Low-temperature-resistant tobacco early growth and fast development microbial community and application thereof

Abstract

The application discloses a low-temperature-resistant tobacco early growth and fast development microbial group and application thereof, the early growth and fast development microbial group is a mixed microbial group of Ensifer morelensis YC2, Pseudomonas inefficax YC8 and Pseudomonas putida YC15; the three kinds of bacteria are preserved in the China General Microbiological Culture Collection Center, and the preservation dates are all March 11, 2024. The specific bacteria are screened from the tobacco root surface at low temperature for the first time, and the synthetic microbial group is obtained by combining the three strains with the most significant growth-promoting effect based on the growth-promoting capacity and growth-promoting characteristics; the synthetic microbial group has a significant early growth and fast development effect on tobacco plants, and has a significant improvement effect on the agronomic characters of the tobacco plants.

Description

Technical Field

[0001] This invention relates to a low-temperature resistant tobacco early-maturing and rapid-growing bacterial strain and its application, belonging to the field of agricultural production technology. Background Technology

[0002] The extensive seedling cultivation methods, deteriorating soil physicochemical properties, and declining biological activity in tobacco-growing areas result in weak early-stage seedlings and prolonged recovery periods, thus hindering the balance between yield and quality in flue-cured tobacco. Currently, domestic cultivation techniques to promote early and rapid growth in tobacco plants mainly include improved seedling raising methods and nutrient soil formulas, selection of suitable transplanting periods and fertilization methods, and mulching. Other techniques include using growth regulators to cultivate strong seedlings and altering rhizosphere pH. However, these individual techniques have not been effectively optimized, assembled, or integrated for large-scale tobacco production. Furthermore, these technologies suffer from poor local suitability, low farmer acceptance, unclear production targets, and time-consuming labor requirements, leading to limited expected results and hindering implementation in tobacco-producing areas. Therefore, conducting research on the application of microorganisms related to early and rapid growth in tobacco is an effective way to ensure healthy flue-cured tobacco production, achieve sustainable soil utilization in tobacco-growing areas, and improve tobacco quality.

[0003] Currently, most microbial research is limited to single growth-promoting bacteria or synthetic microbial communities composed of multiple single bacteria with simple functions. However, neglecting the location and function of synthetic microbial communities within the community can easily lead to low colonization rates and limited functional expression. Core taxa address a major problem with current synthetic microbial communities: their ability to support stable communities that can survive long-term in the soil. Furthermore, traditional research on soil microbial regulation primarily uses functional microorganisms from the soil surface or rhizosphere (0–2 mm from the root surface), with very little research on root-surface growth-promoting microorganisms. Therefore, developing a more efficient and stable artificial synthetic microbial community can provide support for the development of effective growth-promoting agents, thereby fundamentally overcoming the bottleneck of early and rapid growth in 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-maturing and rapid-growing bacterial group and its application, so as to effectively improve the agronomic traits of tobacco plants and the quality of flue-cured tobacco.

[0005] Technical Solution: To solve the above-mentioned technical problems, this invention provides a low-temperature resistant, early-maturing and rapid-growing bacterial community on the root surface of tobacco. The early-maturing and rapid-growing bacterial community is a mixed community of *Ensifer morelensis* YC2, *Pseudomonas inefficax* YC8, and *Pseudomonas putida* YC15. All three bacteria are deposited at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, on March 11, 2024. The *Ensifer morelensis* YC2 has the accession number CGMCC No. 29988 and is classified as *Ensifer morelensis*. The *Pseudomonas inefficax* YC8 has the accession number CGMCC No. 29989 and is classified as *Pseudomonas putida*. inefficax; The Pseudomonas putida YC15 has the accession number CGMCC No. 29990 and is classified as: Pseudomonas putida.

[0006] The present invention also provides a method for screening the proliferative and synthetic bacterial communities, comprising the following steps:

[0007] (1) Obtain a suspension of tobacco root surface microbiome and isolate native bacteria from tobacco root surface at low temperature (20℃);

[0008] (2) The growth-promoting effect of bacteria was verified at low temperature (20℃);

[0009] (3) Determination of growth-promoting characteristics of growth-promoting bacteria at low temperature (20℃);

[0010] (4) Antagonistic / synergistic effects between strains at low temperature (20℃);

[0011] (5) Construction of synthetic communities at low temperature (20℃) and verification by pot experiment.

