Application of nerol in prevention and treatment of tomato bacterial wilt

Abstract

The invention discloses an application of nerol in inhibition of Coreopsis solani. The invention further discloses application of nerol in prevention and treatment of tomato bacterial wilt. It is found for the first time that the low-concentration nerol can effectively inhibit tomato bacterial wilt pathogenic bacteria, namely, the korelelia solanacearum, and the method is an effective method for preventing and treating tomato bacterial wilt.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial prevention and control, and particularly to the application of nerol in preventing and controlling tomato bacterial wilt. Background Art

[0002] Bacterial wilt is an important bacterial soil-borne disease caused by Ralstonia solanacearum ( Ralstonia Solanacearum ). It mainly infects plant roots and enters plant cells, grows in the intercellular spaces, then enters the xylem and multiplies massively throughout the plant, causing plant wilting and ultimately leading to plant death. This pathogen can infect more than 250 plant species in more than 50 families, including tomatoes, eggplants, tobacco, potatoes, peppers, etc., and is an important limiting factor restricting global vegetable production. Ralstonia solanacearum is particularly harmful to solanaceous plants and is the most difficult crop bacterial disease to control.

[0003] Currently, there are mainly three methods for preventing and controlling bacterial wilt of solanaceous plants: physical control, chemical control, and biological control.

[0004] By rotating with non-solanaceous plants for 3 - 4 years, the accumulation of pathogens in the soil can be effectively reduced, and the incidence of bacterial wilt can be decreased; adopting high-ridge cultivation can reduce soil humidity and lower the incidence of the disease. Physical control methods mainly reduce the occurrence of diseases by changing environmental conditions and management measures, but their effects are affected by various factors such as climate, soil conditions, and crop growth status. Therefore, the control effects may not be ideal and stable. Physical control measures require continuous management and maintenance, and a large amount of manpower and material resources need to be invested, increasing the management cost of agricultural production.

[0005] Studies have shown that the application of chemical fungicides can effectively control bacterial wilt. For example, 3-indolebutyric acid (algicide), sodium metasilicate, and trichloronitromethane (fumigant) have been widely used to control bacterial wilt. Although the use of chemical reagents has a relatively direct control effect, it has instability and transience, and there are hazards such as chemical residues, easy damage to the soil ecosystem, and reduction of soil fertility, which is not conducive to the long-term development of the facility industry.

[0006] Biological control refers to using beneficial microorganisms and their metabolites to resist crop diseases, which is a safe and sustainable control method and a hot field for current control of bacterial wilt. There are five types of biological control methods for bacterial wilt: studying environmentally friendly and antibacterial plant-derived fungicides, agricultural antibiotics formed by secondary metabolites produced by microbial fermentation, screening non-pathogenic Ralstonia solanacearum, antagonistic bacteria, and specific phage viruses. These five types of biological control methods are of great significance for controlling bacterial wilt.

[0007] Nerol is an acyclic monoterpenoid alcohol. Natural nerol exists in plants such as orange leaves, lemons, roses, and grapefruits. As a safe and edible spice, nerol is widely used in the fields of medicine, cosmetics, food, etc. due to its unique rose and neroli floral scents. Currently, there is no relevant research on the prevention and control of tomato bacterial wilt with nerol. Summary of the Invention

[0008] The purpose of the present invention is to provide the application of nerol in the prevention and control of tomato bacterial wilt in view of the actual problems and needs in production practice.

[0009] The present invention adopts the following technical solutions: The first aspect of the present invention provides the application of nerol in inhibiting Ralstonia solanacearum.

[0010] Furthermore, when applied, the concentration of nerol is 0.001 mmol / L - 1 mmol / L.

[0011] Even further, when applied, the concentration of nerol is 0.01 mmol / L.

[0012] The second aspect of the present invention provides the application of nerol in the prevention and control of tomato bacterial wilt.

[0013] Compared with the prior art, the present invention has the following advantages: The present invention discovers for the first time that low-concentration nerol can effectively inhibit Ralstonia solanacearum, the pathogen of tomato bacterial wilt, and it is an effective method for preventing and controlling tomato bacterial wilt.

[0014] The present invention uses the plate confrontation method (agar punching method and dichotomous plate culture method) and spectrophotometry (microplate reader) to detect the inhibitory effect of nerol on Ralstonia solanacearum. The results show that nerol has an inhibitory effect on Ralstonia solanacearum. When applying nerol in inhibiting Ralstonia solanacearum, the concentration of nerol is 0.001 mmol / L - 1 mmol / L, and the optimal concentration is 0.01 mmol / L.

