Method for regulating tobacco leaf surface bacterial colony by exogenous hormone and application

Abstract

The invention discloses a method for regulating and controlling tobacco leaf surface bacterial colonies through exogenous hormones and application, and relates to the technical field of tobacco agriculture. Comprising the following steps: 1) preparing a working solution; (2) adding a nonionic surfactant; and 3) field spraying. The invention aims to fill the technical blank of field application of plant hormones through a systematic, normative and scientific method, and provides a reproducible and generalizable solution for quality improvement and disease prevention and control of crops such as tobacco and the like.

Description

Technical Field

[0001] This invention relates to the field of tobacco agricultural technology, and more specifically to a method and application of using exogenous hormones to regulate the microbial community on tobacco leaves. Background Technology

[0002] Tobacco (Nicotiana tabacum L.) is an important economic crop, and its yield and quality directly affect the economic benefits of the tobacco industry chain. To improve the appearance and quality of tobacco leaves and control leaf diseases, plant hormone regulation methods are currently employed, such as cytokinins (e.g., 6-benzyladenine) and jasmonic acids (e.g., methyl jasmonic acid). These methods influence the physiological metabolic processes of tobacco plants through exogenous application, thereby improving quality or enhancing resistance. US2005 / 0034365A1 mentions reducing TSAs in tobacco by spraying substances such as SA, but its disclosure focuses more on SA concentration and spraying time, without providing a unified formulation process. CN108569931A addresses the combination of foliar growth regulators and fertilizers for rice, focusing on specialized spraying devices and formulations. CN113785687A relates to spraying devices for water-soluble fertilizers. These technologies emphasize spraying devices and do not focus on the specific preparation of hormone stock solutions or the regulation of foliar microorganisms. Although several patents and documents (such as CN109090118A "Tobacco Strengthening Sprouting Agent") involve the use of hormones or microorganisms in combination, the current technology still has some shortcomings.

[0003] While there is research on the effects of various plant hormones, few have been applied in large-scale fields, especially in tobacco cultivation. For hormone application techniques that regulate foliar probiotics to enhance disease resistance, few inventions or utility model patents can be found. Most studies and applications have not systematically optimized the dissolution methods, stock solution concentrations, and spraying dosages of hormones such as 6-BA and MeJA, resulting in inaccurate dosages, poor stability, or inconvenience in field application. Most existing technologies do not explicitly address the addition of nonionic surfactants, such as Tween-like agents or silicon carbide surfactants, to plant hormone solutions, thus affecting hormone spread and absorption efficiency on leaves. Particularly in the use of methyl jasmonate (MeJA), current technologies focus primarily on its resistance-inducing effects, lacking in-depth exploration and application strategies regarding how it indirectly combats disease by regulating the structure of plant surface microbial communities (such as probiotic colonization). Many published patents or literature do not provide detailed descriptions of the timing, frequency, concentration, and spraying dosage of hormone treatments, making it difficult to directly guide field practice and limiting practical application. The lack of integrated operational guidelines applicable to different farmers and scales of plant protection has prevented this approach from being widely adopted and promoted.

[0004] While existing technologies have reference value in regulating tobacco quality or disease resistance with plant hormones, there is still a lack of a systematic, repeatable, and field-friendly technical solution for the use of plant hormones plus adjuvants (nonionic surfactants). In particular, there is still significant room for improvement in using hormones to coordinate tobacco quality and enhance resistance (such as through probiotic colonization).

[0005] Therefore, providing a method for regulating foliar microbial communities through exogenous plant hormones to suppress leaf diseases or improve tobacco quality is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a method and application for regulating tobacco leaf microbial colonies using exogenous hormones.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for regulating tobacco leaf microbial community using exogenous hormones includes the following steps:

[0009] 1) Preparation of working solution:

[0010] Dilute the plant hormone stock solution with water to obtain a working solution with a concentration of 10 mg / L to 50 mg / L;

[0011] Adding 1L of 100mg / L stock solution to 9L of water will prepare a 10mg / L (approximately 45μmol / L) working solution; adding 2L of stock solution to 8L of water will produce a 20mg / L working solution.

[0012] The plant hormone stock solution is:

[0013] Any one of the following: 10mM IAA stock solution, 100mg / L 6-BA stock solution, 100mM MeJA stock solution, or 1g / L salicylic acid stock solution;

[0014] 2) Add nonionic surfactants:

[0015] Add a nonionic surfactant to the working solution prepared in step 1), wherein the amount of surfactant added is 0.01%-0.05% (v / v), to obtain a spray solution;

[0016] Adding nonionic surfactants improves the leaf adhesion and absorption efficiency of the plant hormone working solution, enhances leaf wettability, spreadability, and absorption efficiency, while minimizing irritation to plant tissues.