[0012] In this invention, the tobacco is preferably a field tobacco seedling transplanted 30 to 50 days ago, more preferably 30 days ago.

[0013] In this invention, ultrasonication is preferably used to obtain tobacco root surface mycelial cells; the reagent used in the shaking centrifugation method is preferably PBS buffer; and the reagent used in the ultrasonication method is preferably a surfactant. The surfactant used in this invention is preferably 0.5 mmol / L Tween 80.

[0014] In this invention, the isolation method is preferably the plate coating method, which preferably uses 10% TSA medium (containing actinomycete ketone). The preferred culture conditions for the plate coating method are dark incubation at 15-20℃ for 4-6 days, more preferably dark incubation at 20℃ for 5 days. Twenty bacterial strains are selected from the roots of tobacco seedlings grown in four different soil types using the plate coating method. The strains isolated from the roots of tobacco seedlings grown in the four different soil types are purified three times by streak plating in 10% TSA medium to obtain pure colonies. The pure colonies are then cultured in TSA medium with shaking. The preferred shaking conditions are temperature 15-20℃, rotation speed 170-190 r / min, and time 20-28 h, more preferably temperature 20℃, rotation speed 180 r / min, and time 24 h.

[0015] The molecular biological identification method of this invention comprises, in sequence, DNA extraction, PCR amplification, agarose gel electrophoresis detection of PCR amplification products, sequencing, sequence comparison analysis, and homology comparison. The DNA extraction is performed using a DNA extraction kit; the PCR amplification is 16S rDNA PCR amplification; the sequencing is performed by Shanghai Sangon Biotech Co., Ltd.; the sequence comparison analysis is performed using BLAST on NCBI; and the homology comparison is performed in the GenBank database.

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

[0017] The present invention also provides a microbial agent or preparation, characterized in that it contains one or more of the aforementioned early-growing and rapid-growth bacterial groups or their fermentation broth and sterile supernatant.

[0018] This invention also provides the application of the aforementioned early-growth and rapid-development bacterial flora or the aforementioned bacterial agent or preparation in promoting tobacco growth and improving the agronomic traits of tobacco plants. The growth-promoting synthetic bacterial flora of this invention can significantly increase the aboveground dry weight and plant height of tobacco plants.

[0019] This includes the following steps: applying the early-growing and rapid-developing bacterial group or the bacterial agent or preparation to the tobacco.

[0020] The present invention also provides a method for applying the growth-promoting synthetic microbial community: directly pouring the growth-promoting synthetic microbial community onto the roots of tobacco plants; or mixing it with well-rotted organic manure at a weight ratio of 1:10 to 1:15.

[0021] The dosage of the early-growing and rapid-developing bacterial group is 4×10. 9 ~6×10 9 One per plant.

[0022] The concentration of early-growing and rapidly developing bacteria in the bacterial agent or preparation is 4 × 10⁻⁶. 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 screens specific strains from the root surface of tobacco at low temperature, and combines the three strains with the most significant growth-promoting effect based on the growth-promoting test to obtain a growth-promoting synthetic microbial community, which has a significant effect on the early and rapid growth of tobacco plants at low temperature, and at the same time has a significant effect on improving the agronomic traits of tobacco plants; 2. The growth-promoting synthetic microbial community described in the present invention has the effect of early and rapid growth and improves the agronomic traits of tobacco plants, and is easy to promote. Attached Figure Description

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

[0025] Figure 2 Pot experiments were conducted to verify the growth-promoting effects of different bacterial strains;

[0026] Figure 3 Pot experiments were conducted to verify the growth-promoting effect of the synthetic community. Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0028] 1. Obtaining a suspension of tobacco root surface microbiome

[0029] The sampling sites were located in four representative soils in Yongzhou, Hunan Province (26°42'N, 111°70'E), with a history of rice and tobacco rotation. Samples were collected 20 days after tobacco sowing and transplanting. The above-ground parts of the plants were cut off, and the roots were held with sterile forceps and vigorously stirred in an Erlenmeyer flask containing PBS buffer to ensure that all soil on the root surface was submerged in the buffer. The roots were then rinsed with PBS buffer and placed in a test tube containing surfactant (0.5 mmol / L Tween 80), just submerging the roots. The tubes were ultrasonically cleaned twice in an ultrasonic cleaner with ice, 30 seconds each time. The roots were then removed to obtain a suspension of tobacco root surface microbiome.

