Microbacterium T2 for decomposing trichothecin and application of microbacterium T2

Abstract

The invention belongs to the technical field of plant growth-promoting bacteria, and particularly relates to a microbacterium T2 for decomposing trichothecin and application of the microbacterium T2. The invention provides a strain of microbacterium trichiothenolytica T2, and the preservation number of the strain is GDMCC (China General Microbiological Culture Collection Center) No. 64981. The strain of microbacterium trichiothenolytica T2 has the advantages that the preservation number is GDMCC No. 64981; the microbacterium T2 for decomposing the trichothecin provided by the invention has the capabilities of producing IAA (Indoleacetic Acid), producing siderophores, producing ammonia, fixing nitrogen and dissolving iron, and can promote the growth of plants. The result of the embodiment shows that the microbacterium T2 for decomposing the trichothecin can obviously promote iron absorption and growth of arabidopsis thaliana under the iron limiting condition; potting experiments also show that the microbacterium unilaterosporum T2 can improve iron deficiency yellowing of different plants and promote plant growth, and has a good effect.

Description

Technical Field

[0001] This invention belongs to the field of plant growth-promoting bacteria technology, specifically relating to a strain of Trichosporon monotypic bacteria T2 and its applications. Background Technology

[0002] Iron (Fe) is an essential micronutrient for plant growth and development, participating in many important physiological and metabolic processes, such as photosynthesis, respiration, nitrogen fixation, and hormone synthesis. Although iron is abundant in soil, its solubility is very low, especially in calcareous soils where the soluble iron content is often far below the amount required for normal plant growth and development. Iron deficiency in crops hinders chlorophyll synthesis, causing young leaves to yellow first, and in severe cases, the plant to turn white, leading to decreased yield and quality. Iron deficiency in crops also affects iron absorption in humans, leading to anemia, fatigue, adverse pregnancy outcomes, and impaired cognitive development in children, among other health problems. Iron deficiency is one of the most common micronutrient deficiencies globally, affecting the health of over 2 billion people worldwide. Therefore, iron deficiency is a global problem.

[0003] To address this issue, the most common agricultural measure is the application of various inorganic, organic, and chelated iron fertilizers, but the high cost and environmental pollution problems are also obvious. Plant rhizosphere microorganisms are the plant's second genome, playing a crucial role in promoting nutrient absorption and assisting plants in coping with various biotic and abiotic stresses. Increasing evidence suggests that applying plant rhizosphere growth-promoting bacteria to improve crop yield and quality is one of the trends in modern sustainable agriculture. Current research on using microorganisms to promote plant nutrient absorption mainly focuses on microorganisms promoting the absorption of macroelements such as nitrogen, phosphorus, and potassium. Existing technologies include Chinese patent CN116254192A, which reports on a side-carrier-producing strain of *Bacillus paclitaxel* from rice and its application, demonstrating that *Bacillus paclitaxel* promotes plant iron absorption; and Chinese patent CN118755641A, which reports on a highly efficient side-carrier-producing *Dystridium difficile* and its application, demonstrating that *Dystridium difficile* promotes plant iron absorption. There are no existing reports on *Trichoderma solani* promoting plant iron absorption. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Trichoderma solani T2, which can be used to improve iron deficiency chlorosis in plants, promote iron absorption and plant growth.

[0005] To address the aforementioned technical problems, the present invention proposes the following technical solution:

[0006] This invention provides a strain of Microbacterium trichothecenolyticum T2, with accession number GDMCC No. 64981.

[0007] This invention provides a microbial agent comprising *Trichoderma solani* T2 as described in the above technical solution.

[0008] Preferably, the viable count of *Trichoderma solani* T2 in the bacterial agent is ≥1×10⁻⁶. 8 CFU / g.

[0009] Preferably, the application form of the Trichoderma solani T2 includes bacterial suspension.

[0010] The present invention provides a method for preparing a microbial agent, comprising: inoculating Trichosporon monotypic bacteria T2 into a culture medium and culturing it to obtain a fermentation broth, wherein the fermentation broth is centrifuged and resuspended to obtain a microbial agent.

[0011] Preferably, the culture temperature is 28–33°C and the culture time is 1–3 days.

