CN115777722A - Application of alexidine dihydrochloride to inhibition of phytopathogens - Google Patents
Application of alexidine dihydrochloride to inhibition of phytopathogens Download PDFInfo
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- CN115777722A CN115777722A CN202211523679.3A CN202211523679A CN115777722A CN 115777722 A CN115777722 A CN 115777722A CN 202211523679 A CN202211523679 A CN 202211523679A CN 115777722 A CN115777722 A CN 115777722A
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- alexidine dihydrochloride
- dihydrochloride
- alexidine
- tricyclazole
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Abstract
The invention belongs to the technical field of agricultural science and discloses application of alexidine dihydrochloride in inhibition of phytopathogens. Alexidine dihydrochloride can effectively inhibit the growth and the morbidity of phytopathogen at low concentration. Can inhibit the growth of 10 plant pathogenic bacteria including Magnaporthe grisea, and is suitable for industrial crops such as crops, fruits, vegetables, etc. Meanwhile, the pesticide composition can be used as a pesticide adjuvant to synergistically inhibit pathogenic bacteria with tricyclazole.
Description
Technical Field
The invention belongs to the technical field of agricultural science, and mainly relates to application of alexidine dihydrochloride in inhibition of phytopathogens.
Background
Pathogenic bacteria bring great threat to global grain safety and huge economic loss, the most economic and effective method for disease prevention and control is planting of disease-resistant varieties, but the period for breeding new disease-resistant varieties is long, and the resistance is weakened along with the increase of the planting years, so that the breeding of broad-spectrum and durable resistant varieties is always the key point and difficult point in the research of disease-resistant breeding. The prevention and control of plant diseases in actual production still relies on chemical prevention and control in a large number, but most of the bactericidal drugs used in the current production are similar and single, and the research of new bactericides requires long-time investment and cannot meet the actual production requirements. And the problems of pesticide residue, environmental pollution, pathogenic bacteria drug resistance and the like of rice grains are increasingly highlighted due to the large and lasting use of chemical pesticides. Therefore, the discovery of the new bactericide has important significance for the rapid application in the prevention and treatment of plant diseases.
Alexidine dihydrochloride (AXD) was first applied in mouthwashes and was found to prevent oral disease (Spolsky VW, forsythe AB. Effects of alexidine.2HCL morthwash on plain and gingivitis after once Months. J Dent Res.1977Nov;56 (11): 1349-58.). Alexidine dihydrochloride has been found to bind to the PTPMT1 protein and inhibit the growth of a part of human pathogenic fungi as well as cancer cells (dose-spenton, d.et al. Pharmaceutical targeting of the mitochondrial phosphose PTPMT1.J. Pharmaceutical. Exp. Ther.333,584-592, (2010)), but the mechanism of inhibition is not well understood. Whether alexidine dihydrochloride can be used as a bactericide to inhibit the onset of phytopathogens has not been reported, and whether alexidine dihydrochloride can be used as an adjuvant to inhibit the onset of phytopathogens in a synergistic manner with other medicaments has not been known.
The applicant finds that alexidine dihydrochloride can inhibit the growth of 10 plant pathogenic bacteria when being used as an effective component, and the applicant finds that the alexidine dihydrochloride can be used for inhibiting the formation of attached cells of important infection structures of rice blast germs in cooperation with tricyclazole, the alexidine dihydrochloride can inhibit the growth of pathogenic bacteria of crops, fruit trees and vegetables, and the alexidine dihydrochloride has a good broad-spectrum antibacterial effect and is used in laboratory and field tests. Can obviously inhibit the occurrence of rice blast and wheat scab.
Disclosure of Invention
The invention aims to provide application of alexidine dihydrochloride in inhibition of phytopathogen, wherein the phytopathogen is as follows: magnaporthe oryzae (Magnaporthe oryzae), fusarium graminearum (Fusarium graminearum), rhizopus tritici (Bipolaris sokiniana), epinephelus zeae (Bipolaris maydis), phytophthora sojae (Phytophthora sojae), leptosphaeria brasiliensis (Leptosphaeria biglobosa), botrytis cinerea (Botrytis cinerea), pyricularia pyrifera (Valsa pyri), pyrenopsis Malassensis (Valsa mali), and Monilinia fructicola (Monilinia fructicola).
