Method for rapidly screening compounded medicament for preventing and treating colletotrichum gloeosporioides

文档序号：574312 发布日期：2021-05-21 浏览：25次中文

阅读说明：本技术 一种快速筛选防治炭疽病菌复配药剂的方法 (Method for rapidly screening compounded medicament for preventing and treating colletotrichum gloeosporioides ) 是由陈淑宁袁会珠闫晓静杨代斌于 2019-11-20 设计创作，主要内容包括：本发明公开了属于农药技术领域的一种快速筛选防治炭疽病菌复配药剂的方法。本发明的方法通过构建靶标基因敲除子,比较了不同DMI药剂对敲除子和亲本菌株间的EC-(50)差异,从而分析DMI类杀菌剂对靶标结合偏好性,根据靶标结合偏好性对DMI类药剂进行分组,并由此筛选了针对C.nymphaeae菌株具有增效作用的复配药剂组合,验证发现复配组合增效作用明显。本发明的方法缩小了选择复配DMI类药剂的范围,在炭疽病菌的防治中,可选药剂非常有限,DMI类药剂为炭疽病害的防治提供了有力保障,而筛选具有增效作用的DMI类药剂复配组合,可以提供更多的防治方案。(The invention discloses a method for rapidly screening a compound medicament for preventing and treating colletotrichum gloeosporioides, belonging to the technical field of pesticides. The method of the invention compares EC between different DMI medicament pair knockouts and parent strains by constructing target gene knockouts 50 And the difference is analyzed, thereby analyzing the target binding preference of the DMI bactericides, grouping the DMI medicaments according to the target binding preference, screening the compound medicament combination with the synergistic effect aiming at the C.nymphaeae strain, and verifying that the synergistic effect of the compound combination is obvious. The method of the invention reduces the range of selecting the compound DMI medicament, the selectable medicament is very limited in the control of anthrax pathogen, the DMI medicament provides powerful guarantee for the control of anthrax disease, and the screened DMI medicament compound combination with synergistic effect can provide more control schemes.)

1. A method for rapidly screening a compound medicament for preventing and treating colletotrichum gloeosporioides is characterized by comprising the following steps:

(1) respectively constructing CYP51A gene and CYP51B gene knockout strains of the anthrax bacteria, namely delta CYP51A and delta CYP 51B;

(2) respectively measuring EC of colletotrichum gloeosporioides wild type, CYP51A gene knockout strain and CYP51B gene knockout strain on different DMI medicaments₅₀Comparison of CYP51A Gene or CYP51B Gene knock-out strainsWith wild type strain EC₅₀According to the variation of sensitivity to DMI-class agents, the following grouping selections are made:

class I: EC of Δ CYP51B compared to wild type strain₅₀Decrease, and EC of Δ CYP51A₅₀A DMI agent that remains unchanged;

class II: EC of Δ CYP51A compared to wild type strain₅₀Decrease, and EC of Δ CYP51B₅₀A DMI agent that remains unchanged;

class III: EC of Δ CYP51A and Δ CYP51B compared to wild type strains₅₀A DMI agent that is both reduced;

the compounding method comprises the following steps: the three types of medicaments with the change difference are compounded in pairs, so that the synergistic effect is achieved: i.e. class I and II, class I and III, class II and III.

2. The method of claim 1, wherein said reduction is statistically significant, and P is 0.01.

3. The method of claim 1, wherein the unchanged state is that, after comparison, the P is 0.01 without significant difference on a statistical level.

4. The method of claim 2 or 3, wherein the statistical method is analysis of variance (ANOVA) and is calculated by Least Significant Difference (LSD) method.

5. The method of any one of claims 1-4, wherein the DMI-class agent comprises: imidazole amide, propiconazole, imazalil, cyproconazole, difenoconazole or epoxiconazole.

6. The method according to any one of claims 1 to 5, wherein in the compounding method, when the three types of agents are compounded two by two, the ratio is 0.5 to 10: 0.5-10.

7. The method of claim 6, wherein the ratio is 1-5: 1-5.

8. The method of claim 6, wherein the ratio is 1: 1.

Technical Field

The invention belongs to the technical field of pesticides, and particularly relates to a method for rapidly screening a compound DMI medicament for preventing and treating anthracnose pathogen.