[0012] This invention provides the application of the above-described Trichoderma solani T2 or the above-described bacterial agent or the bacterial agent prepared by the above-described preparation method in any one or more of the following 1) to 5);

[0013] 1) Promotes iron absorption in plants;

[0014] 2) Promote plant growth;

[0015] 3) Improves plant yellowing;

[0016] 4) Preparation of microbial fertilizers;

[0017] 5) Prepare plant growth promoters.

[0018] Preferably, the plant includes one or more of Arabidopsis thaliana, cucumber, and maize.

[0019] Preferably, the plant growth promotion includes increasing plant biomass.

[0020] Preferably, the improvement of plant etiolation includes increasing the chlorophyll content of the plant.

[0021] The beneficial effects of this invention: This invention provides a strain of *Microbacterium trichothecenolyticum* T2, with the preservation number GDMCC No. 64981. The *Microbacterium trichothecenolyticum* T2 provided by this invention possesses the abilities to produce IAA, siderophores, ammonia, fix nitrogen, and dissolve iron, thus promoting plant growth. The results of the examples show that *Microbacterium trichothecenolyticum* T2 can significantly promote iron absorption and growth in *Arabidopsis thaliana* under iron-limited conditions; pot experiments also show that *Microbacterium trichothecenolyticum* T2 can improve iron deficiency chlorosis in different plants and promote plant growth, demonstrating good effects.

[0022] In summary, the *Trichoderma solani* T2 of this invention can be used to improve iron deficiency chlorosis in plants, promote iron absorption and growth, and provide theoretical and technical support for the development and application of microbial agents, microbial fertilizers or plant growth promoters. Attached Figure Description

[0023] Figure 1 The image shows the morphology of Trichosporon monotypic bacteria T2, with the left side showing the colony morphology and the right side showing the Gram staining.

[0024] Figure 2 To decipher the phylogenetic tree of the 16S rDNA sequence of Trichosporon microbacterium T2;

[0025] Figure 3 To decipher the growth-promoting characteristics of Trichoderma solani T2, where A represents IAA production capacity, B represents siderophore production capacity, C represents ammonia production capacity, and D represents nitrogen fixation capacity;

[0026] Figure 4 The effect of Trichoderma solani T2 on iron deficiency chlorosis in Arabidopsis thaliana is shown in the plate image.

[0027] Figure 5 Figure 1 shows an experiment using *Trichoderma solani* T2 to improve iron deficiency chlorosis in potted plants of Arabidopsis thaliana.

[0028] Figure 6 Figure 1 shows a pot experiment to promote Arabidopsis thaliana growth using Trichoderma solani T2.

[0029] Figure 7 An experimental diagram illustrating how *Trichoderma solani* T2 can improve iron deficiency chlorosis in cucumbers grown in pots.

[0030] Figure 8 Figure 1. Experimental diagram of potted maize plants to improve iron deficiency chlorosis caused by Trichoderma spore-forming bacteria T2.

[0031] Biological Preservation Instructions

[0032] Microbacterium trichothecenolyticum T2 was deposited on May 8, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No. 64981), located at 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province.

[0033] Bacillus velezensis SQR9 was deposited on February 27, 2012, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNo.5808. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0034] This invention provides a strain of *Microbacterium trichothecenolyticum* T2, with accession number GDMCC No. 64981. The 16S rDNA gene sequence of *Microbacterium trichothecenolyticum* T2 described in this invention is shown in SEQ ID NO. 1.

[0035] The *Trichoderma solani* T2 strain described in this invention was screened from the rhizosphere of maize. *Trichoderma solani* T2 possesses the abilities to produce IAA (intracytoplasmic acid), siderophores, ammonia, fix nitrogen, and dissolve iron, and can be used to improve iron deficiency chlorosis in plants, promote iron absorption and growth, thereby achieving a plant growth-promoting effect. On LB agar plates, *Trichoderma solani* T2 colonies are round, pale yellow, smooth, non-protruding, opaque, and have smooth edges.

