Tobacco growth-promoting drought-resistant drought-enduring strain and application thereof

Abstract

The invention discloses a tobacco growth-promoting, drought-resisting and drought-enduring strain and application thereof, the growth-promoting, drought-resisting and drought-enduring strain Kluyvera intermedia and Pseudomonas lini are screened from tobacco field soil in an arid region by adopting a gradual screening strategy, and the tobacco growth-promoting, drought-resisting and drought-enduring strain has the advantages that the phosphorus dissolving capacity is strong, the yield is high, and the yield is high. And the strain has the advantage of high capability of producing indoleacetic acid, siderophore and 1-aminocyclopropane-1-carboxylic acid deaminase. In the tobacco planting process, a microbial composition containing Kluyvera intermedia and / or Pseudomonas lini is applied, so that the agronomic traits of tobacco are remarkably influenced, the growth-promoting and drought-resisting capabilities of the tobacco are remarkably improved, for example, the plant height, the stem diameter and the leaf area are increased, the biomass is increased, and the growth-promoting and drought-resisting capabilities of the tobacco are also improved. Meanwhile, the drought tolerance of the tobacco, such as the osmotic stress resistance, is remarkably enhanced, the yield and the output value of the tobacco in a drought environment are favorably improved, and the method has wide application potential in drought and semi-drought tobacco planting areas.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular to the field of plant stress resistance research technology. Background Art

[0002] Drought is one of the most serious factors hindering crop growth. The Panxi Plateau in southwestern Sichuan Province, my country, with its perennially high temperatures and low rainfall, is one of the most drought-prone regions in China. This region, home to a large flue-cured tobacco production area in Sichuan, is susceptible to environmental influences during its growth, with drought being a major factor hindering tobacco production in the region. Therefore, there is an urgent need to develop new technologies to alleviate the drought stress faced by tobacco production in the Panxi region.

[0003] Currently, domestic and international efforts to improve tobacco drought tolerance primarily involve altering cultivation techniques, regulating with exogenous chemical additives, and expressing drought-resistant genes. Modifying tobacco cultivation techniques primarily involves establishing irrigation systems and installing mulch films in fields. However, such measures are difficult to implement in mountainous areas where water is scarce, and mulch films can easily contaminate the soil. While exogenous chemicals have been reported to effectively alleviate drought stress, their addition can lead to risks such as changes in soil pH, the production of toxic substances, and the accumulation of heavy metals, potentially disrupting the soil microbiome. There are numerous reports on the application of drought-resistant genes in tobacco. While these transgenic tobacco plants have demonstrated excellent drought tolerance under laboratory conditions, this approach is time-consuming and labor-intensive, and may also result in the loss of other beneficial traits in the tobacco gene pool.

[0004] Research has found that microorganisms can effectively alleviate plant damage and promote plant growth under drought stress, making it a research hotspot. Plant growth-promoting bacteria can help plants enhance their drought resistance by promoting nutrient absorption and soil water flow.

[0005] There are many studies at home and abroad on screening growth-promoting bacteria with functions such as producing plant hormones to promote the growth of plants such as tobacco, but there are few reports on screening growth-promoting bacteria for drought-stressed environments. For example, the growth-promoting bacteria screened from the rhizosphere soil of plateau bluegrass by Zhao Shudong et al. had a phosphate solubility capacity of only 274.30 mg / L and a maximum indoleacetic acid production of 76.16 mg / L; the growth-promoting bacteria screened from the rhizosphere soil of Polygonum multiflorum by Liu Changzheng et al. had an indoleacetic acid production capacity of 38.65±0.45 mg / L and the strongest siderophore production capacity was 0.49±0.01Ar·A0 -1 Lian Faqin et al. screened out bacteria from the rhizosphere soil of rubber trees with the highest ability to produce 1-aminocyclopropane-1-carboxylic acid deaminase of 0.23 U / mg and the ability to produce indoleacetic acid of 19.01 mg / L.

[0006] Liu Yuande et al. applied a microbial agent made from a fusion of tobacco phosphate- and potassium-dissolving bacteria and an indigenous bacteria propagation agent to tobacco, and found that compared with tobacco not applied with the microbial agent made from a fusion of tobacco phosphate- and potassium-dissolving bacteria and an indigenous bacteria propagation agent, the leaf area increased by 12.4%, but there was no significant difference in plant height, and the drought resistance of tobacco was not significantly improved.

