CN114668015B - Application of oxadiazine cinnamamide compound in preventing and treating plant diseases - Google Patents

Abstract

The invention belongs to the technical field of plant disease prevention and control, and specifically relates to the application of oxadiazine cinnamamide compounds in the prevention and control of plant diseases. The present invention proves through research and experiments that (E)-N-[(Z)-3-methyl-4-nitroimine-1,3,5-oxadiazin-1-yl]cinnamic amide compounds can On the basis of not affecting the normal growth and reproduction of bacteria, it inhibits the virulence factors of bacteria from invading plants and achieves a significant effect in preventing and treating plant diseases. At the same time, it is not easy for bacteria to develop drug resistance and can be used for a long time. Further, the combined use of oxadiazine cinnamamide compounds and Ralstonia picketii F20 has a synergistic effect and can improve the effect of preventing and treating plant diseases on the basis of separate use.

Description

Application of oxadiazine cinnamamide compounds in preventing and controlling plant diseases

Technical field

The invention belongs to the technical field of plant disease prevention and control. More specifically, it relates to the application of oxadiazine cinnamamide compounds in preventing and treating plant diseases.

Background technique

Rice bacterial blight is an important bacterial disease on rice caused by Xanthomonas oryzae pv. oryzae (Xoo), which has a serious impact on rice yield and grain quality. Black rot of cruciferous crops is an important bacterial disease on cruciferous vegetables caused by Xanthomonas campestris pv. campestris (Xcc). The diseases caused by the above-mentioned strains infecting plants will seriously affect the growth and reproduction of plants. Therefore, the development of effective plant disease prevention and control drugs is one of the key issues that urgently need to be solved.

For plant diseases caused by bacteria, the traditional prevention and control method is to use antibiotics that target key factors for bacterial survival. This is basically the same idea when it comes to the development of new drugs. For example, the Chinese patent application discloses the application of 2-acetamido-1,5,-anhydro-2-deoxy-D-glucitol in the preparation of active drugs against rice bacterial blight, which has a significant effect on the growth of rice bacterial blight. The inhibitory effect of psoralen; Chinese patent application CN110178849A discloses the use of psoralen in the prevention and treatment of plant bacterial diseases. Experiments have proved that psoralen can effectively inhibit bacterial blight of rice and black rot of cruciferous vegetables. growth. However, targeting the key factors for bacterial survival to inhibit bacterial growth and reproduction can easily lead to the development of bacterial drug resistance due to the selection effect of "survival of the fittest" in nature. Drug resistance is becoming increasingly serious, and commonly used antibiotics and other drugs cannot be effective. Newly developed drugs will soon cause bacteria to produce new drug-resistant genes, making them unable to be used for a long time, eventually leading to the emergence of bacterial drug resistance becoming more common and serious.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the defects and shortcomings of existing commonly used antibiotics and the development of new drugs that easily cause bacterial resistance, and to provide an application of oxadiazine cinnamamide compounds in the prevention and treatment of plant diseases.

The object of the present invention is to provide a composition for preventing and treating plant diseases.

Another object of the present invention is to provide the application of the composition in preventing and controlling plant diseases.

The above objects of the present invention are achieved through the following technical solutions:

The research of the present invention has found that the bacterial type III secretion system (T3SS) is a key virulence factor in Gram-negative pathogenic bacteria. The lack of its function has no obvious impact on the growth of bacteria. It is highly homologous and homologous in Xanthomonas pathogenic bacteria. Conserved, that is, T3SS is a key and conserved virulence factor in plant diseases caused by Gram-negative pathogenic bacteria and can be used as a target for the design of new drug molecules. Harpin protein is a secreted toxic protein in the T3SS of rice bacterial blight. It can assist effector proteins to enter host cells and cause disease. It can also induce allergic reactions in non-host plant tobacco. The gene encoding harpin protein is hpa1, which is located downstream of the T3SS regulatory pathway. The promoter region contains a complete PIP-box, which can be regulated by HrpX and can also be induced to express in hrp induction medium. These are common characteristics of T3SS cascade regulation of downstream gene expression. The inventor found that (E)-N-[(Z)-3-methyl-4-nitroimine-1,3,5-oxadiazine- 1-yl]cinnamamide compounds can inhibit the expression of hpa1, thereby reducing bacterial virulence factors. Inhibiting the expression of the XopN gene has a similar effect.

