A strain of Enterobacter huaxiensis 314A1 and its applications

Abstract

The present invention discloses a strain of Enterobacter huaxiensis 314A1 and its application. The Enterobacter huaxiensis 314A1 was deposited at the China Center for Type Culture Collection on October 31, 2023, with the deposit number: CCTCC NO: M20232090. In the present invention, the bacterial powder is used as a seed dressing agent to dress wheat seeds and then sow them. This bacterial powder can accelerate the germination and early growth rate of wheat, and at the same time can increase the biomass of wheat, specifically manifested as promoting the development of wheat lateral roots, increasing the biomass of wheat, accelerating the germination rate of wheat seeds, and increasing the yield of wheat.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural biotechnology, and specifically relates to a strain of Enterobacter huaxiensis 314A1 and its application. Background Art

[0002] In the actual production of wheat, in order to increase wheat yield and ensure food supply, large-scale application of fertilizers is often adopted. The results of multiple fertilizer efficiency tests show that reasonable application of chemical fertilizers can increase the average wheat yield by 48%. Although the yield increase effect brought by large-scale fertilization is very significant, the environmental pollution and chemical fertilizer waste problems brought about by this method are also very prominent. Tracking the fate of nitrogen after nitrogen fertilizer application found that only 35% was absorbed by crops, and the rest was lost or entered the environment, causing environmental pollution. Therefore, new fertilizers to replace traditional fertilizers are developing rapidly, and various microbial fertilizers (bacterial agents) are emerging in an endless stream.

[0003] Enterobacter huaxiensis ( Enterobacter huaxiensis ) belongs to the genus Enterobacter and the species Enterobacter huaxiensis. It is a facultative aerobic and non-spore-forming Gram-negative bacterium, which widely exists in nature and is distributed in air, soil, feces, water and animals and plants. It has the characteristics of rapid growth and production of various plant hormones. Experiments have shown that Enterobacter huaxiensis can colonize in the rhizosphere soil and roots of plants and play an important plant growth-promoting function. In addition, there are also studies showing that it can alleviate heavy metal cadmium pollution. A large number of studies have pointed out that Enterobacter, as a beneficial root endophyte, has great growth-promoting potential and less side effects on the ecological environment. There have been a large number of studies on the molecular mechanism of Enterobacter promoting plant growth and development. How to further develop it into a green and environmentally friendly microbial fertilizer is of great significance for the development of sustainable agriculture. Summary of the Invention

[0004] Object of the Invention: The technical problem to be solved by the present invention is to provide a strain of Enterobacter huaxiensis ( Enterobacter huaxiensis ) 314A1 isolated from the inner part of vigorously growing wheat roots. This bacterium can be used as a seed dressing agent to play a role in rapid germination and growth promotion during wheat planting.

[0005] Another technical problem to be solved by the present invention is to provide a bacterial agent containing the Enterobacter huaxiensis ( Enterobacter huaxiensis ) 314A1.

[0006] Another technical problem to be solved by the present invention is to provide a preparation method of the above-mentioned bacterial agent, specifically related to the preparation method of live bacteria seed dressing powder.

[0007] Another technical problem to be solved by the present invention is to provide the application of Enterobacter huaxiensis ( Enterobacter huaxiensis ) 314A1 or the bacterial agent in promoting wheat growth.

[0008] Technical solution: To solve the above technical problems, the present invention provides a strain of Enterobacter huaxiensis ( Enterobacter huaxiensis ), 314A1. The Enterobacter huaxiensis ( Enterobacter huaxiensis ), 314A1 was deposited at the China Center for Type Culture Collection on October 31, 2023, with the deposit number ： CCTCC NO: M20232090 。

[0009] The content of the present invention also includes a bacterial agent containing the Enterobacter huaxiensis ( Enterobacter huaxiensis ), 314A1.

[0010] Among them, the dosage form of the bacterial agent is powder. Preferably, the powder is a live bacteria seed dressing powder.

[0011] The content of the live bacteria in the live bacteria seed dressing powder of Enterobacter huaxiensis ( Enterobacter huaxiensis ), 314A1 is 3.58×10 8 cfu / mL.

[0012] The content of the present invention also includes a preparation method of the bacterial agent, which is characterized by including the following steps: After the Enterobacter huaxiensis ( Enterobacter huaxiensis ), 314A1 is expanded in culture, it is inoculated into a fermentation medium, and after cultivation, the cells are obtained by centrifugation. The wet cells are mixed with corn starch, dried and then pulverized and sieved to obtain the live bacteria seed dressing powder.