[0007] Therefore, currently, screening for microorganisms with both growth-promoting and drought-tolerant functions from tobacco rhizospheres is limited, and there are very few examples of their application in actual tobacco production under drought conditions. There is a lack of diversity in tobacco growth-promoting and drought-tolerant strain libraries for production applications, as well as practical application examples. The screening of tobacco growth-promoting and drought-tolerant strains requires consideration of both growth-promoting and drought-tolerant functions. Growth-promoting and drought-tolerant functions primarily include the production of plant hormones or proteases, such as indoleacetic acid, cytokinins, and 1-aminocyclopropane-1-carboxylate deaminase, to promote plant growth; secretion of organic acids to dissolve insoluble salts such as calcium phosphate and silicate aluminum in the soil, promoting crop utilization of nutrients such as phosphorus and potassium; and production of siderophores and hydrogen cyanide to resist pathogens and withstand adverse stresses. Resistance to high osmotic pressure is a prerequisite for the survival of growth-promoting and drought-tolerant bacteria in drought environments. Only bacteria combining these two functions can have practical application value.

[0008] For the above reasons, the inventors screened bacterial strains with drought-resistant and drought-promoting functions in tobacco field soil in arid areas, studied their characteristics, and explored their effects on tobacco growth in arid areas by inoculating the screened strains, thereby developing tobacco growth-promoting, drought-resistant and drought-tolerant bacterial agents with excellent effects to promote the development of the tobacco industry in arid areas. Summary of the Invention

[0009] In order to overcome the above problems, the present inventors conducted in-depth research on microorganisms in tobacco field soil in arid areas, and adopted a step-by-step screening strategy to screen out growth-promoting, drought-resistant and drought-tolerant strains Kluyvera intermedia L5N1 and Pseudomonas lini S2-1 from tobacco field soil in arid areas. The strains have strong phosphate solubilization ability and produce high amounts of indoleacetic acid (IAA), siderophores and 1-aminocyclopropane-1-carboxylic acid deaminase (ACC deaminase). During tobacco cultivation, applying Kluyvera intermedia L5N1 and / or Pseudomonas lini S2-1 or a microbial composition containing them not only has a significant effect on the agronomic traits of tobacco, but also significantly improves the growth-promoting and drought-resistant ability of tobacco, such as increasing tobacco biomass, plant height, stem diameter and leaf area, while significantly enhancing the drought resistance of tobacco, such as resistance to osmotic stress. These strains have broad application potential in arid and semi-arid tobacco-growing areas, thereby completing the present invention.

[0010] Specifically, the purpose of the present invention is to provide the following aspects:

[0011] In a first aspect, the present invention provides an isolated Kluyvera intermedia L5N1, which is deposited in the China Center for Type Culture Collection and is numbered CCTCC NO. M2022531.

[0012] and microorganisms for promoting drought resistance and drought tolerance, comprising Kluyvera intermedia L5N1.

[0013] In a second aspect, the present invention provides an isolated Pseudomonas lini S2-1, which is deposited in the China Center for Type Culture Collection and has a number of CCTCC NO.M2022530.

[0014] and microorganisms for promoting drought resistance and drought tolerance, comprising Pseudomonas linen S2-1.

[0015] In a third aspect, the present invention provides the use of the above-mentioned Kluyvera centrifuga L5N1 and a biological composition comprising the same, or Pseudomonas linen S2-1 and a microbial composition comprising the same, or a microbial composition comprising Kluyvera centrifuga L5N1 and Pseudomonas linen S2-1 in improving the drought resistance and drought tolerance of tobacco growth promotion.

[0016] In a fourth aspect, the present invention provides a method for growing tobacco, comprising: applying a microorganism selected from the following to tobacco seedlings:

[0017] Kluyvera intermedia L5N1 or a microbial composition comprising the same;

[0018] Pseudomonas linensis S2-1 or a microbial composition comprising the same;

[0019] A microbial composition comprising Kluyvera intermedia L5N1 and Pseudomonas linens S2-1.

[0020] In a fifth aspect, an embodiment of the present invention provides a bio-organic fertilizer, which includes: organic fertilizer and the aforementioned microbial composition containing Kluyvera intermedia L5N1 and Pseudomonas linen S2-1 obtained by co-fermentation with the organic fertilizer.

[0021] The beneficial effects of the present invention include:

[0022] (1) The L5N1 and S2-1 strains provided by the present invention have excellent growth-promoting drought resistance and drought tolerance, effectively improve the agronomic traits of tobacco, and help tobacco effectively resist drought.

[0023] (2) The L5N1 and S2-1 strains provided by the present invention have strong phosphate solubility and high ability to produce indoleacetic acid, siderophore and 1-aminocyclopropane-1-carboxylic acid deaminase.

[0024] (3) The L5N1 and S2-1 strains provided by the present invention have a stronger effect when used in combination. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Showing the growth pictures of L5N1 and S2-1 in Example 1;

[0026] Figure 2 Showing the pictures of the phosphorus dissolution circles of L5N1 and S2-1 in Example 1;

[0027] Figure 3 The graph showing the ability of L5N1 and S2-1 to resist osmotic stress in Example 2 is shown;

[0028] Figure 4 Showing the tobacco growth picture in Example 3;

[0029] Figure 5 The changes in stem diameter and leaf area of ​​tobacco plants in potted test using bio-organic fertilizer in Example 5 are shown;

[0030] Figure 6 The results show the changes in high-quality tobacco leaves and total yield when the bio-organic fertilizer was applied to the field test in Example 6. DETAILED DESCRIPTION

[0031] The present invention will be described in further detail below with reference to the accompanying drawings and examples, through which the features and advantages of the present invention will become more clearly understood.