Background

The Colletotrichum anthrachaeae (Colletotrichum nomphaeae) is an important pathogenic bacterium, can infect up to 400 plants, and the host of the Colletotrichum anthrachaeae almost covers all crops, is one of the most important pathogenic bacteria causing plant diseases, and can cause serious pre-harvest and post-harvest harm to important economic crops such as fruits, vegetables, cereals, ornamental plants and the like.

Until now, the use of fungicides has remained the primary means of controlling the disease, but only a few types of fungicides are effective. Lanosterol demethylase inhibitors (DMIs) are widely used for the control of phytopathogens due to their broad-spectrum activity, relatively weak toxicity (compared with other protective fungicides) and postharvest activity. In the protection of a large number of vegetables and fruits, after the resistance of MBCs and QoIs bactericides is generated to cause control failure, the DMIS bactericides become main bactericides for controlling the diseases. DMI drugs are diverse in variety, act on sterol 14-alpha demethylase (coded by CYP51 gene) in the fungal ergosterol biosynthesis pathway, and play a role in sterilization by inhibiting the biosynthesis of ergosterol, a component of bacterial cell membranes.

At present, almost no DMI compound medicament for controlling anthrax germs exists, and a traditional method for screening synergistic effect is to randomly select two medicaments to carry out compound test, so that the required workload is large, the identification cost is high, and the test period is longer. Therefore, the invention is particularly important for the rapid and accurate medicament compound screening method, and can shorten the identification period and improve the screening efficiency.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a method for rapidly screening a compound medicament for preventing and treating colletotrichum gloeosporioides.

The inventor of the application discovers that the anthrax fungi contains two genes of CYP51A and CYP51B through previous research, and the inhibition of the two proteins by different DMI bactericides is different.

Therefore, the technical scheme of the invention is as follows:

a method for rapidly screening a compound medicament for preventing and treating colletotrichum gloeosporioides comprises the following steps:

(1) respectively constructing CYP51A gene and CYP51B gene knockout strains of the anthrax bacteria, namely delta CYP51A and delta CYP 51B;

(2) respectively measuring EC of colletotrichum gloeosporioides wild type, CYP51A gene knockout strain and CYP51B gene knockout strain on different DMI medicaments₅₀Value, comparison of the CYP51A Gene or CYP51B knock-out Strain with the wild-type Strain EC₅₀According to the difference in sensitivity to DMI-type agents, toThe following grouping options:

class I: EC of Δ CYP51B compared to wild type strain₅₀Decrease, and EC of Δ CYP51A₅₀A DMI agent that remains unchanged;

class II: EC of Δ CYP51A compared to wild type strain₅₀Decrease, and EC of Δ CYP51B₅₀A DMI agent that remains unchanged;

class III: EC of Δ CYP51A and Δ CYP51B compared to wild type strains₅₀A DMI agent that is both reduced;

the compounding method comprises the following steps: the three types of medicaments with the change difference are compounded by two, so that the synergistic effect is achieved: i.e. class I and II, class I and III, class II and III.

In the above method, the reduction means that after comparison, the difference is statistically significant, and P is 0.01.

In the above method, the retention means that after comparison, there is no significant difference in statistical level, and P is 0.01.

In the above method, optionally, the statistical method is analysis of variance, and the Least Significant Difference (LSD) method is used for calculation.

In the above method, the DMI-based agent comprises: imidazole amide, propiconazole, imazalil, cyproconazole, difenoconazole or epoxiconazole.

In the method, in the compounding method, when the three types of medicaments are compounded pairwise, the proportion is 0.5-10: 0.5-10, e.g., 0.5-1: 0.5-1; 0.5-5: 0.5-5; 3-7: 3-7; 5-10: 5-10;

in the above method, optionally, the ratio is 0.5-1:0.5-1 or 1-5: 1-5.