[0017] The nonionic surfactant is:

[0018] Any one of Tween-20, Tween-80, Agral 90, or Silwet L-77;

[0019] 3) Field spraying:

[0020] The spray solution prepared in step 2) is evenly sprayed onto the surface of tobacco leaves by foliar spraying to ensure that both the front and back of the leaves are wetted.

[0021] Furthermore, the preparation of the plant hormone stock solution in step 1) includes the following steps:

[0022] Preparation of the IAA (indoleacetic acid) mother liquor:

[0023] Weigh 1.75g ​​IAA (MW≈175.18), add 5–10mL of 95% ethanol to dissolve, shake thoroughly to mix, and dilute to 1L with deionized water to obtain a 10mM IAA stock solution.

[0024] The prepared IAA stock solution should be stored away from light at 4°C and can be kept for 1–2 weeks.

[0025] (Note: IAA is easily degraded by light and high temperatures; avoid prolonged exposure.)

[0026] Preparation of the 6-BA (6-benzyladenine) mother liquor:

[0027] Weigh 1.0 g of 6-BA powder, add 5 mL of 1 mol / L hydrochloric acid (HCl) to promote dissolution, stir thoroughly, transfer to a 10 L volumetric flask, and dilute to 10 L with distilled water to obtain a 6-BA stock solution with a concentration of 100 mg / L (i.e. 100 ppm).

[0028] The prepared 6-BA stock solution should be refrigerated at 4°C and can be stored for 1 week.

[0029] Preparation of the MeJA (methyl jasmonate) mother liquor:

[0030] Weigh 22.43g of high-purity MeJA stock solution (usually in high-purity liquid form), add 10–20mL of anhydrous ethanol to dissolve it completely, and then make up to 1000mL with anhydrous ethanol to prepare a 100mM MeJA anhydrous ethanol stock solution.

[0031] Store in an airtight, light-proof, and refrigerated container; it can be stored for 1-2 months.

[0032] Preparation of the salicylic acid mother liquor:

[0033] Weigh 1g of salicylic acid (MW = 138.12g / mol), add 5–10mL of 95% ethanol, stir thoroughly to dissolve completely, and then slowly dilute to 1000mL with deionized water to obtain a 1g / L salicylic acid stock solution.

[0034] Salicylic acid mother liquor is relatively stable and can be stored at 4°C away from light for 6 months.

[0035] Furthermore, the spraying described in step 3) should be carried out in the early morning or late evening when there is no wind and the sun is shining brightly, in order to reduce evaporation loss and photolysis.

[0036] Furthermore, when used for disease control, preventative spraying should be carried out at the early stage of disease occurrence, before the peak season for disease outbreaks, or during the stage when plants are susceptible to disease.

[0037] Spray once every 7-15 days, for 2-4 consecutive times, depending on the disease occurrence and the plant's growth stage.

[0038] Furthermore, when used to improve quality, it should be sprayed during the vigorous growth period of tobacco leaves, before maturity, or after topping.

[0039] Spray once every 10-15 days, for 1-2 consecutive times.

[0040] For example, spraying after topping flue-cured tobacco can promote the growth and development of tobacco leaves and the formation of quality.

[0041] Furthermore, in step 3), the spraying should be done according to the size of the plant and the density of the leaves, ensuring that the leaf surface is moist but not dripping, with an application rate of 60-120 liters of spray solution per acre.

[0042] Application of a method for regulating tobacco leaf microbial community using exogenous hormones in the prevention and control of tobacco diseases.

[0043] Application of a method for regulating tobacco leaf microbial community using exogenous hormones in improving tobacco quality.

[0044] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:

[0045] 1. A standardized method for preparing mother liquor and working solution is provided, including selection of co-solvent (such as HCl, NaOH, ethanol, DMSO), setting of mother liquor concentration, addition of surfactant and aseptic treatment process, eliminating the problems of inconsistent formulation and poor experimental repeatability in the prior art.

[0046] 2. Optimize foliar spraying technical parameters, clarify the spraying concentration, spray volume, spraying timing and frequency, and incorporate non-ionic surfactants (such as Tween-20 / -80, Agral 90, Silwet L-77) as auxiliary ingredients to significantly improve the spreadability and absorption efficiency of hormones on the leaf surface, and solve the problems of uneven coverage and low absorption rate of existing sprays.