[0015] The present invention uses pot experiments to detect the effect of nerol in preventing and controlling tomato bacterial wilt. The results show that the incidence rate of tomato plants exposed to the nerol environment is 0%, while the incidence rate of tomato plants not exposed to the nerol environment is 30%. Nerol has a preventive effect on the occurrence of tomato bacterial wilt. Brief Description of the Drawings

[0016] Figure 1 It is a plate diagram of the inhibition zone produced by nerol on Ralstonia solanacearum.

[0017] Figure 2 It is a schematic diagram of the coating test design in the dichotomous plate culture detection.

[0018] Figure 3 Nerol inhibits the growth of Ralstonia solanacearum (the suspension of Ralstonia solanacearum was diluted 10 5 times).

[0019] Figure 4 Nerol inhibits the growth of Ralstonia solanacearum (the suspension of Ralstonia solanacearum was diluted 10 6 times).

[0020] Figure 5 Nerol inhibits the growth of Ralstonia solanacearum (the suspension of Ralstonia solanacearum was diluted 10 7 times).

[0021] Figure 6 It is the effect of nerol on the growth of Pseudomonas syringae pv. tomato.

[0022] Figure 7 It is the effect of nerol on the growth of Bacillus. Specific embodiments

[0023] The present invention will be further described below in conjunction with specific embodiments and the accompanying drawings. It should be understood that these embodiments and the drawings are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the description of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present invention.

[0024] The structure of nerol involved in the following examples is as follows:

[0025] Purchased from Shanghai Macklin Biochemical Co., Ltd., with a purity of 97%.

[0026] The diameter of the plates (culture dishes) involved in the following examples is 90 mm.

[0027] The formula of the NB liquid medium involved in the following examples is as follows (1 L system): Tryptone 5 g, Yeast extract 0.5 g, Anhydrous glucose 10 g, Beef extract 3 g.

[0028] The formula of the NA solid medium involved in the following examples is as follows (1 L system): Tryptone 5 g, Yeast extract 0.5 g, Anhydrous glucose 10 g, Beef extract 3 g, Agar powder 17 g.

[0029] The formula of the R2A liquid medium involved in the following examples is as follows (1 L system): Tryptone 0.5 g, Acid-hydrolyzed casein 0.5 g, Yeast extract powder 0.5 g, Soluble starch 0.5 g, Dipotassium hydrogen phosphate 0.3 g, Magnesium sulfate (MgSO4·7H2O) 0.024 g, Sodium pyruvate 0.3 g, Glucose 0.5 g.

[0030] The formulation of the R2A solid medium involved in the following examples is as follows (1 L system): 0.5 g of tryptone, 0.5 g of acid-hydrolyzed casein, 0.5 g of yeast extract powder, 0.5 g of soluble starch, 0.3 g of dipotassium hydrogen phosphate, 0.024 g of magnesium sulfate (MgSO4·7H2O), 0.3 g of sodium pyruvate, 0.5 g of glucose, and 20 g of agar powder.

[0031] Example 1

[0032] In this example, the inhibitory effect of nerol on Ralstonia solanacearum was detected by the plate confrontation method (agar punching method).

[0033] Preparation of the Ralstonia solanacearum suspension: Ralstonia solanacearum ( R. solanacearum QL-Rs1115, GenBank: GU390462; under Koch's postulates, it was confirmed to have strong pathogenic ability, the same below) was cultured overnight by shaking at 150 r / min and 27 °C in NB liquid medium to obtain a bacterial suspension (OD600 = 0.988).

[0034] Preparation of the nerol solution: Nerol was dissolved in a co-solvent (1 v / v% Tween-80) to prepare a stock solution of 400 μL / mL, and then diluted with sterile water to 4 μL / mL, and filtered and sterilized with a 0.22 μm microporous filter membrane.

[0035] Plate confrontation method (agar punching method): The Ralstonia solanacearum suspension was diluted with sterile water to a concentration of 10 8 CFU / mL. 1 μL of the diluted Ralstonia solanacearum suspension was evenly spread on a plate of NA solid medium (16 mL) by the plate coating method. Three holes (each hole with a diameter of 8 mm, directly through to the bottom) were punched in the NA solid medium plate with a puncher, and 70 μL of the nerol solution was added to each hole. The co-solvent (1 v / v% Tween-80) was diluted with sterile water to 6 μL of co-solvent (1 v / v% Tween-80) / mL, and 70 μL was taken and added to the hole as a blank control.

[0036] The NA solid medium plate was cultured overnight at 30 °C, and it was observed whether an inhibition zone was formed. If an inhibition zone was present, it indicated an inhibitory effect; otherwise, there was no inhibitory effect.