The invention also aims to provide the application of the alexidine dihydrochloride in preparing the phytopathogen inhibitor.
In order to achieve the purpose, the invention adopts the following technical measures:
the application of alexidine dihydrochloride in inhibiting phytopathogens comprises using alexidine dihydrochloride as the only effective component or one of the effective components, and is used for inhibiting the phytopathogens;
the plant pathogenic bacteria are as follows: magnaporthe oryzae (Magnaporthe oryzae), fusarium graminearum (Fusarium graminearum), rhizopus tritici (Bipolaris sokiniana), epinephelus zeae (Bipolaris maydis), phytophthora sojae (Phytophthora sojae), leptosphaeria brasiliensis (Leptosphaeria biglobosa), botrytis cinerea (Botrytis cinerea), pyricularia pyricularis (Valsa pyri), malus pumilus (Valsa mali) and/or Monilinia fructicola (Monilinia fructicola).
In the above application, the application process is to spray the agent containing alexidine dihydrochloride on the leaves suffering from plant diseases.
The protection scope of the invention also includes: use of alexidine dihydrochloride for the preparation of a phytopathogen inhibitor.
In the above-mentioned application, it is preferable to prepare the bacteriostatic agent by combining with tricyclazole.
The above bacteriostatic agent, preferably, the ratio of the alexidine dihydrochloride to the tricyclazole is: 1 mu M of 2-6.67mg/L.
Compared with the prior art, the invention has the following advantages:
1. alexidine dihydrochloride can effectively inhibit the growth and the morbidity of phytopathogens at low concentration.
2. Alexidine dihydrochloride can be used as a pesticide adjuvant to synergistically inhibit pathogenic bacteria with other bactericides.
3. Alexidine dihydrochloride can inhibit the growth of pathogenic bacteria of 10 plants including Magnaporthe grisea, and is suitable for industrial crops such as crops, fruits, vegetables, etc.
Drawings
FIG. 1 is a schematic representation of alexidine dihydrochloride inhibiting the growth of phytopathogens;
wherein: a, the alexidine dihydrochloride inhibits the growth of different pathogenic bacteria on a flat plate; and b, the alexidine dihydrochloride inhibits the growth inhibition rate of different pathogenic bacteria.
FIG. 2 shows the results of statistics of inhibition of rice blast by alexidine dihydrochloride;
wherein: a, inhibiting the occurrence of rice blast by using alexidine dihydrochloride with different concentrations, and counting the morbidity result 7 days after inoculation; and b, counting disease spots on the diseased leaves.
FIG. 3 is a graph showing that alexidine dihydrochloride can be used as an adjuvant to synergistically inhibit formation of blast fungus attachment cells with tricyclazole;
wherein: a, after the alexidine dihydrochloride and the tricyclazole are compounded, the formation of rice blast germ attachment cells is inhibited, and the scale =10 μm; b, the inhibition rate of formation of anchorage zone after compounding of the alexidine dihydrochloride and the tricyclazole; c, fractional influence (Fa) -Combination Index (CI) analysis of different alexidine dihydrochloride combinations with tricyclazole. CI <1, synergistic effect; CI =1, additive effect; CI >1, antagonism.
FIG. 4 shows the inhibition of rice blast and head blight by alexidine dihydrochloride in the field;
wherein: a, inhibiting rice blast occurrence in fields by using alexidine dihydrochloride; b, investigating the disease condition index of rice blast fungus; c, inhibiting wheat scab in the field by using alexidine dihydrochloride; d, investigating the wheat scab disease index.
Detailed Description
For a better understanding of the present invention, the present invention will be further described with reference to specific examples, which are, unless otherwise specified, conventional in the art, and reagents or materials commercially available therefrom. The following are merely illustrative of several embodiments of the present invention. It is obvious that the present invention is not limited to the following embodiments, but many variations are possible. Therefore, modifications and improvements based on the disclosure of the present invention should be made by those skilled in the art within the scope of the claims of the present invention.