[0036] This invention extracts the 16S rDNA sequence of *Trichoderma solani* T2, performs a BLAST homology sequence search in GenBank, and constructs a phylogenetic tree, see [link to GenBank documentation]. Figure 2 This indicates that strain T2 belongs to Microbacterium trichothecenolyticum.

[0037] This invention provides a microbial agent comprising *Trichoderma solani* T2 as described in the above technical solution.

[0038] In this invention, the viable count of *Trichoderma solani* T2 in the bacterial agent can be ≥1×10⁻⁶. 8 CFU / g, and can even be 1×10 8 CFU / g.

[0039] In this invention, the application of the Trichosporon monosodium spp. T2 may include bacterial suspension.

[0040] As an optional embodiment, the method for preparing the bacterial suspension of the present invention includes: inoculating the *Trichoderma unicinophilus* T2 in a culture medium and culturing it to obtain a fermentation broth; centrifuging and resuspending the fermentation broth to obtain a bacterial suspension. As an optional embodiment, the culture temperature of the present invention is 28–33°C, or 29–31°C, more preferably 30°C; in the embodiments of the present invention, the culture temperature is 28, 29, 30, 31, 32, or 33°C. As an optional embodiment, the culture time is 1–3 days, more preferably 2 days; in the embodiments of the present invention, the culture time is 1, 2, or 3 days. As an optional embodiment, the culture rotation speed is 150–180 rpm, further preferably 160–170 rpm; in the embodiments of the present invention, the culture rotation speed is 150, 160, 170, or 180 rpm.

[0041] The fermentation broth is obtained, and preferably, the fermentation broth is centrifuged and resuspended to obtain a bacterial suspension. As an optional embodiment, the centrifugation speed and time are not particularly limited, and conventional parameters can be used. After centrifugation, bacterial cells are obtained, and preferably, the bacterial cells are resuspended to obtain a bacterial suspension. The resuspension is preferably performed using a 0.3% (w / w) NaCl solution. As an optional embodiment, the viable count of *Trichoderma solani* T2 in the bacterial suspension is ≥1 × 10⁻⁶. 8 CFU / mL, or 1×10 8 CFU / mL. The culture medium of this invention preferably includes a liquid culture medium; the liquid culture medium of this invention preferably includes LB liquid culture medium. As an optional embodiment, the LB liquid culture medium of this invention comprises: 10g peptone, 5g yeast extract, 5g NaCl, and 1L distilled water.

[0042] This invention provides a method for preparing a microbial agent, comprising: inoculating *Trichoderma solani* T2 in a culture medium and culturing it to obtain a fermentation broth; centrifuging and resuspending the fermentation broth to obtain the microbial agent. The preparation method of the microbial agent of this invention is the same as the preparation method of the bacterial suspension described above, which has been discussed and will not be repeated here. The viable count of *Trichoderma solani* T2 in the microbial agent of this invention can be ≥1×10⁻⁶. 8 CFU / mL, and can even be 1×10 8 CFU / mL.

[0043] This invention provides the application of *Trichoderma solani* T2 or the bacterial agent described in the above-mentioned technical solutions in any one or more of the following 1) to 5):

[0044] 1) Promotes iron absorption in plants;

[0045] 2) Promote plant growth;

[0046] 3) Improves plant yellowing;

[0047] 4) Preparation of microbial fertilizers;

[0048] 5) Prepare plant growth promoters.

[0049] In specific embodiments of the present invention, Arabidopsis thaliana, cucumber, and corn are used as examples to verify the effects. The preferred method for promoting plant growth in the present invention includes increasing plant biomass; the method for improving plant etiolation includes increasing plant chlorophyll content. As an optional implementation, increasing plant biomass includes increasing plant fresh weight.

[0050] As an optional implementation, the application of the present invention includes application to the soil and / or the base of the stem. In an embodiment of the present invention, when the *Trichoderma solani* T2 agent is applied to the soil and / or the base of the stem, the amount of the *Trichoderma solani* T2 agent applied can be 1-5 mL per seedling, more preferably 2 mL; the application time is immediately after transplanting.