[0032] The word "exemplary" is used exclusively herein to mean "serving as an example, example, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments. Although various aspects of the embodiments are shown in the drawings, the drawings are not necessarily drawn to scale unless otherwise indicated.

[0033] The inventors screened out two local strains with excellent growth-promoting drought resistance and drought tolerance from tobacco rhizosphere soil. One is Kluyvera centrum L5N1, which has been deposited in the China Center for Type Culture Collection and is numbered CCTCC NO.M2022531 at the China Center for Type Culture Collection; the other is Pseudomonas linen S2-1, which has been deposited in the China Center for Type Culture Collection and is numbered CCTCC NO.M 2022530 at the China Center for Type Culture Collection.

[0034] In the present invention, the inventors adopted a stepwise screening approach to obtain L5N1 and S2-1. Specifically, strains with strong phosphate solubilization abilities were first selected, using the appearance of a phosphate-solubilizing ring around the colonies as the primary screening criteria. Among the phosphate-solubilizing strains, strains with IAA production were selected. Furthermore, strains with siderophore and ACC deaminase production were further selected. Finally, by adding PEG-6000 to Luria-Bertani medium, strains with growth-promoting, drought-resistant, and drought-tolerant properties were screened.

[0035] Specifically, phosphate-solubilizing bacteria are screened on inorganic phosphorus media, such as the National Botanical Research Institute Phosphate Growth Medium (NBRIP Inorganic Phosphate Medium);

[0036] Next, strains with phosphate solubilization and IAA production were screened using Luria-Bertani liquid medium (LB medium) containing L-tryptophan;

[0037] Afterwards, strains with phosphate solubilization, IAA production and siderophore functions were screened using MKB medium;

[0038] Finally, the ACC deaminase activity was determined from the above strains with the functions of phosphate solubilization, IAA production and siderophore according to the Penrose method, and the target strains were screened out, that is, the strains with high phosphate solubilization, high production of IAA, siderophore and ACC deaminase.

[0039] Furthermore, the above-mentioned strains with efficient phosphate solubility, high IAA production, siderophore and ACC deaminase were used to extract DNA using a DNA extraction kit, and PCR amplification was performed. The amplified products were sequenced for 16S rRNA. The results showed that one strain was Kluyvera intermedia, and its 16S rRNA had a total of 1440 bases. As used in this article, it was named L5N1; the other strain had the closest evolutionary relationship to Pseudomonas lini, with a total of 1506 bases, so it was judged to be Pseudomonas lini and named S2-1.

[0040] Among them, L5N1 and S2-1 were excellent in phosphate solubilization ability, indoleacetic acid production ability, 1-aminocyclopropane-1-carboxylic acid deaminase production ability, and siderophore production ability.

[0041] The effect of mixed application of L5N1 and S2-1 is better.

[0042] Further studies have found that L5N1 and S2-1 can also be used in combination with other strains to further improve the agricultural traits of tobacco under drought stress, such as biomass, plant height, leaf area, stem diameter, etc., that is, drought resistance is further improved.

[0043] According to the present invention, when L5N1 or S2-1 is applied separately to the growing tobacco, the growth-promoting drought resistance and drought tolerance of the tobacco are improved; when L5N1 and S2-1 are applied jointly to the growing tobacco, the growth-promoting drought resistance and drought tolerance of the tobacco are further improved, especially the dry weight of the middle leaves is significantly increased, indicating that the two have a synergistic effect; when L5N1, S2-1 and other strains, such as Bacillus sp., pseudomonas sp., etc., are applied jointly to the growing tobacco, its drought tolerance, for example, the agricultural traits of tobacco under drought stress, such as biomass, plant height, leaf area, stem diameter, etc., are further improved.

[0044] The present invention also provides a method for growing tobacco, comprising: applying L5N1 or a microbial composition containing the same, or S2-1 or a microbial composition containing the same, or a microbial composition consisting of L5N1 and S2-1 or a microbial composition containing L5N1 and S2-1 to growing tobacco seedlings.

[0045] The tobacco seedlings can be processed and planted in a conventional manner without any special requirements.

[0046] Furthermore, the microorganisms or microbial compositions are applied starting from about one month after the tobacco seedlings are planted, and the microorganisms or microbial compositions are optionally applied again after an interval of 5 to 10 days.

[0047] In a preferred embodiment, tobacco seedlings with better growth are selected and planted in March or May, and the above-mentioned microorganisms or microbial compositions are applied in April during spring drought or June during summer drought. At this time, tobacco shows stronger growth-promoting drought resistance and drought tolerance.

