosphere protection system at the most sensitive stage of crop growth. These shortcomings collectively restrict the practical promotion and application of existing microbial inoculants in saline-alkali farmland. Summary of the Invention

[0006] The purpose of this invention is to provide a strain of Agrobacterium tumefaciens DTI9 and its applications to address the shortcomings of existing technologies.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The first aspect of this invention provides a strain of Agrobacterium tumefaciens (… Agrobacterium rubiDTI9 is ​​deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 30, 2026, with accession number CGMCC No. 38525.

[0009] A second aspect of the present invention provides a bacterial agent prepared from the above-mentioned Agrobacterium tumefaciens DTI9, wherein the bacterial cell concentration of the bacterial agent is ≥1×10⁻⁶. 9 cfu / mL.

[0010] Further, the bacterial agent is prepared by the following method: *Agrobacterium spp. DTI9* is inoculated into a liquid culture medium for liquid fermentation to obtain *Agrobacterium spp. DTI9* fermentation broth; the fermentation broth is centrifuged, the supernatant is discarded, the bacterial precipitate is resuspended in sterile water, and the bacterial concentration is adjusted to ≥1 × 10⁻⁶. 9 The bacterial agent was obtained by dispersing cfu / mL.

[0011] Furthermore, the liquid culture medium includes YMA liquid culture medium.

[0012] Furthermore, the liquid fermentation culture conditions are: temperature 27-30℃, rotation speed 170-190 r / min, and time 12-24 h.

[0013] The third aspect of this invention provides the application of the above-mentioned Agrobacterium tumefaciens DTI9 in the stress resistance and growth promotion of pepper seedlings under salt-low nitrogen combined stress.

[0014] The fourth aspect of this invention provides the application of the above-mentioned Agrobacterium tumefaciens DTI9 in promoting the growth of chili seedlings and improving the stress resistance reserves of chili seedlings under normal cultivation conditions.

[0015] The fifth aspect of this invention provides the application of the above-mentioned microbial agent in promoting growth and stress resistance of pepper seedlings under salt-low nitrogen combined stress.

[0016] The sixth aspect of this invention provides the application of the above-mentioned microbial agent in promoting the growth of chili seedlings and improving the stress resistance reserves of chili seedlings under normal cultivation conditions.

[0017] The beneficial effects of this invention are:

[0018] This invention screened a strain of Agrobacterium tumefaciens from saline-alkali land. Agrobacterium rubiDTI9 possesses excellent salt tolerance and also functions as a nitrogen fixation agent, iron carrier, and auxin (IAA) producer, enabling it to adapt to the complex growth environment of soil salinity-low nitrogen stress. Under normal cultivation conditions, the *Agrobacterium suis* DTI9 of this invention significantly promotes pepper seedling growth and increases the levels of antioxidants in pepper seedlings, enhancing their potential stress resistance reserves. Under salinity-low nitrogen stress, it can reduce peroxidation damage to the seedling membrane, increase the activity of antioxidant enzymes and the accumulation of osmotic regulators, improve the stress resistance of pepper seedlings, and improve their growth status, with a more pronounced effect on the underground parts.

[0019] In summary, the Agrobacterium tumefaciens DTI9 of this invention has the dual advantages of promoting growth under normal cultivation conditions and resisting and protecting seedlings under salt-low nitrogen combined stress conditions. It has strong ecological adaptability and stable function, and has important application value and broad prospects for promotion in the fields of green improvement of secondary saline soil and green and efficient cultivation of peppers. Attached Figure Description

[0020] Figure 1 This is a colony morphology diagram of strain DTI9.

[0021] Figure 2 A phylogenetic tree for strain DTI9 constructed based on the 16S rRNA sequence.

[0022] Figure 3 The growth curves of strain DTI9 under different NaCl concentrations are shown.

[0023] Figure 4 The diameter of strain DTI9 under different pH conditions.

[0024] Figure 5 The ability of strain DTI9 to produce siderophores was determined.

[0025] Figure 6 The ability of strain DTI9 to produce auxin (IAA) was determined.

[0026] Figure 7 This is a potted plant illustration of chili seedlings with different treatments as shown in Example 6.

[0027] Figure 8 The image shows chili seedlings treated differently in Example 6.

[0028] Figure 9 Example 6 illustrates the effect of different treatments on the leaf width of chili seedlings.

[0029] Figure 10 Example 6 illustrates the effect of different treatments on the height of chili seedlings.

[0030] Figure 11 Example 6 illustrates the effect of different treatments on the stem diameter of chili seedlings.

[0031] Figure 12 Example 6 illustrates the effect of different treatments on the aboveground fresh weight of chili seedlings.

[0032] Figure 13 Example 6 illustrates the effect of different treatments on the fresh weight of the underground parts of chili seedlings.

[0033] Figure 14 Example 6 illustrates the effect of different treatments on the total fresh weight of chili seedlings.

