CN114668015A - Application of oxadiazine cinnamamide compounds in preventing and treating plant diseases - Google Patents

Abstract

The invention belongs to the technical field of plant disease control, and particularly relates to an application of an oxadiazine cinnamamide compound in plant disease control. The research and experiment prove that the (E) -N- [ (Z) -3-methyl-4-nitroimine-1, 3, 5-oxadiazine-1-yl ] cinnamamide compound can inhibit the invasion of virulence factors of bacteria to plants on the basis of not influencing the normal growth and reproduction of the bacteria, achieves the effect of obviously preventing and treating plant diseases, is not easy to cause the bacteria to generate drug resistance, and can be applied for a long time. Further, the oxadiazine cinnamamide compounds and the Ralstonia pilonii F20 are used in a combined mode, so that the synergistic effect is achieved, and the effect of preventing and treating plant diseases can be improved on the basis of single use.

Description

Application of oxadiazine cinnamamide compounds in preventing and treating plant diseases

Technical Field

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

Background

Bacterial blight of rice is an important bacterial disease in rice caused by fusarium oxysporum f.sp.oryzae pv. oryzae, Xoo, and has a serious influence on both rice yield and grain quality. Cruciferous crop black rot is an important bacterial disease on cruciferous vegetables caused by Xanthomonas campestris pv. campestris, Xcc. Diseases caused by the bacterial strains after infecting plants can seriously affect the growth and the propagation of the plants, so that the research and the development of effective plant disease control medicines are one of the key problems which need to be solved urgently.

For plant diseases caused by bacteria, the traditional control means is to use antibiotics which take key factors of bacterial survival as action targets, and the traditional control means is also the basic idea in the research and development of new drugs. For example, Chinese patent application discloses the application of 2-acetamido-1, 5-anhydro-2-deoxy-D-glucitol in the preparation of active drugs for resisting rice bacterial blight, and the application has obvious inhibition effect on the growth of rice bacterial blight; chinese patent application CN110178849A discloses the use of psoralen in preventing and treating plant bacterial diseases, and experiments prove that psoralen can effectively inhibit the growth of monads such as rice bacterial blight germ and cruciferous vegetable black rot germ. However, the key factor of bacterial survival is taken as an action target to inhibit the growth and reproduction of bacteria, and due to the selection action of 'high-priority and low-priority' in the nature, the drug resistance of bacteria is easy to generate, the drug resistance phenomenon is increasingly serious, common drugs such as antibiotics cannot take effect, newly developed drugs can enable the bacteria to generate new drug resistance genes soon and cannot be used for a long time, and finally the generation of the drug resistance of the bacteria is more and more common and serious.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defect and the defect that the conventional common antibiotics and the research and development of new drugs are easy to cause bacterial drug resistance, and provides the application of the oxadiazine cinnamamide compounds in the prevention and treatment of plant diseases.

The invention aims to provide a composition for controlling plant diseases.

The invention also aims to provide the application of the composition in preventing and treating plant diseases.

The above purpose of the invention is realized by the following technical scheme:

the research of the invention finds that the bacterial III type secretion system (T3SS) is a key virulence factor in gram-negative pathogenic bacteria, the loss of the function of the bacterial III type secretion system has no obvious influence on the growth of the bacteria, and the bacterial III type secretion system is highly homologous and conservative in xanthomonas, namely T3SS is a key and conservative virulence factor in plant diseases caused by the gram-negative pathogenic bacteria and can be used as a target for novel drug molecule design. The Harpin protein is a secreted toxic protein in rice bacterial blight T3SS, can assist effector protein to enter host cells to cause pathogenicity, and can also induce non-host plant tobacco to generate anaphylactic reaction. The gene encoding the harpin protein is hpa1, which is located downstream of the T3SS regulatory pathway, contains the entire PIP-box in the promoter region, is capable of being regulated by HrpX, and is also inducible to be expressed in hrp induction medium. The common characteristics of T3SS cascade regulation and control of downstream gene expression are that (E) -N- [ (Z) -3-methyl-4-nitroimine-1, 3, 5-oxadiazine-1-yl ] cinnamamide compounds can inhibit the expression of hpa1, and further reduce virulence factors of bacteria. Similar effects are obtained by inhibiting the expression of the XopN gene.

