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

Abstract

The invention discloses pseudomonas fluorescens D2-21 and application thereof in promoting wheat seed germination and seedling growth, and belongs to the technical field of microorganism application. The pseudomonas fluorescens D2-21 provided by the invention is preserved in the China General Microbiological Culture Collection Center (CGMCC) on February 13, 2025, and the preservation number is CGMCC No.33522. The pseudomonas fluorescens D2-21 provided by the invention has the advantages that the preservation number is CGMCC No.33522; the pseudomonas fluorescens D2-21 has the capabilities of fixing nitrogen, producing siderophores and producing IAA (Indoleacetic Acid). Experimental results show that when the wheat seeds are cultured by using the pseudomonas fluorescens D2-21 fermentation liquor provided by the invention, the germination index and the vitality index of the wheat seeds are remarkably improved, germinated wheat seedlings have better growth vigor, and the pseudomonas fluorescens D2-21 promotes the germination of the wheat seeds and the growth of the seedlings. The invention provides a novel efficient growth-promoting strain for wheat seedling emergence, and the strain is green and safe and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial applications, and particularly to a Pseudomonas fluorescens D2-21 and its application in promoting wheat seed germination and seedling growth. Background Art

[0002] The smooth germination of wheat seeds and the normal growth of seedlings are decisive factors for plant reproduction, having important economic and ecological significance. Due to the increasing demand for wheat and continuous cropping throughout the year, continuous cropping obstacles occur, including adverse situations such as soil nutrient deficiency and aggravated pests and diseases; coupled with stress caused by different abiotic factors, problems such as low seed germination rate and poor seedling growth often occur.

[0003] Plant growth-promoting rhizo bacteria (PGPR) refer to beneficial bacteria in the rhizosphere soil of plants that have the characteristics of inhibiting plant diseases and promoting plant growth, and are also the most widely used microorganisms in current microbial improvement measures. Existing research has shown that some microbial strains can promote plant seed germination or seedling growth, but there are few strains with related functions, and there are generally problems such as unstable growth-promoting functions 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 object of the present invention is to provide a Pseudomonas fluorescens D2-21 and its application in promoting wheat seed germination and seedling growth to solve the problems existing in the above-mentioned prior art. Culturing wheat seeds with the provided Pseudomonas fluorescens D2-21 can significantly promote wheat seed germination and enhance the growth potential of wheat seedlings after germination.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a Pseudomonas fluorescens D2-21, and the preservation number of the Pseudomonas fluorescens D2-21 is CGMCC No. 33522. It was deposited on February 13, 2025, at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

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

[0009] The present invention also provides an application of the above-mentioned Pseudomonas fluorescens D2-21 or the above-mentioned fermentation broth in preparing a product for promoting plant seed germination and / or promoting 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, which contains the above-mentioned Pseudomonas fluorescens D2-21 or the above-mentioned fermentation broth.

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

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

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

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

[0016] (3) Application in promoting plant seedling growth.

[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, which includes the step of culturing the plant seeds with the above-mentioned fermentation broth or the above-mentioned microbial preparation.

[0019] Preferably, the plant is wheat.

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

[0021] The Pseudomonas fluorescens D2-21 provided by the present invention was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on February 13, 2025, with the deposit number CGMCC No. 33522. Pseudomonas fluorescens D2-21 has the abilities of nitrogen fixation, siderophore production and IAA production. The experimental results show that culturing wheat seeds with the fermentation broth of Pseudomonas fluorescens D2-21 provided by the present invention significantly improves the germination index and vigor index of wheat seeds, and the shoot length and root length of the germinated wheat seedlings are better than those of the control group, with better growth potential, indicating that Pseudomonas fluorescens D2-21 promotes the germination of wheat seeds and the growth of seedlings. The present invention provides a new highly effective growth-promoting strain for the emergence of wheat, which is green and safe and has broad application prospects. Brief Description of the Drawings

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for use in the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0023] Figure 1 It is a colony morphology diagram of Pseudomonas fluorescens D2-21;

[0024] Figure 2 It is a phylogenetic evolution tree diagram of Pseudomonas fluorescens D2-21;

[0025] Figure 3 It is an experimental result diagram of the nitrogen fixation characteristics of Pseudomonas fluorescens D2-21;