In the above method, optionally, the ratio is 1: 1.

the invention has the advantages and effects that:

the invention provides a method for rapidly screening DMI compound medicaments with a synergistic effect on C.nymphaeae. The method compares EC between different DMI medicament pair knockouts and parent strains by constructing target gene knockouts₅₀Difference to analyze the pair of DMI bactericidesAnd (3) target binding preference, grouping the DMI medicaments according to the target binding preference, screening a compound medicament combination with a synergistic effect on the C.nymphaeae strain, and verifying that the compound combination has an obvious synergistic effect.

The method of the invention reduces the range of selecting the compound DMI medicament, the selectable medicament is very limited in the control of anthrax pathogen, the DMI medicament provides powerful guarantee for the control of anthrax disease, and the screened DMI medicament compound combination with synergistic effect can provide more control schemes.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The materials and devices used in the present invention are commercially available unless otherwise specified.

The present invention is further illustrated by, but is not limited to, the following examples.

Example 1 construction of the Strain Δ CYP51A and Δ CYP51B

A. Construction of fragments for knock-out construction

Fusion PCR products for CYP51A and CYP51B knockouts were constructed by the double-join (DJ) PCR method (Chen, S., Yuan, N., Schnabel, G., Luo, C.2017.function of the genetic element 'Mona' associated with genetic resistance in Monilinia fructicola. molecular plant Pathology 18: 90-97.). The primers required are shown in Table 1. Taking Δ CYP51A as an example, the 5 'end and 3' end sequences of CYP51A gene were amplified from the genome of C.nymphaeae by primers Nyma5-FOR/Nyma5-REV and Nyma3-FOR/Nyma 3-REV. The complete sequence of the hygromycin gene (including the promoter, terminator and hygromycin gene of a. nidulans) was amplified from the vector pBHT2 by the primers HypF/HypR. And carrying out second-round fusion on the fragments through Nyma _5_ nest/Hyp-nest-5R and Hyp-nest-3F/Nyma _3_ nest to obtain an upstream fragment and a downstream fragment of the knocked-out CYP51A gene, wherein the upstream fragment and the downstream fragment are used for knocking out the upstream sequence of CYP51A, shown as SEQ ID NO.1 in the sequence table, and the downstream fragment sequence of CYP51A, shown as SEQ ID NO.2 in the sequence table.

And constructing a fusion PCR product for knocking out CYP51B in the same way, wherein the primer is shown in Table 1, so as to obtain an upstream fragment and a downstream fragment for knocking out CYP51B, wherein the upstream fragment for knocking out CYP51B is shown in SEQ ID NO.3 in the sequence table, and the downstream fragment for knocking out CYP51B is shown in SEQ ID NO.4 in the sequence table.

B. Preparation of protoplast of Colletotrichum

Protoplast preparation methods are described by Chen et al (Chen et al, 2017) and include:

preparing enzymolysis liquid, wherein the preparation is carried out on site: weighing 0.08g lysozyme, 0.08g collapse enzyme, 0.08g Snail enzyme, 0.04g cellulase, and dissolving in 8ml 1M NH₄And (3) placing the Cl solution into a 10ml centrifugal tube, shaking the Cl solution in a shaking table for 30min to fully dissolve the enzyme solution, and filtering and sterilizing the solution.

Protoplasts of c.ymphaeae wild parent strain CaPH40 were prepared as follows:

1) inoculating the strain on PDA plate, culturing at 22 deg.C for 4 days, selecting fresh mycelium with inoculating needle, inoculating in 50ml PDB culture solution, and shake culturing at 22 deg.C for 120 r/min.

2) Filtering with double-layer sterilized gauze after 3 days to obtain mycelium, and adding 1M NH₄The mycelia were washed with Cl solution.

3) Adding mycelium into the fully dissolved enzymolysis solution, adding 0.1g of mycelium into about 1ml of enzymolysis solution, shaking for 4h in a shaking table at 27 ℃, and fully performing enzymolysis. After 4h, a large number of individual semitransparent spherical cells can be seen through microscopic examination, and the cells are protoplasts.

4) Centrifuging at 3000rpm for l 0min, and discarding.

5) 5ml of STC solution was added and washed once, centrifuged at 3000rpm for 10min, and the supernatant was discarded.

