Ensifer YC2 for promoting early growth and rapid germination of tobacco plants and application of ensifer YC2

Abstract

The invention discloses an ensifer YC2 for promoting tobacco plants to grow early and fast and application of the ensifer YC2, the ensifer YC2 is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation address is Beijing, China, the preservation number is CGMCC No.29988, the preservation date is March 11, 2024, and the classification name of the ensifer YC2 is Ensifer morelecsis. The specific strain YC2 is separated and screened from the tobacco root surface at low temperature (20 DEG C) for the first time, is remarkably colonized and proliferated by three times on the tobacco root surface, remarkably promotes the growth of the overground part of the tobacco, has a remarkable early-growing and quick-growing effect on the tobacco plants, and also has a remarkable improvement effect on the agronomic traits of the tobacco plants; the growth-promoting synthetic flora provided by the invention has the effects of early growth and quick germination, can improve agronomic characters of tobacco plants, and is easy to popularize.

Description

Technical Field

[0001] The invention relates to a sword fungus YC2 for promoting early growth and rapid development of tobacco plants and application thereof, belonging to the technical field of tobacco production. Background Art

[0002] Research on early growth and rapid development of tobacco has made significant progress in recent years, mainly focusing on genetic improvement, agricultural management and biotechnology. Through genetic engineering, researchers have successfully bred new tobacco varieties with early growth and rapid development characteristics, which show obvious advantages in growth cycle and yield. In addition, the innovation of agricultural management technology has also provided strong support for the early growth and rapid development of tobacco, such as optimizing planting density, reasonable fertilization and irrigation. However, there are still some shortcomings in the current research on early growth and rapid development of tobacco. First, the application of genetic improvement technology is not widespread enough, and the promotion and application of new varieties are subject to certain restrictions. Secondly, the innovation of agricultural management technology cannot meet the needs of tobacco production in all regions, especially in areas with water shortages and harsh climatic conditions. Finally, the research on early growth and rapid development of tobacco lacks interdisciplinary cooperation and communication, which limits the expansion of research depth and breadth.

[0003] Microbial means play a vital role in solving the problem of early growth and rapid development of tobacco, and their importance and necessity cannot be ignored. By participating in soil ecological processes, microorganisms can significantly improve soil structure, increase soil fertility and microbial activity, and provide an ideal soil environment for tobacco root growth. This helps to promote early growth and rapid development of tobacco, shorten the growth cycle, and improve yield and quality. In addition, microorganisms can directly provide tobacco with the required nutrients through biological processes such as nitrogen fixation, phosphorus solubilization, and potassium solubilization, reduce the use of chemical fertilizers, reduce production costs, and reduce environmental pollution. Therefore, using microbial technology to solve the problem of early growth and rapid development of tobacco not only has significant economic benefits, but also has important ecological and social benefits. Therefore, in-depth research on the relationship between microorganisms and early growth and rapid development of tobacco is of great significance to promoting the sustainable development of the tobacco industry. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a sword fungus YC2 that promotes early growth and rapid development of tobacco plants and its application, thereby effectively improving the agronomic traits of tobacco plants and the quality of flue-cured tobacco.

[0005] Technical solution: In order to solve the above technical problems, the present invention provides a strain of Ensifer morelensis YC2, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a storage address of CGMCC No.29988 in Beijing, China, a storage date of March 11, 2024, and a classification name of Ensifer morelensis.

[0006] Wherein, the Ensifer morelensis YC2 is isolated at 20°C.

[0007] Wherein, the YC2 strain is obtained from the tobacco root surface:

[0008] (1) Tobacco seedlings were grown at low temperature (20°C) for 20 days. Well-growing tobacco plants were selected, the aboveground parts of the plants were cut off, and the roots were clamped with sterile tweezers and stirred vigorously in a conical bottle filled with PBS buffer so that all the soil on the root surface entered the buffer. The roots were then rinsed with PBS buffer and placed in a test tube filled with a surfactant (0.5 mmol / L Tween80) to just immerse the roots. The roots were ultrasonically cleaned twice in an ultrasonic cleaner with ice cubes for 30 seconds each time, and the roots were taken out to obtain the tobacco root surface microbiome suspension.

[0009] (2) Take 1 mL of tobacco root microbial suspension and dilute it by 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 For the series of gradients, take 200 μL of 10 -4 , 10 -5 , 10 -6 The suspension of different concentrations was evenly spread on 0.1TSA medium (containing cycloheximide), and each concentration gradient was repeated twice, and cultured in the dark at 20℃ for 5 days. According to the morphology, color and size of bacteria, single colonies were picked from the plates with appropriate concentration gradients, and lines were drawn on the plate separation medium for purification and culture. The strains obtained after purification and culture were used as test strains and inoculated on beef extract peptone slant for temporary storage.

[0010] (3) Extract DNA from the test strain and perform 16S rRNA sequencing to determine the taxonomic status of the strain.

[0011] The 16s rDNA base sequence of the strain is shown in SEQ ID No: 1.