Therefore, the invention claims the application of the oxadiazine cinnamamides compound in preventing and treating plant diseases, wherein the structural formula of the oxadiazine cinnamamides compound is shown as the formula (III):

wherein R is hydrogen or C_1～5Alkyl radical, C_1～5Alkoxy, halogen or nitro.

Preferably, R is hydrogen, C_1～3Alkyl radical, C_1～3Alkoxy, halogen or nitro.

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

More preferably, 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 with gram-negative pathogenic bacteria.

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

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

In addition, the present invention also claims a composition for controlling plant diseases, comprising effective amounts of oxadiazine cinnamamides and Ralstonia pickettii (Ralstonia pickettii) F20;

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

wherein R is as defined above;

the Ralstonia pickettii (Ralstonia pickettii) F20 is preserved in Guangdong province collection center for microorganisms in 2018, 4 and 3 days, and the preservation number is GDMCC No: 60347.

further, the concentration bacterial dose ratio of the oxadiazine cinnamamide compound to the Ralstonia pickettii F20 is 100 mu M: (20X 10)⁷)～(25×10⁷) CFU/mL. Through experimental research, the inventor discovers that when the oxadiazine cinnamamide compounds with obvious T3SS inhibiting effect and the Ralstonia pilei F20 with DSF or DSF signal analog degrading effect are used in combination, plant diseases can be controlled from multiple mechanisms, and the composition shows obvious synergistic effect when used in combination, so that the disease control effect is obviously improved, and a good disease resistance effect is achieved under the condition of low concentration.

Therefore, the invention also claims the application of the composition in controlling plant diseases.

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

The invention has the following beneficial effects:

the research and experiment prove that the (E) -N- [ (Z) -3-methyl-4-nitroimine-1, 3, 5-oxadiazine-1-yl ] cinnamamide compound can inhibit the invasion of virulence factors of bacteria to plants on the basis of not influencing the normal growth and reproduction of the bacteria, achieves the effect of obviously preventing and treating plant diseases, is not easy to cause the bacteria to generate drug resistance, and can be applied for a long time. Further, the oxadiazine cinnamamide compounds and the Ralstonia pilonii F20 are used in a combined mode, so that the synergistic effect is achieved, and the effect of preventing and treating plant diseases can be improved on the basis of single use.

Drawings

FIG. 1 is a graph showing the growth curves of Xoo in M210 medium (a, c, e) and XOM2 medium (b, d, f) supplemented with test compound in example 3.

FIG. 2 is a plot of the growth of Xcc8004 of example 3 in both the rich medium XZM (a) and the induction medium NYG (b) supplemented with the test compound.

FIG. 3 is a graph showing the effect of 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 of example 4 on the HR of Xoo on tobacco.

FIG. 4 is a graph showing the effect of the compounds III-7, III-8, III-10, III-12, III-13, III-14, III-15, III-16, III-17, III-18, III-19 of example 4 on HR on tobacco by Xcc.

FIG. 5 is a photograph of water-stained lesions on rice seedlings of the Xoo strain treated with 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 of example 5.

FIG. 6 is a physical map of lesion length and statistics of lesion length on the leaves of adult rice plants produced by Xoo strains treated with 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 of example 5.

FIG. 7 is a graph showing the effect of treatment with compounds III-7, III-8, III-10, III-12, III-13, III-14, III-15, III-16, III-17, III-18, III-19 of example 6 on the development of black rot disease in radish by the Xcc8004 strain.

FIG. 8 is a graph showing the effect of the combination of the compound III-12 of example 7 and the biocontrol bacterium F20 strain on the development of black rot disease by the Xcc8004 strain on radish.

FIG. 9 is a real map of lesion length and statistics of lesion length on adult rice plant leaves of the Xoo strain after the compound III-19 and the biocontrol bacterium F20 in example 7 were combined.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

TABLE 1 physicochemical Properties of the Compounds used in the examples

Table 1 all compounds were prepared with reference to chinese patent application CN 101774979A.

Test pathogen species: xoo PXO99^AWild type strains and corresponding mutant strains hpa1 in Xoo (pPROBE-AT-hpa1 plasmid insert Xoo bacteria); xcc8004 wild-type strain, andmutant strain Xcc-xopN (pMS402-xopN plasmid insert Xcc8004 strain).