[0047] 3. Establish a hormone-microbe synergistic regulation mechanism. Taking methyl jasmonate (MeJA) as an example, systematically design an application scheme to induce probiotic colonization and thus enhance plant disease resistance. Currently, there is no patented complete technical system covering MeJA to regulate foliar microbial colonization and implement field spraying.

[0048] 4. Construct an integrated field application solution that covers the entire process from mother liquor preparation, working solution ratio, spraying method, environmental condition control, to probiotic colonization and disease resistance effect evaluation. It has clear operational specifications and practical feasibility for promotion, overcoming the significant defects of existing technologies that lack field operability and have poor promotion.

[0049] This invention aims to fill the technological gap in the field application of plant hormones through a systematic, standardized, and scientific approach, and to provide replicable and scalable solutions for improving the quality and controlling diseases of crops such as tobacco. Attached Figure Description

[0050] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0051] Figure 1 Screening for characteristic microorganisms in the control and treatment groups;

[0052] Figure 2 To illustrate the functional differences in leaf microbial communities between the treatment group (T) and the control group (CK). Detailed Implementation

[0053] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0054] Depending on the type of disease to be controlled or the quality indicators to be improved, one or more suitable plant hormones are selected as active ingredients. The plant hormones may be selected from, but are not limited to:

[0055] Auxins: such as indoleacetic acid (IAA), naphthaleneacetic acid (NAA), 2,4-D, etc. Auxins may indirectly affect the colonization and growth of leaf microorganisms by influencing plant cell elongation, root development, and the composition of plant exudates.

[0056] Cytokinins, such as kinetin, 6-benzyladenine (6-BA), and zeatin, may influence the community dynamics of foliar microorganisms by promoting cell division, delaying senescence, and regulating plant immune responses.

[0057] Jasmonates (JAs): such as methyl jasmonate (MeJA). Jasmonates are important signaling molecules in plant defense responses and may induce plants to produce defensive substances, thereby affecting the balance between pathogens and beneficial bacteria.

[0058] Salicylic acid (SA): Salicylic acid is a key signaling molecule for systemically acquired resistance (SAR) in plants. It may directly or indirectly promote the growth of beneficial microorganisms and enhance the plant's resistance to pathogens.

[0059] Example 1:

[0060] Applying methyl jasmonate (MeJA) promotes the colonization of beneficial bacteria and controls tobacco leaf diseases.

[0061] Jasmonic acid (JA) and its derivatives (such as MeJA) are important lipid signaling molecules in plants, playing a central role in plant defense responses to biotic stresses (such as pathogen infection and insect pests). MeJA can induce plants to produce defensive secondary metabolites and activate the expression of disease-resistant genes. Furthermore, MeJA may indirectly affect the composition and function of the phyllosphere microbial community by altering leaf exudates or the leaf surface microenvironment, thereby promoting the proliferation of beneficial microorganisms and forming a biological barrier against pathogens. To investigate the effects of methyl jasmonic acid (MeJA) on the colonization and disease resistance of beneficial bacteria on tobacco leaves, this experiment used a stock solution dilution method to prepare a MeJA spray solution and applied it through multiple foliar sprays.

[0062] 1. Preparation of methyl jasmonate mother liquor

[0063] First, measure an appropriate amount of MeJA stock solution, add a small amount of anhydrous ethanol to dissolve it (accounting for 1-2% of the final volume, about 1-2 mL), and prepare a 100 mM MeJA ethanol solution as a stock solution. Seal and store it in the dark and refrigerated.

[0064] 2. Preparation of working solutions with different dosages

[0065] Before use, dilute the stock solution to the required working concentrations: 0.1 mM (low dose) and 0.5 mM (high dose) MeJA. Use sterile distilled water for dilution, and add 0.02% (v / v) Tween-20 as a nonionic surfactant to the working solution to improve the spreading and absorption of MeJA on the leaf surface. Prepared working solutions should be used immediately to avoid MeJA evaporation and loss of efficacy due to prolonged storage in water.

[0066] 3. Field spraying

[0067] This experiment was conducted in the Changsha tobacco-growing area, where wildfire disease is prevalent. The first treatment was administered one week after transplanting the tobacco seedlings (when the plants had approximately 5 true leaves), followed by spraying every 7 days for a total of 3 applications. Spraying was carried out in the evening on windless, dry, and sunny days, avoiding strong sunlight, rain, and high temperatures that could affect pesticide absorption. Using a manual fine mist sprayer or a pressurized spray bottle, the MeJA working solution was evenly sprayed onto both sides of the tobacco leaves, with a standard application rate of 10 mL per plant, ensuring the leaves were moist but not dripping. Irrigation or washing of the leaves should be avoided shortly after spraying to allow the hormones and surface microorganisms to fully interact.

