Application of hinokitiol in preventing and / or controlling bacterial diseases

Abstract

The present invention belongs to the technical field of plant disease control, and specifically relates to an application in preventing and / or controlling plant bacterial diseases. Through screening, the present invention discovers that hinokitiol has an inhibitory effect on Ralstonia solanacearum LS2020, Pseudomonas syringae pv. tomato DC3000, and Xanthomonas campestris pv. vesicatoria 85-10, thereby effectively preventing and controlling plant bacterial wilt and leaf spot diseases. The present invention discloses the lethal concentration of hinokitiol for inhibiting Ralstonia solanacearum and the pathogen of tomato leaf spot disease, providing a new approach for the control of plant bacterial diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant disease control, and particularly relates to the application of hinokitiol in preventing and / or controlling plant bacterial diseases. Background Art

[0002] Ralstonia solanacearum is a devastating plant pathogenic bacterium worldwide, with a wide host range. It can infect more than 400 plant species in more than 50 families, causing bacterial wilt. The occurrence and damage of this disease are particularly serious in important solanaceous crops in China, such as tomatoes, peppers, eggplants, potatoes, and tobacco, etc., which have caused serious impacts on agricultural production. Ralstonia solanacearum survives in the soil throughout the year, invades through the roots of plants, and finally colonizes in the vascular bundles of plants, causing the whole plant to wilt. Due to the complexity of root infection and vascular bundle colonization, the field control of bacterial wilt is extremely challenging. According to its academic and economic importance, Ralstonia solanacearum is considered the second largest plant pathogenic bacterium in the world and has become an important factor restricting the sustainable development of related industries.

[0003] The onset of bacterial wilt is due to the reproduction of its pathogen in the plant vascular bundles, resulting in the blockage of ducts, the obstruction of plant water transportation, and thus the plant shows wilting symptoms. The main symptoms of bacterial wilt are that the plants quickly wilt and die, and the stems and leaves still remain green. When the brown part of the diseased stem is squeezed by hand, a milky white bacterial liquid will be discharged. Under high temperature and high humidity conditions, this disease is extremely likely to occur. Bacterial wilt has a fast onset, is severe, and is difficult to control, which has attracted wide attention. Therefore, it is very necessary to effectively control bacterial wilt.

[0004] Currently, there are mainly three methods for controlling bacterial wilt: physical control, chemical control, and biological control.

[0005] Currently, the methods for physically controlling Ralstonia solanacearum are as follows: In 2013, Ding Wei et al. proposed in patent CN103190222A to add a modifier to tobacco planting fields. Through soil improvement, the soil nutrients are balanced to control tobacco bacterial wilt. In addition, common physical controls in agricultural production also include timely disinfecting the infected soil and replacing the bed soil, and formulating a reasonable crop rotation plan. There are also ways such as grafting cultivation and burning diseased plants for control. However, such control methods are single, the control effect is average, they only treat the symptoms but not the root cause, and they are easy to cause environmental pollution. Usually, other methods need to be used for synergistic control.

[0006] Methods for controlling bacterial wilt with chemical agents include: In 2000, an article published by Qin Xindao et al. in "Tropical Agricultural Sciences" showed that spraying 50% Qingkuling wettable powder 6 times in the fields of dryland eggplant with bacterial wilt had a control effect of up to about 73% against bacterial wilt, and the residual effect period was as long as 5 months; In 2004, an article published by Kong Yufan et al. in "Chinese Tobacco Science" showed that using 20% Qingkuling wettable powder for controlling tobacco bacterial wilt had a control effect of 62.1%-81.5%, and the effect was significantly better than that of 72% streptomycin sulfate wettable powder. Although the chemical control has good effects, the resulting chemical pollution causes greater damage to the environment.

