CN114258915A - Application of 9-phenanthrol in rice blast prevention and control - Google Patents
Application of 9-phenanthrol in rice blast prevention and control Download PDFInfo
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- CN114258915A CN114258915A CN202210140663.8A CN202210140663A CN114258915A CN 114258915 A CN114258915 A CN 114258915A CN 202210140663 A CN202210140663 A CN 202210140663A CN 114258915 A CN114258915 A CN 114258915A
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Abstract
The invention discloses application of 9-phenanthrol in rice blast prevention and control. Experiments show that: the compound can effectively inhibit hypha growth and spore germination of rice blast fungi, can interfere formation of attachment cells by conidia, has an obvious inhibition effect on rice blast, and inhibits pathogenicity of the rice blast fungi, so the compound has good application value in the aspects of rice blast prevention and control and drug development.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of 9-phenanthrol in rice blast prevention and control.
Background
Rice (Oryza sativa L.) is one of important food crops, occupies a very important position in food production, nearly 50% of the global population takes the rice as main food, and the safe production of the rice is very important for guaranteeing social stability and economic development. Like other crops, the rice is inevitably invaded by a series of pathogenic bacteria during the planting growth and development period, the yield of the rice and the quality of the rice are seriously influenced, wherein the rice blast caused by Magnaporthe oryzae (Magnaporthe oryzae) is one of the most serious diseases of the rice, which is a major problem in the world rice production, the yield of the rice can be reduced by 10-30% every year, the disease can be generated in all rice planting areas and different growth periods of the rice, and the rice has the characteristics of wide distribution and great harm, so that the yield of the rice is seriously reduced, and the food production safety of China and even the whole world is threatened. Therefore, the method has important economic value and social benefit for the research on the control of the rice blast germs.
The rice blast fungus infection process on rice mainly comprises the following steps: (1) conidium spreads with wind and rain and adheres to the surface of rice leaf; (2) germinating conidia to form a germ tube; (3) the germ tube is differentiated to form an attachment cell; (4) the attached cells are differentiated to form infection nails; (5) the infective spikes penetrate the host cell and form infective hyphae within the host cell, which expand from cell to cell. The rice blast fungus mainly infects the overground part tissue of rice by conidium, when the conidium contacts the host epidermis, the conidium germinates to form a germination pipe, the germination pipe is also specifically differentiated to generate an attachment cell, and the attachment cell forms an infection nail which penetrates through the host epidermis and then continuously enters the host cell, so that the host plant has symptoms in about 5-7 days. The key process of successful rice infection by rice blast fungi is to form a highly specialized infection structure, namely an attachment cell. Turgor pressure generated after the attached cells are mature can enable the infection nails to penetrate through the rice cuticle, so that rice cells are successfully infected. When the rice blast fungus can not form complete attachment cells, the pathogenicity of the rice blast fungus is obviously weakened.
At present, the main methods for preventing and treating rice blast are breeding resistant varieties, medicament control and field management, wherein most of the disease-resistant varieties are vertical resistant varieties based on the theory of 'gene to gene', and the varieties easily lose disease resistance. The chemical control has the advantages of economy, high efficiency, convenience, rapidness and the like, and is an indispensable part in a comprehensive rice blast treatment system. The prevention and control of rice blast is a hot problem of global research. The chemical control plays an important role in the comprehensive control of plant diseases and insect pests, and has the characteristics of quick effect, simple and convenient method, strong emergency property and no limitation by regionality and seasonality.
9-Phenanthrol (CAS number 484-17-3) is a fused ring aromatic hydrocarbon compound, a red solid, commonly used as Ca in clinic2+Activated non-selective cation channel (TRPM 4) inhibitor, and can be used for treating myocardial ischemia. At present, the application of 9-phenanthrol in agricultural production is not reported.
Disclosure of Invention
Aiming at the technical problems, the invention provides application of 9-phenanthrol in rice blast prevention and control.
The invention provides application of 9-phenanthrol in preparation of a drug for preventing and controlling rice blast germs.
Further, the 9-phenanthrol is used for preparing medicines for inhibiting hypha growth and spore germination of rice blast fungi.
Further, the application of the 9-phenanthrol in preparing a medicine for interfering the formation of blast fungus attachment cells.
Preferably, the concentration of the 9-phenanthrol is 5ug/m to 30 ug/m.
The invention also provides a method for preventing and controlling rice blast by using 9-phenanthrol, which comprises the following steps: dissolving 9-phenanthrol in dimethyl sulfoxide to prepare a mother solution of 5000ug/ml, sucking the 9-phenanthrol mother solution by a pipette, adding the mother solution into a PDA culture medium cooled to 55 ℃, and adjusting the concentration of the 9-phenanthrol to be 5ug/ml-30 ug/ml; then uniformly spraying the spore suspension on rice leaves to make the rice leaves be full of liquid drops.