[0068] 4. Disease investigation and assessment

[0069] The incidence of wildfire disease in tobacco plants of each treatment group was investigated. The disease severity of each tobacco plant was graded using a scale of 0-9, and the incidence rate and disease index were calculated.

[0070] • Incidence rate (%) = (Number of infected plants / Total number of plants surveyed) × 100%

[0071] Disease index = [Σ(number of diseased leaves at each level × corresponding level value) / (total number of leaves surveyed × highest level value)] × 100

[0072] The results of the morbidity survey are shown in Table 1: the control effect of low concentration of methyl jasmonate was 41.3%, and the control effect of high concentration was 54%.

[0073] Table 1. Results of Wildfire Incidence Survey

[0074]

[0075] 5. Leaf surface microbial community analysis

[0076] Seven days after the last MeJA spray (i.e., before pathogen inoculation), tobacco leaf samples were collected from both the high-concentration MeJA treatment group and the control group. Thirty leaves were randomly collected from each treatment group (from different plants), and the samples were mixed together as one sample. The samples were immediately flash-frozen in liquid nitrogen and stored at -80°C.

[0077] use Total microbial DNA was extracted from leaf surfaces using the Spin Kit for Soil (MP Biomedicals, USA). 16S rRNA gene sequencing: High-throughput sequencing of the V3-V4 region of the bacterial 16S rRNA gene was performed using the Illumina MiSeq platform.

[0078] The primers are:

[0079] 338F: 5'-ACTCCTACGGGAGGCAGCAG-3', as shown in SEQ ID No. 1;

[0080] 806R: 5'-GGACTACHVGGGTWTCTAAT-3', as shown in SEQ ID No. 2.

[0081] Sequencing data underwent quality control, OTU (Operational Taxonomic Unit) clustering, species annotation, alpha diversity analysis (e.g., Shannon index, Chao1 index), and beta diversity analysis (e.g., PCoA, NMDS). The analysis focused on the relative abundance changes of beneficial microorganisms (e.g., Bacillus and Pseudomonas).

[0082] Table 3 shows that leaf microbial diversity increased after application of methyl jasmonate. Figure 1 • By screening the two groups of characteristic microorganisms, it was found that the relative abundance of various beneficial microorganisms, such as Bacillus, Pseudarthrobacter, and Pseudoxanthomonas, was significantly increased in the treatment group, indicating that the applied methyl jasmonate can promote the colonization of beneficial microorganisms on tobacco leaves.

[0083] Table 2. Microbial alpha diversity indices in the treatment with methyl jasmonate and the control group.

[0084]

[0085] Example 2:

[0086] Foliar application of 6-benzyladenine (6-BA) improves the quality of flue-cured tobacco.

[0087] 1.6-BA Mother Liquor Preparation

[0088] First, accurately weigh 1.0 g of 6-BA powder using a precision balance, add about 5 mL of 1 mol / L hydrochloric acid (HCl) to promote its dissolution, stir thoroughly, transfer to a 10 L volumetric flask, and dilute to 10 L with distilled water to obtain a 6-BA stock solution with a concentration of 100 mg / L (i.e., 100 ppm).

[0089] 2. Preparation of working solution

[0090] The spraying concentration used in the experiment was 25 mg / L (approximately 110 μmol / L), which was obtained by mixing 2.5 L of stock solution with 7.5 L of water to obtain 10 L of working solution. 0.01% of a nonionic surfactant (Tween-20) was added to the prepared working solution.

[0091] 3. Field spraying

[0092] This experiment was conducted in the Yunyan 87 tobacco demonstration area in Chenzhou, Hunan Province. Spraying was performed on the day of topping the flue-cured tobacco, choosing a sunny, windless early morning or evening to avoid interference from high temperatures or rainfall. The experiment included a 6-BA treatment group and a control group (sprayed with an equal volume of distilled water). A fine mist sprayer was used to spray the foliage, ensuring approximately 125 mL of solution was applied to each plant, completely wetting the central functional leaves and the entire plant until slight dripping occurred. Each plant was sprayed only once. After spraying, the treatment and control areas were clearly marked for sampling and subsequent data measurement.

[0093] 4. Test Results

[0094] The agronomic traits of tobacco leaves, such as leaf length and width, and fresh leaf weight, were better in the 6-BA treatment group than in the control group. The fresh weight of a single leaf in the control group was 10.85g; the fresh weight of a single leaf in the 6-BA treatment group reached 12.81g, indicating an improvement in leaf traits.