Therefore, the present invention claims the use of oxadiazine cinnamic amide compounds in preventing and treating plant diseases. The structural formula of the oxadiazine cinnamic amide compounds is as shown in formula (III):

Wherein, R is hydrogen, C _1-5 alkyl group, C _1-5 alkoxy group, halogen or nitro group.

Preferably, the R is hydrogen, C _1-3 alkyl, C _1-3 alkoxy, halogen or nitro.

Preferably, R is hydrogen, methyl, methoxy, halogen or nitro.

More preferably, the R is hydrogen, 2-chloro, 3-chloro, 4-chloro, 2-bromo, 3-bromo, 4-bromo, 2-fluoro, 3-fluoro, 4-fluoro, 2-methyl , 3-methyl, 4-methyl, 2-methoxy, 3-methoxy, 4-methoxy, 2-nitro, 3-nitro or 4-nitro.

Further, the plant disease is caused by infection by Gram-negative pathogenic bacteria.

Preferably, the plant diseases are rice bacterial blight and cruciferous black rot.

More preferably, the plant disease is rice bacterial blight or cruciferous black rot.

In addition, the present invention also claims a composition for preventing and treating plant diseases, which includes an effective amount of oxadiazine cinnamamide compounds and Ralstonia pickettii F20;

The structural formula of the oxadiazine cinnamamide compound is shown in formula (III):

Among them, the definition of R is consistent with the above;

The Ralstonia pickettii F20 was deposited in the Guangdong Provincial Microbial Culture Collection Center on April 3, 2018, and the deposit number is GDMCC No: 60347.

Further, the concentration and bacterial count ratio of the oxadiazine cinnamamide compound and Ralstonia pitioni F20 is 100 μM: (20×10 ⁷ ) to (25×10 ⁷ ) CFU/mL. The inventor found through experimental research that the combined use of the oxadiazine cinnamic acid amide compound of the present application with significant T3SS inhibitory effect and Ralstonia peetoni F20 with the ability to degrade DSF or DSF signal analogues can be used from multiple sources. The mechanism direction is to prevent and treat plant diseases, and the combined use of the composition shows a significant synergistic effect, the disease prevention and treatment effect is significantly improved, and a better disease resistance effect is achieved under lower concentration conditions.

Therefore, the present invention also claims the use of the composition in preventing and controlling plant diseases.

Further, the plant disease is caused by infection by Gram-negative pathogenic bacteria. Preferably, the plant diseases are rice bacterial blight and cruciferous black rot.

The invention has the following beneficial effects:

The present invention proves through research and experiments that (E)-N-[(Z)-3-methyl-4-nitroimine-1,3,5-oxadiazin-1-yl]cinnamic acid amide compounds can On the basis of not affecting the normal growth and reproduction of bacteria, it inhibits the virulence factors of bacteria from invading plants and achieves a significant effect in preventing and treating plant diseases. At the same time, it is not easy for bacteria to develop drug resistance and can be used for a long time. Further, the combined use of oxadiazine cinnamamide compounds and Ralstonia picketii F20 has a synergistic effect and can improve the effect of preventing and treating plant diseases on the basis of separate use.

Description of drawings

Figure 1 is the growth curve of Xoo in Example 3 in M210 medium (a, c, e) and XOM2 medium (b, d, f) added with the compound to be tested.

Figure 2 is the growth curve of Xcc 8004 in Example 3 in rich medium XZM (a) and induction medium NYG (b) added with the compound to be tested.

Figure 3 shows compounds III-1, III-2, III-4, III-5, III-7, III-8, III-9, III-10, III-12, III-13, III- in Example 4. 15. Effect diagram of III-16 and III-19 on HR of Xoo on tobacco.

Figure 4 shows compounds III-7, III-8, III-10, III-12, III-13, III-14, III-15, III-16, III-17, III-18, III- Effect diagram of 19 pairs of Xcc on HR in tobacco.

Figure 5 shows compounds III-1, III-2, III-4, III-5, III-7, III-8, III-9, III-10, III-12, III-13, III- in Example 5 15. Picture of water-soaked lesions produced by Xoo strains treated with III-16 and III-19 on rice seedlings.

Figure 6 shows compounds III-1, III-2, III-4, III-5, III-7, III-8, III-9, III-10, III-12, III-13, III- in Example 5. 15. Physical pictures and statistics of the length of lesions produced by Xoo strains treated with III-16 and III-19 on adult rice leaves.