6) Adding STC solution, and performing microscopic counting with blood count plate to adjust the protoplast concentration to 10⁶One per ml. Directly transforming the obtained protoplast or adding 1/10 volume 60% PEG4000, mixing, packaging, and keeping at-80 deg.C for a short timeAnd (4) storing to obtain the protoplast suspension.

PEG-mediated protoplast transformation

1) And (3) adding 160 mu l of the prepared protoplast suspension (obtained in the step B) into a 10ml glass tube, adding 35 mu l of each of the purified upstream fragment and downstream fragment (obtained in the step A), slightly sucking and blowing by using a gun head, uniformly mixing, and standing on ice for 15 min.

2) Gradually adding 200 μ l PEG solution, and 800 μ l PEG solution along the tube wall, rotating the centrifuge tube while each adding, mixing, and standing on ice for 15 min.

3) Add 1ml of STC solution and mix well.

4) Mu.l of the transformation mixture was added to a petri dish (phi 9cm), 20ml of about 45 ℃ regeneration medium was poured into the petri dish, mixed with the transformation mixture, solidified, sealed, and cultured at 22 ℃ in the dark.

5) 1-2 days later, the protoplast just germinates, and a screening culture medium containing hygromycin of 150 mu g/ml is added for culture at 22 ℃.

6) After 6 days, single colonies growing well on the surface of the screening medium grew out.

D. Knockout transformant validation

The wild parent strain CaPH40 of the nyphaeae has no hygromycin resistance gene (Hyp), so the wild parent strain can not grow on a PDA plate containing hygromycin hygB 200 mug/mL, and the vector fragment containing the hygromycin resistance gene constructed in vitro can grow normally on the PDA plate containing hygB 200 mug/mL and has stable resistance to hygromycin if the CYP51 gene is successfully replaced homologously. Thus, knockout transformants were selected based on their growth on hygB 200. mu.g/mL PDA plates for multiple generations.

A single colony which can normally grow on a PDA culture medium containing 200 mu g/ml hygromycin and can still stably grow after being transferred for more than 5 generations is taken as a knockout transformant.

The delta CYP51A and delta CYP51B knockouts were screened and verified by PCR amplification using specific primers. Specifically, DNA extraction kit (Beijing Quanjin biology Co., Ltd.) was used to extract DNA of the parental strains CaPH40, Δ CYP51A, and Δ CYP51B knock-out daughter as templates. Specific primers, namely, Check _ CNA _ For/Check _ Hyg _ Rev and Check _ Hyg _ For/Check _ CNA _ Rev are designed according to the gene sequence after homologous replacement, and 2460bp and 1845bp fragments of upstream and downstream junction regions in a delta CYP51A knockout son are specifically amplified respectively after homologous replacement. Specific primers, namely Check _ CNB _ For/Check _ Hyg _ Rev and Check _ Hyg _ For/Check _ CNB _ Rev, are designed, and 2405bp and 2185bp fragments of upstream and downstream junction regions in the delta CYP51B knockout molecule are respectively and specifically amplified after homologous substitution.

According to the method, 9 strains of the delta CYP51A knockout strain and 10 strains of the delta CYP51B knockout strain are obtained together, and 3 transformants are randomly picked for subsequent reagent sensitivity tests.

TABLE 1 primer information

Example 2

And (3) testing the susceptibility of DMI medicaments of wild type anthracnose pathogen, delta CYP51A strain and delta CYP51B strain.

Wild type anthracnose strain (CaPH40), Δ CYP51A strain thereof and Δ CYP51B strain thereof were tested for sensitivity to the above agents by colony growth assay using imamide, propiconazole, imazalil, cyproconazole, difenoconazole, epoxiconazole. The method comprises the following specific steps:

(1) preparing imidazole amide, propiconazole, imazalil, cyproconazole, difenoconazole and epoxiconazole into 10 parts by using dimethyl sulfoxide respectively⁴Mu.g/ml of mother liquor.

(2) Adding the above mother solution into sterilized PDA culture medium, cooling to 45 deg.C, making into medicated plate with final concentration of 0,0.01,0.03,0.1,0.3,1,3,10,30,100 μ g/ml and diameter of 9cm by using each medicine, and repeating for 3 times for each proportion of medicine.