[0034] Figure 15 Example 6 illustrates the effect of different treatments on the aboveground dry weight of chili seedlings.

[0035] Figure 16 Example 6 illustrates the effect of different treatments on the dry weight of the underground parts of chili seedlings.

[0036] Figure 17 Example 6 illustrates the effect of different treatments on the total dry weight of chili seedlings.

[0037] Figure 18 Example 6 illustrates the effect of different treatments on the MDA content of chili seedlings.

[0038] Figure 19 Example 6 illustrates the effect of different treatments on the POD activity of chili seedlings.

[0039] Figure 20 Example 6 illustrates the effect of different treatments on CAT activity in chili seedlings.

[0040] Figure 21 Example 6 illustrates the effect of different treatments on the SOD activity of chili seedlings.

[0041] Figure 22 Example 6 illustrates the effect of different treatments on the soluble sugar content of chili seedlings.

[0042] Figure 23 Example 6 illustrates the effect of different treatments on the soluble protein content of chili seedlings.

[0043] Information on the preservation of biological materials

[0044] Strain DTI9, classified as Agrobacterium suis. Agrobacterium rubi Latin name Agrobacterium rubi It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on April 30, 2026, with accession number CGMCC No. 38525. Detailed Implementation

[0045] The present invention will be further explained below with reference to embodiments and accompanying drawings. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0046] Unless otherwise specified, the plates (petition dishes) used in the following examples are all 90 mm in diameter.

[0047] Unless otherwise specified, the culture media and reagents used in the following examples have the following formulations:

[0048] Ashby liquid medium (1 L): mannitol 10.0 g, sodium chloride 0.2 g, magnesium sulfate heptahydrate 0.2 g, calcium carbonate 5.0 g, calcium sulfate 0.1 g, potassium dihydrogen phosphate 0.2 g; pH 7.0.

[0049] Ashby solid medium (1 L): mannitol 10.0 g, sodium chloride 0.2 g, magnesium sulfate heptahydrate 0.2 g, calcium carbonate 5.0 g, calcium sulfate 0.1 g, potassium dihydrogen phosphate 0.2 g, agar 18.0 g; pH 7.0.

[0050] YMA liquid medium (1 L): mannitol 10.0 g, yeast extract 1.0 g, dipotassium hydrogen phosphate 0.5 g, magnesium sulfate heptahydrate 0.2 g, sodium chloride 0.1 g, calcium sulfate 0.1 g; pH 7.0.

[0051] YMA solid medium (1 L): mannitol 10.0 g, yeast extract 1.0 g, dipotassium hydrogen phosphate 0.5 g, magnesium sulfate heptahydrate 0.2 g, sodium chloride 0.1 g, calcium sulfate 0.1 g, agar 18.0 g; pH 7.0.

[0052] Chromium Azurite S (CAS) detection medium (1 L): Chromium Azurite S 0.0605 g, cetyltrimethylammonium bromide 0.0729 g, ferric chloride hexahydrate 0.002645 g, disodium hydrogen phosphate dodecahydrate 1.2135 g, ammonium chloride 0.125 g, potassium dihydrogen phosphate 0.375 g, sodium chloride 0.625 g, agar 9.0 g; pH 6.8±0.1.

[0053] Knop nutrient solution (1 L): 0.80 g calcium nitrate, 0.25 g potassium nitrate, 0.14 g potassium dihydrogen phosphate, 0.25 g magnesium sulfate heptahydrate, 0.02 g ferric chloride; pH 5.8±0.1.

[0054] Low-nitrogen Knop nutrient solution (1 L): calcium nitrate 0.03 g, potassium nitrate 0.25 g, potassium dihydrogen phosphate 0.14 g, magnesium sulfate heptahydrate 0.25 g, ferric chloride 0.02 g; pH 5.8±0.1.

[0055] Methyl red-bromocresol green mixed indicator: Dissolve 0.5 g of bromocresol green and 0.1 g of methyl red in 100 mL of ethanol and mix well.

[0056] Boric acid-indicator solution (20 g / L): Dissolve 20 g boric acid (H3BO3) in 1 L of water, add 5 mL of methyl red-bromocresol green mixed indicator, and adjust to a slightly purple-red color (pH 4.8) with dilute sulfuric acid (0.1 M H2SO4) or dilute alkali (0.1 M NaOH). Prepare fresh before use.