[0037] The results were as Figure 1 shown. The inhibition zone of nerol on Ralstonia solanacearum was 0.2 - 0.5 cm, indicating that it had an inhibitory effect on Ralstonia solanacearum.

[0038] Example 2

[0039] In this example, the inhibition effect of nerol on Ralstonia solanacearum was detected by the plate confrontation method (two-part Petri dish method) (as Figure 2 shown). The height inside the two-part Petri dish is 1.4 cm, and the height of the middle partition is 0.8 cm.

[0040] Preparation of Ralstonia solanacearum suspension: Ralstonia solanacearum ( R. solanacearum QL-Rs1115, GenBank: GU390462) was cultured overnight by shaking in NB liquid medium at 150 r / min and 27 °C to obtain a bacterial suspension (OD600 = 1.061).

[0041] Preparation of nerol solution: Nerol was dissolved in a cosolvent (1 v / v% Tween-80) to prepare a stock solution of 400 μL / mL, and then diluted with sterile water to 4 μL / mL. Filtration sterilization was performed using a 0.22 μm microporous membrane.

[0042] Plate confrontation method (two-part Petri dish method): The left half of the two-part Petri dish was NA solid medium (8 mL). The Ralstonia solanacearum suspension was diluted 10 5 , 10 6 and 10 7 times. 1 μL of the diluted Ralstonia solanacearum suspension was evenly spread on the NA solid medium in the left half of the two-part Petri dish. A sterile filter paper was placed in the right half of the two-part Petri dish, and 2 μL, 5 μL, 10 μL, and 20 μL of nerol solution were respectively dropped on the sterile filter paper. A control group (CK) was set: The diluted Ralstonia solanacearum suspension was spread on the NA solid medium in the left half of the two-part Petri dish, but no sterile filter paper was placed in the right half, and no nerol solution was dropped.

[0043] The two-part Petri dish was cultured at 30 °C for 24 h, and the number of colonies on the NA solid medium was observed. Each treatment had 3 replicates.

[0044] The formula for the inhibition rate is as follows:

[0045] The results are shown by Figures 3 - 5 Nerol has an inhibitory effect on the growth of Ralstonia solanacearum.

[0046] Comparative Example 1 In this comparative example, the inhibition effect of nerol on Pseudomonas sp. Pseudoxanthomonas _2 and Bacillus sp. Bacillus T-5 was detected by the plate confrontation method (two-part Petri dish method) (Pseudomonas sp. Pseudoxanthomonas _2 and Bacillus sp. BacillusT-5 from Bio-organic soil amendment promotes the suppression of Ralstonia solanacearum by inducing changes in the functionality and composition of rhizosphere bacterial communities (Deng Xuhui, Zhang Na, Li Yuchan, Zhu Chengzhi, Qu Baoyuan, Liu Hongjun, Li Rong, Bai Yang, Shen Qirong, Salles Joana Falcao. NEW PHYTOLOGIST, 2022, 235(4): 1558-1574). The two-compartment Petri dish is 1.4 cm high, and the middle partition is 0.8 cm high.

[0047] Preparation of Pseudoxanthomonas suspension: Pseudoxanthomonas Pseudoxanthomonas _2 was cultured overnight in R2A liquid medium at 150 r / min and 30 °C with shaking to obtain a bacterial suspension (OD600 = 0.542).

[0048] Preparation of Bacillus suspension: Bacillus Bacillus T-5 was cultured overnight in NB liquid medium at 150 r / min and 30 °C with shaking to obtain a bacterial suspension (OD600 = 0.771).

[0049] Preparation of nerol solution: Nerol was dissolved in a cosolvent (1 v / v% Tween-80) to prepare a stock solution of 400 μL / mL, and then diluted with sterile water to 4 μL / mL. The solution was filtered and sterilized through a 0.22 μm microporous membrane.

[0050] Dual culture method (two-compartment Petri dish method): The left half of the two-compartment Petri dish was filled with 8 mL of R2A solid medium. The Pseudoxanthomonas suspension was diluted 10 5 , 10 6 and 10 7 times with sterile water. 1 μL of the diluted Pseudoxanthomonas suspension was evenly spread on the R2A solid medium in the left half of the two-compartment Petri dish. A sterile filter paper was placed in the right half of the two-compartment Petri dish, and 2 μL and 5 μL of nerol solution were respectively dropped on the sterile filter paper. A control group (CK) was set up: The diluted Pseudoxanthomonas suspension was spread on the R2A solid medium in the left half of the two-compartment Petri dish, but no sterile filter paper was placed in the right half, and no nerol solution was dropped.