(3) The wild-type parent strains CaPH40, delta CYP51A (3 transformants were randomly selected) and delta CYP51B (3 transformants were randomly selected) were inoculated on each plate, and after 5 days of culture on a PDA plate, a cake with a diameter of 0.5cm was punched along the edge of the colony using a punch, and the mycelia were inoculated face down in the drug-and-control (0. mu.g/ml plate) medium in step (2), placed in a 28 ℃ incubator in the dark for culture, and the diameter of the colony was measured after 7 days.

(4) When the diameter of the control colony reaches more than 80% of the diameter of the culture dish, measuring the diameter of the colony by using a cross method, repeating the treatment for 3 times, calculating the inhibition rate (namely the percentage for inhibiting the growth of hyphae compared with the control) of each medicament at different concentrations, obtaining a toxicity regression equation through linear regression analysis between the inhibition rate and the logarithm of the concentration of the series of medicaments, and then calculating the EC of each medicament₅₀。

The results are shown in tables 1 to 3. Wherein, toxicity regression equation and related coefficient of each medicament to wild strain CaPH40, delta CYP51A knockout after CYP51A gene knockout, and delta CYP51B knockout after CYP51B gene knockout are shown in tables 1, 2 and 3, and EC of each medicament for each strain is calculated according to the toxicity regression equation₅₀The value is obtained.

In particular, the EC of the wild type parent strain for each DMI-type agent is shown in table 1₅₀Values, and their virulence regression equations, correlation coefficients; EC (EC)₅₀Values were calculated from the virulence regression equation.

Tables 2 and 3 show the EC of Δ CYP51A and Δ CYP51B knockouts for each DMI-based agent₅₀Values, and their virulence regression equations, correlation coefficients; wherein the virulence regression equation is calculated from the average value of the hypha growth inhibition rates of randomly selected 3 knockout on plates containing different medicament concentrations, and the EC of each medicament for each strain is calculated according to the virulence regression equation₅₀The value is obtained.

TABLE 1 EC of various Agents against wild type Colletotrichum anthracis CAPH40₅₀Value of

Drug treatment	EC₅₀	Regression equation of virulence	Correlation coefficient
				Minamides	0.06	Y＝5.6949+0.3835x	0.99
Propiconazole	1.61	Y＝4.8856+0.9801x	0.99
				Enconazole	1.09	Y＝5.0214+0.5601x	0.99
Cyproconazole	52.13	Y＝1.6215+1.9792x	0.98
				Difenoconazole	3.95	Y＝4.5963+0.4415x	0.99
Epoxiconazole	1.17	Y＝4.9721+1.3865x	0.99

；

TABLE 2 EC of each agent against the Δ CYP51A knockdown₅₀Value of

Drug treatment	EC₅₀	Regression equation of virulence	Correlation coefficient
				Minamides	0.05	Y＝4.9723+0.6493x	0.98
Propiconazole	0.04	Y＝6.0689+0.8050x	0.99
				Enconazole	0.79	Y＝4.8920+1.1832x	0.99
Cyproconazole	0.02	Y＝6.1627+1.9105X	0.99
				Difenoconazole	0.73	Y＝4.9765+0.9045	0.99
Epoxiconazole	0.003	Y＝5.0123+0.3425x	0.98

；

TABLE 3 EC of each agent against the Δ CYP51B knockdown₅₀Value of

According to the EC of each strain for each DMI-type agent₅₀Values, each DMI agent is classified into three classes:

class I: EC of Δ CYP51B compared to wild type strain₅₀Decrease, and EC of Δ CYP51A₅₀A DMI agent that remains unchanged; the drug in group I was more effective on the Δ CYP51B transformants, meaning that it was more potent in inhibiting CYP51A (CYP 51A alone in Δ CYP51B transformants);

class II: EC of Δ CYP51A compared to wild type strain₅₀Decrease, and EC of Δ CYP51B₅₀A DMI agent that remains unchanged; the drug in group I was more effective on the Δ CYP51A transformants, meaning that it was more potent in inhibiting CYP51B (CYP 51A alone in Δ CYP51B transformants);

class III: EC of Δ CYP51A and Δ CYP51B compared to wild type strains₅₀A DMI agent that is both reduced; meaning that the drug has inhibitory activity against both CYP51A and CYP 51B.