[0013] Among them, the fermentation medium is: tryptone 10 g / L, soluble starch 9.5 g / L, yeast extract 5 g / L, ammonium chloride 8.5 g / L, sodium chloride 10 g / L, (NH4)2SO4 5 g / L, and the initial pH is 7.0.

[0014] The content of the present invention also includes the application of the Enterobacter huaxiensis ( Enterobacter huaxiensis ), 314A1 or the bacterial agent in promoting the growth of wheat.

[0015] Among them, the promotion of wheat growth includes promoting wheat germination and the growth of wheat roots.

[0016] Among them, the bacterial agent is a live bacteria seed dressing powder, and the application includes diluting the live bacteria seed dressing powder with water, mixing it with wheat seeds, soaking them and then sowing.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The strain of Enterobacter huaxiensis provided by the present invention can metabolize and produce various phytohormones, thereby promoting the development of wheat lateral roots, wheat germination, and increasing wheat yield. The specific manifestations of promoting the development of wheat lateral roots are the development and growth of lateral roots and the increase in root hairs; the specific manifestation of promoting wheat germination is the accelerated germination rate and vigorous growth vitality of wheat; the specific manifestation of increasing wheat yield is the decrease in the average grain weight of wheat, the increase in the average number of grains per spike, and the increase in the theoretical yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a diagram showing the promotion of wheat seed germination by the 314A1 bacteria.

[0019] Figure 2 It is a diagram showing the promotion of wheat seed germination by the 314A1 bacteria (where A is a diagram showing the germination of wheat seeds at the same period; B is a diagram showing the data of the bud length and root length of wheat seeds treated with different bacterial concentrations at 24, 36, and 48 h).

[0020] Figure 3 It is a comparison diagram showing the promotion of wheat lateral root growth by the 314A1 bacteria.

[0021] Figure 4 It is a data comparison diagram showing the effects of the 314A1 bacteria on wheat root length, root area, average root diameter, number of nodes, number of root tips, and number of branches.

[0022] Figure 5 It is a comparison diagram showing the growth of wheat five days after sowing with the 314A1 bacteria agent for seed dressing treatment.

[0023] Figure 6 It is a comparison diagram showing the ear development of wheat after sowing with the 314A1 bacteria agent for seed dressing treatment. (Where A is a diagram showing the ears of wheat in the experimental group and the control group; B is a data comparison diagram showing the ear length and ear weight of wheat after treatment).

[0024] Figure 7 It is a data comparison diagram showing the total length, fresh weight of the above-ground part, fresh weight of the underground part, and fresh weight of leaves of wheat at different days after sowing with the 314A1 bacteria agent for seed dressing treatment.

[0025] Figure 8 It is a data comparison diagram showing the 100-grain weight, average number of ears, average number of grains, and theoretical yield of wheat after harvesting with the 314A1 bacteria agent for seed dressing treatment. DETAILED DESCRIPTION OF THE INVENTION

[0026] The embodiments of the present invention will be described in detail below. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. For those not specified in the examples, the operations are carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0027] Example 1 Screening of Enterobacter huaxiensis Enterobacter huaxiensis 314A1 of the present invention

[0028] (1) Screening process

[0029] Collect dry plants of Yangmai 15 growing vigorously in Yangzhou City and wash their roots. The specific washing method is as follows: shake off the soil on the wheat roots, soak them in 75% ethanol (v / v) for 60 s after rinsing with pure water, then soak them in 3.125% NaClO (v / v) for 6 min, then soak them in 75% ethanol for 30 s, and rinse them five times with sterile water. Take 50 mL of the sterile water from the last rinse and spread it on an LB plate, and culture it overnight at 38°C. Transfer the washed wheat roots into a sterile mortar in a laminar flow hood, add 3 mL of sterile water, grind them with a sterile pestle, and take the supernatant. Spread it on an LB plate by the gradient dilution method to isolate bacterial isolates from the wheat root samples. Purify them three times by the streak plate method, number the purified bacteria, and preserve them with 30% sterile glycerol (v / v).

[0030] Measure various physiological indexes of the screened bacteria, and preferably select a strain of bacteria with functions such as phosphorus solubilization, nitrogen fixation, and indole production, and name it 314A1.

[0031] (2) Colony characteristics and cell morphology

[0032] Streak 314A1 on an LB solid medium and culture it overnight at 38°C to observe the colony morphology. Stain the 314A1 strain in the logarithmic growth phase by the Gram staining method and then observe it under a microscope to observe the morphological characteristics of 314A1.