[0051] The dichotomous petri dishes were cultured at 30 °C for 24 h, and the number of colonies on the R2A solid medium was observed. There were 3 replicates for each treatment.

[0052] The left half of the dichotomous petri dish was NA solid medium (8 mL). The Bacillus spore suspension was diluted 10 5 、10 6 and 10 7 times. 1 μL of the diluted Bacillus spore suspension was evenly spread on the NA solid medium in the left half of the dichotomous petri dish. A sterile filter paper was placed in the right half of the dichotomous petri dish, and 2 μL and 5 μL of nerol solution were respectively dropped on the sterile filter paper. A control group (CK) was set: the diluted Bacillus spore suspension was spread on the NA solid medium in the left half of the dichotomous petri dish, but no sterile filter paper was placed in the right half, and no nerol solution was dropped.

[0053] The dichotomous petri dishes were cultured at 30 °C for 24 h, and the number of colonies on the NA solid medium was observed. There were 3 replicates for each treatment.

[0054] The formula for the inhibition rate is as follows:

[0055] The results showed that Figure 6 and 7 nerol not only had no inhibitory effect on Pseudomonas syringae and Bacillus sp., but instead had a certain promoting effect on both Pseudomonas syringae and Bacillus sp., among which the promoting effect on Bacillus sp. was significant.

[0056] Example 3

[0057] In this example, the inhibitory effect of nerol on Ralstonia solanacearum was detected by spectrophotometry (microplate reader), and the specific method was as follows: Preparation of Ralstonia solanacearum suspension: Ralstonia solanacearum ( R. solanacearum QL-Rs1115, GenBank: GU390462) was cultured overnight in NB liquid medium at 150 r / min and 27 °C to obtain a bacterial suspension (OD600 = 0.787).

[0058] Preparation of nerol solution: Nerol was dissolved in a co-solvent (1 v / v% Tween-80) to prepare a stock solution of 100 mmol / L, and then diluted with sterile water to solutions with concentrations of 10, 1, 0.1, and 0.01 mmol / L. The solutions were filtered and sterilized with a 0.22 μm microporous filter membrane.

[0059] Add 170 μL of NB liquid medium, 10 μL of a fluorescently labeled Ralstonia solanacearum bacterial suspension, and 20 μL of nerolidol solutions (at concentrations of 10, 1, 0.1, and 0.01 mmol / L) to a 96-well plate. After adding to the 96-well plate, the final concentrations of nerolidol in the nerolidol solutions are 1, 0.1, 0.01, and 0.001 mmol / L, respectively. In the control group wells, add 170 μL of NB liquid medium, 10 μL of a fluorescently labeled Ralstonia solanacearum bacterial suspension, and 20 μL of 1 v / v% Tween-80 at concentrations of 10 μL / mL, 1 μL / mL, 0.1 μL / mL, and 0.01 μL / mL (dilute the co-solvent (1 v / v% Tween-80) with sterile water to 10 μL co-solvent (1 v / v% Tween-80) / mL, 1 μL co-solvent (1 v / v% Tween-80) / mL, 0.1 μL co-solvent (1 v / v% Tween-80) / mL, 0.01 μL co-solvent (1 v / v% Tween-80) / mL). Each treatment has 3 replicates.

[0060] Incubate the 96-well plate at 30 °C for 24 h, and read the values using a microplate reader with excitation at 587 nm and emission at 610 nm.

[0061] The formula for the bacteriostatic rate is as follows:

[0062] Among them, OD 对照 is the absorbance measured for the control, and OD 各处理 is the absorbance measured for each treatment.

[0063]

[0064] The results are shown in Table 1. Nerolidol at different concentrations has an inhibitory effect on Ralstonia solanacearum. The bacteriostatic rate of 1 mmol / L nerolidol against Ralstonia solanacearum is 38.03%, the bacteriostatic rate of 0.1 mmol / L nerolidol against Ralstonia solanacearum is 39.83%, the bacteriostatic rate of 0.01 mmol / L nerolidol against Ralstonia solanacearum is 45.33%, the bacteriostatic rate of 0.001 mmol / L nerolidol against Ralstonia solanacearum is 40.9%. The bacteriostatic effect is optimal when the concentration of nerolidol is 0.01 mmol / L.

[0065] Example 4

[0066] This example is a test of the bacteriostatic effect (against Ralstonia solanacearum) of nerolidol on tomatoes.