Figure 7 shows compounds III-7, III-8, III-10, III-12, III-13, III-14, III-15, III-16, III-17, III-18, III- Effect picture of the Xcc 8004 strain producing black rot disease on radish after 19 treatments.

Figure 8 is a diagram showing the effect of compound III-12 combined with biocontrol bacteria F20 strain on the black rot disease produced by Xcc 8004 strain on radish in Example 7.

Figure 9 is a physical picture of the length of lesions produced by the Xoo strain on adult rice leaves after compound treatment with compound III-19 and biocontrol bacteria F20 in Example 7, as well as statistics on the length of lesions.

Detailed ways

The invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

Table 1 Physical and chemical properties of compounds used in the examples

All compounds in Table 1 were prepared with reference to Chinese patent application CN101774979A.

Pathogenic strains tested: Xoo PXO99 ^A wild-type strain and the corresponding mutant strain hpa1 in Xoo (pPROBE-AT-hpa1 plasmid inserted into Xoo bacteria); Xcc 8004 wild-type strain, and mutant strain Xcc-xopN (pMS402-xopN plasmid Insert Xcc 8004 bacteria).

The compounds to be tested that inhibit the T3SS of Xanthomonas oryzae oryzae and Xanthomonas campestris were dissolved in DMSO and prepared into a test solution with a concentration of 100 μM.

Unless otherwise stated, the reagents and materials used in the following examples were all commercially available.

Example 1 Test of activity of compounds III-1 to III-19 on Xoo hpa1 gene promoter

Streak and activate the mutant strain (hpa1 in Xoo) on the PSA plate. After 2 to 3 days, pick a single colony and place it in M210 liquid culture medium. Add ampicillin and grow until the OD ₆₀₀ is about 2.0. Transfer to In fresh M210 medium, grow until the OD ₆₀₀ is about 0.6. Collect the cells by centrifugation. Wash the cells once with XOM2 medium and resuspend them in XOM2 medium. Add ampicillin and adjust the OD ₆₀₀ to 0.3. Add the final concentration: 100 μM of the compound to be tested, an equal volume of DMSO as a solvent control, and treated at 28°C and 200 r/min for 16 h. Each treatment was repeated three times. The cells were collected by centrifugation and resuspended in PBS buffer for flow cytometry detection. GFP mean fluorescence intensity (MFI). The specific algorithm is: DMSO% = 100 × MFI (compound to be detected) / MFI (DMSO), inhibition rate % = 100% - DMSO%. See Table 2 for the experimental results.

Table 2 Inhibitory effects of compounds on Xoo hpa1 gene promoter

Note: Since compounds III-6, III-11 and III-18 are insoluble in XOM2 medium, activity testing and further research cannot be carried out.

As can be seen from Table 2, most compounds have strong inhibitory effects on hpa1 promoter activity. Except for compounds III-14 (inhibition rate 66.64%) and III-17 (inhibition rate 86.38%), the remaining 14 compounds have strong inhibitory effects on hpa1 promoter activity. The inhibition rates of gene promoters exceeded 90%.

Example 2 Inhibitory effects of compounds III-1 to III-19 on Xcc xopN promoter

Streak and activate the Xcc-xopN strain on the NYG plate. After 2 to 3 days, pick a single colony and place it in the NYG liquid culture medium. Add rifampicin and grow until the OD ₆₀₀ is about 1.0. Transfer it to fresh culture medium at a ratio of 1:100. In NYG medium, grow to an OD ₆₀₀ of about 0.6, collect the cells by centrifugation, wash the cells once with XZM medium, and then resuspend them in XZM medium, adjust the OD ₆₀₀ to 0.05, and then add the compound to be tested to its final concentration. was 100 μM, and an equal volume of DMSO was used as a solvent control. After treatment for 16 hours at 28°C and 200 r/min, a microplate reader was used to detect the luminescence intensity of the luciferase. Each treatment was repeated three times. The specific algorithm for the inhibition rate is: inhibition rate % = 100% - Lux activity (compound/DMSO). See Table 3 for the experimental results.