[0033] It is found through observation that the colonies of this bacterium are yellowish-white, relatively large in shape and irregular at the edges. Microscopic observation shows that this bacterium is short rod-shaped, arranged singly, in pairs or in chains. Gram staining shows that it is a Gram-negative bacterium.

[0034] (3) 16S rDNA sequence homology analysis of the strain

[0035] Inoculate one loop of 314A1 into 10 mL of LB medium and shake culture for 16 h. Take 1 mL of the bacterial solution and centrifuge at 10,000 rpm for 1 min. Discard the supernatant, add 200 - 300 μL of sterile water to the precipitate, place it in a boiling water bath for 10 min, immediately place it at -20 °C for 3 min, then centrifuge at 12,000 rpm at 4 °C for 5 min. The supernatant is the template. Using the extracted genomic DNA as the template, perform 16S rDNA amplification with the universal bacterial primers 27f and 1492r.

[0036] 27f: 5’-AGAGTTTGAT CCTGGCTCAG-3’, SEQ ID No.2;

[0037] 1492r: 5’-GGTTACCTTG TTACGACTT-3’, SEQ ID No.3.

[0038] The high-fidelity DNA polymerase used is 2x Phanta Max Master Mix from Vayme Biotech. The PCR system is 50 μL: ddH2O: 20 μL; 2xPhanta Max Master Mix: 25 μL; 27f: 2 μL; 1492r: 2 μL; template DNA: 1 μL. The PCR conditions are pre-denaturation at 94 °C for 4 minutes, denaturation at 94 °C for 30 seconds, extension at 72 °C for 1.5 minutes, a total of 35 cycles, final extension at 72 °C for 10 minutes, and preservation at 4 °C.

[0039] The amplified product obtained by PCR is purified and recovered and then sent to Tsingke Biotechnology Co., Ltd. for sequencing analysis. The obtained results are compared with the 16S rDNA gene sequences of related strains in the National Center for Biotechnology Information (NCBI) in the United States, and it is found that its sequence has 99.66% similarity with Enterobacter huaxiensis Enterobacter huaxiensis. Combining the colony morphology and its 16S rDNA gene sequence, the strain 314A1 of the present invention is identified as Enterobacter huaxiensis ( Enterobacter huaxiensis) , and its 16S rDNA is as shown in SEQ ID No.1.

[0040] Example 2 Experimental study on the promotion of wheat seed germination by Enterobacter huaxiensis strain 314A1

[0041] Disinfect wheat seeds and soak them overnight. The wheat variety selected is Yangmai 15. The specific disinfection method is to soak them in 50% alcohol for 30 s, then transfer them to a 3% hypochlorous acid solution and soak for 6 min, and rinse with sterile water 5 times. Spread the treated seeds in a petri dish lined with two layers of filter paper. Inoculate the overnight-cultured Enterobacter huaxiensis bacterial solution into the petri dish at dilution multiples of 0 times, 4 times, 8 times, 16 times, 32 times, and 64 times, and record the weight. Subsequently, weigh every 12 h and make up to the original weight with clear water. Record the bud length and root length every 12 h, and take 5 replicates for each group. The results showed that compared with the control, after treatment with 314A1, the wheat seeds showed different performances. Specifically, when the concentration of 314A1 was relatively high, it showed an inhibitory effect, and when the dilution multiple of 314A1 was relatively large, it showed a promoting effect. As Figure 1 、 Figure 2 shown, it indicates that strain 314A1 can promote wheat germination at low concentrations.

[0042] Example 3 Experimental study on the promotion of wheat root development by Enterobacter huaxiensis 314A1

[0043] Disinfect wheat seeds and soak them overnight, and culture them in sterile distilled water until the two-leaf and one-heart stage. The wheat variety selected is Yangmai 15. The specific disinfection method is to soak them in 50% alcohol for 30 s, then transfer them to a 3% hypochlorous acid solution and soak for 6 min, and rinse with sterile water 5 times. Set up a control group (CK) and a live bacteria treatment group (HJ). Culture Enterobacter huaxiensis 314A1 for 15 h, and at this time the bacterial concentration is about 2×10 8 cfu, add it to the experimental group seedling tray, and at the same time add 100 mL of LB culture medium to the control group and the experimental group. End the culture when the wheat grows to the three-leaf and one-heart stage. Take three replicates for each group and scan them in a root scanner. The results showed that compared with the control, after treatment with 314A1 bacterial solution, the main root length of wheat in the experimental group decreased, but the lateral root growth was significantly enhanced. As Figure 3 、 Figure 4 shown, it indicates that strain 314A1 can promote the development of wheat lateral roots, specifically manifested as a slight decrease in root length, an increase in root area, an unchanged average root diameter, an increase in the number of nodes, an increase in the number of root tips, and an increase in the number of branches.

