Low-temperature-resistant tobacco early-growing quick-running bacterial flora and application thereof

Abstract

The invention discloses a low-temperature-resistant tobacco early-growing and quick-running bacterial flora and application of the low-temperature-resistant tobacco early-growing and quick-running bacterial flora. The low-temperature-resistant tobacco early-growing and quick-running bacterial flora is a mixed bacterial flora of Ensifer morellas YC2, Pseudomonas invalescens YC8 and Pseudomonas putida YC15, and the low-temperature-resistant tobacco early-growing and quick-running bacterial flora is a mixed bacterial flora of Ensifer morellas YC2, Pseudomonas invalescens YC8 and Pseudomonas putida YC15. The low-temperature-resistant tobacco early-growing and quick-running bacterial flora and the application of the low-temperature-resistant tobacco early-growing and quick-running bacterial flora. The three strains are all preserved in the China General Microbiological Culture Collection Center on March 11, 2024. Specific strains are separated and screened from tobacco root surfaces at low temperature for the first time, three strains with the most remarkable growth promoting effect are combined to obtain a synthetic flora based on the growth promoting capacity and the growth promoting characteristic, the obvious early-growth and quick-germination effects on tobacco plants are achieved, and meanwhile the obvious improvement effect on the agronomic traits of the tobacco plants is achieved.

Description

Technical Field

[0001] The invention relates to a low-temperature-resistant tobacco early-growing and fast-developing bacterial community and application thereof, belonging to the technical field of agricultural production. Background Art

[0002] Problems such as extensive seedling cultivation in tobacco-growing areas, deterioration of soil physicochemical properties, and gradual degradation of biological activity have resulted in weak early seedlings and a long seedling acclimatization period, which in turn has restricted the uncoordinated production and quality of flue-cured tobacco. At present, the main cultivation technologies for promoting early growth and rapid development of tobacco plants in China include improved seedling cultivation methods and nutrient soil formulas, selection of appropriate transplanting periods and fertilization methods, and mulching. Other technical measures include the use of growth regulators to cultivate strong seedlings and change rhizosphere pH. However, these individual technologies have not been effectively optimized, assembled, or integrated for large-scale tobacco production. In addition, the relevant technologies have poor local suitability and low acceptance by tobacco farmers. In addition, the production problems have unclear targets and are time-consuming, resulting in the expected effects being not obvious, and implementation cannot be promoted in the production areas. Therefore, conducting research on the application of microorganisms related to early growth and rapid development of tobacco is an effective way to ensure the healthy production of flue-cured tobacco, achieve sustainable soil utilization in tobacco-growing areas, and improve the quality of flue-cured tobacco.

[0003] At present, most microbial research is limited to single growth-promoting bacteria or synthetic bacterial communities composed of multiple simple functional single bacteria. However, not considering the position and function of the synthetic bacterial community in the community can easily lead to low colonization rates and limited functional expression. The core taxonomic group solves a major problem of current synthetic bacterial communities, namely the ability to support stable communities that can survive in the soil for a long time. In addition, traditional functional microorganisms in soil microbial regulation research are mainly taken from bulk soil or crop rhizosphere soil (0 to 2 mm away from the root surface), and there is little research on root surface growth-promoting microorganisms. Therefore, the development of a more efficient and stable artificial synthetic bacterial community can provide support for the research and development of effective growth-promoting bacterial agents, thereby fundamentally breaking through the bottleneck of early growth and rapid development of tobacco. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a low-temperature resistant tobacco early-growing and fast-developing bacterial community and its application, so as to effectively improve 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 low-temperature-resistant early-growing and fast-growing bacterial community on the root surface of tobacco, wherein the early-growing and fast-growing bacterial community is a mixed bacterial community of Ensifer morelensis YC2, Pseudomonas inefficax YC8 and Pseudomonas putida YC15; the three bacteria are all deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, the deposit address is: Beijing, China, and the deposit date is March 11, 2024; the Ensifer morelensis YC2 deposit number is CGMCC No.29988, and the classification name is: Ensifer morelensis; the Pseudomonas inefficax YC8 deposit number is CGMCCNO.29989, and the classification name is: Pseudomonas inefficax; the Pseudomonas putida YC15 is deposited with CGMCC No. 29990 and is classified and named as: Pseudomonas putida.