[0057] Example 1: Isolation, purification and screening of functional strains

[0058] Healthy tamarisk trees were selected from saline-alkali land in Dongtai City, Yancheng City, Jiangsu Province. Root samples (including roots and attached soil) were collected and placed in sterile sample bags, then transported to the laboratory in a 4℃ ice pack incubator. The root samples were placed in centrifuge tubes and vortexed at 2000 r / min for 10 min to detach and collect the soil particles attached to the roots. 0.1 g of the collected soil was weighed, added to 10 mL of sterile water, mixed thoroughly, vortexed at 2000 r / min for 5 min, and allowed to stand for 30 s. In a clean bench, 1 mL of the suspension was added to 9 mL of sterile water to prepare 10 mL of the turbid solution. -1 Diluent, and so on, to obtain 10 -2 10 -3 10 -4 and 10 -5 Diluents. Take 200 μL of each graded dilution and spread it evenly on Ashby agar plates containing 1% (w / v, i.e., 1 g NaCl in 100 mL of medium) NaCl. Incubate at 30°C for 72 h. Pick single colonies with different morphologies from the plates and purify them multiple times on Ashby agar plates containing 1% (w / v) NaCl using the three-zone streak method. Inoculate the purified strains onto YMA agar slants and store them at 4°C for short-term use.

[0059] The purified strains were initially screened by inoculating them onto Ashby solid medium containing 1% (w / v) NaCl and culturing at 28°C. Strains were selected based on colony growth rate and colony diameter. The strains obtained from the initial screening were then further screened: the strains obtained from the initial screening were streaked onto Ashby solid medium plates containing 1% (w / v) NaCl for activation and cultured at 28°C for 48 h. Single colonies were picked using an inoculation loop and inoculated into Ashby liquid medium containing 1% (w / v) NaCl, and cultured with shaking at 28°C and 180 r / min for 24 h to obtain a seed culture. The seed culture was then inoculated into Ashby liquid medium containing 1% (w / v) NaCl at a 1% (v / v) inoculation rate and cultured with shaking at 28°C and 180 r / min for 7 days to obtain the strain culture. The total nitrogen content of the bacterial culture medium was determined using the Kjeldahl method: 10 mL of bacterial culture medium and 10 mL of 98% (wt) concentrated sulfuric acid were transferred into a Kjeldahl flask and placed in a graphite digestion furnace in a fume hood. The temperature was raised to 380℃ for digestion. During this process, 200 μL of 30% (wt) hydrogen peroxide was added every 20 min. Heating was stopped when the liquid in the flask turned transparent. The Kjeldahl flask was removed and cooled to room temperature. The digest was transferred to a 50 mL volumetric flask and diluted to 50 mL with deionized water to obtain the digestion solution to be tested. 20 mL of the digestion solution was placed in the sample tube of a K9840 automatic Kjeldahl nitrogen analyzer (Shandong Haineng Scientific Instruments Co., Ltd.). The instrument automatically added 10 mL of deionized water and 25 mL of 40% (wt) sodium hydroxide solution. Distillation was carried out for 5 min. The ammonia gas produced by distillation was introduced into a conical flask containing 10 mL of boric acid-indicator solution. A 0.01 mol·L⁻¹ solution was used. -1 The hydrochloric acid standard solution was titrated with the boric acid-indicator solution in the conical flask to a light gray-red endpoint. The volume of hydrochloric acid standard solution consumed was recorded, and the nitrogen content of the sample (mg·L⁻¹) was calculated. -1 The nitrogen fixation capacity of the strains was evaluated, and the screening results are shown in Table 1.

[0060] Table 1. Nitrogen fixation capacity of strains

[0061] DTI9 15.76±0.22 DTL3 12.25±0.02 DTA8 11.90±0.05 DTI6 10.85±0.18 DTA2 10.50±0.36 DTM5 10.05±0.12 DTM6 9.45±0.28 DTJ7 9.45±0.02 DTA7 7.80±0.15 DTH4 7.00±0.17 DTL4 7.00±0.07 DTA9 6.65±0.08 DTA4 6.30±0.67 DTC7 5.60±0.10 DTJ1 5.60±0.05 DTK2 3.85±0.10 DTG3 3.50±0.02 DTH3 2.80±0.16 DTF4 1.40±0.17

[0062] The results in Table 1 show that strain DTI9 has the strongest nitrogen fixation ability, and subsequent experiments will be conducted using strain DTI9.

[0063] Example 2: Identification of strain DTI9

[0064] Colony morphology identification:

[0065] Strain DTI9 was activated by streaking on Ashby agar plates containing 1% (w / v) NaCl. After incubation at 28°C for 48 h, a single colony was picked up with an inoculation loop and streaked onto Ashby agar plates containing 1% (w / v) NaCl. The colonies were then incubated at 28°C for 72 h for observation. Figure 1 As shown, the colonies are round, with neat edges, transparent, and smooth.