The test compound for inhibiting the rice bacterial blight and Xanthomonas campestris T3SS is dissolved in DMSO to prepare a test solution with a concentration of 100 μ M.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

EXAMPLE 1 testing of the Activity of Compounds III-1 to III-19 on the promoter of the Xoo hpa1 Gene

Streaking and activating a mutant strain (hpa1 in Xoo) on a PSA (pressure swing adsorption) plate, picking a single colony to be placed in an M210 liquid culture medium after 2-3 days, adding ampicillin, and growing to OD₆₀₀Approximately 2.0, transferred to fresh M210 medium at a 1:100 ratio, grown to OD₆₀₀About 0.6, the cells were collected by centrifugation, washed once with XOM2 medium and resuspended in XOM2 medium, ampicillin was added thereto, and OD was adjusted₆₀₀When the concentration reaches 0.3, a test compound with a final concentration of 100 mu M and an equal volume of DMSO (dimethyl sulfoxide) as a solvent control are added, the mixture is treated for 16h at the temperature of 28 ℃ and at the speed of 200r/min, each treatment is repeated for 3 times, the thalli are collected by centrifugation and resuspended in PBS (phosphate buffer solution), and the average fluorescence intensity (MFI) of GFP is detected by a flow cytometer. The specific algorithm is as follows: DMSO% > -100 × MFI (compound to be detected)/MFI (DMSO), inhibition% > -100% -DMSO%, see table 2 for experimental results.

TABLE 2 inhibitory Effect of Compounds on the promoter of the Xoo hpa1 Gene

Note: since compounds III-6, III-11 and III-18 were insoluble in XOM2 medium, activity testing and further investigation were not possible.

As can be seen from Table 2, most of the compounds have strong inhibitory effect on hpa1 promoter activity, except for compounds III-14 (inhibition rate of 66.64%) and III-17 (inhibition rate of 86.38%), the inhibition rate of the hpa1 gene promoter by the other 14 compounds is over 90%.

Example 2 inhibition of the Xcc xopN promoter by Compounds III-1 to III-19

Streak-activating Xcc-xopN strain on NYG plate, picking single colony to be placed in NYG liquid culture medium after 2-3 days, adding rifampicin, growing to OD₆₀₀About 1.0, transferred to fresh NYG medium at a ratio of 1:100, and grown to OD₆₀₀About 0.6, the cells were collected by centrifugation, washed once with XZM medium, resuspended in XZM medium, and OD adjusted₆₀₀And when the concentration is 0.05, adding the compound to be detected to the final concentration of 100 mu M, using equal volume DMSO as a solvent control, processing for 16h at 28 ℃ at 200r/min, then detecting the luminescence intensity of the fluorescent enzyme by using an enzyme-labeling instrument, and repeating the processing for 3 times. The specific algorithm of the inhibition rate is as follows: the inhibition rate was 100% -Lux activity (compound/DMSO), and the experimental results are shown in table 3.

TABLE 3 inhibitory Effect of Compounds on the promoter of the Xcc-xopN Gene

Compound (I)	Lux activity/OD600	Inhibition ratio%
			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 of the compounds (III-7, III-8, III-10, III-12, III-13, III-14, III-15, III-16, III-17, III-18, III-19) had strong inhibitory effects on the promoter activity of xopN.

EXAMPLE 3 Effect of highly active Compounds on Xoo and Xcc growth

Growth curves of Xoo in the presence of test compound in rich medium M210 and T3SS induction medium XOM2 and Xcc8004 in the presence of test compound in rich medium NYG and induction medium XZM were determined, respectively.

1. Effect of highly active Compounds on Xoo growth

Since XOM2 is a lean medium in which Xoo hardly grows, 0.5% sucrose was supplemented as a carbon source. Streaking and activating Xoo wild type strains on a PSA (pressure swing adsorption) plate, picking single colonies after 2-3 days, placing the single colonies in an M210 liquid culture medium, and growing to OD₆₀₀About 2.0, the cells were collected by centrifugation, washed once with sterile water, resuspended in M210 or XOM2, and OD adjusted₆₀₀Is 0.1. Respectively adding small molecular compounds with the final concentration of 100 mu M and taking equal volume DMSO as a solvent control, uniformly blowing and mixing, adding the mixed solution into a growth curve plate, and adding 200 mu L of the mixed solution into each hole. Each treatment was set up in 4 parallel replicates and the experiment was independently repeated three more times. The growth curve of Xoo was measured by a growth curve measuring instrument, and data were read every 1h for 60 h. The experiment was independently repeated 3 times. The results of the effect of 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, III-15, III-16 and III-19) on the growth of Xoo in medium M210 and XOM2 are shown in FIG. 1.