[0006] The present invention also provides a method for screening the growth-promoting synthetic flora, comprising the following steps:

[0007] (1) Obtaining a suspension of tobacco root microbiota and isolating native bacteria on the tobacco root surface at low temperature (20°C);

[0008] (2) Verify the growth-promoting effect of bacteria at low temperature (20°C);

[0009] (3) Determination of the growth-promoting properties of growth-promoting bacteria at low temperature (20°C);

[0010] (4) Antagonism / synergy between bacterial species at low temperature (20°C);

[0011] (5) Construction of synthetic communities at low temperature (20℃) and verification in potted experiments.

[0012] In the present invention, the tobacco is preferably a field tobacco seedling that has been transplanted for 30 to 50 days, more preferably 30 days.

[0013] In the present invention, the ultrasonic method is preferably used to obtain tobacco root surface bacterial cells; the reagent used in the vibration centrifugation method is preferably PBS buffer; the reagent used in the ultrasonic method is preferably a surfactant. The surfactant in the present invention is preferably 0.5mmol / L Tween 80.

[0014] In the present invention, the separation method is preferably a plate coating method, and the plate coating method preferably uses 10% TSA culture medium (containing actinomycin), and the culture conditions of the plate coating method are preferably 15-20°C dark culture for 4-6 days, and more preferably 20°C dark culture for 5 days; the plate coating method picks 20 strains of bacteria from the roots of tobacco seedlings planted in 4 kinds of soils. In the present invention, the strains isolated from the roots of tobacco seedlings planted in the 4 kinds of soils are purified by plate streaking in 10% TSA culture medium 3 times to obtain pure colonies. In the present invention, the pure colonies are shaken and cultured in TSA culture medium, and the shaking culture conditions are preferably 15-20°C, 170-190r / min, and 20-28h, and more preferably 20°C, 180r / min, and 24h.

[0015] The molecular biological identification method of the present invention is DNA extraction, PCR amplification, agarose gel electrophoresis detection of PCR amplification products, sequencing, sequence comparison analysis, and homology comparison. The DNA extraction of the present invention is extracted by a DNA extraction kit; the PCR amplification is 16S rDNA PCR amplification; the sequencing is performed by Shanghai Sangon Biotechnology Technology Service Co., Ltd.; the sequence comparison analysis is performed by Blast comparison analysis on NCBI; and the homology comparison is performed by homology comparison in the Genbank database.

[0016] In the embodiment of the present invention, 20 strains of bacteria were isolated under low temperature; the measurement indicators verified by the potted plant experiment included: fresh weight of tobacco plants, dry weight and plant height increase, and the results screened out 3 strains with the most significant growth-promoting effect under low temperature.

[0017] The present invention also provides a bacterial agent or preparation, characterized in that it contains one or more of the early-growing and fast-developing bacterial flora or their fermentation liquid and sterile supernatant.

[0018] The present invention also provides the use of the early-growing and fast-developing bacterial flora or the bacterial agent or preparation in promoting tobacco growth and improving agronomic traits of tobacco plants. The growth-promoting synthetic bacterial flora of the present invention can significantly increase the aboveground dry weight and plant height of tobacco plants.

[0019] The method comprises the following steps: applying the early-growing and fast-developing bacterial flora or the bacterial agent or preparation to tobacco.

[0020] The present invention also provides an application method of the growth-promoting synthetic bacterial flora: directly pouring the growth-promoting synthetic bacterial flora onto the roots of tobacco plants; or mixing the growth-promoting synthetic bacterial flora with decomposed organic manure at a weight ratio of 1:10 to 1:15.

[0021] The dosage of the early-growing and fast-growing bacterial community is 4×10 9 ~6×10 9 Pieces / plant.