[0066] Molecular biological identification:

[0067] Genomic DNA of strain DTI9 was extracted by Sangon Biotech (Shanghai) Co., Ltd., and PCR amplification and sequencing were performed using universal primers for the bacterial 16S rRNA gene. The obtained 1281 bp 16S rRNA gene sequence (as shown in SEQ ID NO.1) was subjected to BLAST homology comparison in the NCBI database. The results showed that this sequence was similar to that of *Agrobacterium tumefaciens* (…). Agrobacterium rubi Members are highly homologous, especially to Agrobacterium spp. ( Agrobacterium rubi The type strain LMG 156 (NR118991.1) showed a 98.28% similarity. To further confirm its taxonomic position, the 16S rRNA sequences of related type strains were downloaded from the database, and a phylogenetic tree was constructed using MEGA 12 software via neighbor-joining. Figure 2 As shown, strain DTI9 and Agrobacterium rubi LMG 156 clusters in the same highly supported evolutionary branch.

[0068] Based on the colony morphology, sequence similarity, and phylogenetic analysis results of strain DTI9, strain DTI9 was identified as *Agrobacterium spp.* Agrobacterium rubi It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 30, 2026, with accession number CGMCC No. 38525.

[0069] Example 3: Salt tolerance test of strain DTI9

[0070] Strain DTI9 was activated by streaking on YMA solid agar plates and cultured at 28°C for 48 h. Single colonies were then picked using an inoculation loop and inoculated into YMA liquid agar, and cultured with shaking at 28°C and 180 r / min for 24 h to prepare a seed culture. The seed culture was then inoculated at 1% (v / v) into YMA liquid agar with NaCl concentrations of 0% (w / v), 3% (w / v), 6% (w / v), and 9% (w / v), respectively, and cultured with shaking at 28°C and 180 r / min. The OD of the culture medium was measured periodically (every 4 h). 600 The values ​​were calculated, and a growth curve was plotted.

[0071] The results are as follows Figure 3 As shown, strain DTI9 grew well under 0% (w / v) and 3% (w / v) NaCl conditions, with OD... 600 The OD value increased significantly with culture time, reaching a high cell density in the middle and late stages of culture. Under 6% (w / v) NaCl conditions, strain DTI9 could still grow, but the OD value... 600 The values ​​were significantly lower than those under 0% (w / v) and 3% (w / v) NaCl conditions. Under 9% (w / v) NaCl conditions, the cell density showed almost no significant increase, indicating that this concentration exceeded the tolerance limit of strain DTI9, and its growth was severely inhibited.

[0072] Example 4: pH tolerance determination of strain DTI9

[0073] The pH of YMA solid medium was adjusted using 1 mol / L HCl and 1 mol / L NaOH solutions, with five pH gradients (6, 7, 8, 9, and 10) to prepare plates with different pH levels. Strawberry strain DTI9 was activated by streaking on YMA solid medium plates and incubated at 28°C for 48 h. Single colonies were then picked using an inoculation loop and inoculated onto plates with the different pH levels. Each pH treatment was repeated three times, and the plates were incubated at 28°C for 3 days. Colony growth was observed and recorded every 24 h, and the diameter of strain DTI9 (colony diameter) was measured and recorded.

[0074] like Figure 4 As shown, strain DTI9 can grow in the pH range of 6 to 9, with intact colony morphology. It grows particularly well in the pH range of 7 to 9, with larger colony diameters.

[0075] Example 5: Determination of the siderophore-producing and auxin (IAA)-producing abilities of strain DTI9

[0076] (1) Determination of the siderophore production capacity of strain DTI9

[0077] Strain DTI9 was activated by streaking on YMA solid medium plates and incubated at 28°C for 48 h. Single colonies were then picked using an inoculation loop and inoculated onto CAS detection medium plates, where they were incubated at 28°C for 5 days. On the CAS detection medium plates, siderophore-producing colonies showed a distinct orange halo around them. The diameter of the orange halo (D) and the colony diameter (d) were measured, and the D / d ratio was calculated. A higher ratio indicates a stronger siderophore-producing ability of the strain. Figure 5 The orange halo diameter (D) was measured to be 13.39 mm, the colony diameter (d) was 6.43 mm, and the siderophore index (D / d) was 2.08 ± 0.03.

[0078] (2) Determination of the ability of strain DTI9 to produce auxin (IAA)

[0079] Weigh 10 mg of indole-3-acetic acid (IAA), dissolve it in a small amount of ethanol (1-2 mL, just enough to dissolve), and then dilute to 100 mL with deionized water to obtain an IAA standard stock solution with a concentration of 100 mg / L. Prepare a series of IAA standard working solutions with concentrations of 0, 10, 20, 30, 40, and 50 mg / L by diluting with deionized water and store them protected from light. Take 2 mL of each IAA standard working solution of different concentrations, add an equal volume of Salkowski's colorimetric solution (Fuzhou Feijing Biotechnology Co., Ltd., PH1941), and incubate at room temperature in the dark for 30 min. Measure the absorbance at 530 nm using a spectrophotometer. Plot an IAA standard curve with the IAA standard working solution concentration on the x-axis and the absorbance value on the y-axis.