[0095] Table 3 Effects of application of functional microbial agents on tobacco agronomic traits under low temperature stress.

[0096]

[0097] Example 3:

[0098] Verification of the regulatory effects of plant hormones on foliar microbial communities

[0099] To demonstrate the regulatory effect of plant hormones on the microbial community on plant leaves, we performed high-throughput sequencing on tobacco leaves treated with the aforementioned 6-BA (Example 2) and control leaves. Picrust2 function was then predicted from the microbiome data.

[0100] Figure 2Analysis showed that 6-BA treatment significantly reshaped the functional spectrum of leaf microorganisms in the tobacco leaves of the treatment group, specifically by: enhancing microbial metabolic capacity (especially secondary metabolism and signal response); activating signal transduction and transcriptional regulatory pathways related to plant interactions; and enriching secondary metabolic pathways (such as stilbenoid synthesis) and cytochrome P450, which may enhance the microorganisms' ability to inhibit pathogens (such as synthesizing antibacterial substances) or assist plants in degrading their own toxins (such as phenols).

[0101] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for regulating tobacco leaf microbial colonies using exogenous hormones, characterized in that, Includes the following steps: 1) Preparation of working solution: Dilute the plant hormone stock solution with water to obtain a working solution with a concentration of 10 mg / L to 50 mg / L; The plant hormone stock solution is: Any one of the following: 10mM IAA stock solution, 100mg / L 6-BA stock solution, 100mM MeJA stock solution, or 1g / L salicylic acid stock solution; 2) Add nonionic surfactants: Add a nonionic surfactant to the working solution prepared in step 1), wherein the amount of surfactant added is 0.01%-0.05% (v / v), to obtain a spray solution; The nonionic surfactant is: Any one of Tween-20, Tween-80, Agral 90, or Silwet L-77; 3) Field spraying: The spray solution prepared in step 2) is evenly sprayed onto the surface of tobacco leaves by foliar spraying to ensure that both the front and back of the leaves are wetted.

2. The method according to claim 1, characterized in that, Step 1) The preparation of the plant hormone stock solution includes the following steps: Preparation of the IAA (indoleacetic acid) mother liquor: Weigh 1.75g ​​of IAA, add 5-10mL of 95% ethanol to dissolve, shake thoroughly to mix, and dilute to 1L with deionized water to obtain a 10mM IAA stock solution. Preparation of the 6-BA (6-benzyladenine) mother liquor: Weigh 1.0 g of 6-BA powder, add 5 mL of 1 mol / L hydrochloric acid (HCl) to promote dissolution, stir thoroughly, transfer to a 10 L volumetric flask, and dilute to 10 L with distilled water to obtain a 6-BA stock solution with a concentration of 100 mg / L. Preparation of the MeJA (methyl jasmonate) mother liquor: Weigh 22.43g of high-purity MeJA stock solution, add 10-20mL of anhydrous ethanol to dissolve it completely, and then make up to 1000mL with anhydrous ethanol to prepare a 100mM MeJA anhydrous ethanol stock solution. Preparation of the salicylic acid mother liquor: Weigh 1g of salicylic acid, add 5-10mL of 95% ethanol, stir thoroughly to dissolve completely, and then slowly dilute to 1000mL with deionized water to obtain a 1g / L salicylic acid stock solution.

3. The method according to claim 1, characterized in that, Step 3) The spraying should be carried out in the early morning or evening when there is no wind and it is sunny, avoiding high temperature and strong sunlight to reduce evaporation loss and photolysis.

4. The method according to claim 1, characterized in that, When used for disease control, preventative spraying should be carried out at the early stage of disease occurrence, before the peak season for disease, or when plants are susceptible to disease. Spray once every 7-15 days, for 2-4 consecutive times, depending on the disease occurrence and the plant's growth stage.

5. The method according to claim 1, characterized in that, When used to improve quality, spray during the vigorous growth period of tobacco leaves, before maturity, or after topping. Spray once every 10-15 days, for 1-2 consecutive times.

6. The method according to claim 1, characterized in that, Step 3) The spraying should be done according to the size of the plant and the density of the leaves, ensuring that the leaf surface is moist but not dripping. The application rate is 60-120 liters of spray solution per acre.

7. The application of the method for regulating tobacco leaf microbial community by exogenous hormones as described in claim 1 in the prevention and control of tobacco diseases.

8. The application of the method for regulating tobacco leaf microbial community by exogenous hormones as described in claim 1 in improving tobacco quality.

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