CN115777722B - Application of alexidine dihydrochloride in inhibiting plant pathogenic bacteria - Google Patents
Application of alexidine dihydrochloride in inhibiting plant pathogenic bacteria Download PDFInfo
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- CN115777722B CN115777722B CN202211523679.3A CN202211523679A CN115777722B CN 115777722 B CN115777722 B CN 115777722B CN 202211523679 A CN202211523679 A CN 202211523679A CN 115777722 B CN115777722 B CN 115777722B
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- alexidine
- alexidine dihydrochloride
- pathogenic bacteria
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Abstract
The invention belongs to the technical field of agricultural science, and discloses application of alexidine dihydrochloride in inhibiting plant pathogenic bacteria. Alexidine dihydrochloride can inhibit growth and onset of plant pathogenic bacteria at low concentration and high efficiency. Can inhibit the growth of 10 plant pathogenic bacteria including rice blast bacteria, and is suitable for crops, fruits, vegetables and other economic crops. Meanwhile, the compound 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 inhibiting plant pathogenic bacteria.
Background
Pathogenic bacteria pose a great threat to global grain safety and cause great economic loss, and the most economical and effective method for disease prevention and control is to plant disease-resistant varieties, but the period for cultivating new disease-resistant varieties is longer, and the resistance is weakened along with the increase of the planting years, so the cultivation of broad-spectrum and durable resistant varieties is always the key point and the difficulty in disease-resistant breeding research. The prevention and control of plant diseases in actual production are largely dependent on chemical control, but the sterilizing medicines used in the current production are largely similar and single, and the research of new bactericides needs long-time investment and cannot meet the actual production requirements. And the problems of pesticide residue, environmental pollution, pathogen resistance and the like of rice grains are increasingly prominent due to a large amount of persistent chemical pesticides. Therefore, the discovery of new bactericides is of great importance for rapid application in controlling plant diseases.
Alexidine dihydrochloride (Alexidine dihydrochloride, AXD) was first applied in mouthwash, and was found to prevent oral disease (Spolsky VW,Forsythe AB.Effects of alexidine.2HCL mouthwash on plaque and gingivitis after six months.J Dent Res.1977Nov;56(11):1349-58.). alexidine dihydrochloride was found to bind to PTPMT1 protein and inhibit the growth of some human pathogenic fungi and inhibit cancer cells (Doughty-Shenton,D.et al.Pharmacological targeting of the mitochondrial phosphatase PTPMT1.J.Pharmacol.Exp.Ther.333,584-592,(2010).), but its antibacterial mechanism was not yet clear. Whether alexidine dihydrochloride can be used as a bactericide to inhibit the occurrence of plant pathogenic bacteria has not been reported yet, and whether alexidine dihydrochloride can be used as an adjuvant to synergistically inhibit the occurrence of pathogenic bacteria with other agents is not known yet.
The applicant finds that the alexidine dihydrochloride can inhibit the growth of 10 plant pathogenic bacteria when being used alone as an active ingredient, and the applicant finds that the alexidine dihydrochloride can cooperate with tricyclazole to inhibit the formation of attached cells of important infection structures of rice blast bacteria, and the alexidine dihydrochloride can inhibit the growth of pathogenic bacteria including crops, fruit trees and vegetables, and has good broad-spectrum antibacterial effect, and the alexidine dihydrochloride can be used in laboratories and field experiments. Can significantly inhibit occurrence of Pyricularia oryzae occurrence of wheat scab.
Disclosure of Invention
The invention aims to provide an application of alexidine dihydrochloride in inhibiting plant pathogenic bacteria, wherein the plant pathogenic bacteria are as follows: rice blast fungus (Magnaporthe oryzae), fusarium graminearum (Fusarium graminearum), wheat root rot fungus (Bipolaris sorokiniana), corn small spot fungus (Bipolaris maydis), phytophthora sojae (Phytophthora sojae), black shank fungus (Leptosphaeria biglobosa), botrytis cinerea (Botrytis cinerea), pear tree rot fungus (VALSA PYRI), apple tree rot fungus (VALSA MALI), brown rot fungus (Monilinia fructicola).
Another object of the present invention is to provide the use of alexidine hydrochloride for the preparation of a plant pathogenic bacteria inhibitor.