[0080] Strain DTI9 was activated by streaking on YMA solid agar plates and cultured at 28°C for 48 h. Single colonies were then picked using an inoculation loop and inoculated into YMA liquid agar, and cultured with shaking at 28°C and 180 rpm for 24 h to prepare the seed culture. Experimental group: The seed culture was transferred to YMA liquid agar containing 0.1 g / L tryptophan at a 1% (v / v) inoculation rate and cultured at 28°C and 180 rpm for 36 h to obtain the culture medium. 2 mL of the culture medium was added to an equal volume of Salkowski's colorimetric solution and incubated at room temperature in the dark for 30 min. The absorbance was measured at 530 nm using a spectrophotometer. Control group (CK): The seed culture was replaced with an equal volume of YMA liquid agar. Figure 6 As shown, the left side represents the control group, and the right side represents the experimental group. The IAA content produced by strain DTI9 was calculated based on the IAA standard curve. The calculated IAA production by strain DTI9 was 96.26 ± 0.97 μg / mL.

[0081] Example 6: Pot experiment on growth promotion of pepper by strain DTI9

[0082] The seedling substrate used in this embodiment was provided by the Xinjiang Branch of Huaian Chaimihe Agricultural Technology Co., Ltd., with organic matter (on a dry basis) of 22.67% (wt), EC value of 1.8 mS / cm, pH 6.24, total nutrients (N+P2O5+K2O, on a dry basis) of 1.85% (wt) and moisture of 36.51% (wt).

[0083] 1. Preparation of DTI9 bacterial agent

[0084] Strain DTI9 was activated by streaking on YMA solid medium plates and incubated at 28°C for 48 h. Single colonies were then picked using an inoculation loop and inoculated into 250 mL Erlenmeyer flasks containing 100 mL of YMA liquid medium. The flasks were incubated at 28°C with shaking at 180 rpm for 24 h to prepare the seed culture. The seed culture was then inoculated into YMA liquid medium at a ratio of 1 mL seed culture to 300 mL YMA liquid medium and incubated at 28°C with shaking at 180 rpm for 12 h to obtain the fermentation broth. The fermentation broth was centrifuged at 4000 rpm for 20 min, the supernatant was discarded, and the bacterial pellet was resuspended in sterile water and adjusted to OD500 with sterile water. 600 =1.6 to a bacterial concentration of 4.4 × 10⁻⁶ 9 CFU / mL was used to obtain DTI9 bacterial agent.

[0085] 2. Pot experiment on the effect of DTI9 inoculant on pepper growth promotion under salt-low nitrogen combined stress.

[0086] To investigate the growth-promoting effect of DTI9 inoculant on peppers under salt-low nitrogen combined stress, this experiment included four treatments: CK, T1, T2, and T3, with each treatment replicated eight times (i.e., eight potted plants per treatment). The settings for each treatment are as follows:

[0087] CK (control), non-stress treatment: no DTI9 bacterial agent was applied, but Knop nutrient solution was applied.

[0088] T1, Salt-Low Nitrogen Combined Stress Treatment: No DTI9 bacterial agent was applied, but a low-nitrogen Knop nutrient solution containing 150 mmol / L NaCl was applied.

[0089] T2, Salt-Low Nitrogen Combined Stress + DTI9 Bacterial Agent Treatment: DTI9 bacterial agent was applied, along with a low-nitrogen Knop nutrient solution containing 150 mmol / L NaCl.

[0090] T3, non-stress + DTI9 bacterial agent treatment: apply DTI9 bacterial agent and apply Knop nutrient solution.

[0091] The pot experiment was conducted in early December 2025 in a plastic greenhouse at Huai'an Chaimihe Agricultural Technology Co., Ltd. The pots were 12 cm in diameter and 10 cm high, each filled with seedling substrate to about 1 cm from the rim. The chili pepper variety Biyu 888 was selected, and plump, uniformly sized seeds were chosen. The seeds were soaked in a 70% (v / v) ethanol solution for 1 minute, rinsed 3–5 times with sterile water, then soaked in a 1% (w / v) sodium hypochlorite solution for 1 minute, rinsed 3–5 times with sterile water, and then soaked in sterile water for 1 hour. After sterilization and soaking, the seeds were placed in petri dishes lined with two layers of moist filter paper and germinated at 25℃ in the dark for 3 days. Three germinated seeds with uniform growth were transplanted into each pot. For the DTI9 inoculant treatment, 30 mL of DTI9 inoculant was applied to each pot at transplanting (i.e., 10 mL of seedling substrate around each germinating pepper seed). For the treatment without DTI9 inoculant, the same volume of sterile water was applied. Starting from day 2 after transplanting, sterile water was applied every 2 days, with each treatment applying 60 mL of sterile water per pot (i.e., 20 mL of seedling substrate around each plant) until day 19 after transplanting. Starting from day 20 after transplanting, KNOP nutrient solution or a low-nitrogen KNOP nutrient solution containing 150 mmol / L NaCl was applied every 3 days (each treatment was performed according to the settings), with each treatment applying 60 mL of KNOP nutrient solution or a low-nitrogen KNOP nutrient solution containing 150 mmol / L NaCl per pot (i.e., 20 mL of seedling substrate around each plant) until day 61 after transplanting. Specifically: On day 1 after transplanting, apply 30 mL of inoculant or sterile water to each pot; on days 2, 4, 6, 8, 10, 12, 14, 16, and 18 after transplanting, apply 60 mL of sterile water to each pot; on days 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, and 59 after transplanting, apply 60 mL of KNOP nutrient solution or a low-nitrogen KNOP nutrient solution containing 150 mmol / L NaCl to each pot. All other management conditions remained consistent across treatments, with no additional fertilizers or pesticides applied.