[0048] In the present invention, the manner of applying L5N1 or a microbial composition containing the same, or S2-1 or a microbial composition containing the same, or a microbial composition consisting of L5N1 and S2-1 or a microbial composition containing L5N1 and S2-1 is spraying or irrigation, preferably irrigation.

[0049] Preferably, before use, L5N1 is expanded to 1×10 7～9 CFU / mL of bacterial solution, more preferably, L5N1 is expanded to 1×10 8 CFU / mL of bacterial solution; expand S2-1 to 1×10 7～9 CFU / mL of bacterial solution, more preferably S2-1 was expanded to 1×10 8 CFU / mL of bacterial solution, and then apply it by spraying or irrigation.

[0050] Among them, applying 80 to 120 mL of bacterial solution to each tobacco seedling can meet the requirements of promoting growth, resisting drought and endurance of tobacco seedlings.

[0051] According to the present invention, when L5N1 and S2-1 are applied to tobacco seedlings simultaneously, the ratio of their expanded culture solutions is 1-10:1-10, preferably 4:5 (v:v).

[0052] In the present invention, the tobacco obtained by the above-mentioned planting method has a strong drought resistance. Even if the water is cut off for more than 21 days, the tobacco leaves will hardly wilt; its growth-promoting drought resistance, such as plant height, stem thickness, leaf area, and biomass, are significantly increased. Among them, when L5N1 and S2-1 are applied simultaneously during the growth of tobacco, the growth-promoting drought resistance of tobacco, such as plant height, stem thickness, leaf area, and biomass, increases more significantly. This may be because the two strains of L5N1 and S2-1 survive in a drought environment and dissolve phosphorus, which enhances the absorption of nutrients by tobacco. The secretion of ACC deaminase reduces the ethylene concentration in the tobacco body, which plays a positive role in the drought-resistant growth of tobacco. Therefore, the two strains of L5N1 and S2-1 complement each other in function and have stronger capabilities.

[0053] Example

[0054] The present invention is further described below through specific examples, but these examples are merely exemplary and do not constitute any limitation on the scope of protection of the present invention.

[0055] The following reagents and culture medium compositions are as follows:

[0056] Luria-Bertani medium (LB medium): 10.0 g of peptone, 5.0 g of yeast extract powder, 10.0 g of sodium chloride, 1000 mL of distilled water, natural pH, and 10.00 g of agar powder for solid medium.

[0057] National Botanical Research Institute Phosphate (NBRIP) medium: D-glucose 10.0 g, Ca3(PO4)2 5.0 g, MgCl2·6H2O 5.0 g, MgSO4·7H2O 0.25 g, KCl 0.2 g, (NH4)2SO4 0.1 g, distilled water 1000 mL, pH 7, solid medium plus 15.0 g agar powder.

[0058] MKB medium: 15.0 mL of glycerol, 5.0 g of acid-hydrolyzed casein (iron-free), 2.5 g of K2HPO4, 2.5 g of MgSO4·7H2O, 1000 mL of distilled water, natural pH.

[0059] Salkowski's color developer: 10 mL of 0.5 mol / L FeCl3 solution is mixed with 500 mL of 35% HClO4.

[0060] CAS assay solution: First, add 6 mL of 10 mmol / L CTAB (cetyltrimethylammonium bromide) solution to a 100 mL volumetric flask and dilute appropriately with distilled water. This is referred to as Solution I. Next, mix 1.5 mL of 1 mmol / L FeCl3 solution with 7.5 mL of 2 mmol / L CAS (chrome azurol) solution and add this to the volumetric flask containing Solution I. Next, dissolve 4.307 g of anhydrous dimethylamine in 30 mL of water, then add 6.25 mL of 12 mol / L HCl to obtain a pH 5.6 buffer solution. Transfer this to the volumetric flask containing Solution I and dilute to volume with distilled water.

[0061] Example 1 Screening and identification of L5N1 and S2-1

[0062] (1) The tobacco field soil in the arid area of ​​Miyi County, Panzhihua was stored at 4°C and the L5N1 and S2-1 strains were screened according to the following steps.

[0063] Step 1, screening of phosphate-solubilizing bacteria: weigh 5.0 g of the above soil and pour it into a 100 mL conical flask, add 50 mL of sterile water, shake in a constant temperature shaker at 30 ° C for 1 h, and dilute 1 mL of soil suspension to 10 by gradient dilution in a clean bench. -1 , 10 -2 , 10 -3 , 10 -4 and 10 -5 50 μL of each dilution was applied to NBRIP inorganic phosphorus medium and cultured at 30°C for 72 h. Then, colonies with a clear transparent zone on the edge were selected and inoculated onto LB medium and cultured at 30°C for 48 h to obtain phosphate-solubilizing bacteria. Based on this, step 2, i.e., screening for indoleacetic acid (IAA)-producing strains, was carried out.