[0051] In this embodiment of the invention, a bacterial suspension was obtained by culturing *Trichoderma solani* T2. Applying this suspension to the base of the stems of *Arabidopsis thaliana*, cucumber, and maize increased chlorophyll content and biomass, as well as the content of active iron within the plants. Pot experiments further clarified the beneficial effects of *Trichoderma solani* T2 in improving iron deficiency chlorosis and promoting plant growth.

[0052] The *Trichoderma solani* T2 strain described in this invention is of great significance for developing functional microbial fertilizers that improve crop iron nutrition and yield.

[0053] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0054] Example 1

[0055] 1. Identification of strains

[0056] The LB liquid medium consists of 10g peptone, 5g yeast extract, 5g NaCl, and 1L distilled water, with a pH of 7.0. 2% agar is added to the LB solid medium.

[0057] Strain T2 was previously isolated from the rhizosphere of maize in the laboratory. Strain T2 was streaked onto LB agar plates and incubated at 37°C for 3 days. The colony morphology is as follows: Figure 1As shown in the left image, the colonies of strain T2 are round, pale yellow, smooth, non-protruding, opaque, and have smooth edges.

[0058] Strain T2 was Gram-stained using a Gram staining kit (purchased from Beijing Solarbio Science & Technology Co., Ltd.), and observed under an oil immersion microscope. Figure 1 As shown in the right-hand image, strain T2 has rod-shaped cells that are blue-purple, indicating it is a Gram-positive bacterium.

[0059] DNA was extracted from bacterial strain T2 using the OMEGA bacterial genomics kit. The 16S rDNA sequence of strain T2 was then amplified using universal bacterial primers 27F (SEQ ID NO. 2: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID NO. 3: 5'-GGTTACCTTGTTACGACTT-3'). The PCR system consisted of 2 μL DNA template, 2 μL each primer, 25 μL 2×Taq Plus MasterMixⅡ, and 19 μL ultrapure water. PCR conditions were as follows: 94℃ for 3 min; 94℃ for 15 s, 53℃ for 20 s, 72℃ for 1.5 min, 33 cycles; extension at 72℃ for 10 min. The PCR products were stored at 4℃. The PCR products were sequenced by Nanjing Qingke Biotechnology Co., Ltd. The sequencing results (see 16S rDNA sequence) were compared using BLAST in GenBank, and a phylogenetic tree was constructed using MEGA 6.06 (see [link to GenBank profile]). Figure 2 The study found that its 16S rDNA sequence (NCBI accession number PQ298955.1) showed a high similarity to *Microbacterium trichothecenolyticum*. This strain T2 is *Microbacterium trichothecenolyticum*.

[0060]

[0061] 2. Fetal growth promotion function test

[0062] 2.1 Ability to produce indoleacetic acid

[0063] Preparation method of Salkowski colorimetric solution: Dissolve 0.45g of ferric chloride in an appropriate amount of distilled water, slowly add 58.74mL of concentrated sulfuric acid, cool, and then dilute to 100mL with distilled water.

[0064] LB liquid medium containing 0.1 g / L tryptophan is abbreviated as LB medium 1.

[0065] Inoculate Trichosporon monotypic acid bacillus T2 into LB medium 1 and culture at 30℃ and 170 rpm for 2 days. Centrifuge and take the supernatant, mix it with an equal volume of Salkowski colorimetric solution, and let it stand in the dark for 30 minutes. If a pink color appears, it indicates that it has the ability to produce IAA.

[0066] Using LB medium 1 as a control (CK), mix LB medium 1 with an equal volume of Salkowski colorimetric solution, let stand in the dark for 30 minutes, and observe the color.

[0067] Bacillus velezensis SQR9 was used as a positive control, and the assay procedure was the same as that for strain T2.

[0068] The results are as follows Figure 3 As shown in Figure A, the supernatant of strain T2 turns pink when mixed with Salkowski colorimetric solution, indicating that strain T2 has the ability to produce IAA.

[0069] 2.2 Iron production capacity

[0070] Trichoderma solani T2 was inoculated into siderophore detection medium (purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.) and incubated statically at 37℃ for 3 days. If strain T2 produced an orange-yellow halo, it indicated that the strain had the ability to produce siderophores. Bacillus belyssus SQR9 was used as a positive control, and the method for determining siderophore production ability was the same as for strain T2. ​​Bacillus belyssus SQR9 also showed the ability to produce siderophores.