As can be seen, compound III-3 inhibited the growth of Xoo in lean medium compared to the DMSO control, 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 exhibit significant inhibitory effects during each phase of Xoo growth.

2. Effect of highly active Compounds on Xcc growth

Mixing Xcc8004 streaking and activating on NYG plate, 2-3 days later, selecting single colony, placing in NYG liquid culture medium, adding rifampicin, and growing to OD₆₀₀About 2.0, the cells were collected by centrifugation, washed once with sterile water, resuspended in NYG or XZM, and OD adjusted₆₀₀Is 0.1. 100 μ M test compound was added separately, and an equal volume of DMSO was used as a solvent control, with 3 replicates per treatment set. Setting the temperature at 28 ℃, and measuring by a full-automatic growth curve instrument. Data were read every 2h for a total of 48h and the experiment was repeated 3 times independently. The results of the effect of 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) on the growth of Xcc8004 in NYG medium and XZM medium are shown in FIG. 2.

As can be seen, 11 highly active compounds did not show significant inhibition of the growth of Xcc8004 compared to the solvent control (DMSO) and the wild type strain (None) in the lag phase, log phase and stationary phase of the growth of Xcc8004 compared to the DMSO control.

Example 4 Effect of highly active Compounds on the allergic reaction (HR) of Xoo or Xcc on tobacco

1. Effect of highly active Compounds on Xoo allergic reaction (HR) on tobacco

Since Compound III-3 has a significant inhibitory effect on the growth of wild-type strains of Xoo, this example was further investigated for only 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) which had no inhibitory activity on Xoo strains.

Xoo strain is cultured in M210 culture medium overnight, thallus is collected by centrifugation, thallus is resuspended in sterile water, and OD is adjusted₆₀₀To 0.8, the test compound was added to a final concentration of 100. mu.M, and treated at 28 ℃ for 2 hours with an equal volume of DMSO as a control. Then, the treated Xoo strain was inoculated to Nicotiana benthamiana cultured for two months by a syringe without a needle, and the Nicotiana benthamiana was continuously cultured in a greenhouse after inoculation, and the phenomenon was observed by photographing 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-19 on Xoo HR on tobaccoThe results of the effect of (c) are shown in fig. 3.

As can be seen, 13 compounds all had significant inhibitory effect on HR compared to the DMSO control, and therefore, further studies were conducted on the 13 compounds. Where CK represents blank control, WT represents inoculation of wild type strain, and WT + DMSO represents solvent control.

2. Effect of highly active Compounds on the allergic reaction (HR) of Xcc8004 on tobacco

Xcc8004 Strain is cultured to OD in NYG Medium₆₀₀About 1.0 as seed liquid, transferring into fresh NYG culture medium to OD according to 1:100 ratio₆₀₀About 0.5, centrifuging, collecting thallus, resuspending twice with sterilized water, and adjusting OD₆₀₀When the amount of the compound was 0.2, the compound was added thereto, mixed well, incubated at 28 ℃ for 2 hours, and then 5. mu.L of the mixture was injected into tobacco leaves using a syringe with a needle removed, and the HR reaction was observed after 1 to 2 days. The effect of 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) on the anaphylactic reaction (HR) of Xcc8004 on tobacco is shown in FIG. 4.

As can be seen, 11 compounds had a significant inhibitory effect on HR on the non-host plant tobacco, Xcc8004, compared to the solvent control and the wild type strain. Wherein CK represents blank control, None represents wild type strain inoculation, and DMSO represents solvent control.

EXAMPLE 513 Effect of 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 variety: the susceptible variety is IR 24. The Xoo strain can generate water stain-like disease spots on infected rice leaves.

Xoo strains were grown to OD in M210 medium₆₀₀About 2.0, the cells were collected by centrifugation, resuspended in sterile water, and OD adjusted₆₀₀To 0.8, the test compound was added to a final concentration of 100. mu.M, and treated with an equal volume of DMSO as a solvent control at 28 ℃ for 2 h.