[0092] 3. Indicator Measurement

[0093] On day 61 after transplanting, growth indicators such as plant height, fresh weight, dry weight, leaf width, and stem diameter of pepper seedlings in each treatment were measured, as well as physiological indicators such as malondialdehyde content, peroxidase activity, catalase activity, superoxide dismutase activity, soluble sugar content, and soluble protein content.

[0094] The methods for measuring each growth index are as follows:

[0095] Plant height: The vertical height of the chili seedling from the surface of the seedling substrate to the apical bud (growing point) was measured using vernier calipers, and the unit is mm.

[0096] Fresh weight: Remove the chili seedlings completely from the seedling substrate, rinse the roots with clean water, and dry the surface moisture of the chili seedlings with absorbent paper; cut the chili seedlings at the base of the stem to divide them into above-ground and underground parts, and weigh them separately using an electronic balance to obtain the fresh weight of the above-ground part and the fresh weight of the underground part, in g; the total fresh weight is the sum of the fresh weight of the above-ground part and the fresh weight of the underground part.

[0097] Dry weight: The above-ground and underground parts with the measured fresh weight were placed in an oven and blanched at 105℃ for 30 min, and then dried at 75℃ to constant weight; after being removed, they were placed in a desiccator to cool to room temperature, and weighed separately using an electronic balance to obtain the dry weight of the above-ground parts and the dry weight of the underground parts, in g; the total dry weight is the sum of the dry weight of the above-ground parts and the dry weight of the underground parts.

[0098] Leaf width: Select the third fully unfolded functional leaf from the bottom of the chili seedling and measure the lateral width of the widest part of the leaf using vernier calipers. The unit is mm.

[0099] Stem diameter: The transverse diameter of the base of the chili seedling stem is measured using vernier calipers in two mutually perpendicular directions. The average of the two measurements is taken as the stem diameter, in cm.

[0100] The methods for measuring each physiological indicator are as follows:

[0101] The third-to-last fully expanded functional leaf from each treatment of pepper seedlings was collected for the determination of malondialdehyde content, peroxidase activity, catalase activity, superoxide dismutase activity, soluble sugar content, and soluble protein content.

[0102] (1) Determination of malondialdehyde (MDA) content

[0103] The thiobarbituric acid (TBA) method was used. 0.5 g of fresh leaves were weighed and placed in a pre-cooled (4℃) mortar. 5 mL of pre-cooled (4℃) 5% (w / v) trichloroacetic acid (TCA) solution was added, and the mixture was ground in an ice bath until homogenized. The homogenate was centrifuged at 4000 r / min for 10 min at 4℃. 2 mL of the supernatant was collected, and 2 mL of 0.67% (w / v, prepared with 5% (w / v) TCA solution) thiobarbituric acid (TBA) solution was added. After mixing, the mixture was developed in a boiling water bath for 30 min. After cooling to room temperature, it was centrifuged at 4000 r / min for 10 min, and the absorbance of the supernatant at 450 nm, 532 nm, and 600 nm was measured. The MDA content was calculated in μmol / g FW.

[0104] (2) Peroxidase (POD) activity assay

[0105] The guaiacol method was used. 0.5 g of fresh leaves were weighed and placed in a pre-cooled (4℃) mortar. 8 mL of pre-cooled (4℃) 0.1 mol / L phosphate buffer (pH 6.0) was added, and the mixture was ground into a homogenate in an ice bath. The homogenate was centrifuged at 10000 r / min for 10 min at 4℃. The supernatant was the enzyme extract and stored in an ice bath for later use.

[0106] The reaction system consisted of: 2.5 mL of 0.1 mol / L phosphate buffer (pH 6.0), 2 mL of 0.05 mol / L guaiacol, 1 mL of 0.2% (w / v) H2O2, and 0.1 mL of enzyme extraction solution was added to initiate the reaction. OD was immediately measured. 470 The value was measured every 30 seconds, and recorded for a total of 3 minutes. The OD value was expressed as a percentage per minute. 470 A change of 0.01 corresponds to 1 unit of enzyme activity (U). POD activity is calculated in units of u / (g·min).

