Pantoea bacterium 30D3 producing siderophore and application thereof in promoting plant growth and efficient utilization of iron under alkaline low-iron condition

Abstract

The present application relates to a strain of producing siderophore of pantoea bacteria 30D3 and its application in promoting plant growth and efficient utilization of iron under alkaline low iron condition, the pantoea bacteria 30D3 provided is indigenous microorganism isolated and screened from the rhizosphere soil of citrus in Ganzhou, Jiangxi, which still maintains the ability of efficient secretion of siderophore in alkaline low iron environment, and solves the problem of limited activity of the existing siderophore-producing strain under such adversity.Experiments prove that under the condition of alkaline low iron with pH 8.0, applying the strain 30D3 to the rhizosphere of arabidopsis and citrus rootstock trifoliate orange can significantly promote plant growth, increase plant fresh weight, and significantly improve the iron content of plant aboveground part, effectively alleviate the symptoms of iron deficiency.The strain provided by the present application is an environmentally friendly biological resource, and the application method is simple, which can significantly reduce the use amount of chemical iron fertilizer, reduce the cost of agricultural production, and has good application prospect and popularization value.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, specifically to a novel Pantotheca strain 30D3 with highly efficient iron-carrier production capacity, and its application in promoting plant growth and increasing plant iron content under alkaline low-iron stress. Background Technology

[0002] Iron is an essential micronutrient for plants, participating in key physiological processes such as chlorophyll synthesis, photosynthetic electron transport, and respiration. However, in northern my country and some limestone regions in the south, soil pH is generally high, exhibiting alkaline or calcareous reactions. Under alkaline conditions, iron in the soil mainly exists in the form of insoluble ferric oxides or hydroxides, resulting in extremely low levels of available iron that plants can absorb and utilize. This often leads to iron deficiency chlorosis in plants, severely inhibiting crop growth, reducing yield, and decreasing fruit quality.

[0003] Currently, the main measures to alleviate iron deficiency in plants include applying chelated iron fertilizers, foliar spraying of ferrous sulfate solution, and soil acidification improvement. However, chelated iron fertilizers are expensive, foliar spraying has a short-lived effect and does not address the root cause, and long-term, large-scale application of acidic fertilizers may damage soil structure and cause non-point source pollution. Therefore, finding an efficient, economical, and environmentally friendly strategy for iron supplementation in plants is an urgent problem that modern agriculture needs to solve.

[0004] Utilizing siderophore-producing microorganisms to promote iron uptake in plants has become a research hotspot in recent years. Siderophores are small-molecule chelates secreted by microorganisms under iron-limited conditions, possessing a high affinity for ferric ions. They can dissolve and transport insoluble iron in the soil to the rhizosphere for plant absorption and utilization. However, most reported siderophore-producing microorganisms perform well in acidic or neutral environments, and their secretion capacity and growth-promoting effects often decrease significantly under alkaline and low-iron stress. Therefore, discovering new indigenous microbial resources that can still efficiently secrete siderophores and stably promote plant growth and efficient iron utilization under alkaline and low-iron stress has significant application value and practical significance. Summary of the Invention

[0005] This invention aims to provide a pan-based iron-producing bacterium, 30D3, and its application in promoting plant growth and efficient iron utilization under alkaline, low-iron conditions.

[0006] The present invention adopts the following technical solution:

[0007] A pantoea ananatis 30D3 strain that produces siderophores was deposited at the China Center for Type Culture Collection on May 18, 2026, with accession number CCTCC NO: M 2026999.

[0008] The application of Pantotheca 30D3 in promoting plant growth under alkaline, low-iron conditions.

[0009] The application of Pantotheca 30D3 in increasing iron content in plants under alkaline, low-iron conditions.

[0010] The specific application method involves treating the plant rhizosphere with a bacterial agent containing Pantotheca 30D3.

[0011] Furthermore, the preparation method of the bacterial agent containing Pantotheca 30D3 is as follows: Pantotheca 30D3 is inoculated into LB liquid medium, cultured at 28℃ and 200 rpm with shaking until the logarithmic growth phase, the bacterial cells are collected by centrifugation, resuspended in sterile water and the OD600 value is adjusted to 0.2.

