Pseudomonas fluorescens D2-21 and application thereof in promoting seed germination and seedling growth of wheat

Abstract

This invention discloses a strain of *Pseudomonas fluorescens* D2-21 and its application in promoting wheat seed germination and seedling growth, belonging to the field of microbial application technology. The *Pseudomonas fluorescens* D2-21 provided by this invention was deposited on February 13, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 33522. *Pseudomonas fluorescens* D2-21 possesses nitrogen-fixing, siderophore-producing, and IAA-producing abilities. Experimental results show that culturing wheat seeds using the fermentation broth of *Pseudomonas fluorescens* D2-21 provided by this invention significantly improves the germination index and vigor index of wheat seeds, and the germinated wheat seedlings exhibit better growth, indicating that *Pseudomonas fluorescens* D2-21 promotes wheat seed germination and seedling growth. This invention provides a new, highly efficient, and safe strain for promoting wheat emergence, with broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of microbial application technology, and in particular to a strain of fluorescent Pseudomonas D2-21 and its application in promoting wheat seed germination and seedling growth. Background Technology

[0002] Successful seed germination and normal seedling growth are crucial factors in plant reproduction, holding significant economic and ecological importance. However, the increasing demand for wheat and continuous cropping year after year have led to cropping obstacles, including soil nutrient depletion and increased pests and diseases. Furthermore, various abiotic stresses often result in low seed germination rates and poor seedling growth.

[0003] Plant growth-promoting rhizobacteria (PGPR) are beneficial bacteria found in the rhizosphere soil of plants that inhibit plant diseases and promote plant growth. They are also the most widely used microorganisms in current microbial improvement measures. Existing research shows that some microbial strains can promote seed germination or seedling growth, but the number of strains with these functions is relatively small, and they generally suffer from problems such as unstable growth-promoting function and environmental pollution.

[0004] Therefore, there is an urgent need to provide a microorganism that can efficiently, safely, and stably promote wheat seed germination and seedling growth. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of *Pseudomonas fluorescens* D2-21 and its application in promoting wheat seed germination and seedling growth, in order to solve the problems existing in the prior art. The *Pseudomonas fluorescens* D2-21 provided by this invention can significantly promote wheat seed germination and enhance the growth potential of wheat seedlings after germination when used to cultivate wheat seeds.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a strain of Pseudomonas fluorescens D2-21, which has the accession number CGMCC No.33522 and was deposited on February 13, 2025 at the China General Microbiological Culture Collection Center, located at No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0008] The present invention also provides a fermentation broth containing the above-mentioned fluorescent Pseudomonas D2-21.

[0009] The present invention also provides the use of the above-mentioned fluorescent Pseudomonas D2-21 or the above-mentioned fermentation broth in the preparation of products that promote plant seed germination and / or promote plant seedling growth.

[0010] Preferably, the product is a microbial preparation.

[0011] The present invention also provides a microbial preparation for promoting plant seed germination and / or promoting plant seedling growth, containing the above-mentioned Pseudomonas fluorescens D2-21 or the above-mentioned fermentation broth.

[0012] Preferably, the concentration of *Pseudomonas fluorescens* D2-21 in the microbial preparation is 10. 5 -10 7 cfu / mL.

[0013] The present invention also provides the use of the above-mentioned *Pseudomonas fluorescens* D2-21, the above-mentioned fermentation broth, or the above-mentioned microbial preparation in any of the following:

[0014] (1) Application in the production of plant growth regulators;

[0015] (2) Application in promoting plant seed germination;

[0016] (3) Application in promoting the growth of plant seedlings.

[0017] Preferably, the plant is wheat.

[0018] The present invention also provides a method for promoting plant seed germination and / or promoting plant seedling growth, comprising the step of culturing the plant seeds using the above-described fermentation broth or the above-described microbial preparation.

[0019] Preferably, the plant is wheat.

[0020] The present invention discloses the following technical effects:

[0021] The *Pseudomonas fluorescens* D2-21 strain provided by this invention was deposited on February 13, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 33522. *Pseudomonas fluorescens* D2-21 possesses nitrogen-fixing, siderophore-producing, and IAA-producing capabilities. Experimental results show that culturing wheat seeds with the fermentation broth of *Pseudomonas fluorescens* D2-21 provided by this invention significantly improved the germination index and vigor index of wheat seeds. The seedling length and root length of the germinated wheat seedlings were superior to the control group, exhibiting better growth, indicating that *Pseudomonas fluorescens* D2-21 promotes wheat seed germination and seedling growth. This invention provides a new, highly efficient, and safe strain for promoting wheat emergence, with broad application prospects. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a colony morphology diagram of *Pseudomonas fluorescens* D2-21.