Through comparing the sensitivity difference of knocking-out seeds and wild type anthracnose pathogenic bacteria to different DMI bactericides, the tested DMI bactericides are grouped according to the sensitivity change degree, wherein the miamide belongs to the group I, compared with wild type strains, the sensitivity of delta CYP51A is unchanged, and the sensitivity of delta CYP51B is obviously changed; propiconazole and imazalil belong to group II, the sensitivity of delta CYP51B is unchanged, and the sensitivity of delta CYP51A is obviously changed; cyproconazole, difenoconazole and epoxiconazole belong to group III, and the sensitivity of Δ CYP51A and Δ CYP51B are all significantly increased, and the results are shown in table 4.

The drug in group I was more effective on the Δ CYP51B transformants, meaning that it was more potent in inhibiting CYP51A (CYP 51A alone in Δ CYP51B transformants); similarly, the agent in group II has a stronger inhibitory effect on CYP 51B; the agents in group III have inhibitory effects on both CYP51A and CYP 51B.

TABLE 4 EC of groups of DMI Agents on different DMI fungicides₅₀Value of

Mean ± Standard Deviation (SD); ANOVA analysis of the data was performed using SPSS13.0 software (LSD; P is 0.01, and letters in the same column indicate no significant difference between the two.

Further, the EC of knockouts Δ CYP51A and Δ CYP51B was calculated for different DMI fungicides compared to their wild parent strains₅₀The results are shown in Table 5.

TABLE 5 knockout on different DMI-type fungicides compared to the parent anthrax bacteriaEC₅₀Percentage of change

Percent is knockout EC₅₀A more wild type strain EC₅₀The specific calculation method of the change value is as follows: (1-knock-out seed EC₅₀Wild type EC₅₀) % of the total weight of the composition. NS stands for knock-out compared to the wild-type parental strain, EC₅₀There was no significant difference in the values (LSD method, P ═ 0.01).

EXAMPLE 3 synergistic Effect of different combinations of Agents

1. According to the groups, medicaments of the DMI bactericides group I and group II, the medicaments of the group II and group III and the medicaments of the group I and group III are mixed according to the proportion of 1:1, and the synergistic effect is verified.

2. The strain is as follows: to give a certain genetic diversity to the samples, strains 5 from c.nymphaeae were selected, respectively: CaPH40, CaCO3-35, CaCO42, CaCO51 and CaCO 9.

3. The EC for imidazole amide, propiconazole, imazalil, cyproconazole, epoxiconazole and difenoconazole were respectively tested₅₀Value, and EC for compounded combinations₅₀The specific steps are shown in step one, and the compound composition is as follows:

imidyl/propiconazole (group I + group II), imidyl/imazalil (group I + group II);

(ii) imid/cyproconazole (group I + group III), imid/epoxiconazole (group I + group III), imid/difenoconazole (group I + group III);

propiconazole/imazalil (group II + group II);

propiconazole/epoxiconazole (group II + group III), imazalil/difenoconazole (group II + group III).

4. According to Sun cloud method (Sun cloud, Y., Johnson, E.1960.analysis of joint action of antibiotics against houses flies. journal of Econogenic Entomology 53:887-₅₀Calculating the actually measured virulence index (A) of the combinationTI), theoretical virulence index (TTI), and co-virulence coefficient (CTC). The calculation method is as follows:

measured toxicity index (ATI) of compounded agent is single dose EC₅₀Compounded agent EC₅₀×100。

The Theoretical Toxicity Index (TTI) of the compound agent is A toxicity index multiplied by A content (%) in the compound agent + B toxicity index multiplied by B content (%) in the compound agent.

Co-toxicity coefficient (CTC) ═ ATI/TTI × 100.

The antagonism is that CTC is less than or equal to 80, the addition is that 80 CTC is less than 120, and the synergism is that CTC is more than or equal to 120.

5. The results are shown in tables 6-12, and the virulence regression equation is calculated from the average value of the hypha growth inhibition rates of the 5 c.nymphaeae strains on the plates containing different concentrations of the agents in step 2, and each agent and the combination thereof are calculated according to the virulence regression equation, and the EC for the strain is₅₀The value is obtained.