Seedling inoculation: the treated Xoo strain was inoculated with a needle-free syringe onto a two-week old infected rice variety IR24, one sample was inoculated into the middle of each flag leaf, 10 leaves were inoculated into each sample, and the control group was inoculated with an equal amount of DMSO and an equal amount of untreated Xoo strain. After inoculation, the seedlings are continuously placed in a greenhouse for culture, and after 72 hours, the seedlings of the leaves are photographed to observe the occurrence condition of water stain-like disease spots.

As shown in FIG. 5, it can be seen that the Xoo strains treated with 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 exhibited various degrees of reduction in the formation of water-stained lesions on rice seedlings. Wherein WT represents the inoculation of the wild type strain and WT + DMSO represents the solvent control.

Adult plant inoculation: and (4) adopting a leaf-cutting inoculation method on adult rice, and counting the length of the lesion spots 14 days after inoculation. The blank control was untreated Xoo strain.

The results are shown in FIG. 6, which shows that in addition to compounds III-8 and III-9, the Xoo strains treated with 11 compounds (III-1, III-2, III-4, III-5, III-7, III-10, III-12, III-13, III-15, III-16, III-19) produced lesions of different lengths, indicating that the compounds reduced the pathogenicity of the Xoo strain to different extents, compared to the solvent control DMSO. Wherein WT represents the inoculation of the wild type strain and WT + DMSO represents the solvent control.

Example 611 Effect of 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) on the pathogenicity of Xcc8004 on radish

Xcc8004 Strain is cultured to OD in NYG Medium₆₀₀About 1.0 as seed liquid, transferred into fresh NYG medium at a ratio of 1:100 until OD₆₀₀Centrifuging about 0.5, collecting thallus, resuspending twice with sterilized water, and adjusting bacterial liquid OD₆₀₀The screened compound was added thereto and mixed well, followed by incubation at 28 ℃ for 2 hours. Washing radish fleshy root with distilled water for 2 times, wiping the surface with 75% alcohol, cutting into radish slices with thickness of about 0.5cm, placing in a sterilized culture dish, and placing one radish slice in one culture dish.And (3) uniformly coating 100 mu L of the incubated bacterial liquid on radish slices by using a coating rod, slightly airing, sealing a culture dish, culturing in an incubator at 28 ℃, and observing and counting the lesion spots after 10-15 days. Each sample was inoculated with 10 radish pieces and the experiment was repeated 3 times independently.

The pathogenicity of the 11 compounds on the radish Xcc8004 is shown in FIG. 7, and it can be seen from the figure that the 11 compounds have different degrees of reduction of the lesion spots generated on the radish by the Xcc8004 compared with the solvent control and the wild type strain, which indicates that the compounds have obvious inhibition effect on the pathogenicity of Xanthomonas campestris. Wherein None represents the wild type strain inoculation and DMSO represents the solvent control.

Example 7 Effect of biocontrol bacterium F20 on the pathogenicity of Compound III-12 and Compound III-19 paired Xcc8004 on radish and Xoo strains on Rice

The results of the above examples show that the compound III-12 has a remarkable control effect on cruciferous black rot and the compound III-19 has a remarkable control effect on rice bacterial leaf blight, so that the compound III-12 and the compound III-19 are respectively selected to be compounded with the colony quenching biocontrol strain F20, and the application of compounding the cinnamylate oxadiazine derivative and the biocontrol bacteria is explored.

Biocontrol bacteria: ralstonia pickettii F20 (from China patent application CN108611294A, deposited in Guangdong provincial microorganism culture Collection in 2018, 4 and 3 days, and with the deposit number GDMCC No. 60347) can efficiently degrade Xanthomonas group induced DSF signal molecules.

Culturing biocontrol bacteria F20 in LB culture medium to OD₆₀₀About 1.0 as seed liquid, and transferring into fresh LB culture medium according to the proportion of 1: 100; xcc8004 Strain is cultured to OD in NYG Medium₆₀₀About 1.0 as seed liquid, and transferring into fresh NYG culture medium according to the proportion of 1: 100; OD when F20 or Xcc8004₆₀₀When the concentration was about 0.5, the cells were collected by centrifugation and resuspended twice in sterile water. 1mL of OD₆₀₀Equal volumes of 0.5F 20 and Xcc8004 were mixed well, followed by addition of III-12 to a concentration of 100 μ M and thorough mixing. Mixing reduced and compounded sample with 0.5mL of F20 and 1mL of Xcc, supplementing 0.5mL of sterile water, and adding III-12 to reach concentration50 μ M, mix well.