[0071] The results are as follows Figure 3 As shown in Figure B, the T2 colony has an orange-yellow halo around it, indicating that the T2 strain has the ability to produce siderophores.

[0072] 2.3 Ammonia production capacity

[0073] Treatment 1: Inoculate *Trichoderma solani* T2 into peptone aqueous broth (5g peptone, 1L distilled water, pH 7.2), incubate at 30℃ and 170rpm for 2 days, centrifuge, take 1mL of supernatant, add 100μL Nessler's reagent (Aladdin Reagent Co., Ltd.), and let stand for 10min. If a yellow or brownish-red precipitate appears, it indicates that the strain has the ability to produce NH3.

[0074] Treatment 2: Using blank culture medium (i.e., uninoculated peptone aqueous liquid medium) as a control, the specific steps are the same as in Treatment 1, the only difference being that strain T2 is not inoculated.

[0075] Treatment 3: Using Bacillus velezensis SQR9 as a positive control, the specific steps were the same as in Treatment 1, the only difference being that strain T2 was replaced with Bacillus velezensis SQR9.

[0076] The results are as follows Figure 3 As shown in Figure C, the supernatant of strain T2 turns yellow after Nessler's reagent is added, indicating that strain T2 has the ability to produce ammonia.

[0077] 2.4 Nitrogen fixation capacity

[0078] The nitrogen-free culture medium consisted of: 10g sucrose, 0.12g NaCl, 0.5g K2HPO4·3H2O, 1g CaCO3, 0.2g MgSO4·7H2O, 20g agar, and 1L distilled water, with a pH of 7.2.

[0079] *Trichoderma solani* T2 was inoculated onto nitrogen-free medium plates and cultured at 30°C for 5 days. The strain was then transferred from the nitrogen-free medium plates to new nitrogen-free medium plates and passaged three times. *Escherichia coli* was used as a negative control and treated the same way as T2, also passaged three times. Results are as follows... Figure 3 As shown in Figure D, strain T2 can still grow well on nitrogen-free medium, indicating that strain T2 has nitrogen-fixing ability.

[0080] 3. Iron-dissolving capacity test

[0081] Preparation of the Ferrozine assay solution: Dissolve 0.032 g of phenanthridine, 1.93 g of ammonium acetate, and 1.76 g of ascorbic acid in 10 mL of 0.1 M HCl solution to obtain the assay solution.

[0082] MKB medium: 5g casein amino acids, 15mL glycerol, 2.5g MgSO4·7H2O, 2.5g K2HPO4·3H2O and 1L distilled water, pH 7.2.

[0083] Treatment 1: *Trichoderma solani* T2 was inoculated into MKB medium containing 0.5 g / L ferric hydroxide and cultured at 37°C and 170 rpm for 4 days. The supernatant was centrifuged, filtered through a 0.22 μm filter, and then one-tenth of the supernatant volume of Ferrozine assay solution was added. After standing at room temperature in the dark for 30 min, the OD was measured. 562 .

[0084] MKB medium containing 0.5 g / L ferric hydroxide was used as control 1; control 1 was the same as treatment 1, except that it was not inoculated with Trichoderma solani T2.

[0085] Treatment 2: Same as treatment 1, except that Trichoderma solani T2 was inoculated into MKB medium containing 0.5 g / L iron oxide.

[0086] MKB medium containing 0.5 g / L iron oxide was used as control 2. Control 1 was the same as treatment 2, except that it was not inoculated with Trichoderma solani T2.

[0087] OD corresponding to ferric chloride standard solutions of different concentrations 562 A standard curve was plotted, and the concentration of soluble iron in the supernatant was calculated using the standard curve. The results are shown in Table 1. It can be seen that strain T2 can dissolve iron hydroxide and iron oxide. When the MKB medium contains iron hydroxide, the concentration of soluble iron in its supernatant is 54.74 μg / L, and when the MKB medium contains iron oxide, the concentration of soluble iron in its supernatant is 434.65 μg / L, indicating that strain T2 has the ability to dissolve iron.