[0030] 2. Coating separation, culture and purification

[0031] Take 1 mL of tobacco root microbiome suspension and dilute it 10... -2 10 -3 10 -4 10 -5 and 10 -6 A series of gradients, each using 200 μL of 10 -4 10 -5 10-6 Suspensions of various concentrations were evenly spread on 0.1 TSA medium (containing 100 mg / L actinomycin), with each concentration gradient repeated twice. The plates were incubated in the dark at 20°C for 5 days. Single colonies were picked from the plates at the appropriate concentration gradients based on bacterial morphology, color, and size, resulting in 20 bacterial strains. Purification cultures of different bacterial strains are described in [link to purification culture]. Figure 1 .

[0032] Each single colony was purified three times by streaking in 10% TSA medium using an inoculating needle to obtain pure colonies, which were then numbered and stored. Bacterial DNA was extracted using a DNA extraction kit (TIANamp Bacteria DNA Kit, TIANGEN). The extracted DNA was then amplified by PCR using 16S rDNA. The fragment length of the PCR amplification products was detected by 1% agarose gel electrophoresis. The PCR amplification products were then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results were analyzed using BLAST on NCBI and compared for homology with the gene sequences of other strains in GenBank. A total of 20 strains were obtained, and the results are shown in Table 1.

[0033] Table 1. Test bacterial groups used in the potted plant growth-promoting effect test.

[0034]

[0035]

[0036] 3. Pot experiments were conducted to verify the growth-promoting effects of 20 single bacterial strains on tobacco plants.

[0037] After the strain was cultured in TSB medium at 20°C for 48 hours, the bacteria were collected by centrifugation and resuspended in appropriate sterile water until the final density was OD. 600 =1 as the bacterial suspension. Uniformly grown tobacco seedlings were selected and planted in pots containing sterile soil and vermiculite (1:1, v / v). On days 10 and 30, 10 mL of the bacterial suspension was inoculated, while the control group was inoculated with an equal volume of sterile water. The biomass and height increase of tobacco plants were measured 45 days after planting under low temperature conditions. Using the fresh weight, dry weight, and height increase of tobacco plants as indicators, three strains with the most significant growth-promoting effects were screened. These three strains were *Ensifer morelensis*, *Pseudomonas inefficax*, and *Pseudomonas putida*. The screening results are shown below. Figure 2 .

[0038] All three strains were deposited at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, on March 11, 2024. *Ensifer morelensis* YC2 has accession number CGMCC No. 29988 and is classified as *Ensifer morelensis*. *Pseudomonas inefficax* YC8 has accession number CGMCC No. 29989 and is classified as *Pseudomonas inefficax*. *Pseudomonas putida* YC15 has accession number CGMCC No. 29990 and is classified as *Pseudomonas putida*. Homology comparisons of the gene sequences of these strains with other strains in GenBank were performed, and the results are shown in SEQ ID Nos. 1–3. The 16S rDNA sequence of *Ensifer morelensis* YC2 is shown in SEQ ID No. 1.

[0039]

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

[0041]

[0042] The 16S rDNA 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 hours, the bacteria were collected by centrifugation and resuspended in appropriate sterile water to a final density of 102. 8 The bacterial suspension was prepared using cells / mL. 2 μL of the bacterial suspension was inoculated onto nitrogen-free medium, inorganic phosphorus medium, and CAS test medium to detect the nitrogen fixation, phosphorus solubilization, and siderophore production abilities of the strains, and cultured at 20°C for 7 days. A clear hydrolysis zone was observed on nitrogen-free and inorganic phosphorus media, and an orange hydrolysis zone was observed on CAS medium, respectively, as a positive reaction indicating the corresponding growth-promoting ability of the strain. The strength of the bacterial growth-promoting ability was initially determined by the ratio of the diameter of the hydrolysis zone (D) to the colony diameter (d) (D / d), with four parallel experiments per group. The nitrogen fixation, phosphorus solubilization, and siderophore production abilities of YC8 were 2.36, 2.4, and 2.65, respectively, while those of YC15 were 2.00, 1.38, and 2.92, respectively.