[0064] Step 2, screening of IAA-producing bacteria: the screened phosphate-soluble bacteria were inoculated into LB liquid culture medium containing L-tryptophan (500 mg / L), cultured in a shaker at 37°C and 180 r / min for 24 h, 5 mL of bacterial solution was taken, centrifuged at 10,000 r / min for 10 min, 1 mL of supernatant was taken, 50 μL of 10 mM orthophosphoric acid was added thereto, and then 2 mL of Salkowski's color developer was added, mixed thoroughly, and reacted at 25°C for 30 min under dark conditions to obtain a strain, and based on this, step 3, i.e., screening of siderophore-producing bacteria, was carried out.

[0065] Step 3, screening of siderophore-producing bacteria: The strain obtained in step 2 above was inoculated into MKB medium and cultured in a shaker at 28°C and 170 rpm for 48 h. 2 mL of the supernatant was then mixed thoroughly with 2 mL of CAS detection solution. After reacting for 1 h, the absorbance at 630 nm was measured and recorded as Ar.

[0066] The supernatant of uninoculated MKB culture medium was reacted with CAS detection solution as a control, and its absorbance value at 630 nm was recorded as A0.

[0067] The ability of a strain to produce siderophores is represented by Ar / A0. A strain with a ratio less than 1 indicates that it has the ability to produce siderophores, and the lower the ratio, the stronger the siderophore production ability.

[0068] Step 4, screening of ACC deaminase-producing bacteria: ACC deaminase activity was determined from the strains obtained in step 3 that had phosphate solubilization, IAA production, and siderophore activity. The determination method was based on the method of Penrose, see: PENROSE DM, GLICK BRJ PP. Methods for isolating and characterizing ACC deaminase-containing plantgrowth-promoting rhizobacteria[J]. 2010, 118(1): 10-5.

[0069] (2) Identification of bacterial species

[0070] DNA from the strain obtained in step 4 was extracted using a DNA extraction kit (Sigma-Aldrich). PCR amplification was performed using the extracted DNA as a template. Primer sequences used for amplification were synthesized by the Chengdu Synthesis Department of Beijing Qingke Biotechnology Co., Ltd.: universal bacterial primers: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'. 16S rRNA sequencing was performed on the amplified products.

[0071] Final determination: There were two strains obtained in step 4, one of which was Kluyvera intermedia of Kluyvera, with a total of 1440 bases of 16S rRNA, as shown in SEQ ID NO.1, and was named L5N1; the other strain had the closest evolutionary relationship to Pseudomonas lini, with a total of 1506 bases, as shown in SEQ ID NO.2, and was therefore determined to be Pseudomonas lini and named S2-1.

[0072] SEQ ID NO.1:

[0073]

[0074] SEQ ID NO.2:

[0075]

[0076] The 16S rDNA sequences of S2-1 and L5N1 were uploaded to NCBI with accessions ON533882 and ON533881, respectively. The strains were deposited in the China Center for Type Culture Collection with accessions CCTCC NO.M 2022530 and CCTCC NO.M2022531, respectively. The address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0077] The growth-promoting and drought-resistance indices of S2-1 and L5N1 are shown in Table 1 below.

[0078] Table 1 Growth-promoting and drought-resistance indexes

[0079]

[0080] Example 2 Determination of the ability of L5N1 and S2-1 to resist osmotic stress

[0081] (1) Activation of bacterial strains: L5N1 and S2-1 were activated. The activation process was as follows: the bacterial strains stored at -80°C were taken out and thawed, and inoculated onto LB or beef extract peptone medium slant or plate. After 2 to 3 passages, the rejuvenated colonies were selected for subsequent drought resistance experiments.

[0082] (2) LB culture medium with four different gradients of PEG-6000 concentrations of 0%, 10%, 15%, and 20% was prepared. L5N1 and S2-1 activated in step (1) were inoculated into 150 mL of the LB culture medium at a 1% inoculum, respectively. The culture was shaken at 28°C and 160 rpm. The OD600 value was measured every two hours for 72 hours. The results were as follows: Figure 3 shown.

[0083] It can be seen that L5N1 grew well in LB medium without PEG-6000. As the concentration of PEG-6000 increased, the growth of L5N1 strain was gradually inhibited. When the concentration of PEG-6000 reached 20%, its maximum OD 600 The value is only 0.84, which is lower than the maximum OD under normal growth conditions. 600 Value 1.42.

[0084] The growth of S2-1 in 20% PEG-6000 was slower than that of L5N1. It hardly grew in the first 20 hours and reached its maximum growth at around 48 hours with an OD value of 0.81.

[0085] The above results show that the L5N1 and S2-1 strains can grow normally under 0%, 10%, 15% and 20% PEG-6000. Although the L5N1 and S2-1 strains gradually show obvious growth inhibition with the increase of PEG-6000 concentration, it means that they have the feasibility of surviving in normal to severe drought environments and have more advantages.