[0012] The present invention also provides a bacterial agent or preparation, which contains one or more of the Ensifer morelensis YC2 or its fermentation liquid and sterile supernatant.

[0013] The present invention also provides the use of the Ensifer morelensis YC2 or the bacterial agent or preparation in promoting tobacco growth.

[0014] The method comprises the following steps: applying the Ensifer morelensis YC2 or the bacterial agent or preparation to tobacco.

[0015] Wherein, the dosage of Ensifer morelensis YC2 is 1×10 6 ~2×10 6 Piece / strain.

[0016] Wherein, the concentration of Ensifer morelensis YC2 in the bacterial agent or preparation is 1×10 5 cfu / ml~2×10 5 cfu / ml.

[0017] The present invention also provides the method for determining the root surface colonization effect of the strain through the sterile seedling colonization test:

[0018] (1) Prepare 1 / 2 strength MS medium: Dissolve 2.2 g Murashige Skoog medium (containing vitamins) dry powder, 10 g sucrose and 8 g agar powder in 1 L sterile deionized water, adjust pH to 5.8, and sterilize by autoclave at 121°C for 15 minutes. Dispense into 13 cm square culture dishes in a clean bench, 50 ml per dish.

[0019] (2) Tobacco seedling culture: Tobacco seeds were surface sterilized with 75% ethanol (30 s) and 3% NaClO (15 min, 3 times), and then placed in 1 / 2 strength MS medium. After germination for 7 days, the seedlings were transferred to MS medium, with 3 seedlings per medium. The medium was maintained at 20°C for 16 h of light and 8 h of darkness for 7 days.

[0020] (3) Preparation of YC2 fermentation broth: YC2 was fermented at 20°C and 170 rpm for 2 days, and the concentration of the bacterial broth was adjusted to OD 600 =0.1. Inoculate each strain with 50 μL of bacterial suspension. 14-28 days after inoculation, collect bacteria once a week, for a total of 3 times. Rinse the tobacco roots with sterile water to remove floating bacteria. Then place the roots in PBS buffer for 5 minutes to extract bacteria on the tobacco root surface. After removing the roots, dilute the suspension and plate it. The YC2 strain grows on the plate, indicating that it can colonize the tobacco root surface.

[0021] The present invention also provides verification of the early growth and rapid development effect of the YC2 strain. When the YC2 bacterial solution is inoculated into the roots of the sterile tobacco seedlings, compared with no inoculation, YC2 significantly increases the aboveground biomass and plant height of the tobacco plants.

[0022] The invention provides a screening system for fast-growing bacterial strains. The culture temperature for separating the bacterial strains is 20° C., and the tobacco is a tobacco seedling transplanted for 30 to 50 days.

[0023] The content of Ensifer morelensis YC2 is 1×10 5 cfu / ml.

[0024] The present invention also provides the use of the growth-promoting strain in promoting the growth of tobacco seedlings and improving the agronomic traits of tobacco plants. The method of the present invention has strong specialization and stable effect.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention is the first to isolate and screen out a specific strain YC2 from the tobacco root surface at low temperature (20°C); 2. YC2 significantly colonizes on the tobacco root surface and proliferates 3 times, which has a significant early growth and rapid development effect on the tobacco plants, and at the same time has a significant improvement effect on the agronomic traits of the tobacco plants; 3. The strain of the present invention has an early growth and rapid development effect and improves the agronomic traits of the tobacco plants, and is easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The colonization effect of YC2 in sterile tobacco seedlings after 4 weeks of inoculation;

[0027] Figure 2 Potted experiment to verify the growth-promoting effect of growth-promoting strains on tobacco plant biomass and plant height: (A) plant height; (B) aboveground fresh weight; (C) aboveground dry weight. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0029] Embodiment 1:

[0030] (1) Soil sample acquisition: The sampling site is located in 4 representative soils in Yongzhou, Hunan Province (26°42'N, 111°70'E), which has a history of rice and tobacco rotation. Samples were collected 20 days after tobacco sowing and transplanting. The aboveground part of the plant was cut off, and the roots were clamped with sterile tweezers and stirred vigorously in a conical bottle filled with PBS buffer so that all the soil on the root surface entered the buffer. The roots were then rinsed with PBS buffer and placed in a test tube filled with a surfactant (0.5mmol / LTween80) so that the surfactant just submerged the roots. The roots were ultrasonically cleaned twice in an ultrasonic cleaner with ice cubes for 30 seconds each time, and the roots were taken out to obtain the tobacco root surface microbiome suspension.

[0031] (2) Spreading, separation, cultivation and purification: Take 1 mL of tobacco soil suspension and dilute it by 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 For the series of gradients, take 200 μL of 10 -4 , 10 -5, 10 -6 The suspension of different concentrations was evenly spread on 0.1TSA medium (containing 100 mg / L cycloheximide), and each concentration gradient was repeated twice, and cultured in the dark at 20°C for 5 days. Single colonies were picked from the plates with appropriate concentration gradients according to bacterial morphology, color, and size.