Table 3 Inhibitory effects of compounds on Xcc-xopN gene promoter

compound Lux activity/OD ₆₀₀ Inhibition rate% DMSO 37015.46±453.62 / Ⅲ-1 14779.30±485.12 58.73 Ⅲ-2 9907.09±417.37 73.32 Ⅲ-3 16573.91±130.13 55.43 Ⅲ-4 10677.82±378.39 70.33 Ⅲ-5 11132.12±597.02 68.60 III-6 23669.54±931.69 37.04 Ⅲ-7 7033.71±163.70 80.25 Ⅲ-8 6877.86±262.00 80.99 Ⅲ-9 10380.25±378.18 72.34 Ⅲ-10 7047.30±122.75 78.87 III-11 11517.55±227.98 65.77 Ⅲ-12 9373.13±246.85 73.88 Ⅲ-13 7213.30±23.62 76.25 Ⅲ-14 9315.44±15.70 73.05 Ⅲ-15 8340.15±462.34 78.95 Ⅲ-16 6033.08±268.59 82.63 Ⅲ-17 8565.45±51.20 74.86 III-18 8475.61±45.79 76.13 Ⅲ-19 8898.15±167.56 75.56

As can be seen from Table 3, 11 compounds (III-7, III-8, III-10, III-12, III-13, III-14, III-15, III-16, III-17, III-18, III-19) has a strong inhibitory effect on xopN promoter activity.

Example 3 Effect of highly active compounds on the growth of Xoo and Xcc

The growth curves of Xoo in the presence of the compound to be tested in the rich medium M210 and T3SS induction medium XOM2, and the growth curves of Xcc 8004 in the presence of the compound to be tested in the rich medium NYG and induction medium XZM were measured respectively.

1. Effect of highly active compounds on the growth of Xoo

Since XOM2 is a poor medium in which Xoo hardly grows, 0.5% sucrose was supplemented as a carbon source. Streak and activate the Xoo wild-type strain on the PSA plate. After 2 to 3 days, pick a single colony and place it in M210 liquid culture medium. Grow it until the OD ₆₀₀ is about 2.0. Collect the cells by centrifugation. Wash the cells once with sterile water and then repeat them. Suspended in M210 or XOM2, adjust OD ₆₀₀ to 0.1. Add small molecule compounds with a final concentration of 100 μM, and an equal volume of DMSO as a solvent control. After mixing by pipetting, add the mixture to the growth curve plate, and add 200 μL to each well. Four parallel replicate groups were set up for each treatment, and the experiment was repeated three or more times independently. Use a growth curve meter to measure the growth curve of Xoo, and read the data every 1 hour for a total of 60 hours. The experiment was repeated three times independently. 14 highly active compounds (Ⅲ-1, Ⅲ-2, Ⅲ-3, Ⅲ-4, Ⅲ-5, Ⅲ-7, Ⅲ-8, Ⅲ-9, Ⅲ-10, Ⅲ-12, Ⅲ-13, The results of the effects of III-15, III-16 and III-19) on the growth of Xoo in culture medium M210 and XOM2 are shown in Figure 1.

As can be seen from the figure, compared with the DMSO control, compound III-3 inhibits the growth of Xoo in poor medium, while the other 13 compounds (III-1, III-2, III-4, III-5, III-7, Ⅲ-8, Ⅲ-9, Ⅲ-10, Ⅲ-12, Ⅲ-13, Ⅲ-15, Ⅲ-16 and Ⅲ-19) did not show obvious antibacterial effects in each stage of Xoo growth.

2. Effect of highly active compounds on the growth of Xcc

Streak and activate Xcc 8004 on the NYG plate. After 2 to 3 days, pick a single colony and place it in NYG liquid culture medium. Add rifampicin and grow until the OD ₆₀₀ is about 2.0. Collect the cells by centrifugation and wash the cells once with sterile water. , then resuspend in NYG or XZM, and adjust OD ₆₀₀ to 0.1. 100 μM of the compound to be tested was added respectively, and an equal volume of DMSO was used as a solvent control. Three replicates were set for each treatment. Set the temperature to 28°C and measure with a fully automatic growth curve meter. Data were read every 2 hours for a total of 48 hours, and the experiment was repeated three times independently. 11 highly active compounds (III-7, III-8, III-10, III-12, III-13, III-14, III-15, III-16, III-17, III-18, III-19) The results of the effects on the growth of Xcc 8004 in NYG medium and XZM medium are shown in Figure 2.