[0024] Figure 2 Phylogenetic tree of Pseudomonas fluorescens D2-21;

[0025] Figure 3 Figure showing the experimental results of nitrogen fixation characteristics of Pseudomonas fluorescens D2-21;

[0026] Figure 4 Figure 1 shows the experimental results of siderophore production characteristics of Pseudomonas fluorescens D2-21.

[0027] Figure 5 Figure 1 shows the experimental results of IAA production characteristics of *Pseudomonas fluorescens* D2-21; where a represents 24h; b represents 48h; c represents 72h; and d represents 96h.

[0028] Figure 6 The concentration of IAA produced by *Pseudomonas fluorescens* D2-21 in NA and NA+Trp;

[0029] Figure 7 Statistical graphs of root length and seedling length of wheat seedlings in different treatment groups;

[0030] Figure 8 This is an observation chart of wheat seedling growth; the scale bar is 5cm long. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] Example 1

[0037] 1. Experimental Materials

[0038] NA medium: 10g peptone, 3g beef extract, 5g NaCl, 15g agar, 1L water, pH 7. The medium without agar is used as the NA liquid medium.

[0039] Ashube medium: glucose 10g, K2HPO4·3H2O 0.2g, K2SO4 0.2g, NaCl 0.2g, CaCO3 5.0g, MgSO4·7H2O 0.2g, agar 16g, distilled water 1000mL.

[0040] CAS medium: CAS 60.50 mg, cetyltrimethylammonium bromide (HDTMA) 72.90 mg, 1 mmol / L FeCl3·6H2O (prepared with 10 mmol / L HCl) 10 mL, 0.1 mol / L phosphate buffer 50 mL, agar 9.0 g, deionized water 940 mL.

[0041] Salkowski colorimetric reagent: Add 1 mL of 0.5 mol / L ferric chloride to 50 mL of 35% HClO4 and shake well. Prepare and use immediately, and store away from light.

[0042] Wheat seeds: "Jimai 22" purchased from the market, provided by Shandong Yinxing Seed Industry Co., Ltd.

[0043] 2. Strains Isolation

[0044] The D2-21 strain of this invention was isolated from rhizosphere soil samples of *Corydalis yanhusuo* plants in the Heilihe National Nature Reserve, Ningcheng County, Chifeng City (geographical coordinates: N41.27°, W118.21°). The isolation method is as follows:

[0045] Mix the collected soil samples with sterile water, weigh 1.0 g into 99 mL of sterile water, and shake for about 30 minutes. Separately, add 10 g of the sample to sterile water. -4 10 -5 10 -6 The diluted soil suspension was evenly spread on NA medium and incubated at 35°C for 1-3 days. Single colonies of actinomycetes were selected and purified using the streak plating method. The purified strains were then inoculated into NA slant medium and stored at 4°C for later use.

[0046] 3. Strain identification

[0047] 3.1 Morphological characteristics

[0048] Referring to the *Handbook of Systematic Identification of Common Bacteria*, the preserved D2-21 strain was inoculated onto NA solid medium and incubated at 37°C for 1-2 days. The D2-21 strain grew well in NA medium, forming round, pale yellow, moist, opaque colonies with neat edges. The bacteria are rod-shaped and form spores. The optimal growth pH is 7, and it can grow in the range of 15-40°C, with an optimal growth temperature of 37°C. See the attached image for the colony morphology of the D2-21 strain. Figure 1 .

[0049] 3.2 Physiological and Biochemical Identification

[0050] The conventional methods for bacterial identification were used (refer to the "Handbook for Identification of Common Bacteria"). Gram staining, gelatin hydrolysis, catalase, arginine double hydrolysis, nitrate reduction and starch hydrolysis tests were performed on strain D2-21.

[0051] Experimental results showed that D2-21 was a Gram-negative bacterium. The results of the gelatin hydrolysis test, catalase test, arginine double hydrolysis test and nitrate reduction test were positive, while the result of the starch hydrolysis test was negative.