In order to achieve the above object, the present invention adopts the following technical measures:
Use of alexidine dihydrochloride for inhibiting plant pathogenic bacteria, comprising the alexidine dihydrochloride as the only active ingredient or one of the active ingredients, for inhibiting plant pathogenic bacteria;
The plant pathogenic bacteria are as follows: pyricularia oryzae (Magnaporthe oryzae), fusarium graminearum (Fusarium graminearum), rhizopus oryzae (Bipolaris sorokiniana), rhizoctonia cerealis (Bipolaris maydis), phytophthora sojae (Phytophthora sojae), cercospora brassicae (Leptosphaeria biglobosa), botrytis cinerea (Botrytis cinerea), pyricularia pomace (VALSA PYRI), malus pumila (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 subjected to plant diseases.
The protection scope of the invention also comprises: the application of alexidine hydrochloride in preparing plant pathogenic bacteria inhibitor is provided.
In the above application, it is preferable to prepare the bacteriostatic agent in combination with tricyclazole.
The above-mentioned bacteriostat, preferably, the ratio of alexidine hydrochloride to tricyclazole is: 1 mu M2-6.67 mg/L.
Compared with the prior art, the invention has the following advantages:
1. Alexidine dihydrochloride can inhibit growth and onset of plant pathogenic bacteria at low concentration and high efficiency.
2. The 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 10 plant pathogenic bacteria including Pyricularia oryzae, including commercial crops such as crops, fruits, vegetables, etc.
Drawings
FIG. 1 is a schematic representation of alexidine dihydrochloride inhibiting plant pathogen growth;
Wherein: a, alexidine hydrochloride inhibits the growth of different pathogenic bacteria on a flat plate; b, the alexidine hydrochloride inhibits the growth inhibition rate of different pathogenic bacteria.
FIG. 2 shows the results of alexidine dihydrochloride inhibiting the occurrence and statistics of rice blast;
Wherein: a, inhibiting rice blast occurrence by alexidine hydrochloride with different concentrations, and counting the incidence result after inoculation for 7 days; and b, counting the disease spots on the diseased leaves.
FIG. 3 shows that alexidine dihydrochloride acts as an adjuvant in combination with tricyclazole to inhibit the formation of Pyricularia oryzae attachment cells;
Wherein: a, inhibiting the formation of rice blast bacteria attachment cells after the alexidine hydrochloride and the tricyclazole are compounded, wherein the scale=10μm; b, the inhibition rate of the formation of attachment cells after the alexidine hydrochloride and the tricyclazole are compounded; c, fractional impact (Fa) -Combination Index (CI) analysis of different alexidine hydrochloride and tricyclazole combinations. CI <1, synergy; ci=1, additive effect; CI >1, antagonism.
FIG. 4 shows the inhibition of rice blast and scab in alexidine dihydrochloride fields;
wherein: a, inhibiting rice blast occurrence in alexidine hydrochloride fields; b, investigation of the disease index of the rice blast disease; c, inhibiting wheat scab occurrence in alexidine hydrochloride fields; d, investigation of 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 conventional in the art, and the reagents or materials are commercially available, unless otherwise specified. The following list is merely a few specific embodiments of the present invention. Obviously, the invention is not limited to the following embodiments, but can be varied in many ways. Modifications and improvements will readily occur to those skilled in the art based upon this disclosure, and are therefore intended to be within the scope of the invention as claimed.
The alexidine dihydrochloride has a relative molecular mass of 581.7, a molecular formula of C 26H58Cl2N10 and a structural formula of:
Example 1:
use of alexidine dihydrochloride in the preparation of a plant pathogenic bacteria fungicide:
Treatment of Pyricularia oryzae (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):e1002869.)、 Fusarium graminearum (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).)、 Rhizoctonia cerealis (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).)、 corn small spot pathogen (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 parasitica (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).)、 Botrytis cinerea (Leptosphaeria biglobosa)(Deng,Y.et al.Viral cross-class transmission results in disease of a phytopathogenic fungus.The ISME journal,1-12,(2022).)、 tomato gray mold (Botrytis cinerea)(Liu,S.et al.Resistance to boscalid in Botrytis cinerea from greenhouse-grown tomato.Plant Dis 105,628-635,(2021).)、 pear tree rot pathogen (Valsa pyri)(Yuan,H.et al.Biocontrol activity and action mechanism of Paenibacillus polymyxa strain Nl4 against pear Valsa canker caused by Valsa pyri.Front.Microbiol.13,950742,(2022).)、 apple tree rot pathogen (Valsa mali)(Zhang,M.et al.Hce2 domain-containing effectors contribute to the full virulence of Valsa mali in a redundant manner.Mol Plant Pathol 20,843-856,(2019).)、 peach brown rot pathogen with alexidine dihydrochloride respectively (Monilinia fructicola)(Chen,S.,Yuan,N.,Schnabel,G.&Luo,C.Function of the genetic element'Mona'associated with fungicide resistance in Monilinia fructicola.Mol Plant Pathol 18,90-97,(2017).).