The alexidine dihydrochloride has the relative molecular mass of 581.7 and the molecular formula of C 26 H 58 Cl 2 N 10 The structural formula is as follows:
example 1:
the application of alexidine dihydrochloride in preparing a plant pathogenic bacteria bactericide comprises the following steps:
<xnotran> (Magnaporthe oryzae P131) (Xue M, yang J, li Z, hu S, yao N, dean RA, zhao W, shen M, zhang H, li C, liu L, cao L, xu X, xing Y, hsiang T, zhang Z, xu JR, peng YL.Comparative analysis of the genomes of two field isolates of the rice blast fungus Magnaporthe oryzae.PLoS Genet.2012;8 (8): e 1002869.), (Fusarium graminearum PH-1) (Xie, L.et al.Combinatorial biosynthesis of sulfated benzenediol lactones with a phenolic sulfotransferase from Fusarium graminearum PH-1.mSphere 5, (2020).), (Bipolaris sorokiniana) (Zhang, W.et al.A novel effector, csSp1, from Bipolaris sorokiniana, is essential for colonization in wheat and is also involved in triggering host immunity.Mol Plant Pathol 23,218-236, (2022).), (Bipolaris maydis) (Wang, H.et al.Ateosinte-derived allele of a MYB transcription repressor confers multiple disease resistance in maize.Mol Plant 14,1846-1863, (2021).), (Phytophthora sojae) (Si, J.et al.Phytophthora sojae leucine-rich repeat receptor-like kinases: diverse and essential roles in development and pathogenicity.iScience 24,102725, (2021).), (Leptosphaeria biglobosa) (Deng, Y.et al.Viral cross-class transmission results in disease of a phytopathogenic fungus.The ISME journal,1-12, (2022).), </xnotran> Botrytis cinerea (Liu, S.et al. Resistance to Botrytis in Botrytis cinerea from green house-growing tomato Plant Dis 105,628-635, (2021.), pear rot pathogen (Valsa pyr) (Yuan, H.et al. Biocontrol activity and action mechanism of Paenibacillus polymyxa strain N4 agains var tar calcium sulfate by Valsa pyr. Front. Microbiol.13,950742, (2022); apple rot pathogen (Valsa mali) (Zhang, M. et al. Hce2 domain-containing reactors polyol 20,843-856, (2019.), monilinia fructicola (Monilinia fructicola) (Chen, S., yuan, N., schnabel, G. & Luo, C.function of the genetic element 'a' organized with a fungal Plant reactant in Monilinia fructicola 18,90-97, (2017)).
Rice blast germs: the Pyricularia oryzae was inoculated on a Complete Medium (CM) plate at 28 ℃, the concentration of the added alexidine dihydrochloride was 6. Mu.M, the culture was performed for 5 days, the colony size was measured, and the growth rate of the strain was calculated.
Fusarium graminearum: inoculating fusarium graminearum on a PDA culture medium plate at 28 ℃, adding alexidine dihydrochloride at the concentration of 6 mu M, culturing for 3 days, measuring the colony size and calculating the growth speed of the strain.
Wheat root rot: inoculating Rhizopus tritici to PDA culture medium plate at 28 deg.C, adding alexidine dihydrochloride at concentration of 6 μ M, culturing for 5 days, measuring colony size, and calculating strain growth rate.
Corn leaf spot bacteria: inoculating corn microsporum to a PDA culture medium plate at 28 ℃, adding alexidine dihydrochloride with the concentration of 6 mu M, culturing for 6 days, measuring the colony size, and calculating the growth speed of the strain.
Phytophthora sojae: and (3) inoculating phytophthora sojae on a PDA culture medium plate at 28 ℃, adding alexidine dihydrochloride with the concentration of 6 mu M, culturing for 5 days, measuring the size of a bacterial colony, and calculating the growth speed of the bacterial strain.
Rape black shank fungus: the phytophthora parasitica var nigricosa is inoculated on a PDA culture medium plate at 28 ℃, the concentration of the added alexidine dihydrochloride is 6 mu M, the culture is carried out for 5 days, the size of a bacterial colony is measured, and the growth speed of the bacterial strain is calculated.
Tomato botrytis cinerea: the botrytis cinerea is inoculated on a PDA culture medium plate at 28 ℃, the concentration of the added alexidine dihydrochloride is 6 mu M, the culture is carried out for 4 days, the size of a colony is measured, and the growth speed of the strain is calculated.
Pear tree rot germs: inoculating the pear tree canker on a PDA culture medium plate at 28 ℃, adding alexidine dihydrochloride with the concentration of 6 mu M, culturing for 3 days, measuring the colony size and calculating the growth speed of the strain.