[0088] Table 1 Iron-solubilizing capacity of strain T2

[0089] deal with Iron-dissolving capacity (μg / L) <![CDATA[Fe(OH)3]]> 54.74±9.66 <![CDATA[Fe2O3]]> 434.65±22.18

[0090] Example 2

[0091] 1. Plate test to promote iron absorption in Arabidopsis thaliana

[0092] 1.1 Seed disinfection

[0093] Arabidopsis seeds were disinfected with a sodium hypochlorite solution containing 1% available chlorine for 10 minutes, rinsed 5 times with sterile water, and then sown on 1 / 2 MS solid medium (Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.) plates. The plates were then placed vertically in a 22℃ light incubator (day / night ratio of 16h / 8h) for 6 days of activation culture before use.

[0094] 1.2 Preparation of iron-limiting culture medium

[0095] Add 0.5% sucrose and 1% agar to iron-free MS medium. Before pouring the plates, add 50 μM FeCl3 and 10 mM HEPES to the medium and adjust the pH to 8.0 with sodium hydroxide. At this time, the high pH will reduce the availability of iron, thus causing iron deficiency chlorosis in Arabidopsis.

[0096] 1.3 Evaluation of the effect of root inoculation of bacterial strains on agar plates

[0097] T2 treatment: The activated strain T2 was first streaked horizontally onto iron-limited medium. Then, 6-day-old Arabidopsis seedlings were transferred onto the streaked lines, with the stem base positioned 0.5 cm above the lines. The seedlings were then cultured in a light incubator for 9 days. The uninoculated control (CK) was used as the standard treatment. All other treatments were the same as for T2 treatment.

[0098] After 9 days of T2 treatment and CK culture, strain T2 significantly improved iron deficiency chlorosis in Arabidopsis thaliana (see...). Figure 4 Compared with the control, Arabidopsis thaliana inoculated with strain T2 showed increases in chlorophyll content, active iron content, and fresh weight of 221.43%, 44.82%, and 92.87%, respectively (see Table 2).

[0099] Table 2. Effects of strain T2 on Arabidopsis growth in iron-limited medium.

[0100] deal with Chlorophyll (mg / g fresh weight) Active iron (μg / g fresh weight) Fresh weight (mg) CK 0.28±0.02 3.86±0.12 5.47±0.53 T2 0.9±0.06 5.59±0.39 10.55±0.89

[0101] 2. Pot experiment on the effect of strains on the growth of Arabidopsis thaliana

[0102] 2.1 Test Soil

[0103] The tested soil was an artificially simulated calcareous soil, i.e., CaO was added to the peat soil. The mass concentration of CaO in the peat soil was 0.7%. Adding CaO can reduce the availability of iron in the peat soil. Peat soil without added CaO was used as a control.

[0104] 2.2 Preparation of T2 strain suspension

[0105] Strain strain T2 was inoculated into LB liquid medium and cultured at 30°C and 170 rpm for 2 days. The culture was then centrifuged and resuspended in a 0.3% NaCl solution to obtain a resuspension containing 10 viable bacteria. 8 CFU / mL. Take a portion of the bacterial suspension and sterilize it at 121℃ for 20 min to obtain an inactivated bacterial suspension.

[0106] 2.3 Pot Experiment Inoculation Treatment

[0107] Treatment 1: Transplant 6-day-old Arabidopsis thaliana seedlings with uniform growth into artificial calcareous soil, add 2 mL of bacterial suspension to the base of each seedling stem, and incubate in a light incubator for 10 days.

[0108] Control 1: 6-day-old Arabidopsis thaliana seedlings with uniform growth were transplanted into artificial calcareous soil. 2 mL of inactivated bacterial suspension was added to the base of each seedling stem, and the seedlings were cultured in a light incubator for 10 days.

[0109] Treatment 2: Transplant 6-day-old Arabidopsis thaliana seedlings with uniform growth into peat moss, add 2 mL of bacterial suspension to the base of each seedling stem, and incubate in a light incubator for 10 days.

[0110] Control 2: 6-day-old Arabidopsis thaliana seedlings with uniform growth were transplanted into peat moss. 2 mL of inactivated bacterial suspension was added to the base of each seedling stem, and the seedlings were cultured in a light incubator for 10 days.