Rice blast bacteria: the rice blast bacteria were inoculated on Complete Medium (CM) plates at 28℃and the concentration of alexidine dihydrochloride added was 6. Mu.M, cultured for 5 days, and the colony size was measured to calculate the strain growth rate.
Fusarium graminearum: fusarium graminearum 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 3 days, the colony size is measured, and the growth speed of the strain is calculated.
Wheat root rot fungi: wheat root rot is inoculated on a PDA culture medium plate at 28 ℃, alexidine dihydrochloride is added at the concentration of 6 mu M, the culture is carried out for 5 days, the colony size is measured, and the growth speed of the strain is calculated.
Leptosphaeria zea: the corn small spot bacteria are 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 6 days, the colony size is measured, and the growth speed of the strain is calculated.
Phytophthora sojae: the phytophthora sojae 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 colony size is measured, and the strain growth speed is calculated.
Black shank germ of rape: the black shank bacteria of rape are 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 colony size is measured, and the growth speed of the strain is calculated.
Botrytis cinerea: the tomato gray mold 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 colony size is measured, and the growth speed of the strain is calculated.
Pear rot pathogen: the pear rot germ is inoculated on a PDA culture medium plate at 28 ℃, the concentration of the added alexidine dihydrochloride is 6 mu M, the pear rot germ is cultured for 3 days, the colony size is measured, and the growth speed of the strain is calculated.
Apple tree rot pathogen: apple tree rot germs are inoculated on a PDA culture medium plate at 28 ℃, the concentration of the added alexidine dihydrochloride is 6 mu M, the apple tree rot germs are cultured for 2 days, the colony size is measured, and the growth speed of the strain is calculated.
Brown rot of peach: the brown rot of peach 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 8 days, the colony size is measured, and the growth speed of the strain is calculated.
The above treated control group is pathogenic bacteria normally cultured on a plate. Each strain was biologically replicated 5 times, each experiment was repeated three times, colony growth and inhibition rate were as shown in fig. 1 and table one, and inhibition rate calculation formula was: the results of inhibition ratio= (colony diameter of control group-colony diameter of treatment group)/colony diameter of control group show that alexidine dihydrochloride has obvious inhibition effect on the plant pathogenic bacteria, and the alexidine dihydrochloride can be used as broad-spectrum bactericide for inhibiting the growth of the plant pathogenic bacteria.
Table one: alexidine hydrochloride inhibits the growth inhibition rate of different pathogenic bacteria.
Example 2:
alexidine dihydrochloride inhibition rice blast infection assay:
Fresh conidia (5X 10 4/ml) of Pyricularia oryzae P131 are collected from a CM culture medium, sprayed and inoculated to LTH rice seedlings growing for about 1 month, alexidine dihydrochloride (1 mu M,2 mu M,4 mu M) with different final concentrations is added into a spore suspension, tween water with 0.025% concentration is used as a control group, 10ml of spore suspension is sprayed on each treatment, the rice disease condition is observed after illumination culture for 7 days after dark moisturizing culture for 24 hours, and the number of lesions on leaves is counted.
As a result, as shown in FIG. 2, the effect of inhibiting the occurrence of rice blast became more remarkable as the alexidine dihydrochloride concentration was increased, and at 4. Mu.M, rice blast bacteria had almost lost the infecting ability, indicating that alexidine dihydrochloride can inhibit rice blast bacteria infection.
Example 3:
Alexidine dihydrochloride acts synergistically with tricyclazole as an adjuvant:
fresh conidia of Pyricularia oryzae P131 (5X 10 4/mL) were collected from CM medium, and then alexidine dihydrochloride (3. Mu.M), alexidine dihydrochloride (5. Mu.M), tricyclazole (10 mg/L), tricyclazole (15 mg/L), tricyclazole (20 mg/L), alexidine dihydrochloride+tricyclazole (3. Mu.M+10 mg/L), alexidine dihydrochloride+tricyclazole (3. Mu.M+15 mg/L), alexidine dihydrochloride+tricyclazole (3. Mu.M+20 mg/L), alexidine dihydrochloride+tricyclazole (5. Mu.M+10 mg/L), alexidine dihydrochloride+tricyclazole (5. Mu.M+20 mg/L), and 0.1% DMSO control group (CK) were set. The above groups were repeated 3 times.