[0046] Inoculate the seed solution (6×10⁻⁶) at a rate of 1%. 8 The strain was inoculated with cfu / ml into TSA liquid medium containing 5 mmol / L tryptophan and cultured at 20℃ with shaking at 170 rpm for 48 h. The supernatant was then obtained by centrifugation at 10000 rpm for 5 min. An equal volume of Salkawski chromogenic reagent was added to 1 mL of the supernatant, and the mixture was incubated in the dark for 30 min to perform the IAA colorimetric reaction. A pink color change was considered a positive reaction indicating that the strain could produce IAA hormone. Standard curves were prepared at IAA concentrations of 0, 20, 40, 60, 80, and 100 μg / mL. OD values ​​were measured at 530 nm using a Cytation 5 (BioTek) microplate reader. The results were analyzed using the standard curve (y = 0.0183x + 0.023, R0). 2 The calculated IAA yield of YC8 (=0.998) was 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 auxin.

[0047] 5. Antagonistic / synergistic effects between strains

[0048] Three bacterial cultures were prepared in pairs: each of the three bacteria was subjected to liquid shaking, and the supernatant of bacterial culture a was added to a shake flask containing bacterial culture b. The cultures were incubated on a shaker for 24 hours, and the OD value of bacterial culture b was measured. Based on the OD value changes, the antagonistic and synergistic interactions between the bacterial cultures were determined. The results are shown in Table 2. YC2 significantly promoted the growth of YC8 and YC15.

[0049] Table 2. Percentage change (%) of OD value in bacterial culture after pairwise antagonistic / synergistic effects among different bacterial species.

[0050]

[0051] 6. Pot experiment to verify the growth-promoting effect of growth-promoting synthetic microorganisms on tobacco plants.

[0052] After the mixed bacterial strains (equal amounts of the three strains) were cultured in TSB medium at 20°C for 48 hours, the bacteria were collected by centrifugation and resuspended in appropriate sterile water to a final density of 10⁻⁶. 8 The bacterial suspension was prepared using cells / mL. Uniformly grown tobacco seedlings were selected and planted in pots containing sterile soil and vermiculite (1:1, v / v) at 20℃. On days 10 and 30, 10 mL of the bacterial suspension was inoculated, while the control group was inoculated with an equal volume of sterile water. The plants were harvested after 45 days of low-temperature cultivation. The measured indicators included the increase in dry weight, fresh weight, and plant height. Results are shown in [Figure number missing]. Figure 3 The results showed that the synthetic microbial community significantly promoted tobacco growth.

Claims

1. A low-temperature resistant, early-maturing, and rapid-growing bacterial community on the surface of tobacco roots, characterized in that, The early-growing and rapidly developing bacterial group is *Cymbidium* (…). Ensifer morelensis YC2, Ineffective Pseudomonas ( Pseudomonas inefficax YC8 and Pseudomonas putida ( Pseudomonas putida YC15 is a mixed bacterial community composed of three strains in a volume ratio of 1:1:1; all three strains are deposited at the China General Microbiological Culture Collection Center, with a deposit date of March 11, 2024; the *Schizophyllum commune* ( Ensifer morelensis YC2 has the accession number CGMCC NO.29988 and is classified as follows: Ensifer morelensis The ineffective pseudomonas ( Pseudomonas inefficax YC8 has the accession number CGMCC NO.29989 and is classified as follows: Pseudomonas inefficax The aforementioned *Pseudomonas putida* ( Pseudomonas putida YC15 has the accession number CGMCC No. 29990 and is classified as: *Pseudomonas putida*. Pseudomonas putida ).

2. The early-maturing and rapidly developing bacterial flora according to claim 1, characterized in that, The early-maturing and rapidly developing bacterial group was isolated at 20°C.

3. A microbial agent or preparation, characterized in that, The bacterial agent or preparation contains the early-maturing and rapid-growing bacterial group as described in claim 1.

4. The application of the early-growing and rapid-developing bacterial group of claim 1 or the bacterial agent or preparation of claim 3 in promoting tobacco growth.

5. The application according to claim 4, characterized in that, Includes the following steps: Apply the early-growing and rapid-developing bacterial flora of claim 1 or the bacterial agent or preparation of claim 3 to tobacco.

6. The application according to claim 4, characterized in that, The dosage of the early-growing and rapid-developing bacterial group is 4×10. 9 ~6×10 9 One per plant.

7. The application according to any one of claims 4 to 6, characterized in that, The concentration of early-maturing and rapidly growing bacteria in the bacterial agent or preparation is 4 × 10⁻⁶. 8 cfu / ml ~6×10 8 cfu / ml.

8. The application according to claim 4, characterized in that, The application temperature of the aforementioned early-growing and rapid-developing bacterial groups, bacterial agents, or preparations is 20-25 °C.

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