[0022] The concentration of the early-growing and fast-growing bacterial flora in the bacterial agent or preparation is 4×10 8 cfu / ml~6×10 8 cfu / ml.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention isolates and selects specific bacterial species from the root surface of tobacco at low temperature, and based on the growth promotion experiment, combines the three bacterial species with the most significant growth promotion effects to obtain a growth-promoting synthetic bacterial community, which has a significant early growth and rapid development effect on tobacco plants under low temperature, and at the same time has a significant improvement effect on the agronomic traits of tobacco plants; 2. The growth-promoting synthetic bacterial community of the present invention has an early growth and rapid development effect and improves the agronomic traits of tobacco plants, and is easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Purification and cultivation of different bacterial strains;

[0025] Figure 2 Pot experiments verified the growth-promoting effects of different bacterial strains;

[0026] Figure 3 Pot experiments verified the growth-promoting effect of synthetic communities. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0028] 1. Obtaining tobacco root microbiome suspension

[0029] The sampling site was 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 root system was 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 root system was then rinsed with PBS buffer and placed in a test tube filled with a surfactant (0.5mmol / L Tween 80) so that the surfactant just submerged the root system. The root system was ultrasonicated twice in an ultrasonic cleaner with ice cubes for 30 seconds each time, and the root system was taken out to obtain the tobacco root surface microbiome suspension.

[0030] 2. Coating, separation, cultivation and purification

[0031] Pipette 1 mL of tobacco root microbiome suspension and dilute 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 the morphology, color, and size of the bacteria. A total of 20 strains of bacteria were picked. The purification and culture of different bacterial strains can be found in Figure 1 .

[0032] Each single colony was streaked and purified three times in 10% TSA medium using an inoculation needle to obtain pure colonies, which were numbered and stored. DNA extraction kit (TIANamp Bacteria DNA Kit, TIANGEN) was used to extract bacterial DNA. The extracted DNA was subjected to PCR amplification of 16S rDNA. The length of the PCR amplification product was detected by 1% agarose gel electrophoresis, and then the PCR amplification product was sent to Shanghai Shenggong Bioengineering Technology Service Co., Ltd. for sequencing. The sequencing results were subjected to Blast comparative analysis on NCBI and compared with the homology of other strain gene sequences in Genbank. A total of 20 strains were obtained, and the results are shown in Table 1.

[0033] Table 1 Bacterial groups tested for potted plant growth promotion effect test

[0034]

[0035]

[0036] 3. Potted plant experiment verifies the growth-promoting effect of 20 single strains on tobacco plants

[0037] 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 OD 600 =1 as the bacterial suspension. Select tobacco seedlings with uniform growth and plant them in pots containing sterile soil and vermiculite (1:1, v / v). Inoculate 10 mL of bacterial suspension on the 10th and 30th days, respectively, and the control group is inoculated with an equal amount of sterile water. The biomass and plant height increase of the tobacco plants were measured 45 days after planting at low temperature. Using the fresh weight, dry weight and plant height increase of the tobacco plants as the measurement indicators, three strains with the most significant growth-promoting effects were screened out. The three bacteria are Ensifer morelensis, Pseudomonas inefficax, and Pseudomonas putida. The screening results are shown in Figure 2 .

[0038] Among them, the three bacteria are all deposited in the General Microbiology Center of China National Committee for the Preservation of Microbiological Cultures, with the deposit address in Beijing, China, and the deposit date is March 11, 2024; Ensifer morelensis YC2 is deposited with CGMCC No.29988, and is classified as Ensifer morelensis; Pseudomonas inefficax YC8 is deposited with CGMCC No.29989, and is classified as Pseudomonas inefficax; Pseudomonas putida YC15 is deposited with CGMCC No.29990, and is classified as Pseudomonas putida. The above strains were compared for homology with the gene sequences of other strains in Genbank, and the results are shown in SEQ ID No.1 to 3, respectively. The 16s rDNA base sequence of Ensifer morelensis YC2 is shown in SEQID No.1:

[0039]

[0040] The 16s rDNA base sequence of Pseudomonas inefficax YC8 is shown in SEQ ID No. 2:

[0041]

[0042] The 16s rDNA base sequence of Pseudomonas putida YC15 is shown in SEQ ID No.3:

[0043]

[0044] 4. Determination of the growth-promoting ability of growth-promoting bacteria

[0045] 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. 2 μL of bacterial solution was inoculated in nitrogen-free medium, inorganic phosphorus medium, and CAS detection medium to detect the nitrogen fixation, phosphorus solubilization, and siderophore production abilities of the strains, and cultured at 20°C for 7 days. The observation of obvious hydrolysis circles on nitrogen-free medium and inorganic phosphorus medium, and the appearance of orange hydrolysis circles on CAS medium were used as positive reactions indicating that the strains had the corresponding promotion abilities. The strength of bacterial growth promotion ability was preliminarily determined by the ratio (D / d) of the hydrolysis circle diameter (D) to the colony diameter (d), with 4 parallel experiments in each group. The nitrogen fixation, phosphorus solubilization, and siderophore production abilities of YC8 were 2.36, 2.4, and 2.65, respectively, and those of YC15 were 2.00, 1.38, and 2.92, respectively.