Compared with the prior art, the invention has the following beneficial effects: the research of the invention finds that 9-phenanthrol has an inhibiting effect on the growth of rice blast germs, can interfere conidiospores to form attachment cells, has a remarkable delaying effect on the formation of the attachment cells of the spores, and has an obvious inhibiting effect on rice blast, so that the 9-phenanthrol has a good application value in the aspects of rice blast prevention and control and medicament development.
Drawings
FIG. 1 shows the effect of different concentrations of 9-phenanthrol on the growth of the hypha of Pyricularia oryzae;
FIG. 2 is a graph showing the inhibition rate of 9-phenanthrol at different concentrations against different strains of Pyricularia oryzae;
FIG. 3 is a graph showing the effect of different concentrations of 9-phenanthrol on the germination of conidia of Pyricularia oryzae;
FIG. 4 is a graph showing the effect of different concentrations of 9-phenanthrol on the formation of blast fungus appressorium;
FIG. 5 is a graph showing the effect of different concentrations of 9-phenanthrol on the virulence of Pyricularia oryzae;
FIG. 6 shows the effect of different concentrations of 9-phenanthrol on the biomass of Pyricularia grisea.
Detailed Description
Example 19 Effect of Phenanthrol on the growth of Magnaporthe grisea hyphae
A method for inhibiting the growth of rice blast fungus hypha by using 9-phenanthrol comprises the following steps:
s1: dissolving 9-phenanthrol in dimethyl sulfoxide to prepare mother liquor (5000ug/ml), adding the mother liquor into a PDA culture medium to ensure that the final concentration of 9-phenanthrol is 1.25 mu g/ml, 2.5 mu g/ml, 5 mu g/ml, 10 mu g/ml and 15 mu g/ml respectively, and controlling the temperature of the PDA culture medium to be 55 ℃; meanwhile, a PDA culture medium without 9-phenanthrol is set as a blank control group.
The preparation method of the PDA culture medium comprises the following steps:
(1) weighing 10g of sucrose and 15g of agar powder in a balance by using weighing paper;
(2) peeling and cleaning potatoes, weighing 200g of peeled potatoes, and cutting the peeled potatoes into cubes of about 1cm on clean gauze by using a small knife;
(3) weighing 1000mL of distilled water, adding the distilled water and 200g of peeled potato pieces into a pot, boiling the water for 20-30min, and filtering potato residues in a 1L beaker by using 4 layers of clean gauze;
(4) adding 10g of sucrose into the filtered liquid, uniformly stirring the mixture by using a glass rod, pouring the filtrate into a pot, adding 15g of agar powder while stirring, and quickly stirring to prevent agar from caking and sticking to the bottom of the pot;
(5) stopping heating when the liquid is transparent, pouring into a 1L beaker, adding distilled water to a constant volume of 1000mL and natural pH, subpackaging in a conical flask which cannot exceed one third of the conical flask volume, and sterilizing at 121 deg.C for 20min in a high-pressure steam sterilizing pot to obtain PDA culture medium.
The specific method for adding 9-phenanthrol into the PDA culture medium comprises the following steps: and (3) sucking 9-phenanthrol by using a pipette, adding the sucked 9-phenanthrol into the PDA culture medium cooled to 55 ℃ to ensure that the concentrations of the 9-phenanthrol in the final culture medium are respectively 1.25 mu g/ml, 2.5 mu g/ml, 5 mu g/ml, 10 mu g/ml and 15 mu g/ml, uniformly mixing the culture medium, pouring the uniformly mixed culture medium into a sterile culture dish, and avoiding the influence of bubbles on the experimental result in the plate-pouring process as much as possible to obtain the PDA culture medium with different concentrations of the 9-phenanthrol.
S2: culturing test bacteria rice blast germs;
the specific method for culturing test fungus Magnaporthe grisea is as follows
2.1 in a sterilized clean bench, igniting an alcohol lamp, and cooling the PDA culture medium sterilized by high-pressure steam to about 55 ℃;
2.2 pouring about 22mL of culture medium in each culture dish with the diameter of 90mm to ensure that no air bubbles exist in the pouring process as much as possible;
2.3 after the culture medium is completely solidified, taking out the filter paper sheet for testing rice blast germs from the refrigerator;
2.4 putting the tweezers outside the alcohol burner, after the tweezers are cooled, clamping the filter paper sheets with the rice blast germs preserved by the tweezers and putting the filter paper sheets on a PDA culture medium;
2.5 finally, the petri dish is sealed with a sealing film and placed upside down in a constant temperature incubator at 28 ℃ for culturing the test strain.