[0026] Figure 4 It is an experimental result diagram of the siderophore production characteristics of Pseudomonas fluorescens D2-21;

[0027] Figure 5 It is an experimental result diagram of the IAA production characteristics of Pseudomonas fluorescens D2-21; where a is 24h; b is 48h; c is 72h; d is 96h;

[0028] Figure 6 It is the IAA concentration of Pseudomonas fluorescens D2-21 in NA and NA+Trp;

[0029] Figure 7 It is a statistical chart of the root length and shoot length of wheat seedlings in different treatment groups;

[0030] Figure 8 It is an observation diagram of the growth of wheat seedlings; where the scale length is 5 cm. Detailed implementation manners

[0031] Now, the various exemplary implementation manners of the present invention will 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, characteristics, and implementation schemes of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0033] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention has been described only in terms of preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0034] Without departing from the scope or spirit of this invention, various modifications and variations can be made to the specific embodiments of the specification of this invention, which will be apparent to those skilled in the art. Other embodiments obtained from the specification of this invention will be apparent to those skilled in the art. The specification and examples of this invention are merely exemplary.

[0035] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0036] Example 1

[0037] 1. Experimental materials

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

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

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

[0041] Salkowski color reagent: Take 1 mL of 0.5 mol / L ferric chloride and add it to 50 mL of 35% HClO4, shake well, prepare it freshly before use, and store it in the dark.

[0042] Wheat seeds: Commercially available "Jimai 22", provided by Shandong Yinxing Seed Industry Co., Ltd.

[0043] 2. Strain isolation

[0044] The D2-21 strain of the present invention was isolated from the rhizosphere soil sample of Corydalis speciosa in the Heilihe National Nature Reserve, Ningcheng County, Chifeng City (geographical coordinates: N41.27°, W 118.21°). The isolation method is as follows:

[0045] Mix the collected soil sample with sterile water, weigh 1.0 g and add it to 99 mL of sterile water, and shake for about 30 min. Respectively, 10 -4 、10 -5 、10 -6 diluted soil suspensions were evenly spread on NA medium, cultured at 35 °C for 1-3 d, actinomycete single colonies were selected therefrom, purified by the streaking method, and the purified strains were inoculated on NA slant medium and stored at 4 °C for later use.

[0046] 3. Strain identification

[0047] 3.1 Morphological characteristics

[0048] Referring to the "Manual of Systematic Identification of Common Bacteria", the preserved D2-21 strain was inoculated on NA solid medium and cultured at 37 °C for 1-2 d. The D2-21 strain grew well in NA medium, forming colonies that were round, light yellow, moist, opaque, and had neat edges. The cells were rod-shaped, formed spores, the optimal growth pH was 7, and it could grow within the range of 15-40 °C, and the optimal growth temperature was 37 °C. The colony morphology of the D2-21 strain is shown in Figure 1 .

[0049] 3.2 Physiological and biochemical identification

[0050] The conventional method for bacterial identification (referring to the "Manual of Bacterial Identification") was used to conduct Gram staining experiment, gelatin hydrolysis test, catalase test, arginine dihydrolase test, nitrate reduction and starch hydrolysis test on the D2-21 strain respectively.

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

[0052] 3.3 DNA extraction and molecular identification

[0053] After culturing D2-21 on NA medium at 37°C for 2 days, genomic DNA was extracted using the Tsingke TSP101-200 TSINGKE DNA Extraction Kit (universal type). The total volume of the PCR reaction system was 20.0 μL, including 2.0 μL of each upstream and downstream primer, 1.0 μL of DNA template, and 15.0 μL of ddH2O. The PCR reaction program was 98°C for 2 min; 98°C for 10 s, 57°C for 10 s, 72°C for 45 s, for 35 cycles; 72°C for 5 min. The amplified PCR product was subjected to agarose gel electrophoresis (2 μL sample + 6 μL bromophenol blue) at 300 V for 12 min, and a single band was obtained and sent to Tsingke Biotechnology Co., Ltd. in Beijing for purification and sequencing. The 16S rDNA of strain D2-21 was amplified, and the nucleotide sequences of the amplification primers were 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 length of the 16S rDNA gene sequence of strain D2-21 obtained by PCR amplification was 1371 bp. The measured sequence was subjected to Blast alignment analysis in the NCBI database, and it was found that the strains with high homology all belonged to the genus Pseudomonas. A phylogenetic tree was constructed based on the 16S rDNA gene sequence as shown in Figure 2 , and the results showed that strain D2-21 and Pseudomonas fluorescens were in the same branch. Combining with the morphological characteristics identification, it was shown that strain D2-21 belonged to Pseudomonas fluorescens, and was named Pseudomonas fluorescens D2-21.