[0067] Prepare 24 small plastic square pots with dimensions of 7.5 cm × 7.5 cm × 9 cm. Fill each pot with 200 g of soil (the tested soil was collected from a newly reclaimed Chinese cabbage vegetable garden in Libao Town, Haian County, Nantong City, Jiangsu Province, and chicken manure organic fertilizer has been applied to the tested soil all year round) for standby. The tested tomato variety is micro tom. First, soak the tomato seeds in a 3.5 v / v% sodium hypochlorite solution (prepared with sodium hypochlorite stock solution, with available chlorine (calculated as Cl - ) of 5.0 - 10.0% and free alkali (calculated as NaOH) of 0.1 - 1.0%) for 10 min. Subsequently, rinse three times with sterilized distilled water. Spread the treated tomato seeds on filter paper moistened with sterile water and place them in an incubator for germination. The germination temperature is 30 °C, the humidity is maintained at about 60%, there is no light (dark), and no watering is required. After the tomato seeds germinate, transplant the tomato seedlings into a seedling tray equipped with a seedling substrate based on imported peat and coconut coir. The seedling tray is 54 cm long, 28 cm wide, with a hole depth of 4 cm, a hole diameter of 3.8 cm, and a bottom of 2.2 cm. Each hole is filled with 50 g of seedling substrate, and 1 tomato seedling is transplanted into each hole. When the tomato seedlings grow to three true leaves, transplant the tomato seedlings with similar growth conditions into the small plastic square pots already filled with 200 g of soil. One tomato seedling is planted in each small plastic square pot. This experiment was conducted in the greenhouse facilities of Nanjing Agricultural University. During the experiment, the greenhouse temperature was controlled at 25 ± 1 °C, the relative humidity was 50 ± 5%, the light cycle was 16 h of light and 8 h of darkness. Tap water was used for water replenishment, and the soil moisture content in the small plastic square pots was maintained between 65% - 70% by watering. The influence of environmental heterogeneity on the experimental results was eliminated by regularly and randomly adjusting the positions of the potted plants, and the subsequent experiments were under the same conditions.

[0068] Five days after transplantation, place 4 small plastic square pots (7.5 cm × 7.5 cm × 9 cm) into an acrylic transparent cover with dimensions of 30 cm × 20 cm × 40 cm. At the same time, place a 50 mL triangular flask containing 1 mL of nerol (purity 97%, filtered and sterilized with a 0.22 μm microporous membrane) (seal the triangular flask with a tissue culture bottle sealing film to slow down its volatilization rate, and the air permeability of the sealing film is 85%) into the acrylic transparent cover. In the acrylic transparent cover of the control group, place 4 small plastic square pots without placing the triangular flask containing nerol. There are 12 replicates for each treatment (3 acrylic transparent covers for each treatment, and 4 small plastic square pots in each acrylic transparent cover).

[0069] Preparation of Ralstonia solanacearum bacterial suspension: Ralstonia solanacearum ( R. solanacearumQL-Rs1115, GenBank: GU390462) was cultured overnight in NB liquid medium at 150 r / min and 27 °C with shaking to obtain a bacterial suspension (OD600 = 1.321).

[0070] After 3 days of transferring the small plastic square pots into the acrylic transparent cover, first dilute the Ralstonia solanacearum bacterial suspension with sterile water to 10 7 CFU / mL, add 20 mL of the diluted Ralstonia solanacearum bacterial suspension to each pot of tomato seedlings. Pour the Ralstonia solanacearum bacterial suspension onto the roots of the tomato seedlings. Seven days after pouring the Ralstonia solanacearum bacterial suspension on the tomato seedlings, start recording the disease incidence of tomato plants in each treatment until the tomato harvest. Count the tomato plants showing symptoms of bacterial wilt such as green withering of leaves, brown stem cross-sections, and necrosis of vascular tissues as diseased plants. Calculate the incidence of bacterial wilt in tomato plants according to the following method: Incidence of bacterial wilt in tomato plants in each treatment (%) = Number of tomato plants with bacterial wilt in each treatment / Total number of tomato plants in each treatment × 100%

[0071] The results are shown in Table 2. The incidence of tomato plants exposed to nerol was 0%, while the incidence of tomato plants not exposed to nerol was 30%, indicating that nerol has a preventive effect on the incidence of bacterial wilt in tomatoes.

Claims

1. Use of nerol in inhibiting Ralstonia solanacearum.

2. The application according to claim 1, characterized in that When in use, the concentration of nerol is 0.001 mmol / L - 1 mmol / L.

3. The application according to claim 2, characterized in that, When in use, the concentration of nerol is 0.01 mmol / L.

4. Use of nerol in preventing and controlling tomato bacterial wilt.

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