[0007] In recent years, methods for controlling bacterial wilt with biological agents include: In 2020, Yang Liang et al. proposed that hydroxycoumarin compounds can inhibit the growth of Ralstonia solanacearum by regulating the synthesis of lipopolysaccharide in Ralstonia solanacearum and destroying the cell membrane structure of Ralstonia solanacearum; In 2016, Li et al. found that protocatechuic aldehyde can reduce the motility of Ralstonia solanacearum, inhibit the formation of biofilm of Ralstonia solanacearum, destroy the cell membrane structure, and show strong antibacterial activity against Ralstonia solanacearum. The control effect of root irrigation treatment on tobacco bacterial wilt was 92.01%; In 2013, Kiirika et al. used the combined use of chitosan and silicon to control tomato bacterial wilt; In 2011, Li et al. found that Paenibacillus polymyxa and Paenibacillus badius are antagonistic bacteria of Ralstonia solanacearum in tomato, which can inhibit the formation of biofilm of Ralstonia solanacearum; In 2008, an article published by Jiang Ning et al. in "Plant Protection" showed that the extract of Magnolia officinalis Rehd. var. biloba (Rehd.) Law can effectively inhibit Ralstonia solanacearum in potato. Although the use of the above biological agents can effectively inhibit bacterial wilt, their dosages are all large and the control costs are high. In summary, the main means of green prevention and control of Ralstonia solanacearum in the future will be economically effective biological control.

[0008] Bacterial spot diseases of tomatoes, including bacterial speck and bacterial scab of tomatoes, are collectively referred to as bacterial spot in tomato production, generally causing a 20%-43% reduction in production. Pseudomonas syringae pv. tomato (Pst) Pto DC3000 is a saprophyte in the soil that can invade through plant wounds or hydathodes, causing bacterial leaf spot on tomato leaves. Pto DC3000 of Pseudomonas syringae mainly infects plant leaves, petioles, flowers and fruits, with the leaf margins and immature fruits being most significantly affected. After infection with this pathogen, plant growth is delayed, and black lesions appear in the vascular bundles of the stems and leaves, seriously affecting the yield and quality of tomatoes. Xanthomonas campestris pv. Vesicatoria (Xcv) 85-10 is a Gram-negative bacterium that can overwinter on seeds and weeds in the soil, survive for more than 9 months and become an infection source in the following year. This pathogen can also be spread through media such as rainwater, wind, farmyard manure and insects, causing bacterial scab of tomatoes and peppers again. Xcv 85-10 of Xanthomonas mainly harms the leaves, stems and fruits of peppers and tomatoes, especially commonly occurring on the leaves, causing irregular or round yellow lesions on the plant leaves, namely bacterial spot. The invention patent CN201210105218.4 discloses the use of 2-bromo-2-nitro-1,3-propanediol to control bacterial scab of tomatoes; the invention patent CN202111524823.0 discloses the use of boron-tolerant lysine bacillus bacteriocin analogues to control bacterial speck of tomatoes.

[0009] Green prevention and control is an extension of integrated control, which refers to using integrated pest and disease management and green pest management to select pest and disease control materials with the least risk. And green pest management has higher requirements for pest and disease control materials than integrated control, requiring the use of organic materials (plants) or materials of natural origin.

[0010] Guaiacol, CAS No. 499-44-5, also known as 2-hydroxy-4-isopropyl-2,4,6-cycloheptatriene-1-one, is a natural compound of monoterpenoids with a phenolic ketone skeleton, belonging to the tropolone family of compounds. It has good antibacterial properties, moisturizing properties and pest repellent effects. It is a plant component with high safety, has relatively extensive biological activities, and has a fragrant smell and good effects. Generally, guaiacol is applied to the control of fungal diseases, and there is no disclosure that guaiacol can be used to control plant bacterial diseases. Summary of the Invention

[0011] In view of the above problems, the present invention discloses that hinokitiol can prevent and control bacterial diseases of plants and the lethal concentration of hinokitiol for inhibiting Ralstonia solanacearum. This method can effectively inhibit Ralstonia solanacearum with a relatively low concentration of hinokitiol, which is an effective method for preventing and controlling bacterial wilt, and has no pollution to the environment. In addition, hinokitiol is also used to inhibit or kill Pseudomonas syringae pathogens and Xanthomonas campestris.