[0107] (3) Assay of catalase (CAT) activity

[0108] Potassium permanganate titration was used. 0.5 g of fresh leaves were weighed and placed in a pre-cooled (4℃) mortar. 5 mL of pre-cooled (4℃) 0.1 mol / L phosphate buffer (pH 7.0) was added, and the mixture was ground into a homogenate in an ice bath. The homogenate was centrifuged at 10000 r / min for 10 min at 4℃. The supernatant was the enzyme extract and stored in an ice bath for later use.

[0109] The reaction system consisted of 2.5 mL of 0.1 mol / L phosphate buffer (pH 7.0), 1 mL of deionized water, and 1 mL of 0.1 mol / L H₂O₂. 0.5 mL of enzyme extract was added to initiate the reaction, which was then incubated in a 30°C water bath for 10 min. Immediately after the reaction, 2 mL of 10% (w / v) H₂SO₄ was added to terminate the reaction. The remaining H₂O₂ was titrated with 0.02 mol / L KMnO₄ standard solution until a faint pink color appeared that did not fade within 30 seconds. The enzyme extract, deactivated by boiling (10 min), served as a blank control. CAT activity was calculated in mg H₂O₂ / (g·min).

[0110] (4) Superoxide dismutase (SOD) activity assay

[0111] The nitroblue tetrazolium (NBT) photoreduction method was used. 0.5 g of fresh leaves were weighed and placed in a pre-cooled (4℃) mortar. 1 mL of pre-cooled (4℃) 0.05 mol / L phosphate buffer (pH 7.8) was added, and the mixture was ground in an ice bath until homogenized. The homogenate was poured into a centrifuge tube, and the volume was adjusted to 5 mL with 0.05 mol / L phosphate buffer (pH 7.8). The tube was centrifuged at 10000 r / min for 10 min at 4℃. The supernatant was the enzyme extract and stored in an ice bath for later use.

[0112] The reaction system consisted of: 1.5 mL of 0.05 mol / L phosphate buffer (pH 7.8), 0.3 mL of 130 mmol / L methionine (Met), 0.3 mL of 750 μmol / L NBT, 0.3 mL of 100 μmol / L EDTA-Na2, 0.3 mL of 20 μmol / L riboflavin, 0.1 mL of enzyme extraction buffer, and 0.2 mL of deionized water. After mixing, the mixture was incubated at room temperature under 4000 lx light for 20 min, and the OD was measured. 560 Value. One unit of enzyme activity (U) is defined as the inhibition of NBT photoreduction by 50%. SOD activity is calculated in U / g FW.

[0113] (5) Determination of soluble sugar content

[0114] The anthrone colorimetric method was used. Fresh leaves were taken, chopped and mixed, and 0.2 g was weighed and placed in a graduated test tube. 8 mL of deionized water was added, the tube was sealed with plastic film, and extracted in a boiling water bath for 30 min (repeated twice). The extract was filtered into a 50 mL volumetric flask, the test tube and residue were repeatedly rinsed, and the volume was adjusted to the mark to obtain the sample extract.

[0115] Pipette 0.5 mL of the sample extract into a 20 mL graduated test tube, add 1.5 mL of deionized water, then add 0.5 mL of anthrone ethyl acetate reagent (1 g of anthrone dissolved in 50 mL of ethyl acetate, stored in a brown bottle protected from light) and 5 mL of 98% (wt) concentrated sulfuric acid. Shake thoroughly, immediately incubate in a boiling water bath for 1 min, and then allow to cool naturally to room temperature. Using a blank as a reference, measure the absorbance at 630 nm. Plot a standard curve using sucrose as a standard and calculate the soluble sugar content in mg / g FW.

[0116] (6) Determination of soluble protein content

[0117] Coomassie Brilliant Blue G-250 staining method was used. 0.5 g of fresh leaves were weighed and placed in a pre-cooled (4℃) mortar, along with 5 mL of 0.1 mol / L phosphate buffer (pH 7.0). The mixture was then homogenized in an ice bath. The homogenate was centrifuged at 10000 r / min for 15 min at 4℃. 0.1 mL of the supernatant was collected, and 5 mL of Coomassie Brilliant Blue G-250 reagent was added. After mixing and standing for 2 min, the OD was measured. 595 Value. A standard curve was plotted using bovine serum albumin (BSA) as a standard, and the soluble protein content was calculated in mg / g FW.

[0118] 4. Experimental Results

[0119] The results are as follows Figures 7-23 As shown.