S3: cutting a bacterial block on the test bacterium magnaporthe grisea by using a sterile puncher after the test bacterium magnaporthe grisea is cultured for 7 days, wherein the diameter of the bacterial block is 6mm, and placing the bacterial block into a PDA culture medium containing 9-phenanthrol and a blank control PDA culture medium;
s4: placing the PDA culture medium containing 9-phenanthrol and blank control PDA culture medium into a constant temperature incubator, and culturing at 28 deg.C for 7 days. The diameters of the colonies were measured with a vernier caliper, and the growth rate and inhibition rate of the hyphae of the experimental group and the blank control group were calculated.
The calculation formula is as follows
Inhibition rate ═ 0.7 for control strain growth diameter (0.7 for treated strain growth diameter-0.7))/(0.7 for treated strain growth diameter) (note: 0.7 diameter of inoculated block)
Experimental groups prepared 9-phenanthrol PDA culture media with different concentrations of 1.25ug/ml, 2.5ug/ml, 5ug/ml, 10ug/ml and 15ug/ml respectively, inoculated with rice blast fungus blocks respectively, and counted the colony diameter after 7 days to calculate the inhibition rate of 9-phenanthrol on rice blast fungus, and the results are shown in figure 1:
as a result: the results of inoculating 7 different rice blast fungi in a culture medium containing 9-phenanthrol show that different concentrations of 9-phenanthrol have influence on the growth of rice blast fungi hyphae, and the inhibition rate is remarkably increased with the increase of the concentration (fig. 1 and 2).
Example 29 Effect of Phenantrol on spore formation and appressorium formation of Pyricularia oryzae
The implementation steps are as follows:
s1 placing the PDA culture medium containing 9-phenanthrol and blank control PDA culture medium in the fungus block into a constant temperature incubator for 7 days, cutting fungus block on Magnaporthe grisea by using a sterile puncher, wherein the diameter of the fungus block is 6mm, selecting three fungus blocks, placing the three fungus blocks into a PDB culture medium,
the preparation method of the PDB culture medium comprises the following steps:
(1) weighing 10g of sucrose in a balance by using weighing paper;
(2) peeling and cleaning potatoes, weighing 200g of peeled potatoes, and cutting the peeled potatoes into cubes of about 1cm on clean gauze by using a small knife;
(3) weighing 1000mL of distilled water, adding the distilled water and 200g of peeled potato pieces into a pot, boiling the water for 20-30min, and filtering potato residues in a 1L beaker by using 4 layers of clean gauze;
(4) adding 10g of sucrose into the filtered liquid, uniformly stirring the mixture by using a glass rod, pouring the mixture into a 1L beaker, adding distilled water to a constant volume of 1000mL, naturally keeping the pH value, subpackaging the mixture in a conical flask which cannot exceed one third of the volume of the conical flask, and then sterilizing the mixture in an autoclave at 121 ℃ for 20min to obtain the PDB culture medium.
S2: placing the fungus blocks in PDB culture medium, placing in 120rpm, shaking and culturing at 28 deg.C for 4-5 days, respectively sucking 400 μ L mycelium suspension into oat culture medium, and uniformly distributing the bacterial liquid. Culturing in 28 deg.C incubator for about 6 days, scraping mycelia with sterile cotton swab, culturing in light-illumination incubator with light and shade alternation (12h light and 12h dark) for about 5 days, cleaning spores with 0.1% Tween water, making into conidium suspension, and adjusting spore concentration to 105Per mL; respectively adding 9-phenanthrol mother liquor into conidium suspension liquid to the final concentration of 1 mug/ml, 10 mug/ml and 20 mug/ml, dripping the conidium suspension liquid mixed with 9-phenanthrol on a hydrophobic dial plate, keeping moisture in the dark at 28 ℃ for 12 hours, and observing the formation condition of adherent cells by a microscope;
s3: preparing a water agar culture medium, and putting cellophane on the water agar; and (3) cleaning the spores cultured by the oat culture medium with sterile water, filtering, uniformly coating on glass paper, marking, and observing the spore germination and appressorium formation conditions in 2h, 4h, 6h and 8h respectively. 50 spores were observed each time, and 3 times were observed in total.
The preparation method of the oat culture medium comprises the following steps:
(1) weighing 40g of oat and 15g of agar powder in a balance by using weighing paper;
(2) adding a proper amount of water into the weighed oat, soaking for about 10 minutes, and uniformly crushing by using a wall breaking machine;
(3) pouring the uniformly crushed oat into a 1L beaker;
(4) adding 15g of agar powder into a 1L beaker filled with oat, adding distilled water to a constant volume of 1000mL, naturally adjusting pH, subpackaging in a blue-cover bottle until the volume is not more than one third of the volume of the blue-cover bottle, and then sterilizing in an autoclave at 121 ℃ for 30min to obtain the oat culture medium.