Apple tree canker: inoculating apple tree canker on PDA culture medium plate at 28 deg.C, adding alexidine dihydrochloride at concentration of 6 μ M, culturing for 2 days, measuring colony size, and calculating strain growth rate.
Brown rot of peach: inoculating Monilinia fructicola on a PDA culture medium plate at 28 deg.C, adding alexidine dihydrochloride at concentration of 6 μ M, culturing for 8 days, measuring colony size, and calculating strain growth rate.
The control group was a plate of normally cultured pathogenic bacteria. Each strain has 5 biological replicates, each experiment has three replicates, the colony growth condition and the inhibition rate are shown in figure 1 and table one, and the inhibition rate is calculated by the formula: the inhibition rate = (control group colony diameter-treatment group colony diameter)/control group colony diameter shows that alexidine dihydrochloride has obvious inhibition effect on the plant pathogenic bacteria, and the result shows that the alexidine dihydrochloride can be used as a broad-spectrum bactericide to inhibit the growth of the plant pathogenic bacteria.
Table one: alexidine dihydrochloride inhibits the growth inhibition rate of different pathogenic bacteria.
Example 2:
test for inhibiting rice blast pathogen infection by alexidine dihydrochloride:
fresh conidia (5X 10) of Pyricularia oryzae P131 were collected from CM medium 4 One/ml) was spray inoculated to LTH rice seedlings growing for about 1 month, and the treatment group was spore suspension added with alexidine dihydrochloride (1. Mu.M, 2. Mu.M, 4. Mu.M) at various final concentrationsAnd the control group is 0.025% Tween water, 10ml of spore suspension is sprayed on each treatment, the rice morbidity is observed after 24 hours of dark moisture-preserving culture and 7 days of illumination culture, and the number of the disease spots on the leaves is counted.
As shown in FIG. 2, the effect of inhibiting the occurrence of rice blast was more pronounced as the concentration of alexidine dihydrochloride increased, and the rice blast germs were almost lost at 4. Mu.M, indicating that alexidine dihydrochloride inhibited the infection of rice blast germs.
Example 3:
alexidine dihydrochloride acts synergistically with tricyclazole as an adjuvant:
fresh conidia (5X 10) of Pyricularia oryzae P131 were collected from CM medium 4 one/mL), then the spore suspension was added with alexidine dihydrochloride (3 μ M), alexidine dihydrochloride (5 μ M), tricyclazole (10 mg/L), tricyclazole (15 mg/L), tricyclazole (20 mg/L), alexidine dihydrochloride + tricyclazole (3 μ M +10 mg/L), alexidine dihydrochloride + tricyclazole (3 μ M +15 mg/L), alexidine dihydrochloride + tricyclazole (3 μ M +20 mg/L), alexidine dihydrochloride + tricyclazole (5 μ M +10 mg/L), alexidine dihydrochloride + tricyclazole (5 μ M +15 mg/L), alexidine dihydrochloride + tricyclazole (5 μ M +20 mg/L), and a 0.1 dmso Control (CK) was set. Each set of 3 replicates above.
10 mu L of spore suspension is dropped on a hydrophobic slide, and the forming rate of rice blast germ attachment cells is counted after 24h, and the result is shown in figure 3:
from fig. 3, it can be seen that the inhibition ratio of the alexidine dihydrochloride and the tricyclazole combination to the adhered cells is significantly higher than that of the alexidine dihydrochloride and the tricyclazole which are used alone, the effect of the alexidine dihydrochloride and the tricyclazole combination is better than the sum of the effects of the drugs which are used alone, and the inhibition on the formation of the bacteria adhered cells is more and more obvious along with the increase of the concentration of the alexidine dihydrochloride and the tricyclazole combination, and the results show that the alexidine dihydrochloride can cooperate with the tricyclazole to improve the bacteriostatic effect of the alexidine dihydrochloride and the tricyclazole. Meanwhile, the alexidine dihydrochloride + tricyclazole drug combination has synergistic effect calculated according to CompuSyn software, and in the synergistic effect in figure 4 and the table II, CI <1 indicates synergistic effect, CI =1 indicates no synergistic effect, and CI >1 indicates antagonistic effect.