[0012] Furthermore, the alkaline low-iron conditions refer to a growth environment with a pH ≥ 8.0 and an effective iron concentration of less than 10 μmol / L.

[0013] Furthermore, the plants include Arabidopsis thaliana and citrus rootstock trifoliata.

[0014] The beneficial effects of this invention are as follows:

[0015] (1) The Pantotheca 30D3 provided by the present invention is an indigenous microorganism isolated and screened from the rhizosphere soil of citrus in Ganzhou, Jiangxi Province. It maintains the ability to secrete siderophores efficiently in an alkaline and low-iron environment, which solves the problem that the activity of existing siderophore-producing strains is limited under such adverse conditions.

[0016] (2) Experiments have shown that under alkaline low-iron conditions at pH 8.0, applying strain 30D3 to the rhizosphere of Arabidopsis thaliana and citrus rootstock trifoliate orange can significantly promote plant growth, increase plant fresh weight, and significantly increase the iron content in the aboveground parts of the plant, effectively alleviating iron deficiency symptoms.

[0017] (3) The strain provided by this invention is an environmentally friendly biological resource. The application method is simple and can significantly reduce the amount of chemical iron fertilizer used, reduce agricultural production costs, and has good application prospects and promotion value. Attached Figure Description

[0018] Figure 1 Photograph of the siderophore hydrolysis zone produced by strain 30D3 on CAS solid medium.

[0019] Figure 2 This is a table showing the relative siderophore yield (SU value) of strain 30D3 under liquid culture conditions.

[0020] Figure 3 Photographs showing the growth of Arabidopsis thaliana seedlings inoculated with and uninoculated strain 30D3 in alkaline low-iron 1 / 2 MS medium at pH 8.0.

[0021] Figure 4 A bar chart comparing the fresh weight of Arabidopsis seedlings inoculated with and uninoculated with strain 30D3 in alkaline low-iron 1 / 2 MS medium at pH 8.0.

[0022] Figure 5 Photographs showing the growth of trifoliate orange seedlings under alkaline, low-iron sand culture conditions at pH 8.0, with and without the application of strain 30D3 inoculant.

[0023] Figure 6 A bar chart comparing the fresh weight of trifoliate orange seedlings treated with and without the 30D3 bacterial agent under alkaline, low-iron sand culture conditions at pH 8.0.

[0024] Figure 7 A bar chart comparing the aboveground iron content of trifoliate orange seedlings irrigated and unirrigated under alkaline, low-iron sand culture conditions at pH 8.0. Detailed Implementation

[0025] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0026] 1: Isolation, identification and preservation of strain 30D3

[0027] I. Strains Isolation

[0028] Rhizosphere soil samples were collected from citrus rhizosphere soil in Ganzhou, Jiangxi Province. 5 g of soil sample was weighed and added to an Erlenmeyer flask containing 45 mL of sterile phosphate buffer. The mixture was shaken at 150 rpm for 15 minutes and allowed to stand for 10 minutes. The supernatant was then serially diluted to prepare 10⁻¹ to 10⁻¹ samples. 5 Diluted bacterial suspensions. Take 0.1 mL of each dilution of bacterial suspension and spread it evenly on LB agar plates. Incubate upside down in a 28°C incubator for 2–3 days. Pick single colonies with different morphologies and streak them repeatedly for purification at least 3 times to obtain pure cultures, labeled 30D3.

[0029] II. Strain Identification

[0030] The 16S rRNA gene of strain 30D3 was amplified by PCR and sequenced using the universal bacterial primer 27F / 1492R. The obtained sequence was submitted to the NCBI GenBank database for BLAST alignment analysis. The results showed that strain 30D3 had the highest homology (100%) with the 16S rRNA gene sequence of the type strain of Pantoea sp. Based on morphological characteristics, strain 30D3 was identified as a Pantoea bacterium.