[0060] 3.4 Strain preservation

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

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

[0063] 4.1 Nitrogen fixation

[0064] Pseudomonas fluorescens D2-21 was inoculated on the Ashby medium petri dish and cultured at 37 °C for 5 days, and its growth situation was observed (see Figure 3 ). The results showed that Pseudomonas fluorescens D2-21 could grow normally on the Ashby medium and produce a transparent circle, indicating that Pseudomonas fluorescens D2-21 had nitrogen fixation ability and could promote the absorption of nitrogen source by plants.

[0065] Table 1 Transparent circle of nitrogen fixation of Pseudomonas fluorescens D2-21

[0066]

[0067] 4.2 Siderophore production characteristics

[0068] The activated Pseudomonas fluorescens D2-21 was inoculated on the CAS plate and cultured at 35 °C for 5 days. When the sample contained siderophores, the siderophores could chelate with iron ions efficiently, releasing the red CAS and forming a yellow or orange-red halo in the medium. Observe the color change of each plate (see Figure 4 ).

[0069] Table 2 Transparent circle of siderophore production of Pseudomonas fluorescens D2-21

[0070]

[0071] 4.3 Production of the plant growth hormone indole-3-acetic acid (IAA)

[0072] Inoculate a loopful of Pseudomonas fluorescens D2-21 from the slant medium into 50 mL / 250 mL NA liquid medium, and culture at 37 °C and 180 r / min for 24 h to obtain a seed solution of Pseudomonas fluorescens D2-21 with a strain concentration of 10 8 cfu / mL for standby.

[0073] Inoculate 1 mL of the seed solution into 50 mL / 250 mL NA liquid medium and NA liquid medium containing 100 mg / L L-tryptophan (Trp) respectively. Set 3 parallels for each treatment, with the blank medium as the control. Culture at 37 °C and 180 r / min. Take 50 μL of the fermentation broth and place it in a porcelain reaction plate at 24 h, 48 h, 72 h, and 96 h respectively. After adding 50 μL of Salkowski reagent and reacting for 5 minutes, observe the color of the colorimetric solution (see Figure 5 ).

[0074] Another 50 mL of the fermentation broth was placed in a centrifuge tube and centrifuged at 10000 r / min for 10 min. The absorbance of the supernatant was measured according to the method of Glickmann et al (1995). At the same time, an absorbance-concentration standard curve was drawn with analytical pure IAA, and the equation was y = 0.0291x + 0.0543 (R 2 = 0.9991) to calculate the IAA content in the fermentation broth. It can be seen from Figure 6 that after culturing for 24 h, 48 h, 72 h, and 96 h, the IAA yields of Pseudomonas fluorescens D2-21 fermented in NA medium were 2.35 mg / L, 2.82 mg / L, 4.98 mg / L, and 4.71 mg / L respectively; the IAA yields of Pseudomonas fluorescens D2-21 fermented in NA+Trp medium were 4.22 mg / L, 6.30 mg / L, 6.18 mg / L, and 6.47 mg / L respectively. It can be seen that Pseudomonas fluorescens D2-21 has the ability to produce the plant growth hormone indole-3-acetic acid (IAA), which has a promoting effect on the growth of plant plants. Adding the substrate L-tryptophan has an enhancing effect on the IAA-producing ability of Pseudomonas fluorescens D2-21, which is more obvious before 48 h.