[0111] It can be seen that in the artificially simulated calcareous soil of treatment 1, inoculation with strain T2 can significantly improve iron deficiency chlorosis in Arabidopsis thaliana (see...). Figure 5 Compared with control 1, the chlorophyll content, active iron content, and fresh weight of Arabidopsis thaliana inoculated with strain T2 in treatment 1 increased by 74.07%, 81.99%, and 229.17%, respectively (see Table 3). In normal soil treatment 2, inoculation with strain T2 also significantly promoted Arabidopsis growth (see Table 3). Figure 6 Compared with control 2, the fresh weight of treatment 2 increased by 154.57% (see Table 4).

[0112] Table 3. Effects of strain T2 on Arabidopsis growth in simulated calcareous soil.

[0113]

[0114] Table 4. Effects of strain T2 on Arabidopsis growth in normal soil.

[0115] deal with Fresh weight (mg) CK (Control 2) 18.05±1.11 T2 (Process 2) 45.95±6.45

[0116] Example 3

[0117] 1. Test soil

[0118] The tested soil was a calcareous soil from Dangshan County, Anhui Province. Its physicochemical properties were as follows: pH 8.1, CaCO3 361.6 g / kg, organic matter 10.5 g / kg, available nitrogen 107 mg / kg, available phosphorus 13.9 mg / kg, available potassium 156 mg / kg, and available iron 8.4 mg / kg. To further reduce the available iron content, 0.3% CaO was added to the soil.

[0119] 2 Seed disinfection

[0120] Cucumber (Jinchun 4) and corn (Zhengdan 958) seeds were disinfected with a 1% sodium hypochlorite solution for 10 minutes, rinsed 5 times with sterile water, and then evenly spread on moist filter paper and placed in a 30℃ incubator for germination. After the seeds showed signs of germination, they were transferred to moist vermiculite and then placed in a 30℃ light incubator (day / night ratio of 16h / 8h, i.e., 16h / day of light and 8h / day of darkness) for 5 days before use.

[0121] Preparation of suspension of 3 strains of T2 bacteria

[0122] Strain strain T2 was inoculated into LB liquid medium and cultured at 30°C and 170 rpm for 2 days. The culture was then centrifuged and resuspended in a 0.3% NaCl solution to obtain a resuspension containing 10 viable bacteria. 8 CFU / mL. Take a portion of the bacterial suspension and sterilize it at 121℃ for 20 min to obtain an inactivated bacterial suspension.

[0123] 4. Cucumber pot experiment inoculation treatment

[0124] Treatment 1: Transplant 5-day-old cucumber seedlings with uniform growth into calcareous soil, and add 5 mL of strain T2 bacterial suspension to the base of each seedling stem;

[0125] Control 1: Transplant 5-day-old cucumber seedlings with uniform growth into calcareous soil, and add 5 mL of inactivated bacterial suspension to the base of each seedling stem;

[0126] Control 2: Five-day-old cucumber seedlings with uniform growth were transplanted into calcareous soil. 5 mL of 0.5 g / L ferric ethylenediamine di-o-hydroxyphenylacetate (FeEDDHA) was added to the base of each seedling stem as a positive control.

[0127] From the start of cultivation, cucumber plants in treatment 1, control 1, and control 2 were re-inoculated every 10 days. After growing in a greenhouse at 30℃ with 16 hours of light and 8 hours of darkness for 30 days, photos were taken and records were recorded, and chlorophyll content, active iron content, and biomass were measured.

[0128] like Figure 7 It can be seen that inoculation with strain T2 can significantly improve iron deficiency chlorosis in cucumbers. Compared with control 1, the chlorophyll content (SPAD), active iron content and dry weight of cucumbers in treatment 1 inoculated with strain T2 increased by 123.51%, 253.23% and 251.43% respectively, and the effect was comparable to that of control 2 (see Table 5).

[0129] Table 5. Effects of strain T2 on cucumber growth in calcareous soil.