[0032] (3) Determination of taxonomic status: The bacterial genetic material DNA was extracted using a DNA extraction kit (TIANamp Bacteria DNA Kit, TIANGEN). For these DNA samples, 16S rDNA PCR amplification was performed. In order to verify the fragment size of the amplified product, 1% agarose gel electrophoresis technology was used for detection. The PCR-amplified DNA fragments were entrusted to Shanghai Shenggong Biotechnology Technology Service Co., Ltd. for sequencing. After sequencing, the Blast tool on the NCBI platform was used to compare and analyze the obtained sequences in order to explore their homology with the gene sequences of other strains in the Genbank database. Based on the homologous sequences, MEGA 5.0 was used to construct a phylogenetic tree. The sequencing results are shown in SEQ ID No: 1:

[0033]

[0034] (4) This strain belongs to the Proteobacteria, γ-Proteobacteria, Pseudomonasales, Pseudomonadaceae, and Pseudomonas genus. It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on March 11, 2024, with the deposit number CGMCC No.29988, and its classification name is: Ensifer morelensis.

[0035] Example 2: Colonization ability of YC2 on tobacco root surface

[0036] Tobacco seeds were surface sterilized with 75% ethanol (30s) and 3% NaClO (15min, 3 times), respectively, and then placed in 1 / 2 strength Murashige and Skoog (MS) medium (Solarbio) containing 2% (wt / vol) sucrose and 0.8% (wt / vol) agar. After 7 days of germination, the seedlings were transferred to 1 / 2 strength MS medium, with 3 seedlings per medium. The light was 16h at 20℃ and the dark was 8h for 7 days. Ten strains of growth-promoting bacteria YC2 were selected and fermented at 20℃ and 170r / min. The bacterial solution concentration was adjusted to OD with sterile water. 600 =0.1. Each strain was inoculated with 50 μL of bacterial suspension. After 14 days of inoculation, bacteria were recovered once a week for a total of 3 times. The tobacco roots were rinsed with sterile water to remove floating bacteria. The roots were then placed in PBS buffer for 5 min to extract bacteria on the tobacco root surface. After removing the roots, the suspension was diluted and plated to determine the bacteria colonizing on the root surface. The results of Table 1 and Figure 1 It can be seen that YC2 proliferated significantly on the root surface 2 weeks after inoculation. The colonization rate stabilized 3 weeks after inoculation, and the bacteria on the root surface increased 3 times. The results showed that YC2 could colonize on the tobacco root surface and promote the growth of tobacco seedlings.

[0037] Table 1 Colonization rate of YC2 on tobacco root surface

[0038] deal with 2 weeks 3 weeks 4 weeks YC2 92％ 191％ 335％

[0039] Example 3: Potted plant experiment to verify the growth-promoting effect of the strain on tobacco plants

[0040] After the strain was cultured in TSB medium at 20°C for 48 h, the bacteria were collected by centrifugation and resuspended in appropriate sterile water to a final density of 10 8 Cells / mL were used as bacterial suspension. Uniformly grown tobacco seedlings were selected and planted at low temperature (20°C) in pots containing sterile soil and vermiculite (1:1, v / v). 10 mL of bacterial suspension was inoculated on the 10th and 30th days, respectively, and the control group was inoculated with the same amount of sterile water. The biomass and plant height increment of tobacco plants were measured 45 days after planting at low temperature. The results are shown in Figure 2The results showed that YC2 significantly increased tobacco biomass and plant height and promoted tobacco growth.

Claims

1. A strain of Ensifer morelensis YC2, characterized in that: It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit address: Beijing, China, the deposit number is CGMCC No.29988, the deposit date is March 11, 2024, and its classification name is: Ensifer morelensis.

2. Ensifer morelensis YC2 according to claim 1, characterized in that The Ensifer morelensis YC2 was isolated at 20°C.

3. Ensifer morelensis YC2 according to claim 1, characterized in that The 16s rDNA base sequence is shown in SEQ ID No.

1.

4. A bacterial agent or preparation, characterized in that: It contains one or more of the Ensifer morelensis YC2 or its fermentation liquid and sterile supernatant as described in any one of claims 1 to 3.

5. Use of Ensifer morelensis YC2 according to any one of claims 1 to 3 or the bacterial agent or preparation according to claim 4 in promoting tobacco growth at low temperatures.

6. The use according to claim 5, characterized in that: The following steps are involved: The Ensifer morelensis YC2 according to any one of claims 1 to 3 or the bacterial agent or preparation according to claim 4 is applied to tobacco.

7. The use according to claim 5 or 6, characterized in that: The dosage of Ensifer morelensis YC2 is 1×10 6 ~2×10 6 Pieces / plant.

8. The use according to claim 5 or 6, characterized in that: The concentration of Ensifer morelensis YC2 in the bacterial agent or preparation is 1×10 5 cfu / ml~2×10 5 cfu / ml.

9. The use according to any one of claims 5 to 8, characterized in that: The application temperature of the Ensifer morelensis YC2 or the bacterial agent or preparation is 15-20°C.

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