10. Mu.L of spore suspension was dropped on a hydrophobic slide, and after 24 hours, the formation rate of rice blast fungus attachment cells was counted, and the results are shown in FIG. 3:
As can be seen from fig. 3, the inhibition rate of the attachment cells of the alexidine dihydrochloride and tricyclazole combination is significantly higher than that of alexidine dihydrochloride and tricyclazole alone, and the effect of the alexidine dihydrochloride and tricyclazole combination is better than the sum of the effects of the agents alone, as the concentration of the alexidine dihydrochloride and tricyclazole combination increases, the inhibition of the formation of the attachment cells of bacteria becomes more and more obvious, and the results indicate that the alexidine dihydrochloride can cooperate with the tricyclazole to improve the antibacterial effect of the alexidine dihydrochloride and the tricyclazole. Meanwhile, according to CompuSyn software, the alexidine dihydrochloride and tricyclazole drug combination has a synergistic effect, in the synergistic effect in fig. 4 and table two, CI <1 represents that the drug combination has a synergistic effect, ci=1 represents that the drug combination does not have a synergistic effect, and CI >1 represents that the drug combination has an antagonistic effect.
And (II) table: inhibition rate of adhesion cell formation after compounding alexidine hydrochloride and tricyclazole and CI value
Example 4:
Alexidine dihydrochloride can be directly used as a bactericide for directly preventing and controlling rice blast and wheat scab:
The rice blast field test was performed in the Yangjiang city of Guangdong in a rice blast disease nursery with 10M 2 each, 0.1% DMSO as a test control group, 6. Mu.M alexidine dihydrochloride as a treatment group, and three biological replicates were set respectively. The first 2022, 4 and 29 days of rice blast disease is sprayed, and the second spraying date is 5, 3 days. The disease index is investigated on day 5 and 19, 3 points are sampled on the diagonal line of each test cell, 1 is counted from each point, and the disease index of the statistical rice blast is referred to the international rice resistance evaluation grading standard:
Level 0: no disease; stage 1: such as a needle point size brown point; 2 stages: a large brown spot; 3 stages: small and round to slightly longer brown necrotic gray spots, 1-2mm in diameter; 4 stages: typical rice blast spots or ellipsoids with the diameter of 1-2cm and the disease area of the spots is less than 2% of the leaf area; 5 stages: typical rice blast spots have a disease area of less than 10%; stage 6: typical rice blast spots with the incidence area of 10-25%;7 stages: typical rice blast spots with an incidence area of 26-50%;8 stages: typical rice blast spots with an onset area of 51-75%; stage 9: all the leaves die.
Through field experiments, the statistical morbidity index found that 6 μm alexidine dihydrochloride could significantly inhibit the occurrence of rice blast in the field (fig. 4a, b and table three).
And thirdly, inhibiting the occurrence of field test morbidity of rice blast by alexidine dihydrochloride.
In addition, wheat scab antibacterial test was performed on a test base of northwest agriculture and forestry science and technology university of Shaanxi province, 6 mu M alexidine dihydrochloride was added to fresh fusarium graminearum PH-1 spore suspension with a concentration of 10 5 pieces/mL in proportion, the mixture was inoculated, the inoculation period was a wheat flowering period (2022, 5 months, 5 days), 10 mu L of spore suspension was inoculated to wheat ears, and the mixture was subjected to bagging and moisture-retaining culture for 7 days after water spraying. 10 ears of wheat were inoculated each time, 3 biological replicates were set, and the onset was counted after 14 days. The scab disease index statistics were calculated starting at the inoculation point, with a pair of side-by-side kernel onset scored as 1 and a kernel onset statistics of 0.5.
Through field experiments, the statistical morbidity index shows that 6 mu M alexidine dihydrochloride can significantly inhibit wheat scab (c, d in figure 4). By combining the field test results, the alexidine dihydrochloride can well inhibit the occurrence of field plant diseases
Table four: alexidine dihydrochloride inhibits wheat scab from developing in field test disease conditions.
Claims (2)
1. The application of the composition in inhibiting the rice blast fungus Magnaporthe oryzae, the effective components of the composition are alexidine dihydrochloride and tricyclazole, and the ratio of the alexidine dihydrochloride to the tricyclazole is as follows: 1 mu M2-6.67 mg/L.
2. The application of the composition in preparing the Pyricularia oryzae Magnaporthe oryzae inhibitor comprises the following effective components of alexidine dihydrochloride and tricyclazole: 1 mu M2-6.67 mg/L.
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