[0052] 3.3 DNA extraction and molecular identification

[0053] After D2-21 was cultured in NA medium at 37℃ for 2 days, genomic DNA was extracted using the TSINGKE TSP101-200 DNA Extraction Kit (universal type). The total volume of the PCR reaction system was 20.0 μL, including: 2.0 μL each of forward and reverse primers, 1.0 μL of DNA template, and 15.0 μL of ddH2O. The PCR reaction program was: 98℃ for 2 min; 98℃ for 10 s, 57℃ for 10 s, 72℃ for 45 s, 35 cycles; 72℃ for 5 min. The amplified PCR product was subjected to agarose gel electrophoresis (2 μL sample + 6 μL bromophenol blue) at 300V for 12 min, and a single band was obtained and sent to Beijing TSINGKE Biotechnology Co., Ltd. for purification and sequencing. The nucleotide sequences of the primers for PCR amplification of the 16S rDNA of strain D2-21 are as follows:

[0054] Primer 27F (SEQ ID NO.1): 5'-AGTTTGATCMTGGCTCAG-3';

[0055] Primer 1492R (SEQ ID NO.2): 5'-GGTTACCTTGTTACGACTT-3'.

[0056] The 16S rDNA sequence of strain D2-21 is shown in SEQ ID NO.3.

[0057] SEQ ID NO.3:

[0058]

[0059] The 16S rDNA gene sequence of strain D2-21, obtained by PCR amplification, was 1371 bp in length. Blast alignment analysis of the sequence in the NCBI database revealed that strains with high homology all originated from the genus *Pseudomonas*. A phylogenetic tree was constructed based on the 16S rDNA gene sequence as follows: Figure 2 The results showed that strain D2-21 and Pseudomonas fluorescens belong to the same branch. Based on morphological characteristics, strain D2-21 was identified as belonging to Pseudomonas fluorescens and was named Pseudomonas fluorescens D2-21.

[0060] 3.4 Preservation of bacterial strains

[0061] Pseudomonas fluorescens D2-21 was deposited on February 13, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33522. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0062] 4. Growth-promoting characteristics of the strain

[0063] 4.1 Nitrogen fixation

[0064] Fluorescent Pseudomonas D2-21 was inoculated into Assumption medium plates and cultured at 37°C for 5 days. Its growth was then observed (see [link]). Figure 3 The results showed that *Pseudomonas fluorescens* D2-21 could grow normally on Assumption medium and produce a clear zone, indicating that *Pseudomonas fluorescens* D2-21 has nitrogen-fixing ability and can promote the absorption of nitrogen sources by plants.

[0065] Table 1. Nitrogen-fixing transparent zone of *Pseudomonas fluorescens* D2-21

[0066]

[0067] 4.2 Characteristics of iron-producing carriers

[0068] Activated *Pseudomonas fluorescens* D2-21 was inoculated onto CAS plates and cultured at 35°C for 5 days. When the sample contained siderophores, the siderophores could efficiently complex with iron ions, releasing red CAS, which formed a yellow or orange-red halo on the culture medium. The color change of each plate was observed (see...). Figure 4 ).

[0069] Table 2. Clear zones of siderogenic carriers in *Pseudomonas fluorescens* D2-21

[0070]

[0071] 4.3 Production of the plant growth hormone indoleacetic acid (IAA)

[0072] One loopful of *Pseudomonas fluorescens* D2-21 from agar slant culture medium was inoculated into 50 mL / 250 mL NA liquid medium and cultured at 37°C and 180 rpm for 24 h to obtain a bacterial concentration of 10. 8 Seed culture of *Pseudomonas fluorescens* D2-21 with cfu / mL was prepared for use.

[0073] One mL of seed culture was inoculated into 50 mL / 250 mL NA liquid medium and NA liquid medium containing 100 mg / L L-tryptophan (Trp), respectively. Each treatment was performed in triplicate, with a blank medium as a control. The culture was carried out at 37 °C and 180 rpm. At 24 h, 48 h, 72 h, and 96 h, 50 μL of fermentation broth was placed in a porcelain reaction plate, and 50 μL of Salkowski's reagent was added. After reacting for 5 minutes, the color of the solution was observed (see...). Figure 5 ).

[0074] The fermentation broth was then centrifuged in a 50 mL centrifuge tube at 10,000 rpm for 10 min. The supernatant was collected, and the absorbance was measured according to the method of Glickman et al. (1995). Simultaneously, an absorbance-concentration standard curve was plotted using analytical grade IAA, with the equation y = 0.0291x + 0.0543(R²). 2 =0.9991) Calculate the IAA content in the fermentation broth, from Figure 6 It was found that after 24h, 48h, 72h, and 96h of cultivation, the IAA production of *Pseudomonas fluorescens* D2-21 in NA medium was 2.35 mg / L, 2.82 mg / L, 4.98 mg / L, and 4.71 mg / L, respectively; while the IAA production in NA+Trp medium was 4.22 mg / L, 6.30 mg / L, 6.18 mg / L, and 6.47 mg / L, respectively. This indicates that *Pseudomonas fluorescens* D2-21 has the ability to produce the auxin indoleacetic acid (IAA), which promotes plant growth. The addition of the substrate L-tryptophan enhanced the IAA production capacity of *Pseudomonas fluorescens* D2-21, with a more pronounced effect before 48h.