[0012] The technical solution of the present invention is as follows:

[0013] Hinokitiol is applied in the prevention and / or control of bacterial diseases of plants.

[0014] Hinokitiol is applied in the preparation of drugs for preventing and / or controlling bacterial diseases of plants.

[0015] Preferably, the plant bacterial pathogen is one or more of the pathogens of bacterial wilt, Pseudomonas syringae pathogens, and Xanthomonas campestris.

[0016] Preferably, the plant is tomato or tobacco.

[0017] Preferably, when the plant bacterial pathogen is the pathogen of bacterial wilt LS2020, the concentration of hinokitiol is 30 μM - 700 μM.

[0018] Preferably, when the plant bacterial pathogen is Pseudomonas syringae pathogen Pto DC3000, the concentration of hinokitiol is 200 μM - 300 μM.

[0019] Preferably, when the plant bacterial pathogen is Xanthomonas campestris Xcv 85-10, the concentration of hinokitiol is 200 μM - 300 μM.

[0020] The beneficial effects of the present invention:

[0021] Through screening, the present invention finds that hinokitiol has an inhibitory effect on Ralstonia solanacearum LS2020, Pseudomonas syringae Pto DC3000, and Xanthomonas campestris Xcv 85-10, thereby effectively preventing and controlling bacterial wilt and leaf spot of plants. The present invention discloses the lethal concentration of hinokitiol for inhibiting Ralstonia solanacearum and the pathogen of tomato leaf spot, providing a new way for the prevention and control of plant bacterial diseases. Description of the Drawings

[0022] Figure 1 It is a result diagram of the lowest concentration range of the inhibitory effect of hinokitiol on Ralstonia solanacearum LS2020.

[0023] Figure 2 It is a result diagram of the lowest concentration range of the bactericidal effect of hinokitiol on Ralstonia solanacearum LS2020.

[0024] Figure 3Results graph of the lowest concentration range of hinokitiol against Pseudomonas syringae pv. tomato DC3000

[0025] Figure 4 Results graph of the lowest concentration range of hinokitiol against Xanthomonas campestris pv. vesicatoria 85-10

[0026] Figure 5 Results graph of the inhibitory effect of hinokitiol on the occurrence of bacterial wilt in soil

[0027] Figure 6 Results graph of the test for inhibiting the proliferation of Ralstonia solanacearum on Nicotiana benthamiana leaves by hinokitiol Detailed implementation manners

[0028] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] Preparation method of hinokitiol solution, comprising the following steps:

[0030] Weigh a small amount of hinokitiol, dissolve it in dimethyl sulfoxide (DMSO) solution to prepare a stock solution, and its dosage and concentration can be diluted with water as needed.

[0031] The bacteriostatic effect of hinokitiol is determined by spectrophotometry.

[0032] Spectrophotometry: Use a microplate reader to measure the absorbance of the bacterial solution added with different concentrations of hinokitiol, and accurately determine its inhibitory effect on Ralstonia solanacearum. The calculation formula for the inhibition rate is formula (I):

[0033] Inhibition rate = ((OD 阴性空白对照 - OD 桧木醇 ) / OD 阴性空白对照 ) × 100% (I)

[0034] Example 1

[0035] In this example, spectrophotometry was used to detect the inhibitory effect of hinokitiol at the lowest concentration range on Ralstonia solanacearum LS2020.