TABLE 6 indoor toxicity assay for anthrax bacteria with Midamide and propiconazole combinations

Drug treatment	EC50	Co-toxicity coefficient
			Miamide (A)	0.043	-
Propiconazole (B)	1.31	-
			A:B＝1:1	0.063	132

；

TABLE 7 indoor toxicity assay for anthrax bacteria with Midamide and Enconazole combinations

Drug treatment	EC50	Co-toxicity coefficient
			Miamide (A)	0.043	-
Enconazole (B)	1.42	-
			A:B＝1:1	0.07	120

；

TABLE 8 indoor toxicity assay for anthrax bacteria with Midamide and Cycloprofezolidol combinations

Drug treatment	EC50	Co-toxicity coefficient
			Miamide (A)	0.043	-
Cyproconazole (B)	53.14	-
			A:B＝1:1	0.013	649

；

TABLE 9 indoor toxicity assay for anthrax bacteria with Midamide and Epoxiconazole

TABLE 10 indoor toxicity assay for propiconazole and imazalil against anthrax

Drug treatment	EC50	Co-toxicity coefficient
			Propiconazole (A)	1.31	-
Enconazole (B)	1.42	-
			A:B＝1:1	1.75	77

；

TABLE 11 indoor toxicity assay for propiconazole and epoxiconazole on anthrax bacteria

Drug treatment	EC50	Co-toxicity coefficient
			Propiconazole (A)	1.31	-
Epoxiconazole (B)	1.66	-
			A:B＝1:1	0.32	457

；

TABLE 12 indoor toxicity assay for anthrax bacteria with imazalil and difenoconazole

Drug treatment	EC50	Co-toxicity coefficient
			Enconazole (A)	1.42	-
Difenoconazole (B)	2.50	-
			A:B＝1:1	0.44	419

。

Results and analysis

It can be seen from the above table that different groups of DMI agents when mixed at a ratio of 1:1 all have synergistic effects, and that Minamide/Difenoconazole (group I + group III) and Minamide/cyproconazole (group I + group III) have similar virulence coefficients and are also synergistic. When the same group of DMI medicaments are mixed, such as propiconazole/imazalil (group II + group II), the co-toxicity coefficient is 77 at the ratio of 1:1, and the addition/synergy effect is not shown.

Therefore, when two DMI medicaments in different groups are compounded, the compounding has a synergistic effect because the two medicaments have different preferences on the combination of CYP51A and CYP51B targets. However, when the same group of agents were compounded, no synergistic effect was exhibited due to the insignificant preference for CYP51A and CYP51B target binding.

The above examples are merely illustrative for clarity and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Sequence listing

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atctgcatac tggcgactgc atcccaccat agtcggacac gctttatgaa acgcggtaat 420