As can be seen from the figure, compared with the DMSO control, in the slow phase, logarithmic phase and stable phase of the growth of Xcc 8004, compared with the solvent control (DMSO) and the wild-type strain (None), none of the 11 highly active compounds showed any Significantly inhibits the growth of Xcc8004.

Example 4 Effect of highly active compounds on allergic response (HR) of Xoo or Xcc on tobacco

1. Effect of highly active compounds on Xoo’s allergic reaction (HR) on tobacco

Since compound III-3 has an obvious inhibitory effect on the growth of the wild-type strain of Xoo, this example only contains 13 compounds (III-1, III-2, III-4, III-5, Ⅲ-7, Ⅲ-8, Ⅲ-9, Ⅲ-10, Ⅲ-12, Ⅲ-13, Ⅲ-15, Ⅲ-16 and Ⅲ-19) for further research.

The Xoo strain was cultured in M210 culture medium overnight, centrifuged to collect the cells, resuspended in sterile water, and adjusted OD ₆₀₀ to 0.8. The compounds to be tested were added to a final concentration of 100 μM, and an equal volume of DMSO was used as a control, 28 ℃ conditions for 2 hours. Then use a needle-free syringe to inoculate the treated Xoo strain onto Nicotiana benthamiana that has been cultured for two months. After the tobacco is inoculated, it is continued to be cultured in the greenhouse, and the phenomenon is photographed after 24 hours. Compounds III-1, III-2, III-4, III-5, III-7, III-8, III-9, III-10, III-12, III-13, III-15, III-16 and III The results of the effect of -19 on HR of Xoo on tobacco are shown in Figure 3.

It can be seen from the figure that compared with the DMSO control, the 13 compounds have a significant inhibitory effect on HR. Therefore, further research will be conducted on the above 13 compounds. Among them, CK represents the blank control, WT represents the wild-type strain inoculation, and WT+DMSO represents the solvent control.

2. Effect of highly active compounds on allergic reaction (HR) of Xcc 8004 in tobacco

The Xcc 8004 strain is cultured in NYG medium until the OD ₆₀₀ is about 1.0 as the seed liquid. Transfer it to fresh NYG medium at a ratio of 1:100 until the OD ₆₀₀ is about 0.5. Centrifuge and collect the cells, and resuspend them in sterilized water. Twice, adjust OD ₆₀₀ = 0.2, then add the selected compounds, mix thoroughly, and after incubation at 28°C for 2 hours, inject 5 μL into the tobacco leaves with a syringe with the needle removed, and observe the phenomenon of HR reaction after 1 to 2 days. 11 highly active compounds (III-7, III-8, III-10, III-12, III-13, III-14, III-15, III-16, III-17, III-18, III-19) The effect of Xcc 8004 on allergic reaction (HR) in tobacco is shown in Figure 4.

It can be seen from the figure that compared with the solvent control and wild-type strains, 11 compounds have a significant inhibitory effect on the HR of Xcc 8004 on the non-host plant tobacco. Among them, CK represents the blank control, None represents the wild-type strain inoculation, and DMSO represents the solvent control.

Example 5 13 highly active compounds (Ⅲ-1, Ⅲ-2, Ⅲ-4, Ⅲ-5, Ⅲ-7, Ⅲ-8, Ⅲ-9, Ⅲ-10, Ⅲ-12, Ⅲ-13, Ⅲ -15, III-16 and III-19) on the pathogenicity of Xoo strains on rice

Rice varieties: The susceptible variety is IR24. The Xoo strain can produce water-soaked lesions on susceptible rice leaves.

The Xoo strain was cultured in M210 medium until the OD ₆₀₀ was approximately 2.0. The cells were collected by centrifugation, resuspended in sterile water, and adjusted to OD ₆₀₀ to 0.8. The compounds to be tested were added to a final concentration of 100 μM in equal volumes. DMSO was used as the solvent control group and treated at 28°C for 2 hours.

Seedling inoculation: Use a needleless syringe to inoculate the treated Xoo strain onto the susceptible rice variety IR24 that has been cultured for two weeks. Inoculate one sample into the middle of each flag leaf, and inoculate 10 leaves per sample. The control group is inoculated with the same amount. of DMSO and inoculated with equal amounts of untreated Xoo strain. After inoculation, continue to be cultured in the greenhouse, and take photos after 72 hours to observe the occurrence of water-soaked lesions on the leaf seedlings.