[0120] Compared with the control (CK) treatment, the T1 treatment showed lower leaf width, plant height, stem diameter, aboveground fresh weight, underground fresh weight, total fresh weight, aboveground dry weight, underground dry weight, and total dry weight of pepper seedlings. The underground fresh weight and underground dry weight showed significant differences. The MDA content of the T1 treatment was significantly higher than that of the CK treatment. The POD activity, CAT activity, SOD activity, soluble sugar content, and soluble protein content of the T1 treatment were all lower than those of the CK treatment. The soluble sugar content and soluble protein content showed significant differences. This indicates that the salt-low nitrogen combined stress treatment inhibited pepper seedling growth, induced membrane peroxidation damage, and reduced the antioxidant capacity and osmotic regulation substance accumulation of pepper seedlings.

[0121] Compared with the CK treatment, the T2 treatment showed no significant differences in leaf width, plant height, stem diameter, aboveground fresh weight, underground fresh weight, total fresh weight, aboveground dry weight, underground dry weight, and total dry weight of pepper seedlings. The T2 treatment had significantly higher MDA, POD, CAT, and SOD contents than the CK treatment. However, the T2 treatment had no significant differences in soluble sugar and soluble protein contents compared to the CK treatment. This indicates that applying DTI9 inoculant under salt-low nitrogen combined stress can alleviate the inhibitory effect of stress on pepper seedling growth, resulting in no significant difference in growth phenotype between the pepper seedlings and the normal control. However, the pepper seedlings still maintained high levels of antioxidant enzymes, indicating a certain degree of physiological stress response.

[0122] Compared with the CK treatment, the T3 treatment significantly increased leaf width, plant height, stem diameter, aboveground fresh weight, underground fresh weight, total fresh weight, aboveground dry weight, underground dry weight, and total dry weight of pepper seedlings. There was no significant difference in MDA content between the T3 and CK treatments. However, the T3 treatment showed higher levels of POD, CAT, SOD activities, soluble sugar content, and soluble protein content compared to the CK treatment, with POD, CAT, and soluble sugar content showing significant differences. This indicates that under normal culture conditions, application of DTI9 inoculant can significantly promote pepper seedling growth, increase antioxidant levels, and enhance the potential stress resistance reserves of pepper seedlings.

[0123] Compared with treatment T2, treatment T2 showed significantly higher leaf width, plant height, stem diameter, aboveground fresh weight, underground fresh weight, total fresh weight, aboveground dry weight, underground dry weight, and total dry weight of chili seedlings, with the underground fresh weight and underground dry weight showing significant differences. The MDA content of chili seedlings in treatment T2 was significantly lower than that in treatment T1, while the POD activity, CAT activity, SOD activity, soluble sugar content, and soluble protein content of chili seedlings in treatment T2 were significantly higher than those in treatment T1. This indicates that under salt-low nitrogen combined stress, application of DTI9 inoculant can alleviate membrane peroxidation damage in chili seedlings, increase antioxidant enzyme activity and osmotic regulator accumulation, enhance stress resistance, and improve the growth status of chili seedlings, with a more pronounced effect on the underground parts.

[0124] The above specific embodiments are only used to illustrate the technical solution and core features of the present invention, and are intended to enable those skilled in the art to understand and practice the present invention. They do not constitute a limitation on the scope of the present invention. All equivalent modifications or alterations made in accordance with the essential spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A strain of Agrobacterium tumefaciens ( Agrobacterium rubi DTI9, characterized in that, It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 30, 2026, with accession number CGMCC No. 38525.

2. The bacterial agent prepared from Agrobacterium tumefaciens DTI9 according to claim 1, characterized in that, The bacterial cell concentration of the bacterial agent is ≥1×10⁻⁶. 9 cfu / mL.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent was prepared by the following method: Agrobacterium spp. DTI9 was inoculated into a liquid culture medium for liquid fermentation to obtain Agrobacterium spp. DTI9 fermentation broth; the fermentation broth was centrifuged, the supernatant was discarded, the bacterial precipitate was resuspended in sterile water, and the bacterial concentration was adjusted to ≥1 × 10⁻⁶. 9 The bacterial agent was obtained by dispersing cfu / mL.

4. The microbial agent according to claim 3, characterized in that, The liquid culture medium includes YMA liquid culture medium.

5. The microbial agent according to claim 3, characterized in that, The liquid fermentation culture conditions are: temperature 27-30℃, rotation speed 170-190 r / min, and time 12-24 h.

6. The application of Agrobacterium tumefaciens DTI9 as described in claim 1 in stress resistance and growth promotion of pepper seedlings under salt-low nitrogen combined stress.

7. The application of Agrobacterium tumefaciens DTI9 as described in claim 1 in promoting the growth of chili seedlings and improving the stress resistance reserves of chili seedlings under normal cultivation conditions.

8. The application of the microbial agent according to any one of claims 2-4 in the stress resistance and growth promotion of pepper seedlings under salt-low nitrogen combined stress.

9. The application of the microbial agent according to any one of claims 2-4 in promoting the growth of chili seedlings and improving the stress resistance reserves of chili seedlings under normal cultivation conditions.

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