The influence of the compound III-12 and the biocontrol bacterium F20 on the pathogenicity of the Xcc8004 on the radish is shown in figure 8, and it can be seen from the figure that the mixture of III-12 and F20 with the concentration reduced by half can obviously prevent and treat the black rot disease of the Xcc8004 on the radish, and the prevention and treatment effect is similar to twice that of single III-12 or F20, which shows that the compound of the compound III-12 and the biocontrol bacterium F20 has the application prospect of reducing the compound dosage and realizing the multi-target synergistic prevention and treatment of the black rot disease of cruciferae. Wherein None represents the wild type strain inoculation and DMSO represents the solvent control.

Xoo strains Xoo wild-type strains were grown overnight in M210 rich medium. The next day, the seed solution was added to M210 fresh medium at a ratio of 1:100, and the culture was continued until OD was reached₆₀₀When the concentration is about 0.6, the thalli is centrifugally collected, biocontrol bacteria liquid is added into the thalli, the thalli is centrifugally collected again, the thalli is washed twice by using sterile water and then is suspended in 1mL of sterile water, corresponding compounds are respectively added into the mixture, the final concentration is 100 mu M, the same volume of DMSO is used as a control, and the mixture is incubated for 2 hours in an incubator at the temperature of 28 ℃. The leaf blade of the adult rice plant is processed by a leaf cutting method. 10-15 leaves were inoculated per treatment group and after half a month the length of the lesion in the rice was observed and recorded.

As shown in FIG. 9, it can be seen that the biocontrol bacterium F20 alone can produce good control effect on bacterial blight of rice, but compared with the biocontrol bacterium F20 alone or the biocontrol bacterium III-19 alone, the toxicity of Xoo on adult rice plants can be more effectively inhibited after the biocontrol bacterium III-19 and the biocontrol bacterium are combined. Wherein WT represents the inoculation of the wild type strain and WT + DMSO represents the solvent control.

In conclusion, the above examples show that the compounds of the present invention significantly reduce the pathogenicity of rice bacterial blight and Xanthomonas campestris without affecting the growth of pathogenic bacteria. The plant diseases caused by pathogenic bacteria can be prevented and/or treated without influencing the growth of the pathogenic bacteria, so that the generation of drug resistance of the pathogenic bacteria is avoided, and the effective service life of the compound is prolonged.

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims (10)

1. The application of the oxadiazine cinnamamide compounds in controlling plant diseases is characterized in that the structural formula of the oxadiazine cinnamamide compounds is shown as the formula (III):

wherein R is hydrogen or C_1～5Alkyl radical, C_1～5Alkoxy, halogen or nitro.

2. The use of claim 1, wherein R is hydrogen, C_1～3Alkyl radical, C_1～3Alkoxy, halogen or nitro.

3. The use of claim 2, wherein R is hydrogen, methyl, methoxy, halogen or nitro.

4. The use of claim 2, wherein 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.

5. The use according to any one of claims 1 to 4, wherein the plant disease is caused by an infestation with gram-negative pathogenic bacteria.

6. The use of claim 5, wherein the plant disease is bacterial blight of rice, and black rot of cruciferae.

7. A composition for controlling plant diseases, which comprises an effective amount of oxadiazine cinnamamide compounds and Ralstonia pickettii F20;

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

wherein R is as defined in any one of claims 1 to 4;

the Ralstonia pickettii F20 is preserved in Guangdong province microorganism strain collection center in 2018, 4 and 3 days, and the preservation number is GDMCC No: 60347.

8. the composition according to claim 7, wherein the concentration ratio of oxadiazine cinnamamide compound to Ralstonia pilferi F20 is 100 μ M: (20-25). times.10⁷CFU/mL。

9. Use of the composition according to claim 7 or 8 for controlling plant diseases.

10. Use according to claim 9, characterized in that the plant diseases are caused by an infestation with bacteria of gram-negative pathogens.

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