[0086] Example 3 Potted plant test

[0087] The pot experiment was conducted in the Environmental Biotechnology Laboratory of Southwest Jiaotong University. The tobacco seedling variety was Zhongchuan 208, provided by the Panzhihua Branch of Sichuan Tobacco Company. The potting soil was the topsoil of the tobacco fields in Miyi County, Panzhihua City. The base values ​​of the test soil are shown in Table 2.

[0088] Table 2 Physical and chemical properties of the tested soil

[0089]

[0090] Tobacco seedlings of similar growth potential were randomly divided into five groups, each containing four plants. Each pot was planted in a 19 cm high, 23.7 cm top diameter, and 17 cm bottom diameter plastic pot. Each pot was filled with 4 kg of soil and contained one tobacco plant. During the experiment, the plants were maintained under a 16-hour light / 8-hour dark cycle, with daytime temperatures of 30-35°C and nighttime temperatures of 25-30°C. The humidity was maintained at 70-90%. Drought was simulated by withholding water for 21 consecutive days. After the 21-day drought, the soil was rehydrated to 70% of its maximum water holding capacity. 50-200 mL of water was then added daily for 7 days.

[0091] Afterwards, the five groups of tobacco seedlings were treated as follows without watering:

[0092] In the first group, 100 mL of sterile culture medium was applied to each tobacco seedling, which was recorded as the blank control group (CK);

[0093] The second group used irrigation to inoculate each tobacco seedling with 100 mL of 1×10 8 CFU / mL L5N1 bacterial solution, recorded as T1;

[0094] The third group used irrigation to inoculate each tobacco seedling with 100 mL of 1×10 8 CFU / mL S2-1, recorded as T2;

[0095] The fourth group used irrigation to inoculate each tobacco seedling with 100 mL of 1×10 8 CFU / mL L5N1 and S2-1 mixed bacterial solution, of which 1×10 8 CFU / mL of L5N1 and 1×10 8 The ratio of S2-1 in CFU / mL was 4:5 (v:v), which was recorded as T3;

[0096] The fifth group used irrigation to inoculate each tobacco seedling with 100 mL of 1×10 8 CFU / mL QA (QA is a nitrogen-retaining microbial agent, see: Study on nitrogen-retaining microbial agent to reduce nitrogen loss during chicken manure composting and nitrogen transformation mechanism, Zhongping Qiu etc., Journal of Cleaner Production 285(2021)124813), L5N1 and S2-1 mixed bacterial solution, of which 1×10 8 CFU / mL QA, 1×10 8 CFU / mL of L5N1 and 1×10 8 The ratio of S2-1 in CFU / mL was 3:4:5 (v:v:v), which was recorded as T4.

[0097] Seven days later, the second bacterial agent was applied to the first to fifth groups of tobacco seedlings in the same manner as above.

[0098] After 21 days, the tobacco plants to be tested were obtained, and the plant height, stem diameter, leaf area and biomass of the tobacco plants were measured. The results are shown in Table 3.

[0099] Table 3 Tobacco pot experiment results

[0100] Group <![CDATA[Biomass / g·plant -1 > Plant height / cm <![CDATA[Leaf area / cm 2 > Stem diameter / mm CK 16.51±1.27d 19.90±0.67c 106.99±11.14b 4.34±0.14b T1 28.36±1.47b 24.07±0.33b 134.55±15.95b 4.74±0.28ab T2 23.26±1.39c 22.70±0.24b 146.05±7.94b 4.30±0.50b T3 34.45±1.26a 26.90±1.85a 195.32±28.27a 5.13±0.50ab T4 36.02±1.38a 29.17±1.28a 196.44±19.72a 5.40±0.41a

[0101] Note: a, b, c, and d in Table 2 are differential analyses, and the measurement object is each tobacco plant.

[0102] Each group randomly selected a tobacco plant, such as Figure 4 As shown, it can be seen that the degree of wilting of tobacco leaves in the CK group is the greatest, the leaves are curled, and the thickness becomes smaller and thinner. Although the groups T1 to T4 have different degrees of leaf wilting, the degree is lighter than that of the blank control CK. Among them, the degree of wilting in the T3 and T4 groups is the lowest, and some areas of the tobacco leaves are wilted.

[0103] Combine Figure 4 As shown in Table 3, the plant height, leaf area, and biomass of tobacco in the T1 and T2 groups were significantly higher than those in the CK group, demonstrating the robustness of the selected tobacco rhizosphere growth-promoting bacteria and their significant improvement in agronomic traits. Inoculation with either L5N1 or S2-1 had some growth-promoting effects on drought-grown tobacco, but the effect was weaker than the combined effect of L5N1 and S2-1.