[0130] deal with SPAD Active iron (μg / g fresh weight) Dry weight (g) CK (Control 1) 18.29±2.31 4.34±0.51 0.35±0.09 FeEDDHA (Control 2) 40.1±1.97 15.03±3.95 1.41±0.19 T2 (Process 1) 40.88±1.96 15.33±4.07 1.23±0.25

[0131] 5. Inoculation treatment in maize pot experiments

[0132] Treatment 1: Transplant 5-day-old corn seedlings with uniform growth into calcareous soil, and add 5 mL of strain T2 bacterial suspension to the base of each seedling stem;

[0133] Control 1: Transplant 5-day-old corn seedlings with uniform growth into calcareous soil, and add 5 mL of inactivated bacterial suspension to the base of each seedling stem;

[0134] Control 2: Five-day-old corn seedlings with uniform growth were transplanted into calcareous soil, and 5 mL of 0.5 g / L FeEDDHA was added to the base of each seedling stem as a positive control.

[0135] From the start of cultivation, maize plants in each treatment group were re-inoculated every 10 days. After growing for 30 days in a greenhouse at 30°C with 16 hours of light and 8 hours of darkness, photographs were taken and chlorophyll content, active iron content, and biomass were measured.

[0136] like Figure 8 It can be seen that inoculation with strain T2 can significantly improve iron deficiency chlorosis in maize. Compared with control 1, maize treated with strain T2 showed increases of 79.36% in chlorophyll content (SPAD), 211.48% in active iron content, and 65.67% in dry weight, and the effects were comparable to those of control 2 (see Table 6).

[0137] Table 6. Effects of strain T2 on maize growth in calcareous soil.

[0138] deal with SPAD Active iron (μg / g fresh weight) Dry weight (g) CK (Control 1) 20.64±2.99 2.7±0.65 0.67±0.14 FeEDDHA (Control 2) 39.73±3.49 9.8±1.6 1.04±0.13 T2 (Process 1) 37.02±2.64 8.41±0.71 1.11±0.24

[0139] In summary, *Microbacterium trichothecenolyticum* T2 not only possesses various plant growth-promoting abilities (IAA production, siderophore production, ammonia production, and nitrogen fixation), but also exhibits a certain iron-solubilizing capacity. *Microbacterium trichothecenolyticum* T2 significantly improves iron deficiency chlorosis in Arabidopsis thaliana under iron-limited conditions, increases chlorophyll content, enhances active iron content in plants, and promotes plant growth. Furthermore, it demonstrates significant growth-promoting effects on different plants in pot experiments in calcareous soil, indicating that this strain has great potential to improve plant iron nutrition. This invention is of great significance for developing functional microbial agents, microbial fertilizers, or plant growth promoters to improve crop iron nutrition and yield.

[0140] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Microbacterium trichothecenolyticum T2, with accession number GDMCC No. 64981.

2. A microbial agent, characterized in that, Includes the Trichosporonobacter T2 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The viable count of *Trichoderma solani* T2 in the bacterial agent is ≥1×10⁻⁶. 8 CFU / g.

4. The microbial agent according to claim 2 or 3, characterized in that, The application of the Trichosporon monosodium globulus T2 includes bacterial suspension.

5. A method for preparing a microbial agent, characterized in that, include: Trichoderma solani T2 was inoculated into a culture medium and cultured to obtain a fermentation broth. The fermentation broth was centrifuged and resuspended to obtain a bacterial agent.

6. The preparation method according to claim 5, characterized in that, The culture temperature is 28–33°C, and the culture time is 1–3 days.

7. The use of the *Trichoderma solani* T2 according to claim 1, or the bacterial agent according to any one of claims 2 to 4, or the bacterial agent prepared by the preparation method according to claim 5 or 6, in any one or more of the following 1) to 5); 1) Promotes iron absorption in plants; 2) Promote plant growth; 3) Improves plant yellowing; 4) Preparation of microbial fertilizers; 5) Prepare plant growth promoters.

8. The application according to claim 7, characterized in that, The plants include one or more of Arabidopsis thaliana, cucumber, and maize.

9. The application according to claim 7, characterized in that, The plant growth promotion includes increasing plant biomass.

10. The application according to claim 7, characterized in that, The improvement of plant etiolation includes increasing the chlorophyll content of plants.

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