[0036] The Ralstonia solanacearum LS2020 was cultured with shaking at 150 r / min and 28 °C for 24 h in B liquid medium (10 g of tryptone, 1.0 g of yeast extract, 1.0 g of casein hydrolyzate, made up to 1000 mL with ddH2O, sterilized at 121 °C for 20 min) to obtain a bacterial suspension. Guaiacol was dissolved in dimethyl sulfoxide to prepare a 10 mM solution, and the influence of miscellaneous bacteria was removed by filtration through a 0.22 μm microporous filter membrane. 3 mL of B liquid medium was added to each test tube, and different concentrations of guaiacol (30 μM, 40 μM, 50 μM) and dimethyl sulfoxide were set as negative controls. The test tubes were inoculated with Ralstonia solanacearum LS2020, with 3 replicates, and cultured with shaking at 150 r / min and 28 °C overnight. Finally, 200 μL of the bacterial suspension in the test tube was added to a 96-well plate, and the absorbance was measured at 600 nm with a spectrophotometer. The antibacterial rate was calculated according to formula (Ⅰ).

[0037] Figure 1 The results showed that the lowest concentration range of guaiacol with inhibitory effect on Ralstonia solanacearum LS2020 was 30 μM–50 μM (0.000005 g / mL - 0.00008 g / mL), and the antibacterial rate was 85%–100%.

[0038] Example 2

[0039] This example detected the bactericidal effect of guaiacol in the lowest concentration range on Ralstonia solanacearum LS2020.

[0040] LS2020 was cultured with shaking at 150 r / min and 28 °C for 24 h in B liquid medium to obtain a bacterial suspension. Guaiacol was dissolved in dimethyl sulfoxide to prepare a 100 mM solution, and the influence of miscellaneous bacteria was removed by filtration through a 0.22 μm microporous filter membrane. 3 mL of Ralstonia solanacearum LS2020 with an OD600 of 0.1 was added to each test tube, and different concentrations of guaiacol (250 μM, 300 μM) and dimethyl sulfoxide were set as negative controls. With 3 replicates, when cultured with shaking at 150 r / min and 28 °C for 1 h, 2 h, 6 h, and 12 h, the bacterial solution was diluted to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 respectively, and 5 μL of each was taken for colony counting.

[0041] Figure 2 The results showed that the lowest concentration range of guaiacol with bactericidal effect on LS2020 was 200 μM–300 μM (0.000032 g / mL - 0.00048 g / mL), and the antibacterial rate was 60%–90% after 12 hours of drug treatment.

[0042] Example 3

[0043] This example detects the bactericidal effect of hinokitiol within the lowest concentration range on Pseudomonas syringae pv. tomato DC3000.

[0044] Pseudomonas syringae pv. tomato DC3000 was cultured with shaking at 150 r / min and 28 °C for 24 h in LB liquid medium (10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, made up to 1000 mL with ddH2O, sterilized at 121 °C for 20 min) to obtain a bacterial suspension. Hinokitiol was dissolved in dimethyl sulfoxide to prepare a 100 mM solution, and the influence of miscellaneous bacteria was removed by filtration through a 0.22 μm microporous membrane. 3 mL of Pseudomonas syringae pv. tomato DC3000 with an OD600 of 0.1 was added to each test tube, and different concentrations of hinokitiol (200 μM, 300 μM) and dimethyl sulfoxide were set as negative controls. Three replicates were made, and when cultured with shaking at 150 r / min and 28 °C for 1 h, 4 h, 10 h, and 20 h, the bacterial liquid was diluted to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 , and 5 μL of each was taken for colony counting.

[0045] Figure 3 The results showed that the lowest concentration range of hinokitiol with bactericidal effect on Pseudomonas syringae pv. tomato DC3000 was 200 μM–300 μM (0.000032 g / mL - 0.00048 g / mL), and the antibacterial rate was 75%–85% after 10 hours of drug treatment.

[0046] Example 4

[0047] This example detects the bactericidal effect of hinokitiol within the lowest concentration range on Xanthomonas campestris pv. vesicatoria 85-10.