[0031] III. Strains Preservation

[0032] A single colony of the purified strain 30D3 was inoculated into LB liquid medium and cultured at 28°C and 200 rpm for 12 hours with shaking. 600 μL of the bacterial culture was then mixed with an equal volume of 50% (v / v) sterile glycerol and stored at -80°C for long-term preservation. Simultaneously, the strain was sent to the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 2026999.

[0033] 2: Qualitative and quantitative determination of the siderophore production capacity of strain 30D3

[0034] I. Qualitative determination (CAS plate method)

[0035] The strain 30D3, stored at -80℃, was activated on LB agar plates and incubated overnight at 28℃. Single colonies were picked with sterile toothpicks and inoculated onto CAS (Chrome Azurol S) detection plates, and incubated at 28℃ for 5 days. Results are as follows: Figure 1 As shown, strain 30D3 produces a distinct orange-yellow hydrolysis zone around its colonies, indicating that it has a strong ability to secrete siderophores.

[0036] II. Quantitative Determination (Liquid Fermentation Method)

[0037] A single colony of the activated strain 30D3 was inoculated into LB liquid medium and cultured at 28°C with shaking at 200 rpm for 24 hours as a seed culture. A 1% inoculum was then transferred to iron-limited MKB liquid medium and cultured at 28°C with shaking at 200 rpm for 48 hours. The fermentation broth was centrifuged at 4000 rpm for 15 minutes, and the supernatant was filtered through a 0.22 μm filter. The filtrate was mixed with an equal volume of CAS detection solution, and after reacting at room temperature for 1 hour, the absorbance at 630 nm was measured, with uninoculated medium serving as a control. The relative siderophore yield (SU) was calculated.

[0038] SU = (Ar- As) / Ar, where Ar is the reference absorbance and As is the sample absorbance.

[0039] The results are as follows Figure 2 As shown, the siderophore production (SU value) of strain 30D3 reached a relatively high level of about 0.56, confirming that it has the function of efficiently synthesizing and secreting siderophores.

[0040] 3: The effect of strain 30D3 on promoting Arabidopsis growth and iron absorption under alkaline and low-iron conditions.

[0041] I. Preparation of Microbial Agent

[0042] Strawberry strain 30D3, stored at -80℃, was streaked onto LB agar plates and activated overnight at 28℃. A single colony was picked and inoculated into a 250 mL Erlenmeyer flask containing 100 mL of LB liquid medium, and incubated at 28℃ with shaking at 200 rpm for 24 hours. The culture was centrifuged at 4000 rpm for 15 minutes to collect the bacterial cells, washed twice with sterile water, resuspended, and the OD600 value was adjusted to 0.2. This was the experimental bacterial agent.

[0043] II. Arabidopsis thaliana culture and treatment

[0044] After surface sterilization, Arabidopsis thaliana Col-0 seeds were sown on 1 / 2 MS solid medium (normal iron-containing medium, pH=7.2) and vernalized at 4℃ in the dark for 2 days. Then, they were placed in a tissue culture room (22℃, 16h / 8h light / dark cycle) for germination and growth for 3 days. Subsequently, 8 seedlings with uniform growth were selected from the tissue culture table and transferred to 1 / 2 MS solid medium (iron-free formula, pH adjusted to 8.0, with EDTA-Fe³⁺ added to a final concentration of 10 μmol / L). 3 μL of 30D3 bacterial agent with OD600=0.2 was applied to the surface of the medium approximately 1 cm from the seedling roots (treatment group). The control group received an equal volume of sterile water. After 10 days of further culture, observations were conducted and data were collected.

[0045] III. Results and Analysis

[0046] Processing photos taken 10 days later, such as Figure 3 As shown, compared with the control group, Arabidopsis plants inoculated with 30D3 inoculant exhibited vigorous growth, larger and greener leaves, and more developed root systems. The fresh weight of the entire plant in each group was measured, and the results are as follows: Figure 4 As shown, the fresh weight of plants in the treatment group was significantly higher than that in the control group (p<0.05, an increase of approximately 287%). This result indicates that under alkaline, low-iron conditions (pH 8.0, iron 10 μmol / L), Pantotheca 30D3 can significantly promote the growth of Arabidopsis seedlings.