[0075] 5. Experiment on wheat seed germination and seedling growth

[0076] Uniform and plump wheat seeds were selected and treated sequentially with 75% (v / v) alcohol for 1 min, 1% (v / v) NaClO solution for 10 min, and rinsed 5 times with sterile water. Thirty sterilized and plump wheat seeds were selected and neatly arranged in sterilized petri dishes (9 cm in diameter) lined with two layers of filter paper. *Pseudomonas fluorescens* D2-21 seed culture solution diluted 10, 100, and 1000 times (named T1, T2, and T3, respectively) were used as the culture medium, with water as the control (CK). The seeds were cultured at a constant temperature of 22℃, relative humidity of 60%, and 8 h light / 16 h dark conditions. Water loss was replenished periodically using a gravimetric method. Each treatment was repeated in triplicate. Germination data were recorded 3 days after germination, and the seeds were further cultured at a constant temperature of 25℃, relative humidity of 60%, and 14 h light / 10 h dark conditions. Observe and record the number of germinated seeds 24 hours after germination (seeds showing white sprouts are the criterion for germination). Calculate germination vigor on day 3 and germination rate on day 7, and then calculate the germination index and vigor index. The formulas are as follows:

[0077] Germination potential (%) = (Number of germinated seeds in the initial count / Total number of seeds tested) × 100%;

[0078] Germination rate (%) = (Number of seeds germinated in 7 days / Total number of seeds tested) × 100%;

[0079] Germination index = ∑Gt / Dt;

[0080] Vitality index = seedling length × germination index.

[0081] In the formula: Gt is the number of seeds that germinate on day t; Dt is the number of days the seeds have been germinating.

[0082] On the 7th day of the experiment, 10 germinated seedlings were randomly selected from each treatment, and their root length, shoot length, fresh weight and dry weight were measured to assess the growth status of the seedlings.

[0083] The results, as shown in Table 3, indicate that the germination rate and germination index of the T1 treatment group were lower than those of the CK group; the germination potential, germination rate, germination index, and vigor index of the T3 treatment group were all higher than those of the CK group, with the vigor index being 2.72 times that of the CK group. In conclusion, this indicates that high concentrations of *Pseudomonas fluorescens* D2-21 seed solution have a certain inhibitory effect on wheat seed germination, while low concentrations of *Pseudomonas fluorescens* D2-21 seed solution diluted 1000 times have a significant promoting effect on wheat seed germination.

[0084] Table 3. Effects of different concentrations of D2-21 on wheat seed germination.

[0085]

[0086] Note: Different letters indicate significant differences (p<0.05).

[0087] Depend on Figure 7 and Figure 8 It can be seen that the root length of the treatment groups was 2.72 cm, 5.4 cm, and 3.88 cm longer than that of the control group, respectively, and the seedling length of the treatment groups was 3.9 cm, 4.16 cm, and 4.84 cm longer than that of the control group, respectively. In conclusion, the *Pseudomonas fluorescens* D2-21 seed solution promoted the growth of both roots and seedlings in wheat seedlings, and the seedlings treated with lower concentrations showed better growth.

[0088] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of *Pseudomonas fluorescens* D2-21, characterized in that, The fluorescent pseudomonas D2-21 has the accession number CGMCC No.33522 and was deposited on February 13, 2025 at the China General Microbiological Culture Collection Center, located at No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. A fermentation broth containing the fluorescent Pseudomonas D2-21 as described in claim 1, characterized in that, The fermentation broth was obtained by fermenting the fluorescent Pseudomonas D2-21.

3. The use of the *Pseudomonas fluorescens* D2-21 as described in claim 1 or the fermentation broth as described in claim 2 in the preparation of a product that promotes wheat seed germination.

4. The application as described in claim 3, characterized in that, The product is a microbial preparation.

5. A microbial preparation for promoting wheat seed germination, characterized in that, Contains the fluorescent Pseudomonas D2-21 as described in claim 1 or the fermentation broth as described in claim 2.

6. The use of the *Pseudomonas fluorescens* D2-21 as described in claim 1, the fermentation broth as described in claim 2, or the microbial preparation as described in claim 5 in any of the following: (1) Application in the production of indoleacetic acid; (2) Application in promoting wheat seed germination.

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