The experimental results are shown in Figure 5. It can be seen from the figure that compounds III-1, III-2, III-4, III-5, III-7, III-8, III-9, III-10, III-12, The water-soaked lesions produced by Xoo strains treated with III-13, III-15, III-16 and III-19 on rice seedlings were reduced to varying degrees. Among them, WT represents wild-type strain inoculation, and WT+DMSO represents solvent control.

Adult plant inoculation: Use the leaf cutting method to inoculate adult rice plants, and count the length of the lesions 14 days after inoculation. The blank control group was untreated Xoo strain.

The experimental results are shown in Figure 6. It can be seen from the figure that compared with the solvent control DMSO, except for compounds III-8 and III-9, the other 11 compounds (III-1, III-2, III-4, III-5, The length of lesions produced by Xoo strains treated with Ⅲ-7, Ⅲ-10, Ⅲ-12, Ⅲ-13, Ⅲ-15, Ⅲ-16, Ⅲ-19) has varying degrees of reduction, indicating that the compounds Reduced pathogenicity of Xoo strains. Among them, WT represents wild-type strain inoculation, and WT+DMSO represents solvent control.

Example 6 11 highly active compounds (III-7, III-8, III-10, III-12, III-13, III-14, III-15, III-16, III-17, III-18, III -19) Effect on the pathogenicity of Xcc 8004 on radish

Xcc 8004 strain is cultured in NYG medium until OD ₆₀₀ is about 1.0 as seed liquid. Transfer it to fresh NYG medium at a ratio of 1:100 until OD ₆₀₀ = about 0.5. Centrifuge and collect the cells, and resuspend both in sterilized water. times, adjust the OD ₆₀₀ of the bacterial solution = 0.2, then add the selected compounds, mix thoroughly, and incubate at 28°C for 2 hours. Rinse the fleshy root of radish twice with distilled water, wipe its surface clean with 75% alcohol, cut into radish slices with a thickness of about 0.5cm, and place them in a sterilized petri dish, one piece of radish in a petri dish. Take 100 μL of the incubated bacterial solution, apply it evenly on the radish slices with a coating stick, dry it slightly, seal the petri dish, place it in a 28°C incubator for culture, and observe and count the lesions after 10 to 15 days. Each sample was inoculated with 10 pieces of radish, and the experiment was repeated three times independently.

The results of the pathogenicity of 11 compounds against Xcc 8004 on radish are shown in Figure 7. It can be seen from the figure that compared with the solvent control and wild-type strains, the 11 compounds have different degrees of pathogenicity against the lesions produced by Xcc 8004 on radish. reduction, indicating that the compound has a significant inhibitory effect on the pathogenicity of Xanthomonas campestris. Among them, None represents the wild-type strain inoculation, and DMSO represents the solvent control.

Example 7 Effects of biocontrol bacteria F20 compounded with compound III-12 and compound III-19 on the pathogenicity of Xcc 8004 on radish and Xoo strain on rice

From the results of the above examples, it can be seen that compound III-12 has a more obvious control effect on cruciferous black rot, and compound III-19 has a more obvious control effect on rice bacterial blight, so III-12 and III- were selected respectively. 19 was compounded with the population quenching biocontrol strain F20 to explore the application of compounding cinnamic acid oxadiazine derivatives with biocontrol bacteria.

Biocontrol bacteria: Ralstonia pickettii F20 (derived from Chinese patent application CN108611294A, deposited in the Guangdong Microbial Culture Collection Center on April 3, 2018, with the preservation number GDMCC No: 60347), It can efficiently degrade Xanthomonas quorum sensing DSF signaling molecules.

Cultivation of biocontrol bacteria F20 in LB medium until OD ₆₀₀ is about 1.0 is used as seed liquid, and transferred to fresh LB medium at a ratio of 1:100; Xcc 8004 strain is cultured in NYG medium until OD ₆₀₀ is about 1.0 as seed. liquid, transfer to fresh NYG medium at a ratio of 1:100; when the OD ₆₀₀ of F20 or Xcc 8004 = about 0.5, centrifuge and collect the cells, and resuspend twice in sterilized water. Mix 1 mL of F20 with OD ₆₀₀ = 0.5 and Xcc 8004 in equal volumes, then add III-12 to a concentration of 100 μM, and mix thoroughly. For the reduced-volume compounded sample, mix 0.5 mL of F20 and 1 mL of Xcc, add 0.5 mL of sterile water, then add III-12 to a concentration of 50 μM, and mix thoroughly.