[0075] 5. Wheat seed germination and seedling growth test

[0076] Select uniform and plump wheat seeds, and treat them successively with 75% (v / v) alcohol for 1 min, 1% (v / v) NaClO solution for 10 min, and rinse with sterile water 5 times. Select 30 disinfected and plump wheat seeds and place them neatly in a disinfected petri dish (9 cm in diameter) lined with two layers of filter paper. Use the seed solutions of Pseudomonas fluorescens D2-21 diluted 10, 100, and 1000 times as culture media (named T1, T2, and T3 respectively), and use water as the control (CK). Cultivate them at a constant temperature of 22 °C, a relative humidity of 60%, and under the condition of 8 h light / 16 h darkness. During this process, use the weighing method to supplement the lost water at regular intervals, with 3 replicates for each treatment. After germinating for 3 days, count the germination data, and continue to cultivate under the conditions of a constant temperature of 25 °C, a relative humidity of 60%, and 14 h light / 10 h darkness. After 24 h of seed germination, observe and record the number of germinated seeds on time (the criterion for germination is that the seeds show white tips). Count the germination potential on the 3rd day and the germination rate on the 7th day, and calculate the germination index and vigor index. The formulas are as follows:

[0077] Germination potential (%) = (Number of germinated seeds counted initially / Total number of tested seeds) × 100%;

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

[0079] Germination index = ∑Gt / Dt;

[0080] Vigor index = Seedling length × Germination index.

[0081] In the formula: Gt is the number of germinated seeds on the t-th day; Dt is the number of days for seed germination.

[0082] On the 7th day of the experiment, randomly select 10 germinated seedlings from each treatment to measure their root length, shoot length, fresh weight, and dry weight to evaluate the growth status of the seedling.

[0083] The results show that as can be seen from Table 3, the germination rate and germination index in the T1 treatment group are both lower than those in the CK group; the germination potential, germination rate, germination index, and vigor index in the T3 treatment group are all higher than those in the CK group, and the vigor index is 2.72 times that of the CK group. In summary, it shows that the high-concentration seed solution of Pseudomonas fluorescens D2-21 has a certain inhibitory effect on wheat seed germination, while the low-concentration seed solution of Pseudomonas fluorescens D2-21 diluted 1000 times has a promoting effect on wheat seed germination, and the effect is significant.

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

[0085]

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

[0087] It can be seen from Figure 7 and Figure 8 that the root lengths of the treatment groups were 2.72 cm, 5.4 cm, and 3.88 cm longer than those of the CK group, respectively, and the shoot lengths of the treatment groups were 3.9 cm, 4.16 cm, and 4.84 cm longer than those of the CK group, respectively. In summary, the seed liquid of Pseudomonas fluorescens D2-21 can promote the growth of wheat seedling roots and shoots, and the growth uniformity of seedlings treated with low concentrations is better.

[0088] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A Pseudomonas fluorescens D2-21, characterized in that, The preservation number of the Pseudomonas fluorescens D2-21 is CGMCC No. 33522. It was preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on February 13, 2025, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A fermentation broth containing the Pseudomonas fluorescens D2-21 described in claim 1.

3. Use of the Pseudomonas fluorescens D2-21 described in claim 1 or the fermentation broth described in claim 2 in the preparation of a product for promoting plant seed germination and / or promoting plant seedling growth.

4. The application according to claim 3, wherein The product is a microbial preparation.

5. A microbial preparation for promoting plant seed germination and / or promoting the growth of plant seedlings, characterized in that, Contains the Pseudomonas fluorescens D2-21 described in claim 1 or the fermentation broth described in claim 2.

6. The microbial agent according to claim 5, characterized in that, In the microbial agent, the concentration of Pseudomonas fluorescens D2-21 is 10 5 -10 7 cfu / mL.

7. Use of the Pseudomonas fluorescens D2-21 described in claim 1 or the fermentation broth described in claim 2 or the microbial preparation described in claim 5 or 6 in any one of the following: (1) Use in the production of plant auxin; (2) Use in promoting plant seed germination; (3) Use in promoting plant seedling growth.

8. The application according to claim 7, wherein The plant is wheat.

9. A method for promoting plant seed germination and / or promoting the growth of plant seedlings, characterized in that, It includes the step of culturing the plant seeds using the fermentation broth described in claim 2 or the microbial preparation described in claim 5 or 6.

10. The method according to claim 9, characterized in that, The plant is wheat.

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