[0104] Among them, the biomass of the CK group was significantly different from that of the other groups. There was no significant difference in the biomass of the T3 and T4 groups, with an average plant weight of 34.45g and 36.02g, respectively, which were 108.7% and 118.2% higher than the biomass of the CK group, respectively. The biomass of the T1 and T2 groups were 71.8% and 40.9% higher than that of the CK group, respectively. The highest plant heights of the T3 and T4 groups were 26.90cm and 29.17cm, respectively, which were significantly higher than those of the other groups. The lowest plant height of the CK group was 19.90cm. The plant heights of the T3 and T4 groups increased by 35.2% and 46.6% compared with the CK group, respectively. The plant heights of the T1 and T2 groups were 30% and 14.1% higher than those of the CK group, respectively. The leaf areas of the T3 and T4 groups were the largest, at 195.32cm, respectively. 2 and 196.44cm 2 The difference was not significant, and the leaf area of ​​CK group was the smallest at 106.99 cm 2 The leaf areas of T3 and T4 groups increased by 82.6% and 83.6% respectively compared with the CK group, and the leaf areas of T1 and T2 groups were 25.8% and 36.5% larger than those of the CK group, respectively; among the stem diameters of each group, the largest stem diameters of T3 and T4 groups were 5.13 mm and 5.40 mm, which were significantly higher than those of CK and T2. The stem diameters of T3 and T4 groups increased by 18.1% and 24.3% compared with those of the CK group, and the stem diameters of T1 and T2 groups increased by 9.2% and -0.9% respectively compared with the fresh stem diameter of the CK group.

[0105] Example 4 Field Test

[0106] The field trial was conducted in Wanqiu Township, Miyi County, Panzhihua City, at longitude 102.147119, latitude 27.1070707, and an altitude of 1710.26 meters. The region has a hot, dry climate with little rainfall, with the rainy season concentrated in August and September, and average daily temperatures exceeding 34°C in summer. The inoculant was applied in April during the spring drought, during the transplanting phase of tobacco seedlings, and in June during the summer drought, during the plant formation phase.

[0107] The field trial consisted of five treatment groups, or five plots, randomly arranged, measuring 5.0 m long and 4.0 m wide, with 1.2 m spacing between protected rows. Each plot had four ridges, each planted with five tobacco seedlings, for a total of 20 plants per plot. Planting began in March, and one month later, the five treatment groups were treated with a microbial inoculant, using the same treatment method used in Example 3. Fresh and dry weights of the mid-leaf and total leaf were measured after harvest. The results are shown in Table 4.

[0108] Table 4 Tobacco field test results

[0109]

[0110] Note: a, b, and c in Table 3 are differential analysis, and the measurement object is each cell.

[0111] As can be seen from Table 3, there are obvious differences between the T3 and T4 groups and the CK group. The average biomass of the middle leaves of each tobacco plant in the T3 group is 0.30 kg, and the dry weight is 0.05 kg. The average biomass of the middle leaves of each tobacco plant in the T4 group is slightly lower than that of the T3 group, which is 0.27 kg and the dry weight is 0.04 kg. The blank control group CK has the lowest biomass and dry weight of the middle leaves, which are 0.14 kg and 0.02 g, respectively, which are 52.1% and 62.9%, 46.7% and 54.1% lower than those of the T3 and T4 groups, respectively. There were significant differences in the total biomass and dry weight of tobacco leaves between the T3 and T4 groups and the CK group. The average total biomass and dry weight of each tobacco leaf were the largest in T3, which were 0.54kg and 0.088kg; T4 was smaller than the T3 group, with fresh and dry weights of 0.48kg and 0.075kg respectively; the CK group was the smallest, with fresh and dry weights of 0.29kg and 0.038kg respectively, which were 47.0% and 57.6%, 36.4% and 47.2% lower than the T3 and T4 groups respectively.

[0112] In Examples 3 and 4, the plant height, stem diameter, leaf area and biomass of tobacco in the T1 and T2 groups were significantly higher than those in the CK group, demonstrating that the screened L5N1 and S2-1 had strong growth-promoting and drought-resistance abilities, and had a significant improvement effect on the agricultural traits of tobacco; the characterization of the osmotic stress resistance of L5N1 and S2-1 in Example 2 and the wilting photos of tobacco leaves after 21 days of water deprivation in Example 3 showed that the screened L5N1 and S2-1 had drought resistance.

[0113] Examples 2, 3, and 4 demonstrate that the addition of L5N1 and / or S2-1 has a certain growth-promoting effect on tobacco and increases tobacco leaf yield. On the other hand, L5N1 and / or S2-1 can effectively improve tobacco survival rate during field application, thereby promoting yield increase.

[0114] Example 5: Potted Plant Application of L5N1 and S2-1 Compounded with Organic Fertilizer

[0115] The method for obtaining L5N1 and S2-1 is shown in Example 1; L5N1 and S2-1 are mixed in a ratio of 1:1 and added to the organic fertilizer in a mass ratio of 1:9, that is, the total mass ratio of L5N1 and S2-1 to the bio-organic fertilizer is 1:9, and fermented at room temperature for 5 days, turning the mixture every 24 hours. After fermentation, the moisture content is kept below 30%, and the number of viable bacteria is >2×10 7 cfu / g, and obtain biological organic fertilizer.