[0047] 4: The effect of strain 30D3 on promoting the growth of trifoliate orange seedlings and iron absorption under alkaline and low-iron conditions.

[0048] I. Preparation of Microbial Agent

[0049] The preparation method of the bacterial agent is the same as in Example 3, except that the OD600 value of the bacterial solution is adjusted to 0.2.

[0050] II. Cultivation and Treatment of Trifoliate Orange Seedlings

[0051] Selected 45-day-old, uniformly growing Poncirus trifoliata rootstock seedlings were transplanted into plastic pots filled with quartz sand for sand cultivation. Two seedlings were planted in each pot, and Hoagland's nutrient solution was applied every 7 days. The nutrient solution was adjusted to alkaline, low-iron conditions: the pH was adjusted to 8.0 with NaOH solution, and the EDTA-Fe ratio was adjusted accordingly. 3+ The final concentration is 10 μmol / L. Apply 50 mL of nutrient solution to each plant per application.

[0052] The experiment was conducted in two groups: the treatment group received 5 mL of 30D3 bacterial solution with an OD600 of 0.2 per seedling through a ring-root irrigation, while the control group received an equal volume of sterile water. The inoculum application was performed at 7-day intervals, for a total of 3 applications. After 2 months of routine management following the initial inoculum application, various indicators were observed and measured.

[0053] III. Results and Analysis

[0054] Comparison photos of trifoliate orange seedlings taken two months after fungal application are shown below. Figure 5 As shown, the trifoliate orange seedlings irrigated with 30D3 inoculant exhibited significantly better growth than the control group, characterized by increased plant height, darker green leaves, and reduced yellowing. The fresh weight of the whole plant, above-ground parts, and underground parts of each group were measured, and the results are as follows: Figure 6 As shown, the fresh weight of the treatment group was significantly higher than that of the control group (p<0.05). Further collection of young aerial leaves, drying, pulverizing, and determination of iron content yielded the following results: Figure 7 As shown, the iron content in the aboveground parts of the treatment group was significantly higher than that in the control group.

[0055] In summary, the results indicate that Pantotheca 30D3 has a significant growth-promoting effect on trifoliate orange seedlings under alkaline low iron stress, and can effectively improve the plant's iron absorption and upward transport efficiency, thereby improving the plant's iron nutrition status and alleviating iron deficiency symptoms.

[0056] The above description provides examples of the preferred embodiments of the present invention. Parts not detailed herein are common knowledge to those skilled in the art. The scope of protection of the present invention is determined by the claims. Any equivalent modifications based on the technical teachings of the present invention are also within the scope of protection of the present invention.

Claims

1. A pantothenic bacterium 30D3 that produces siderophores, characterized in that, The bacteria is Pantoeaananatis 30D3, which was deposited at the China Center for Type Culture Collection on May 18, 2026, with accession number CCTCCNO: M 2026999.

2. The application of Pantotheca 30D3 according to claim 1 in promoting plant growth under alkaline and low-iron conditions.

3. The application of Pantotheca bacteria 30D3 according to claim 1 in increasing the iron content of plants under alkaline and low-iron conditions.

4. The application according to claim 3, characterized in that, The specific method involves treating the plant rhizosphere with a bacterial agent containing Pantotheca 30D3.

5. The application according to claim 4, characterized in that, The preparation method of the bacterial agent containing Pantotheca 30D3 is as follows: Pantotheca 30D3 is inoculated into LB liquid medium and cultured at 28℃ and 200 rpm with shaking until the logarithmic growth phase. The bacterial cells are collected by centrifugation, resuspended in sterile water and the OD600 value is adjusted to 0.

2.

6. The application according to claim 2 or 3, characterized in that, The alkaline low-iron conditions refer to a growth environment with a pH ≥ 8.0 and an effective iron concentration below 10 μmol / L.

7. The application according to claim 2 or 3, characterized in that, The plants mentioned include Arabidopsis thaliana and trifoliate orange rootstock.

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