A second table: inhibition rate of formation of adhesion cells and CI value after compounding of alexidine dihydrochloride and tricyclazole
Example 4:
alexidine dihydrochloride can be directly used as a bactericide for directly preventing and treating rice blast and wheat scab:
the field test of rice blast is carried out in rice blast disease nursery of Yangjiang city, guangdong province, and each test cell is 10m 2 0.1% DMSO in the control group and 6. Mu.M alexidine dihydrochloride in the treatment group, in each case three biological replicates were set. The pesticide is sprayed on 2022 at the early stage of rice blast disease, 4 months and 29 days, and the second spraying date is 5 months and 3 days. Disease index investigation is carried out on 19 days in 5 months, 3 points of diagonal lines of each test cell are sampled, 1 point is counted at each point, and the rice blast disease incidence index is counted according to the resistance evaluation grading standard of international rice:
stage 0: no disease exists; level 1: such as pinpoint size brown spots; stage 2: large onset brown spots; and 3, level: small, round or slightly long brown necrotic gray spots with a diameter of 1-2mm;4, level: typical rice blast scab or ellipse type with diameter of 1-2cm, and scab area less than 2% of leaf area; and 5, stage: typical rice blast scab, the disease area is less than 10%; and 6, level: typical rice blast scab, the disease area is 10-25%; and 7, stage: typical rice blast scab, the disease area is 26-50%; and 8, stage: typical rice blast scab, the disease area is 51-75%; and 9, stage: all leaves were dead.
Through field experiments, the incidence index is counted, and the occurrence of field rice blast can be remarkably inhibited by 6 mu M of alexidine dihydrochloride (a, b and table three in figure 4).
In the third aspect, alexidine dihydrochloride inhibits the rice blast from occurring in the field test.
In addition, wheat scab bacteriostasis test is carried out in test base of northwest agriculture and forestry science and technology university in Shaanxi province, and fresh wheat scab is 10 in concentration 5 Adding 6 mu M alexidine dihydrochloride into per mL fusarium graminearum PH-1 spore suspension according to a proportion, uniformly mixing, inoculating, wherein the inoculation period is a wheat flowering period (5 months and 5 days in 2022), inoculating 10 mu L of spore suspension into wheat ears, spraying water, bagging, and performing moisture preservation culture for 7 days. 10 ears were inoculated each time, 3 biological replicates were set, and the disease was counted after 14 days. The scab disease index statistics is calculated according to the inoculation point, the disease of a pair of side-by-side grains is recorded as 1, and the disease of one grain is counted as 0.5.
Through field experiments, the incidence index is counted, and the incidence of wheat scab can be obviously inhibited by 6 mu M alexidine dihydrochloride (c and d in figure 4). According to the results of field experiments, alexidine dihydrochloride is considered to be capable of well inhibiting field plant diseases
Table four: alexidine dihydrochloride inhibits the wheat scab from occurring in field tests.
Claims (5)
1. Application of alexidine dihydrochloride to inhibition of phytopathogens: magnaporthe oryzae (Magnaporthe oryzae), fusarium graminearum (Fusarium graminearum), rhizopus tritici (Bipolaris sokiniana), epinephelus zeae (Bipolaris maydis), phytophthora sojae (Phytophthora sojae), leptosphaeria brasiliensis (Leptosphaeria biglobosa), botrytis cinerea (Botrytis cinerea), pyricularia pyricularis (Valsa pyri), malus pumilus (Valsa mali) and/or Monilinia fructicola (Monilinia fructicola).
2. Use according to claim 1, wherein the application is carried out by spraying the leaf leaves affected by the plant disease with a medicament containing alexidine dihydrochloride.
3. Use of alexidine dihydrochloride in the preparation of a phytopathogen inhibitor, wherein the phytopathogen is: magnaporthe oryzae (Magnaporthe oryzae), fusarium graminearum (Fusarium graminearum), rhizopus tritici (Bipolaris sokiniana), epinephelus zeae (Bipolaris maydis), phytophthora sojae (Phytophthora sojae), leptosphaeria brasiliensis (Leptosphaeria biglobosa), botrytis cinerea (Botrytis cinerea), pyricularia pyricularis (Valsa pyri), malus pumilus (Valsa mali) and/or Monilinia fructicola (Monilinia fructicola).
4. The use of claim 3, wherein the inhibitor comprises alexidine dihydrochloride and tricyclazole.
5. The use of claim 4, wherein the ratio of the alaoxetine dihydrochloride to the tricyclazole is: 1 mu M of 2-6.67mg/L.
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