[0048] Xanthomonas campestris pv. vesicatoria 85-10 was cultured with shaking at 150 r / min and 28 °C for 24 h in LB liquid medium to obtain a bacterial suspension. Hinokitiol was dissolved in dimethyl sulfoxide to prepare a 100 mM solution, and the influence of miscellaneous bacteria was removed by filtration through a 0.22 μm microporous membrane. 3 mL of Xanthomonas campestris pv. vesicatoria 85-10 with an OD600 of 0.1 was added to each test tube, and different concentrations of hinokitiol (200 μM, 300 μM) and dimethyl sulfoxide were set as negative controls. Three replicates were made, and when cultured with shaking at 150 r / min and 28 °C for 1 h, 6 h, 10 h, and 20 h, the bacterial liquid was diluted to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5, and 5 μL of each was taken for colony counting.

[0049] Figure 4 The results showed that the lowest concentration range of hinokitiol with bactericidal effect on Xanthomonas campestris pv. vesicatoria Xcv 85-10 was 200 μM–300 μM (0.000032 g / mL - 0.00048 g / mL), and the antibacterial rate was 50%–85% after 20 hours of drug treatment.

[0050] Example 5

[0051] This example illustrates the preventive effect test of hinokitiol on the inoculation of Ralstonia solanacearum LS2020 in greenhouse potted plants.

[0052] Ralstonia solanacearum LS2020 was cultured overnight by shaking at 150 r / min and 28 °C in B liquid medium (10 g of tryptone, 1.0 g of yeast extract, 1.0 g of casein hydrolysate, made up to 1000 mL with ddH2O, sterilized at 121 °C for 20 min) to obtain a bacterial solution. Tomato plants about 3 weeks old were irrigated with hinokitiol (700 μM, 5 mL) and Ralstonia solanacearum LS2020 (5 mL) with an OD600 of 0.2. Dimethyl sulfoxide was set as the control group, and 10 replicates were set.

[0053] Four days after irrigation, the tomato plants began to show symptoms of the disease. The first disease investigation was carried out on the 4th day, and the second disease investigation was carried out on the 6th day. The results are as follows Figure 5 . The results showed that after adding hinokitiol with an effective concentration of 700 μM (0.000112 g / mL) to the tomato soil with Ralstonia solanacearum, the incidence of tomato disease was reduced, indicating that hinokitiol has a preventive effect on Ralstonia solanacearum in the soil.

[0054] Example 6

[0055] This example illustrates the test of hinokitiol on inhibiting the proliferation of Ralstonia solanacearum LS2020 on the leaves of Nicotiana benthamiana.

[0056] Ralstonia solanacearum LS2020 was cultured overnight by shaking at 150 r / min and 28 °C in B liquid medium to obtain a bacterial solution. Nicotiana benthamiana leaves about 3 weeks old with good growth conditions were selected, and hinokitiol (700 μM) and Ralstonia solanacearum LS2020 with an OD600 of 0.0001 were injected into the leaves using a disposable sterile syringe. Dimethyl sulfoxide was set as the control group, and 3 replicates were set.

[0057] Figure 6 The results showed that hinokitiol with an effective concentration of 700 μM (0.000112 g / mL) could significantly inhibit the proliferation of Ralstonia solanacearum LS2020 on the leaves of Nicotiana benthamiana.

Claims

1. Use of hinokitiol in preventing and / or controlling plant bacterial diseases, characterized in that, The pathogenic bacteria of the plant bacterial disease are Ralstonia solanacearum LS2020, Pseudomonas syringae pv. tomato DC3000, or Xanthomonas campestris pv. vesicatoria 85-10. When the pathogenic bacteria are Ralstonia solanacearum LS2020, the concentration of hinokitiol is 200 μM – 300 μM. When the pathogenic bacteria are Pseudomonas syringae pv. tomato DC3000, the concentration of hinokitiol is 200 μM – 300 μM. When the pathogenic bacteria are Xanthomonas campestris pv. vesicatoria 85-10, the concentration of hinokitiol is 200 μM – 300 μM.

2. The application according to claim 1, wherein The plant is tomato or tobacco.

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