[0116] The obtained organic fertilizer was used in a potted plant test. Each pot (21cm×19cm×17cm) was loaded with 3.5kg of soil. Before transplanting tobacco seedlings, 50g of bio-organic fertilizer was added to the tobacco pot in the experimental group, while no bio-organic fertilizer was added to the control group (CK). All treatment groups were subjected to a 15-day continuous water shortage treatment. The results showed that the stem diameter of tobacco in the bio-organic fertilizer group was 75.76% higher than that in the blank control group; the leaf area increased by 25.56% ( Figure 5 ).

[0117] Example 6 Field application of L5N1 and S2-1 compounded with organic fertilizer

[0118] L5N1 and S2-1 were obtained as described in Example 1. L5N1 and S2-1 were mixed at a ratio of 2:3, and added to the organic fertilizer at a total mass to organic fertilizer mass ratio of 1:99. The mixture was fermented at room temperature for 7 days, and stirred every 24 hours. After fermentation, the moisture content was kept below 30%, and the number of viable bacteria was >2×10 7 cfu / g, and obtain bio-organic fertilizer. The prepared bio-organic fertilizer was subjected to field test. After 50g of bio-organic fertilizer was placed in the tobacco nest, tobacco seedlings were transplanted. The control group (CK) did not add bio-organic fertilizer, and other conditions remained unchanged. The growth of tobacco plants under different treatments was observed. The results showed that the total yield of tobacco leaves in the bio-organic fertilizer group increased by 19.32% compared with the blank control group, and the yield of high-quality tobacco leaves increased by 17.78% ( Figure 6 The application of bio-organic fertilizer increased the reducing sugar and potassium ion contents of tobacco leaves by 82.25% and 238.03% respectively.

[0119] The above research results show that L5N1, S2-1 combined with organic fertilizer and applied in the field can significantly promote tobacco growth, increase the biomass of tobacco plants, increase the yield of upper tobacco leaves, and effectively improve tobacco quality.

[0120] The present invention has been described in detail above with reference to preferred embodiments and exemplary examples. However, it should be noted that these specific embodiments are merely illustrative explanations of the present invention and do not constitute any limitation on the scope of protection of the present invention. Various improvements, equivalent substitutions, or modifications may be made to the technical content of the present invention and its embodiments without departing from the spirit and scope of protection of the present invention, and all of these fall within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the appended claims.

Claims

1. An isolated Kluyvera intermedia L5N1, deposited in the China Center for Type Culture Collection, with the number of the China Center for Type Culture Collection being CCTCC NO. M2022531.

2. Microorganisms for promoting drought resistance and drought tolerance, containing Kluyvera intermedia L5N1.

3. An isolated Pseudomonas lini S2-1, deposited in the China Center for Type Culture Collection, with the number of the China Center for Type Culture Collection being CCTCC NO.M 2022530.

4. Microorganisms for promoting drought resistance and tolerance, containing Pseudomonas linen S2-1.

5. A microbial composition for promoting drought resistance and drought tolerance, comprising: Kluyvera L5N1 and Pseudomonas linens S2-1.

6. Use of Kluyvera intermedia L5N1 and biological compositions comprising the same, or Pseudomonas linen S2-1 and microbial compositions comprising the same, or microbial compositions comprising Kluyvera intermedia L5N1 and Pseudomonas linen S2-1 in improving tobacco growth-promoting drought resistance and drought tolerance.

7. The use according to claim 4, wherein the growth-promoting drought resistance comprises an increase in tobacco plant height, stem thickness, leaf area, and biomass, and the drought tolerance comprises an improvement in resistance to osmotic stress.

8. A method for growing tobacco, comprising: Applying microorganisms selected from the following to the tobacco seedlings: Kluyvera intermedia L5N1 or a microbial composition comprising the same; Pseudomonas linensis S2-1 or a microbial composition comprising the same; A microbial composition comprising Kluyvera intermedia L5N1 and Pseudomonas linens S2-1.

9. The method according to claim 7, wherein Before use, Kluyvera spp. L5N1 was expanded to 1×10 7～9 CFU / mL of bacterial solution; expand the culture of Pseudomonas linen S2-1 to 1×10 8～10 CFU / mL of bacterial solution.

10. Bio-organic fertilizer, characterized in that, include: The Kluyvera intermedia L5N1 described in claim 1 and the Pseudomonas linen S2-1 described in claim 3, and organic fertilizer; the mass ratio of the Kluyvera intermedia L5N1 and Pseudomonas linen S2-1 is 1-10:1-10; the total mass of the Kluyvera intermedia L5N1 and Pseudomonas linen S2-1 is prepared with organic fertilizer at a mass ratio of 1:9-99 and then naturally fermented for 5-7 days.

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