[0046] The seed solution (6×10 8 cfu / ml) was inoculated into TSA liquid medium containing 5mmol / L tryptophan, and cultured at 20℃170r / min for 48h. The supernatant was obtained by high-speed centrifugation at 10000r / min for 5min. 1mL of supernatant was added with an equal amount of Salkawski colorimetric reagent and placed in the dark for 30min for IAA colorimetric reaction. The color of the solution turned pink as a positive reaction for the strain to produce IAA hormone. At the same time, a standard curve with IAA concentrations of 0, 20, 40, 60, 80 and 100μg / mL was prepared. The OD value was measured at 530nm by an enzyme reader (Cytation 5, BioTek). The standard curve (y=0.0183x+0.023, R 2 =0.998) The IAA yield of YC8 was calculated to be 47.23 mg / L, indicating that YC8 has the ability to produce IAA auxin. YC15 and YC2 do not have the ability to produce IAA.

[0047] 5. Antagonism / synergy between bacterial species

[0048] The bacterial liquids of the three bacterial strains were prepared in pairs: the three bacterial strains were shaken separately, the supernatant of strain a was added to the shaking bottle of strain b, and the shaking culture was carried out for 24 hours. The OD value of strain b was measured, and the antagonistic and synergistic interaction relationship between the strains was obtained based on the OD change value. The results are shown in Table 2. YC2 significantly promoted the growth of YC8 and YC15.

[0049] Table 2 Percentage change in OD value of bacterial solution after antagonism / synergy between strains (%)

[0050]

[0051] 6. Potted plant experiment verifies the growth-promoting effect of synthetic flora on tobacco plants

[0052] After the mixed strains (three strains in equal amounts) were 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 in pots containing sterile soil and vermiculite (1:1, v / v) at a low temperature of 20°C. 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 plants were harvested after 45 days of planting at low temperature. The measured indicators included: dry weight, fresh weight and plant height increment of tobacco plants. The results are shown in Figure 3 The results showed that the synthetic flora significantly promoted tobacco growth.

Claims

1. A low temperature resistant early growing and fast developing bacterial community on the tobacco root surface, characterized in that: The early-growing and fast-growing bacterial community is a mixed bacterial community composed of Ensifer morelensis YC2, Pseudomonas inefficax YC8 and Pseudomonas putida YC15 in a volume ratio of 1:1:1; the three bacteria are all deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, the deposit address is: Beijing, China, and the deposit date is March 11, 2024; the Ensifer morelensis YC2 deposit number is CGMCC No.29988, and the classification name is: Ensifermorelensis; the Pseudomonas inefficax YC8 deposit number is CGMCCNO.29989, and the classification name is: Pseudomonas inefficax; the Pseudomonas putida YC15 deposit number is CGMCC No.29990, classified and named as: Pseudomonas putida.

2. The early-growing and fast-growing bacterial flora according to claim 1, characterized in that: The early-growing and fast-developing bacterial flora is isolated at 20°C.

3. A bacterial agent or preparation, characterized in that: The bacterial agent or preparation contains one or more of the early-growing and fast-developing bacterial flora or its fermentation liquid and sterile supernatant as described in claim 1.

4. Use of the early-growing and fast-growing bacterial flora according to claim 1 or the bacterial agent or preparation according to claim 3 in promoting tobacco growth.

5. The application according to claim 4, characterized in that: The following steps are involved: The early-growing and fast-developing bacterial flora of claim 1 or the bacterial agent or preparation of claim 3 is applied to tobacco.

6. The use according to claim 4, characterized in that: The dosage of the early-growing and fast-growing bacterial community is 4×10 9 ~6×10 9 Pieces / plant.

7. The use according to any one of claims 4 to 6, characterized in that: The concentration of the early-growing and fast-growing bacterial flora in the bacterial agent or preparation is 4×10 8 cfu / ml~6×10 8 cfu / ml.

8. The use according to any one of claims 4 to 6, characterized in that: The application temperature of the early-growing and fast-developing bacterial flora or bacterial agent or preparation is 20-25°C.

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