tgtgcattta tcaacaaaga tgttttagta gatcacagtg atttcctagt gcttatgtat 480

ttcctaggcc cactgatctc aactgaaatc agcagcgcct gcagcttaat aggagtgggt 540

acgtacctaa tacgaatacg aaccgccttt gcgctccagg gtcatcaagg aaagcaggga 600

actcgatgtg gacatcgccg tcactaacac ctcgaaggct tgtaggtcca gccgaatacg 660

aattcgaaag acattcgagt ttttgtcact caggttgcta gacttggctc caaatcaccg 720

acggagtttt ggatatttgc cattgcctac tgtacacgtc tgaattgaag agaaatatcg 780

tcgtctttcc tggctgagct atcgaaatgt ctactttact tatgttgtac tggtcctgac 840

acagctctcg tcctcttatt aactacaaat gttttttgat cgttgtacaa aatcttgaca 900

aaacccaacc actgaaattc gacagaagat gatattgaag gagcattttt gggcttggct 960

ggagctagtg gaggtcaaca catcaatgct attttggttt agtcgtccag gcggatcaca 1020

aaatttgtgt cgtttgacaa gatggttcat ttaggcaact ggtcagatca gcccacttgt 1080

aagcagtagc ggcggcgctc gaagtgtgac tcttattagc agacaggaac gaggacatta 1140

ttatcatctg ctgcttggtg cacgataact tgtgcgtttg tcaagcaagg taagtgaacg 1200

acccggtcat accttcttaa gttcgccctt cctcccttta tttcagattc aatctgactt 1260

acctattcta cccaagcatc gatatgaaaa agcctgaact caccgcgacg tctgtcgaga 1320

agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag 1380

aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct 1440

gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc 1500

cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc 1560

gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc 1620

agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt 1680

tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg 1740

cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt 1800

ccgtcgcgca ggctctcgat gagctgatgc 1830

<210> 2

<211> 1729

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg 60

taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg 120

ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc ctgacctatt 180

gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg 240

ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga 300

cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt 360

tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg 420

tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg 480

aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc 540

gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg 600

ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg 660

agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg 720

gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc 780

agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg 840

cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa 900

accgacgccc cagcactcgt ccgagggcaa aggaatagag tagatgccgg atccacttaa 960

ccaatgtttg ttcctctcca tgaggtgaaa taaaagtgat gatacgcact ttcaatcggg 1020

attctaagcg ctattacctg gttgtcttgt attgagcgga ctggcttatt cgaaatacta 1080

catgcttgtc cccagctagc aaagttgagt tgatcaagat ctagaatacg gtatttgaag 1140

tcggttaaca cagctacatg atgcttgttc tacatcctaa attcagaaga tgtctgcggt 1200

agaacgttga agctaatcat cgtgtcaaat tgaagctttg aagtttggtc taagcgaaag 1260

gcaacaagcg ccaacacttt attattaaac cttgttgcga agcgcaagga cccatccgag 1320

ctggaacatg ttcctaaaaa ggcaacatct ggatgatcat ccgaagcttt gccgactcaa 1380

ctctcgagcc ctgaaaccgt taataatcac tgacaccctc catttcaagc cgtcaactta 1440

ccccaatgtc atagtcaagg gagctacata ccagatgctc ttcgttcaca gcgaccccaa 1500

cagcacggat tatggcgcct ctcacttcat cacctcattt caatgatgca acagaatcag 1560

aacggttgct tggcactaga agtgctgatt cgaatatcga gccaacgtca tctatcaacg 1620

gacaagaccg agaagccatc gcattgaagc ttgccgcggc agcttactct ttcgtagtcg 1680

cgggtctttt cgtggctaca attggagtta cactaccgca ccagcaaac 1729

<210> 3

<211> 1305

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca 60

atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg 120

gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg 180

agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc 240

gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt 300

tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga 360

ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag 420

aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaataga 480

gtagatgccg gatccactta acgttactga aatcatcaaa cagcttgacg gaggcaattt 540

tgaaatggct tgatcgcgtt caaaggtggt ttccagagtc tggggaattg gagcacacat 600

cgaatcattc ccatctccca ttgcttgaat aggcagggag ggtctccaga tacgaatacg 660

aacggcccgt cattggatga gctgctggtg ccaccgattg gtccctcccc tgttcccgat 720

tttctggcgc aacgtccaac atggaccaac agggacgcac aggtgcttcc gagtggaaag 780

ggggccactg gggggcacct agcatttcat ggaagcaaac atgggaaaga aagggcagaa 840

aacaaaagga agaagagctt gcggtgcagc attctctgcc acaatgcact aggtaccctg 900

tgacagacgc ctcaaaatcg attcgggtgg ccagatgtca cagattcttg gctccacgac 960

aatcttttga cgtgcaaaat gcttgaccgc ctacccagcc atggcagtca tgcaaccttc 1020

cttactttgg atactttggt acattagctt agctgctggc tggctaaggt acctaggtat 1080

ggtgaggtac ctaaggtagg cgcacacaga cgtaaggtaa ggtagtggtg ctccttggtt 1140

acgcaaagaa aatgtggaac ccaacaaaac gcggcaggta tctaatctca ttaatggata 1200

atttaaaacc actgctggac cccccttctc gatgccttcc actttcttcg tgccactgat 1260

attcgtcttg tgcgtgcggt cactactctc gaaaacacag gcaga 1305

<210> 4

<211> 1717

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4