The effect of compound III-12 and biocontrol bacteria F20 on the pathogenicity of Xcc8004 on radish is shown in Figure 8. It can be seen from the figure that the mixture of III-12 and F20 with half the concentration can significantly prevent and control the pathogenicity of Xcc8004 on radish. black rot, and the control effect is similar to twice that of single III-12 or F20, indicating that the combination of compound III-12 and biocontrol bacteria F20 can reduce the dosage of compounds and has the application of multi-target synergy in the prevention and treatment of cruciferous black rot. prospect. Among them, None represents the wild-type strain inoculation, and DMSO represents the solvent control.

Xoo strain Xoo wild-type strain was grown overnight in M210 rich medium. The next day, add the seed liquid to M210 fresh medium at a ratio of 1:100, continue to culture until the OD ₆₀₀ is about 0.6, centrifuge to collect the cells, add biocontrol bacteria liquid to it, and centrifuge again to collect the cells. , rinse the cells with sterile water twice, resuspend them in 1 mL of sterilized water, add the corresponding compounds respectively, with a final concentration of 100 μM, and an equal volume of DMSO as a control, and incubate in a 28°C incubator for 2 hours. Use the leaf-cutting method to treat adult rice plant leaves. Inoculate 10-15 leaves in each treatment group, and observe and record the length of rice lesions half a month later.

The experimental results are shown in Figure 9. It can be seen from the figure that the use of biocontrol bacteria F20 alone can produce good control effects on rice bacterial blight. However, compared with the use of biocontrol bacteria F20 alone or the use of compound III-19 alone, the compound The combination of III-19 and biocontrol bacteria can more effectively inhibit the toxicity of Xoo on adult rice plants. Among them, WT represents wild-type strain inoculation, and WT+DMSO represents solvent control.

In summary, the above examples all demonstrate that the compounds of the present invention significantly reduce the pathogenicity of Xanthomonas oryzae oryzae and Xanthomonas campestris without affecting the growth of pathogenic bacteria. It can prevent and/or treat plant diseases caused by pathogenic bacteria without affecting the growth of pathogenic bacteria, thereby avoiding the development of drug resistance of pathogenic bacteria and extending the effective use period of the compound.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not limit the protection scope of the present invention. For those of ordinary skill in the art, based on the above descriptions and ideas, they can also make There are other variations or modifications in different forms, and it is not necessary and impossible to exhaustively enumerate all implementations here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the claims of the present invention.

Claims (7)

1. The application of oxadiazine cinnamic acid amide compounds in preventing and treating plant diseases, which is characterized in that the structural formula of the oxadiazine cinnamic acid amide compound is as shown in formula (III): Wherein, R is hydrogen, C _1~5 alkyl, C _1~5 alkoxy, halogen or nitro; The plant diseases are rice bacterial blight and cruciferous black rot. 2. Application according to claim 1, characterized in that, the R is hydrogen, C _1~3 alkyl, C _1~3 alkoxy, halogen or nitro. 3. The application according to claim 2, characterized in that R is hydrogen, methyl, methoxy, halogen or nitro. 4. Application according to claim 2, characterized in that, the R is hydrogen, 2-chloro, 3-chloro, 4-chloro, 2-bromo, 3-bromo, 4-bromo, 2-fluoro, 3- Fluorine, 4-fluoro, 2-methyl, 3-methyl, 4-methyl, 2-methoxy, 3-methoxy, 4-methoxy, 2-nitro, 3-nitro or 4 -Nitro. 5. A composition for preventing and treating plant diseases, characterized by comprising an effective amount of oxadiazine cinnamamide compounds and Ralstonia pickettii F20; The structural formula of the oxadiazine cinnamamide compound is shown in formula (III): Wherein, the definition of R is consistent with any one of claims 1 to 4; The Ralstonia pickettii F20 was deposited in the Guangdong Provincial Microbial Culture Collection Center on April 3, 2018, and the deposit number is GDMCC No: 60347. 6. The composition according to claim 5, characterized in that the concentration bacteria ratio of the oxadiazine cinnamamide compound and Ralstonia piti F20 is 100 μM: (20 ~ 25) × 10 ⁷ CFU /mL. 7. Application of the composition of claim 5 or 6 in preventing and treating plant diseases, characterized in that the plant diseases are rice bacterial blight and cruciferous black rot.