As a result: the results of spore germination and appressorium formation are shown in FIGS. 3 and 4: after adding different concentrations of 9-phenanthrol to the conidial suspension, the results show that: the 9-phenanthrol with the concentration of 10ug/ml has an inhibiting effect on the germination and the formation of anchorage cells of the blast fungus conidium, and the germination and the formation of the anchorage cells of the blast fungus conidium are obviously inhibited at the concentration of 20ug/ml (as shown in figures 3 and 4).
Example 39 Effect of phenanthrol on the pathogenicity of Pyricularia oryzae
The experimental procedure was as follows:
adding 9-phenanthrol mother liquor into the conidium suspension of the rice blast fungi to a final concentration of 10ug/ml and 30ug/ml, uniformly spraying the conidium suspension on rice leaves to enable the leaves to be full of liquid drops, carrying out dark moisturizing culture at 28 ℃ for 24 hours, transferring to a greenhouse, carrying out rice blast fungi disease investigation 7 days after inoculation and counting hypha biomass by a fluorescence quantitative method, wherein the specific formula 2 isCT(Os-UBQ)-C T (Mo-Pot2)。
As a result: the rice was inoculated with the conidium suspension containing 9-phenanthrol, and the disease condition of the rice was observed 7 days after inoculation, and the results showed that the disease spots on the rice leaves were significantly reduced after the rice was inoculated with the 9-phenanthrol-treated conidium suspension containing 10ug/ml, and that only sporadic small disease spots were observed on the rice leaves after inoculation with the 9-phenanthrol-treated conidium suspension containing 30ug/ml (fig. 5). The hyphal biomass of the lesions formed by the spore suspensions treated with different concentrations of 9-phenanthrol was found to be significantly reduced by counting the hyphal biomass at the lesions (FIG. 6). It was demonstrated that 9-phenanthrol reduced the occurrence of rice blast by inhibiting conidium germination and appressorium formation.
Claims (5)
1.9 application of phenanthrol in preparing medicines for preventing and controlling rice blast germs.
2. Use according to claim 1, characterized in that: the 9-phenanthrol is used for preparing medicines for inhibiting hypha growth and spore germination of rice blast germs.
3. Use according to claim 1, characterized in that: the application of the 9-phenanthrol in preparing a medicine for interfering the formation of blast fungus appressorium.
4. Use according to any one of claims 1 to 3, wherein: the concentration of the 9-phenanthrol is 5ug/m-30 ug/m.
5. A method for preventing and controlling rice blast by using 9-phenanthrol comprises the following steps:
dissolving 9-phenanthrol in dimethyl sulfoxide to prepare a mother solution of 5000ug/ml, sucking the 9-phenanthrol mother solution by a pipette, adding the mother solution into a PDA culture medium cooled to 55 ℃, and adjusting the concentration of the 9-phenanthrol to be 5ug/ml-30 ug/ml; then uniformly spraying the spore suspension on rice leaves to make the rice leaves be full of liquid drops.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63190804A (en) * | 1987-02-03 | 1988-08-08 | Sumitomo Chem Co Ltd | Agricultural and horticultural germicide |
| EP0282893A2 (en) * | 1987-03-20 | 1988-09-21 | Bayer Ag | Pesticides based on 1,10-phenanthrolines |
| CN1074443A (en) * | 1992-01-15 | 1993-07-21 | 纳幕尔杜邦公司 | The heterocyclic fungicides of bridging |
| JP2001253804A (en) * | 2000-03-10 | 2001-09-18 | Ibaraki Prefecture | Biological control for plant disease damage with fungi on rice plant leave |
| CN112602717A (en) * | 2020-12-15 | 2021-04-06 | 华南农业大学 | Medicine for preventing and controlling rice blast germs |
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- 2022-02-16 CN CN202210140663.8A patent/CN114258915B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63190804A (en) * | 1987-02-03 | 1988-08-08 | Sumitomo Chem Co Ltd | Agricultural and horticultural germicide |
| EP0282893A2 (en) * | 1987-03-20 | 1988-09-21 | Bayer Ag | Pesticides based on 1,10-phenanthrolines |
| CN1074443A (en) * | 1992-01-15 | 1993-07-21 | 纳幕尔杜邦公司 | The heterocyclic fungicides of bridging |
| JP2001253804A (en) * | 2000-03-10 | 2001-09-18 | Ibaraki Prefecture | Biological control for plant disease damage with fungi on rice plant leave |
| CN112602717A (en) * | 2020-12-15 | 2021-04-06 | 华南农业大学 | Medicine for preventing and controlling rice blast germs |
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