[0044] Table 1

[0045]

[0046] Example 4 Preparation of 314A1 powder and its effects on wheat germination, growth and development after seed dressing

[0047] The preparation method of the 314A1 bacterial powder is as follows: Inoculate a loop of the strain preserved in glycerol into an LB culture test tube and culture for 8 h, then inoculate it into a large Erlenmeyer flask at an inoculation amount of 2% and culture for 15 h. Centrifuge the bacterial liquid at 4000 rpm to obtain wet bacterial cells, and then perform seed dressing according to the mass ratio of wet bacterial cells to sterile corn starch = 1:1.2 (m / m). After the seed dressing is completed, grind it with a sterile mortar until there is no agglomeration, and the 314A1 bacterial powder is obtained. The prepared bacterial powder needs to be stored in a refrigerator at 4°C.

[0048] Disinfect the wheat seeds and soak them overnight. The wheat variety used is Yangmai 15. The specific disinfection method is to soak them in 50% alcohol for 30 s, then transfer them to a 3% hypochlorous acid solution and soak for 6 min, and rinse with sterile water 5 times. Prepare the bacterial powder and water according to the ratio of 1 g:2 L, place the seeds soaked overnight in the bacterial powder water and soak for 30 min, and then sow. After sowing, carry out field management. Each time when watering, the treatment group (HJ) uses the prepared bacterial powder water (ratio of bacterial powder: water = 1 g:2 L), and the control group uses clean water. Sample once every ten days to measure the biomass. According to Figure 6 As shown, after treatment, the ear length and ear weight of wheat increase on different days (where A is the display diagram of wheat ears of the experimental group and the control group). According to Figure 7 As shown, on different days, the total length of wheat, the fresh weight of the above-ground part, and the fresh weight of leaves all increase, while the fresh weight of the underground part of wheat increases in the early growth stage and decreases in the later stage. Harvest the control group and the experimental group, respectively count the 100-grain weight, the average number of ears per plant, and the number of grains per ear, and calculate the theoretical yield (total grain weight per mu) through the following formula.

[0049] Total grain weight per mu = number of plants per square meter (about 300) × 667 × average number of ears per plant × average number of grains per ear ÷ 100 × average 100-grain weight

[0050] According to Figure 8 As shown, the average 100-grain weight decreases slightly, and the average number of ears per plant and the number of grains per ear both increase. It can be calculated that the theoretical yield increases after sowing with the 314A1 bacterial powder.

Claims

1. A strain of Enterobacter huaxiensis ( Enterobacter huaxiensis ), namely 314A1, the Enterobacter huaxiensis ( Enterobacter huaxiensis ) 314A1 was deposited at the China Center for Type Culture Collection on October 31, 2023, with the deposit number ： CCTCC NO: M20232090.

2. A microbial agent containing *Enterobacter huaxiensis* Enterobacter huaxiensis Enterobacter huaxiensis 314A1 described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The dosage form of the microbial agent is powder.

4. The microbial agent according to claim 3, wherein The powder is a live bacteria seed dressing powder.

5. The microbial agent according to claim 4, characterized in that, The concentration of *Enterobacter huaxiensis* Enterobacter huaxiensis 314A1 is 3.58×10 8 cfu / mL.

6. The preparation method of the microbial agent according to claim 4, characterized in that, It includes the following steps: After the expanded cultivation of Enterobacter huaxiensis ( Enterobacter huaxiensis ), it is inoculated into the fermentation medium. After cultivation, the cells are obtained by centrifugation. The wet cells are mixed with corn starch, dried and then crushed and sieved to obtain the live bacteria seed dressing powder.

7. The preparation method according to claim 6, wherein The fermentation medium is: tryptone 10 g / L, soluble starch 9.5 g / L, yeast extract 5 g / L, ammonium chloride 8.5 g / L, sodium chloride 10 g / L, (NH4)2SO4 5 g / L, initial pH 7.

0.

8. Use of Enterobacter huaxiensis as described in claim 1 ( Enterobacter huaxiensis ) 314A1 or the microbial agent as described in claim 2 or 3 in promoting the growth of wheat, wherein the promotion of the growth of wheat includes promoting wheat germination and promoting wheat root development.

9. The application according to claim 8, wherein The microbial agent is a live bacteria seed dressing powder, and the application includes diluting the live bacteria seed dressing powder with water, mixing it with wheat seeds, soaking and then sowing.

Images